BioMed Research International: Biotechnology The latest articles from Hindawi Publishing Corporation © 2016 , Hindawi Publishing Corporation . All rights reserved. Molecular Identification of Sex in Phoenix dactylifera Using Inter Simple Sequence Repeat Markers Thu, 26 May 2016 09:31:45 +0000 Early sex identification of Date Palm (Phoenix dactylifera L.) at seedling stage is an economically desirable objective, which will significantly increase the profits of seed based cultivation. The utilization of molecular markers at this stage for early and rapid identification of sex is important due to the lack of morphological markers. In this study, a total of two hundred Inter Simple Sequence Repeat (ISSR) primers were screened among male and female Date palm plants to identify putative sex-specific marker, out of which only two primers (IS_A02 and IS_A71) were found to be associated with sex. The primer IS_A02 produced a unique band of size 390 bp and was found clearly in all female plants, while it was absent in all male plants. Contrary to this, the primer IS_A71 produced a unique band of size 380 bp and was clearly found in all male plants, whereas it was absent in all the female plants. Subsequently, these specific fragments were excised, purified, and sequenced for the development of sequence specific markers further in future for the implementation on dioecious Date Palm for sex determination. These markers are efficient, highly reliable, and reproducible for sex identification at the early stage of seedling. Abdulhafed A. Al-Ameri, Fahad Al-Qurainy, Abdel-Rhman Z. Gaafar, Salim Khan, and M. Nadeem Copyright © 2016 Abdulhafed A. Al-Ameri et al. All rights reserved. A Drosophila Model for Screening Antiobesity Agents Mon, 09 May 2016 14:24:29 +0000 Although triacylglycerol, the major component for lipid storage, is essential for normal physiology, its excessive accumulation causes obesity in adipose tissue and is associated with organ dysfunction in nonadipose tissue. Here, we focused on the Drosophila model to develop therapeutics for preventing obesity. The brummer (bmm) gene in Drosophila melanogaster is known to be homologous with human adipocyte triglyceride lipase, which is related to the regulation of lipid storage. We established a Drosophila model for monitoring bmm expression by introducing the green fluorescent protein (GFP) gene as a downstream reporter of the bmm promoter. The third-instar larvae of Drosophila showed the GFP signal in all tissues observed and specifically in the salivary gland nucleus. To confirm the relationship between bmm expression and obesity, the effect of oral administration of glucose diets on bmm promoter activity was analyzed. The Drosophila flies given high-glucose diets showed higher lipid contents, indicating the obesity phenotype; this was suggested by a weaker intensity of the GFP signal as well as reduced bmm mRNA expression. These results demonstrated that the transgenic Drosophila model established in this study is useful for screening antiobesity agents. We also report the effects of oral administration of histone deacetylase inhibitors and some vegetables on the bmm promoter activity. Tran Thanh Men, Duong Ngoc Van Thanh, Masamitsu Yamaguchi, Takayoshi Suzuki, Gen Hattori, Masayuki Arii, Nguyen Tien Huy, and Kaeko Kamei Copyright © 2016 Tran Thanh Men et al. All rights reserved. Production and Partial Characterization of an Alkaline Xylanase from a Novel Fungus Cladosporium oxysporum Tue, 26 Apr 2016 13:53:08 +0000 A new fungus Cladosporium oxysporum GQ-3 producing extracellular xylanase was isolated from decaying agricultural waste and identified based on the morphology and comparison of internal transcribed spacer (ITS) rDNA gene sequence. C. oxysporum produced maximum xylanase activity of 55.92 U/mL with wheat bran as a substrate and NH4Cl as a nitrogen source. Mg2+ improved C. oxysporum xylanase production. Partially purified xylanase exhibited maximum activity at 50°C and pH 8.0, respectively, and showed the stable activity after 2-h treatment in pH 7.0–8.5 or below 55°C. Mg2+ enhanced the xylanase activity by 2% while Cu2+ had the highest inhibition ratio of 57.9%. Furthermore, C. oxysporum xylanase was resistant to most of tested neutral and alkaline proteases. Our findings indicated that Cladosporium oxysporum GQ-3 was a novel xylanase producer, which could be used in the textile processes or paper/feed industries. Guo-Qiang Guan, Peng-Xiang Zhao, Jin Zhao, Mei-Juan Wang, Shu-Hao Huo, Feng-Jie Cui, and Jian-Xin Jiang Copyright © 2016 Guo-Qiang Guan et al. All rights reserved. Biosimilars in Developed and Developing East and Southeast Asian Countries: Japan, South Korea, and Malaysia—Overview, Evolution, and Regulations Assessment Sun, 24 Apr 2016 09:19:04 +0000 The development of biological products has experienced continuous growth over the past three decades. The expiration of patent protection for many biological medicines has led to the development of biosimilars in many countries around the world. This paper reviews the literature on biosimilar drugs and covers their therapeutic status, clinical trials, approved biosimilars, and regulatory guidelines in Japan, South Korea, and Malaysia. The literature suggests that biosimilars are comparable but not identical to the reference product. They are not a generic version of an innovative product and do not ensure therapeutic equivalence. Biosimilars present more challenges than conventional generics and their marketing approval is also much more complicated. Guidelines for biosimilars were published in Japan in July 2009 by the Ministry of Health, Labour and Welfare (MHLW), in South Korea in March 2009 by the Ministry of Food and Drug Safety (MFDS), and in Malaysia in July 2008 by the National Pharmaceutical Control Bureau (NPCB). Tomas Gabriel Bas and Carolina Oliu Castillo Copyright © 2016 Tomas Gabriel Bas and Carolina Oliu Castillo. All rights reserved. Improve the Anaerobic Biodegradability by Copretreatment of Thermal Alkali and Steam Explosion of Lignocellulosic Waste Wed, 20 Apr 2016 13:57:31 +0000 Effective alteration of the recalcitrance properties like crystallization of cellulose, lignin shield, and interlinking of lignocellulosic biomass is an ideal way to utilize the full-scale potential for biofuel production. This study exhibited three different pretreatment effects to enhance the digestibility of corn stover (CS) for methane production. In this context, steam explosion (SE) and thermal potassium hydroxide (KOH-60°C) treated CS produced the maximal methane yield of 217.5 and 243.1 mL/, which were 40.0% and 56.4% more than untreated CS (155.4 mL/), respectively. Copretreatment of thermal potassium hydroxide and steam explosion (CPTPS) treated CS was highly significant among all treatments and improved 88.46% (292.9 mL/) methane yield compared with untreated CS. Besides, CPTPS also achieved the highest biodegradability up to 68.90%. Three kinetic models very well simulated dynamics of methane production yield. Moreover, scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and X-ray diffraction (XRD) analyses declared the most effective changes in physicochemical properties after CPTPS pretreatment. Thus, CPTPS might be a promising approach to deconstructing the recalcitrance of lignocellulosic structure to improve the biodegradability for AD. Muhammad Abdul Hanan Siddhu, Jianghao Li, Jiafu Zhang, Yan Huang, Wen Wang, Chang Chen, and Guangqing Liu Copyright © 2016 Muhammad Abdul Hanan Siddhu et al. All rights reserved. KvLEA, a New Isolated Late Embryogenesis Abundant Protein Gene from Kosteletzkya virginica Responding to Multiabiotic Stresses Wed, 30 Mar 2016 09:06:57 +0000 The LEA proteins are a kind of hydrophilic proteins, playing main functions in desiccation tolerance. However, their importance as a kind of stress proteins in abiotic stress is being clarified little by little. In this study we isolated, cloned, and identified the first KvLEA gene in Kosteletzkya virginica. Bioinformatic analysis showed that the protein encoded by this gene had common properties of LEA proteins and the multiple sequences alignment and phylogenetic analysis further showed that this protein had high homology with two Arabidopsis LEA proteins. Gene expression analysis revealed that this gene had a higher expression in root and it was induced obviously by salt stress. Moreover, the transcripts of KvLEA were also induced by other abiotic stresses including drought, high temperature, chilling, and ABA treatment. Among these abiotic stresses, ABA treatment brought about the biggest changes to this gene. Collectively, our research discovered a novel LEA gene and uncovered its involvement in multiabiotic stresses in K. virginica. This research not only enriched studies on LEA gene in plant but also would accelerate more studies on K. virginica in the future. Xiaoli Tang, Hongyan Wang, Liye Chu, and Hongbo Shao Copyright © 2016 Xiaoli Tang et al. All rights reserved. Next Generation Sequencing Identifies Five Major Classes of Potentially Therapeutic Enzymes Secreted by Lucilia sericata Medical Maggots Mon, 28 Mar 2016 06:33:48 +0000 Lucilia sericata larvae are used as an alternative treatment for recalcitrant and chronic wounds. Their excretions/secretions contain molecules that facilitate tissue debridement, disinfect, or accelerate wound healing and have therefore been recognized as a potential source of novel therapeutic compounds. Among the substances present in excretions/secretions various peptidase activities promoting the wound healing processes have been detected but the peptidases responsible for these activities remain mostly unidentified. To explore these enzymes we applied next generation sequencing to analyze the transcriptomes of different maggot tissues (salivary glands, gut, and crop) associated with the production of excretions/secretions and/or with digestion as well as the rest of the larval body. As a result we obtained more than 123.8 million paired-end reads, which were assembled de novo using Trinity and Oases assemblers, yielding 41,421 contigs with an N50 contig length of 2.22 kb and a total length of 67.79 Mb. BLASTp analysis against the MEROPS database identified 1729 contigs in 577 clusters encoding five peptidase classes (serine, cysteine, aspartic, threonine, and metallopeptidases), which were assigned to 26 clans, 48 families, and 185 peptidase species. The individual enzymes were differentially expressed among maggot tissues and included peptidase activities related to the therapeutic effects of maggot excretions/secretions. Zdeněk Franta, Heiko Vogel, Rüdiger Lehmann, Oliver Rupp, Alexander Goesmann, and Andreas Vilcinskas Copyright © 2016 Zdeněk Franta et al. All rights reserved. Advances in Biotechnology for Sustainable Development Sun, 27 Mar 2016 12:02:27 +0000 Weiqi Fu, Basel Khraiwesh, Hongbing Liu, and Lei Kai Copyright © 2016 Weiqi Fu et al. All rights reserved. Mutation Detection in an Antibody-Producing Chinese Hamster Ovary Cell Line by Targeted RNA Sequencing Sun, 20 Mar 2016 13:47:13 +0000 Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined. Siyan Zhang, Jason D. Hughes, Nicholas Murgolo, Diane Levitan, Janice Chen, Zhong Liu, and Shuangping Shi Copyright © 2016 Siyan Zhang et al. All rights reserved. Silicon Mitigates Salinity Stress by Regulating the Physiology, Antioxidant Enzyme Activities, and Protein Expression in Capsicum annuum ‘Bugwang’ Sun, 20 Mar 2016 10:15:59 +0000 Silicon- (Si-) induced salinity stress resistance was demonstrated at physiological and proteomic levels in Capsicum annuum for the first time. Seedlings of C. annuum were hydroponically treated with NaCl (50 mM) with or without Si (1.8 mM) for 15 days. The results illustrated that saline conditions significantly reduced plant growth and biomass and photosynthetic parameters and increased the electrolyte leakage potential, lipid peroxidation, and hydrogen peroxide level. However, supplementation of Si allowed the plants to recover from salinity stress by improving their physiology and photosynthesis. During salinity stress, Si prevented oxidative damage by increasing the activities of antioxidant enzymes. Furthermore, Si supplementation recovered the nutrient imbalance that had occurred during salinity stress. Additionally, proteomic analysis by two-dimensional gel electrophoresis (2DE) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed that Si treatment upregulated the accumulation of proteins involved in several metabolic processes, particularly those associated with nucleotide binding and transferase activity. Moreover, Si modulated the expression of vital proteins involved in ubiquitin-mediated nucleosome pathway and carbohydrate metabolism. Overall, the results illustrate that Si application induced resistance against salinity stress in C. annuum by regulating the physiology, antioxidant metabolism, and protein expression. Abinaya Manivannan, Prabhakaran Soundararajan, Sowbiya Muneer, Chung Ho Ko, and Byoung Ryong Jeong Copyright © 2016 Abinaya Manivannan et al. All rights reserved. Cloning and Expression of the γ-Polyglutamic Acid Synthetase Gene pgsBCA in Bacillus subtilis WB600 Thu, 17 Mar 2016 06:46:00 +0000 To clone and express the γ-polyglutamic acid (γ-PGA) synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications. Biaosheng Lin, Zhijuan Li, Huixia Zhang, Jiangwen Wu, and Maochun Luo Copyright © 2016 Biaosheng Lin et al. All rights reserved. Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection Wed, 16 Mar 2016 09:06:38 +0000 Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed in E. coli strain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection. Yi Ma, Chao Ni, Emmanuel E. Dzakah, Haiying Wang, Keren Kang, Shixing Tang, Jihua Wang, and Jufang Wang Copyright © 2016 Yi Ma et al. All rights reserved. Improved Production of Aspergillus usamii endo-β-1,4-Xylanase in Pichia pastoris via Combined Strategies Tue, 15 Mar 2016 13:36:23 +0000 A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase from Aspergillus usamii (A. usamii) in Pichia pastoris (P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene from A. usamii was optimized for P. pastoris and expressed in P. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression of xynB-opt, the Vitreoscilla hemoglobin (VHb) gene was transformed to the recombinant strain containing xynB-opt. The results showed that recombinant strain harboring the xynB-opt and VHb (named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHb in 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHb was optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHb reached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins in P. pastoris. Jianrong Wang, Yangyuan Li, and Danni Liu Copyright © 2016 Jianrong Wang et al. All rights reserved. Improved Stability of a Model IgG3 by DoE-Based Evaluation of Buffer Formulations Thu, 03 Mar 2016 12:55:18 +0000 Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potential storage buffer due to significant visible precipitate formation. An additional 24 full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Thus, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules. Brittany K. Chavez, Cyrus D. Agarabi, Erik K. Read, Michael T. Boyne II, Mansoor A. Khan, and Kurt A. Brorson Copyright © 2016 Brittany K. Chavez et al. All rights reserved. Biochemical and Metabolic Changes in Arsenic Contaminated Boehmeria nivea L. Sun, 28 Feb 2016 13:51:12 +0000 Arsenic (As) is identified by the EPA as the third highest toxic inorganic contaminant. Almost every 9th or 10th human in more than 70 countries including mainland China is affected by As. Arsenic along with other toxins not only affects human life but also creates alarming situations such as the deterioration of farm lands and desertion of industrial/mining lands. Researchers and administrators have agreed to opt for phytoremediation of As over costly cleanups. Boehmeria nivea L. can soak up various heavy metals, such as Sb, Cd, Pb, and Zn. But the effect of As pollution on the biology and metabolism of B. nivea has been somewhat overlooked. This study attempts to evaluate the extent of As resistance, chlorophyll content, and metabolic changes in As-polluted (5, 10, 15, and 20 mg L−1 As) B. nivea in hydroponics. Toxic effects of As in the form of inhibited growth were apparent at the highest level of added As. The significant changes in the chlorophyll, electrolyte leakage, and H2O2, significant increases in As in plant parts, catalase (CAT), and malondialdehyde (MDA), with applied As revealed the potential of B. nivea for As decontamination. By employing the metabolic machinery of B. nivea, As was sustainably removed from the contaminated areas. Hussani Mubarak, Nosheen Mirza, Li-Yuan Chai, Zhi-Hui Yang, Wang Yong, Chong-Jian Tang, Qaisar Mahmood, Arshid Pervez, Umar Farooq, Shah Fahad, Wajid Nasim, and Kadambot H. M. Siddique Copyright © 2016 Hussani Mubarak et al. All rights reserved. Overexpression of AaWRKY1 Leads to an Enhanced Content of Artemisinin in Artemisia annua Tue, 23 Feb 2016 09:46:59 +0000 Artemisinin is an effective component of drugs against malaria. The regulation of artemisinin biosynthesis is at the forefront of artemisinin research. Previous studies showed that AaWRKY1 can regulate the expression of ADS, which is the first key enzyme in artemisinin biosynthetic pathway. In this study, AaWRKY1 was cloned, and it activated ADSpro and CYPpro in tobacco using dual-LUC assay. To further study the function of AaWRKY1, pCAMBIA2300-AaWRKY1 construct under 35S promoter was generated. Transgenic plants containing AaWRKY1 were obtained, and four independent lines with high expression of AaWRKY1 were analyzed. The expression of ADS and CYP, the key enzymes in artemisinin biosynthetic pathway, was dramatically increased in AaWRKY1-overexpressing A. annua plants. Furthermore, the artemisinin yield increased significantly in AaWRKY1-overexpressing A. annua plants. These results showed that AaWRKY1 increased the content of artemisinin by regulating the expression of both ADS and CYP. It provides a new insight into the mechanism of regulation on artemisinin biosynthesis via transcription factors in the future. Weimin Jiang, Xueqing Fu, Qifang Pan, Yueli Tang, Qian Shen, Zongyou Lv, Tingxiang Yan, Pu Shi, Ling Li, Lida Zhang, Guofeng Wang, Xiaofen Sun, and Kexuan Tang Copyright © 2016 Weimin Jiang et al. All rights reserved. Optimization of Cultural Conditions for Antioxidant Exopolysaccharides from Xerocomus badius Grown in Shrimp Byproduct Mon, 22 Feb 2016 12:49:30 +0000 To optimize the production conditions for exopolysaccharides with higher antioxidant activities from Xerocomus badius cultured in shrimp byproduct medium, Plackett-Burman design, path of steepest ascent, and response surface methodology were explored. Based on the results of Plackett-Burman design and path of steepest ascent, a Box-Behnken design was applied to optimization and the regression models. The optimal cultural condition for high yield and antioxidant activity of the exopolysaccharides was determined to be 10.347% of solid-to-liquid ratio, a 4.322% content of bran powder, and a 1.217% concentration of glacial acetic acid. Culturing with the optimal cultural conditions resulted in an exopolysaccharides yield of  g/L and a total antioxidant activity of  U/mg. These values are consistent with the values predicted by the corresponding regression models (RSD < 5%). Xiujun Gao, Peisheng Yan, Xin Liu, Jianbing Wang, and Jiajia Yu Copyright © 2016 Xiujun Gao et al. All rights reserved. Optimized Biotransformation of Icariin into Icariside II by β-Glucosidase from Trichoderma viride Using Central Composite Design Method Sun, 14 Feb 2016 09:27:33 +0000 A crude -glucosidase has been produced from Trichoderma viride and used to explore a simple method to prepare icariside II from icariin. The crude enzyme has been studied by zymography method and used for hydrolysis of ICA. To achieve a high conversion rate of ICA, various factors have been studied including pH, reaction time, temperature, initial concentration of enzyme, and initial concentration of ICA through central composite design experiments. In the condition of the optimum hydrolysis parameters with pH 4.0, 41°C, 1.0 mg/mL ICA, and 9.8 U/mL crude -glucosidase, the conversion rate of ICA reached 95.03% at 1 h. Moreover, the cytotoxicity test showed that ICA II performed inhibition effects on proliferation of A549 cell, while ICA has no cytotoxicity. It indicated that the hydrolysis transformation study of ICA is valuable for exploration of active new drugs. Tao Cheng, Jun Yang, Tong Zhang, Yi-Shun Yang, and Yue Ding Copyright © 2016 Tao Cheng et al. All rights reserved. Upscaling Arbuscular Mycorrhizal Symbiosis and Related Agroecosystems Services in Smallholder Farming Systems Thu, 28 Jan 2016 12:57:55 +0000 Smallholder farming systems form unique ecosystems that can protect beneficial soil biota and form an important source of useful genetic resources. They are characterized by high level of agricultural diversity mainly focused on meeting farmers’ needs. Unfortunately, these systems often experience poor crop production mainly associated with poor planning and resource scarcity. Soil fertility is among the primary challenges faced by smallholder farmers, which necessitate the need to come up with affordable and innovative ways of replenishing soils. One such way is the use of microbial symbionts such as arbuscular mycorrhizal fungi (AMF), a beneficial group of soil microbiota that form symbiotic associations with majority of cultivated crops and play a vital role in biological soil fertility, plant nutrition, and protection. AMF can be incorporated in smallholder farming systems to help better exploit chemical fertilizers inputs which are often unaffordable to many smallholder farmers. The present review highlights smallholder farming practices that could be innovatively redesigned to increase AMF symbiosis and related agroecosystem services. Indeed, the future of global food security depends on the success of smallholder farming systems, whose crop productivity depends on the services provided by well-functioning ecosystems, including soil fertility. Marjorie Bonareri Oruru and Ezekiel Mugendi Njeru Copyright © 2016 Marjorie Bonareri Oruru and Ezekiel Mugendi Njeru. All rights reserved. Synergy between Rhizobium phaseoli and Acidithiobacillus ferrooxidans in the Bioleaching Process of Copper Thu, 28 Jan 2016 06:39:44 +0000 This study investigates the synergy of Rhizobium phaseoli and Acidithiobacillus ferrooxidans in the bioleaching process of copper. The results showed that additional R. phaseoli could increase leaching rate and cell number of A. ferrooxidans. When the initial cell number ratio between A. ferrooxidans and R. phaseoli was 2 : 1, A. ferrooxidans attained the highest final cell number of approximately 2 × 108 cells/mL and the highest copper leaching rate of 29%, which is 7% higher than that in the group with A. ferrooxidans only. R. phaseoli may use metabolized polysaccharides from A. ferrooxidans, and organic acids could chelate or precipitate harmful heavy metals to reduce their damage on A. ferrooxidans and promote its growth. Organic acids could also damage the mineral lattice to increase the leaching effect. Xuecheng Zheng and Dongwei Li Copyright © 2016 Xuecheng Zheng and Dongwei Li. All rights reserved. Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples Wed, 27 Jan 2016 10:11:16 +0000 Given the importance of protease’s worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL−1 of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise. Diego F. Coêlho, Thais Peron Saturnino, Fernanda Freitas Fernandes, Priscila Gava Mazzola, Edgar Silveira, and Elias Basile Tambourgi Copyright © 2016 Diego F. Coêlho et al. All rights reserved. Treatment of Copper Contaminated Municipal Wastewater by Using UASB Reactor and Sand-Chemically Carbonized Rubber Wood Sawdust Column Wed, 20 Jan 2016 12:13:58 +0000 The performance of a laboratory scale upflow anaerobic sludge blanket (UASB) reactor and its posttreatment unit of sand-chemically carbonized rubber wood sawdust (CCRWSD) column system for the treatment of a metal contaminated municipal wastewater was investigated. Copper ion contaminated municipal wastewater was introduced to a laboratory scale UASB reactor and the effluent from UASB reactor was then followed by treatment with sand-CCRWSD column system. The laboratory scale UASB reactor and column system were observed for a period of 121 days. After the posttreatment column the average removal of monitoring parameters such as copper ion concentration (91.37%), biochemical oxygen demand () (93.98%), chemical oxygen demand (COD) (95.59%), total suspended solid (TSS) (95.98%), ammonia (80.68%), nitrite (79.71%), nitrate (71.16%), phosphorous (44.77%), total coliform (TC) (99.9%), and fecal coliform (FC) (99.9%) was measured. The characterization of the chemically carbonized rubber wood sawdust was done by scanning electron microscope (SEM), X-ray fluorescence spectrum (XRF), and Fourier transforms infrared spectroscopy (FTIR). Overall the system was found to be an efficient and economical process for the treatment of copper contaminated municipal wastewater. Swarup Biswas and Umesh Mishra Copyright © 2016 Swarup Biswas and Umesh Mishra. All rights reserved. Multipurpose, Integrated 2nd Generation Biorefineries Tue, 19 Jan 2016 06:26:32 +0000 Alberto Scoma, Lorenzo Bertin, Maria A. M. Reis, Michael Kornaros, and Marta Coma Copyright © 2016 Alberto Scoma et al. All rights reserved. Organogenesis and Ultrastructural Features of In Vitro Grown Canna indica L. Mon, 18 Jan 2016 16:29:33 +0000 An efficient protocol for micropropagation of Canna indica L., an economically and pharmaceutically important plant, was standardized using rhizome explants, excised from two-month-old aseptic seedlings. Complete plant regeneration was induced on MS medium supplemented with 3.0 mg/L BAP plus 1.5 mg/L NAA, which produced the highest number of shoots (73.3 ± 0.5%) and roots (86.7 ± 0.4%) after 2 weeks. Furthermore, the optimum media for multiple shoots regeneration were recorded on MS enriched with 7.0 mg/L BAP (33.0 ± 0.5%). Plantlets obtained were transplanted to pots after two months and acclimatized in the greenhouse, with 75% survival. In addition, ultrastructural studies showed that rhizomes of in vitro grown specimens were underdeveloped compared to the in vivo specimens, possibly due to the presence of wide spaces. Meanwhile, the leaves of in vivo specimens had more open stomata compared to in vitro specimens, yet their paracytic stomata structures were similar. Hence, there were no abnormalities or major differences between in vitro regenerants and mother plants. Sharifah Nurashikin Wafa, Rosna Mat Taha, Sadegh Mohajer, Noraini Mahmad, and Bakrudeen Ali Ahmed Abdul Copyright © 2016 Sharifah Nurashikin Wafa et al. All rights reserved. Preparation of Microencapsulated Bacillus subtilis SL-13 Seed Coating Agents and Their Effects on the Growth of Cotton Seedlings Thu, 14 Jan 2016 15:56:31 +0000 Inoculation of the bacterial cells of microbial seed coating agents (SCAs) into the environment may result in limited survival and colonization. Therefore, the application efficacy of an encapsulated microbial seed coating agent (ESCA) was investigated on potted cotton plants; the agent was prepared with polyvinyl alcohol, sodium dodecyl sulfate, bentonite, and microencapsulated Bacillus subtilis SL-13. Scanning electron micrography revealed that the microcapsules were attached to ESCA membranes. The ESCA film was uniform, bubble-free, and easy to peel. The bacterial contents of seeds coated with each ESCA treatment reached 106 cfu/seed. Results indicated that the germination rate of cotton seeds treated with ESCA4 (1.0% (w/v) sodium alginate, 4.0% polyvinyl alcohol, 1.0% sodium dodecyl sulfate, 0.6% acacia, 0.5% bentonite, and 10% (v/v) microcapsules) increased by 28.74%. Other growth factors of the cotton seedlings, such as plant height, root length, whole plant fresh weight, and whole plant dry weight, increased by 52.70%, 25.13%, 46.47%, and 33.21%, respectively. Further analysis demonstrated that the peroxidase and superoxide dismutase activities of cotton seedlings improved, whereas their malondialdehyde contents decreased. Therefore, the ESCA can efficiently improve seed germination, root length, and growth. The proposed ESCA exhibits great potential as an alternative to traditional SCA in future agricultural applications. Liang Tu, Yanhui He, Chunhui Shan, and Zhansheng Wu Copyright © 2016 Liang Tu et al. All rights reserved. Extraction of Oleic Acid from Moroccan Olive Mill Wastewater Sun, 10 Jan 2016 07:47:55 +0000 The production of olive oil in Morocco has recently grown considerably for its economic and nutritional importance favored by the country’s climate. After the extraction of olive oil by pressing or centrifuging, the obtained liquid contains oil and vegetation water which is subsequently separated by decanting or centrifugation. Despite its treatment throughout the extraction process, this olive mill wastewater, OMW, still contains a very important oily residue, always regarded as a rejection. The separated oil from OMW can not be intended for food because of its high acidity of 3.397% which exceeds the international standard for human consumption defined by the standard of the Codex Alimentarius, proving its poor quality. This work gives value addition to what would normally be regarded as waste by the extraction of oleic acid as a high value product, using the technique of inclusion with urea for the elimination of saturated and unsaturated fatty acids through four successive crystallizations at 4°C and 20°C to have a final phase with oleic acid purity of 95.49%, as a biodegradable soap and a high quality glycerin will be produced by the reaction of saponification and transesterification. Reda Elkacmi, Noureddine Kamil, Mounir Bennajah, and Said Kitane Copyright © 2016 Reda Elkacmi et al. All rights reserved. Application of Innovative Technologies for Improved Food Quality and Safety Mon, 04 Jan 2016 12:58:15 +0000 Yiannis Kourkoutas, Nikos Chorianopoulos, Aspasia Nisiotou, Vasilis P. Valdramidis, and Kimon A. G. Karatzas Copyright © 2016 Yiannis Kourkoutas et al. All rights reserved. Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor by Bacillus subtilis SCK6 Thu, 31 Dec 2015 13:20:36 +0000 This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant of Bacillus subtilis SCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transform B. subtilis SCK6 supercompetent cells. Expression of GCSF was monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as revealed by the regeneration of neutrophils in mice treated with ifosfamine, was also similar to the commercial preparation of GCSF. This, to our knowledge, is the first study that reports secretory expression of human GCSF in B. subtilis SCK6 with final recovery of up to 96 mg/L of the culture supernatant, without involvement of any chemical inducer. Shaista Bashir, Saima Sadaf, Sajjad Ahmad, and Muhammad Waheed Akhtar Copyright © 2015 Shaista Bashir et al. All rights reserved. Impact of pH Management Interval on Biohydrogen Production from Organic Fraction of Municipal Solid Wastes by Mesophilic Thermophilic Anaerobic Codigestion Sun, 27 Dec 2015 13:42:19 +0000 The biohydrogen productions from the organic fraction of municipal solid wastes (OFMSW) were studied under pH management intervals of 12 h (PM12) and 24 h (PM24) for temperature of °C and °C. The OFMSW or food waste (FW) along with its two components, noodle waste (NW) and rice waste (RW), was codigested with sludge to estimate the potential of biohydrogen production. The biohydrogen production was higher in all reactors under PM12 as compared to PM24. The drop in pH from 7 to 5.3 was observed to be appropriate for biohydrogen production via mesophilic codigestion of noodle waste with the highest biohydrogen yield of 145.93 mL/g under PM12. When the temperature was increased from 37°C to 55°C and pH management interval was reduced from 24 h to 12 h, the biohydrogen yields were also changed from 39.21 mL/g to 89.67 mL/g , 91.77 mL/g to 145.93 mL/g , and 15.36 mL/g to 117.62 mL/g for FW, NW, and RW, respectively. The drop in pH and VFA production was better controlled under PM12 as compared to PM24. Overall, PM12 was found to be an effective mean for biohydrogen production through anaerobic digestion of food waste. Chaudhry Arslan, Asma Sattar, Ji Changying, Abdul Nasir, Irshad Ali Mari, and Muhammad Zia Bakht Copyright © 2015 Chaudhry Arslan et al. All rights reserved. Production of Monomeric Aromatic Compounds from Oil Palm Empty Fruit Bunch Fiber Lignin by Chemical and Enzymatic Methods Tue, 22 Dec 2015 06:20:05 +0000 In this study, oil palm empty fruit bunch (OPEFBF) was pretreated with alkali, and lignin was extracted for further degradation into lower molecular weight phenolic compounds using enzymes and chemical means. Efficiency of monomeric aromatic compounds production from OPEFBF lignin via chemical (nitrobenzene versus oxygen) and enzymatic [cutinase versus manganese peroxidase (MnP)] approaches was investigated. The effects of sodium hydroxide concentration (2, 5, and 10% wt.) and reaction time (30, 90, and 180 minutes) on the yield of aromatic compounds were studied. The results obtained indicated that nitrobenzene oxidation produced the highest yield ( ppm hydroxybenzoic acid,  ppm p-hydroxybenzaldehyde,  ppm vanillic acid,  ppm vanillin,  ppm syringic acid,  ppm syringaldehyde,  ppm p-coumaric acid, and  ppm ferulic acid), among the tested methods. High sodium hydroxide concentration (10% wt.) was needed to promote efficient nitrobenzene oxidation. However, less severe oxidation condition was preferred to preserve the hydroxycinnamic acids (p-coumaric acid and ferulic acid). Cutinase-catalyzed hydrolysis was found to be more efficient than MnP-catalyzed oxidation in the production of aromatic compounds. By hydrolyzed 8% wt. of lignin with 0.625 mL cutinase g−1 lignin at pH 8 and 55°C for 24 hours, about  ppm hydroxybenzoic acid,  ppm syringaldehyde,  ppm vanillin,  ppm p-coumaric acid, and  ppm ferulic acid were produced. Pei-Ling Tang, Osman Hassan, Mohamad Yusof Maskat, and Khairiah Badri Copyright © 2015 Pei-Ling Tang et al. All rights reserved. Calcium Mitigates Arsenic Toxicity in Rice Seedlings by Reducing Arsenic Uptake and Modulating the Antioxidant Defense and Glyoxalase Systems and Stress Markers Sun, 20 Dec 2015 06:33:38 +0000 The effect of exogenous calcium (Ca) on hydroponically grown rice seedlings was studied under arsenic (As) stress by investigating the antioxidant and glyoxalase systems. Fourteen-day-old rice (Oryza sativa L. cv. BRRI dhan29) seedlings were exposed to 0.5 and 1 mM Na2HAsO4 alone and in combination with 10 mM CaCl2 (Ca) for 5 days. Both levels of As caused growth inhibition, chlorosis, reduced leaf RWC, and increased As accumulation in the rice seedlings. Both doses of As in growth medium induced oxidative stress through overproduction of reactive oxygen species (ROS) by disrupting the antioxidant defense and glyoxalase systems. Exogenous application of Ca along with both levels of As significantly decreased As accumulation and restored plant growth and water loss. Calcium supplementation in the As-exposed rice seedlings reduced ROS production, increased ascorbate (AsA) content, and increased the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), and the glyoxalase I (Gly I) and glyoxalase II (Gly II) enzymes compared with seedlings exposed to As only. These results suggest that Ca supplementation improves rice seedlings tolerance to As-induced oxidative stress by reducing As uptake, enhancing their antioxidant defense and glyoxalase systems, and also improving growth and physiological condition. Anisur Rahman, Mohammad Golam Mostofa, Md. Mahabub Alam, Kamrun Nahar, Mirza Hasanuzzaman, and Masayuki Fujita Copyright © 2015 Anisur Rahman et al. All rights reserved. Environmental Biotechnology: Current Advances, New Knowledge Gaps, and Emerging Issues Mon, 14 Dec 2015 09:14:22 +0000 Abd El-Latif Hesham, T. Komang Ralebitso-Senior, Yu Zhang, and Qing X. Li Copyright © 2015 Abd El-Latif Hesham et al. All rights reserved. Towards a Universal Approach Based on Omics Technologies for the Quality Control of Food Sun, 13 Dec 2015 07:21:08 +0000 In the last decades, food science has greatly developed, turning from the consideration of food as mere source of energy to a growing awareness on its importance for health and particularly in reducing the risk of diseases. Such vision led to an increasing attention towards the origin and quality of raw materials as well as their derived food products. The continuous advance in molecular biology allowed setting up efficient and universal omics tools to unequivocally identify the origin of food items and their traceability. In this review, we considered the application of a genomics approach known as DNA barcoding in characterizing the composition of foodstuffs and its traceability along the food supply chain. Moreover, metabolomics analytical strategies based on Nuclear Magnetic Resonance (NMR) and Mass Spectroscopy (MS) were discussed as they also work well in evaluating food quality. The combination of both approaches allows us to define a sort of molecular labelling of food that is easily understandable by the operators involved in the food sector: producers, distributors, and consumers. Current technologies based on digital information systems such as web platforms and smartphone apps can facilitate the adoption of such molecular labelling. Emanuele Ferri, Andrea Galimberti, Maurizio Casiraghi, Cristina Airoldi, Carlotta Ciaramelli, Alessandro Palmioli, Valerio Mezzasalma, Ilaria Bruni, and Massimo Labra Copyright © 2015 Emanuele Ferri et al. All rights reserved. Cadmium Removal from Aqueous Systems Using Opuntia albicarpa L. Scheinvar as Biosorbent Wed, 09 Dec 2015 13:08:16 +0000 The aim of this research was to investigate the use of a natural adsorbent like nopal (Opuntia albicarpa L. Scheinvar) for removing cadmium from aqueous solutions with low concentrations of this metal. Two treatments were applied to the cladodes: a dehydration to get dehydrated nopal (DHN) and heating up to 90°C to obtain a thermally treated nopal (TN). After examining the effect of various pH values (2–7), the capacity of each biosorbent was examined in batch sorption tests at different dosages (0, 500, 1000, 1500, 2000, and 3000 mg L−1). The results indicated that adsorption of cadmium to biomass of DHN and TN was highly dependent on pH and biosorbent dosage. The best removal of cadmium (53.3%, corresponding to of 0.155 mg g−1) was obtained at pH 4.0 by using the TN sorbent. Infrared and Raman spectra confirmed that cadmium removal occurred via adsorption to –OH functional groups. Rosa Icela Beltrán-Hernández, Gabriela Alejandra Vázquez-Rodríguez, Luis Felipe Juárez-Santillán, Ivan Martínez-Ugalde, Claudia Coronel-Olivares, and Carlos Alexander Lucho-Constantino Copyright © 2015 Rosa Icela Beltrán-Hernández et al. All rights reserved. Improvement of D-Ribose Production from Corn Starch Hydrolysate by a Transketolase-Deficient Strain Bacillus subtilis UJS0717 Wed, 02 Dec 2015 13:21:17 +0000 D-Ribose is a five-carbon sugar and generally used as an energy source to improve athletic performance and the ability. The culture conditions for maximum D-ribose production performance from cheap raw material corn starch hydrolysate were improved by using one-factor-at-a-time experiments and a three-level Box-Behnken factorial design. The optimal fermentation parameters were obtained as 36°C culture temperature, 10% inoculum volume, and 7.0 initial pH. The mathematical model was then developed to show the effect of each medium composition and their interactions on the production of D-ribose and estimated that the optimized D-ribose production performance with the concentration of 62.13 g/L, yield of 0.40 g/g, and volumetric productivity of 0.86 g/L·h could be obtained when the medium compositions were set as 157 g/L glucose, 21 g/L corn steep liquor, 3.2 g/L (NH4)2SO4, 1 g/L yeast extract, 0.05 g/L MnSO4·H2O, and 20 g/L CaCO3. These findings indicated the D-ribose production performance was significantly improved compared to that under original conditions. Zhuan Wei, Jue Zhou, WenJing Sun, FengJie Cui, QinHua Xu, and ChangFeng Liu Copyright © 2015 Zhuan Wei et al. All rights reserved. Indirect Manganese Removal by Stenotrophomonas sp. and Lysinibacillus sp. Isolated from Brazilian Mine Water Tue, 01 Dec 2015 12:56:46 +0000 Manganese is a contaminant in the wastewaters produced by Brazilian mining operations, and the removal of the metal is notoriously difficult because of the high stability of the Mn(II) ion in aqueous solutions. To explore a biological approach for removing excessive amounts of aqueous Mn(II), we investigated the potential of Mn(II) oxidation by both consortium and bacterial isolates from a Brazilian manganese mine. A bacterial consortium was able to remove 99.7% of the Mn(II). A phylogenetic analysis of isolates demonstrated that the predominant microorganisms were members of Stenotrophomonas, Bacillus, and Lysinibacillus genera. Mn(II) removal rates between 58.5% and 70.9% were observed for Bacillus sp. and Stenotrophomonas sp. while the Lysinibacillus isolate 13P removes 82.7%. The catalytic oxidation of Mn(II) mediated by multicopper oxidase was not properly detected; however, in all of the experiments, a significant increase in the pH of the culture medium was detected. No aggregates inside the cells grown for a week were found by electronic microscopy. Nevertheless, an energy-dispersive X-ray spectroscopy of the isolates revealed the presence of manganese in Stenotrophomonas sp. and Lysinibacillus sp. grown in K medium. These results suggest that members of Stenotrophomonas and Lysinibacillus genera were able to remove Mn(II) by a nonenzymatic pathway. Natália Rocha Barboza, Soraya Sander Amorim, Pricila Almeida Santos, Flávia Donária Reis, Mônica Mendes Cordeiro, Renata Guerra-Sá, and Versiane Albis Leão Copyright © 2015 Natália Rocha Barboza et al. All rights reserved. Growth Inhibition and Membrane Permeabilization of Candida lusitaniae Using Varied Pulse Shape Electroporation Tue, 01 Dec 2015 12:03:03 +0000 Candida lusitaniae is an opportunistic yeast pathogen, which can readily develop resistance to antifungal compounds and result in a complex long-term treatment. The efficient treatment is difficult since structure and metabolic properties of the fungal cells are similar to those of eukaryotic host. One of the potential methods to improve the inhibition rate or the cell permeability to inhibitors is the application of electroporation. In this work we investigated the dynamics of the growth inhibition and membrane permeabilization of C. lusitaniae by utilizing the various pulse shape and duration electric field pulses. Our results indicated that single electroporation procedure using 8 kV/cm electric field may result in up to % inhibition rate. Also it has been experimentally shown that the electroporation pulse shape may influence the inhibitory effect; however, the amplitude of the electric field and the pulse energy remain the most important parameters for definition of the treatment outcome. The dynamics of the cell membrane permeabilization in the 2–8 kV/cm electric field were overviewed. V. Novickij, A. Grainys, E. Lastauskienė, R. Kananavičiūtė, D. Pamedytytė, A. Zinkevičienė, L. Kalėdienė, J. Novickij, A. Paškevičius, and J. Švedienė Copyright © 2015 V. Novickij et al. All rights reserved. Safety Evaluation of the Coagulase-Negative Staphylococci Microbiota of Salami: Superantigenic Toxin Production and Antimicrobial Resistance Mon, 30 Nov 2015 13:08:47 +0000 The risks of contracting staphylococci food poisoning by the consumption of improperly manufactured salami and the possibility of this food being reservoirs for antibiotic resistance were evaluated. Nineteen coagulase-negative staphylococci (CNS) strains were found in commercial and artisanal salami. The species in commercial salami were S. saprophyticus, S. sciuri, S. xylosus, and S. carnosus. Artisanal salami showed S. succinus, S. epidermidis, and S. hominis but no S. carnosus. Phylogenetic analyses grouped the strains into three major staphylococcal species groups, comprised of 4 refined clusters with similarities superior to 90%. Fifteen strains harbored multiple enterotoxin genes, with high incidence of seb/sec and sea, 57% and 50%, respectively, intermediate incidence of sed/seh/selm and sei/seln/tst-H, 33% and 27%, correspondingly, and low incidence of see/selj/selo and seg, of respectively 13% and 1%. Real time RT-PCR and enzyme-linked-immunosorbent assays confirmed the enterotoxigenicity of the strains, which expressed and produced enterotoxins in vitro. The CNS strains showed multiresistance to several antimicrobials of therapeutic importance in both human and veterinarian medicine, such as β-lactams, vancomycin, and linezolid. The effective control of undue staphylococci in fermented meat products should be adopted to prevent or limit the risk of food poisoning and the spread of antimicrobial-resistant strains. Raquel Soares Casaes Nunes, Eduardo Mere Del Aguila, and Vânia Margaret Flosi Paschoalin Copyright © 2015 Raquel Soares Casaes Nunes et al. All rights reserved. Ethanol and Protein from Ethanol Plant By-Products Using Edible Fungi Neurospora intermedia and Aspergillus oryzae Mon, 23 Nov 2015 12:07:41 +0000 Feasible biorefineries for production of second-generation ethanol are difficult to establish due to the process complexity. An alternative is to partially include the process in the first-generation plants. Whole stillage, a by-product from dry-mill ethanol processes from grains, is mostly composed of undegraded bran and lignocelluloses can be used as a potential substrate for production of ethanol and feed proteins. Ethanol production and the proteins from the stillage were investigated using the edible fungi Neurospora intermedia and Aspergillus oryzae, respectively. N. intermedia produced 4.7 g/L ethanol from the stillage and increased to 8.7 g/L by adding 1 FPU of cellulase/g suspended solids. Saccharomyces cerevisiae produced 0.4 and 5.1 g/L ethanol, respectively. Under a two-stage cultivation with both fungi, up to 7.6 g/L of ethanol and 5.8 g/L of biomass containing 42% (w/w) crude protein were obtained. Both fungi degraded complex substrates including arabinan, glucan, mannan, and xylan where reductions of 91, 73, 38, and 89% (w/v) were achieved, respectively. The inclusion of the current process can lead to the production of 44,000 m3 of ethanol (22% improvement), around 12,000 tons of protein-rich biomass for animal feed, and energy savings considering a typical facility producing 200,000 m3 ethanol/year. Veronika Bátori, Jorge A. Ferreira, Mohammad J. Taherzadeh, and Patrik R. Lennartsson Copyright © 2015 Veronika Bátori et al. All rights reserved. In Vitro Propagation, Phytochemical Analysis, and Evaluation of Free Radical Scavenging Property of Scrophularia kakudensis Franch Tissue Extracts Mon, 16 Nov 2015 08:25:56 +0000 The current study deals with in vitro propagation, antioxidant property estimation, and assessment of acacetin content in Scrophularia kakudensis Franch. Adventitious shoot induction was achieved from the nodal explant with the highest number of adventitious shoots per explant (17.4) on Murashige and Skoog’s (MS) medium fortified with 2.0 mg·L−1 6-benzyladenine (BA) and 0.5 mg L−1 indole-3-acetic acid (IAA). Maximum number of roots per plant (16.5) was noted in half strength MS medium supplemented with 0.5 mg·L−1 IAA. The regenerated plants displayed successful survival ratio (95%) in the greenhouse. The highest content of acacetin, a pharmaceutically important flavonoid, was observed in the shoot extracts (in vitro: 32.83 µg·g−1 FW; in vivo: 30.05 µg·g−1 FW) followed by root extracts. Total phenol and flavonoid contents along with free radical scavenging assays revealed the occurrence of larger amount of antioxidants in shoot extract in comparison with callus and root extracts of S. kakudensis. Thus, the outcome of the present study can be highly beneficial for the germplasm conservation and commercial cultivation of S. kakudensis for therapeutic purposes. Abinaya Manivannan, Prabhakaran Soundararajan, Yoo Gyeong Park, and Byoung Ryong Jeong Copyright © 2015 Abinaya Manivannan et al. All rights reserved. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1 Sun, 15 Nov 2015 11:35:40 +0000 Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications. Emanuele Sasso, Rolando Paciello, Francesco D’Auria, Gennaro Riccio, Guendalina Froechlich, Riccardo Cortese, Alfredo Nicosia, Claudia De Lorenzo, and Nicola Zambrano Copyright © 2015 Emanuele Sasso et al. All rights reserved. Optimization of Extraction Parameters for Enhanced Production of Ovotransferrin from Egg White for Antimicrobial Applications Tue, 10 Nov 2015 13:10:25 +0000 Ovotransferrin is the second most abundant protein (~12-13% of the total egg protein) in egg white after ovalbumin. Ovotransferrin is a potent natural antimicrobial agent as it possesses antibacterial, antifungal, and antiviral properties and is also the major metal binding protein found in egg, which makes it an industrially important protein. Ovotransferrin was extracted from egg white using its metal (iron) binding properties. In the present study, eggs from two different sources were used (fresh local eggs from domestic household source and poultry eggs from shops) to compare the results and Response Surface Methodology was used for the experiment design and data analysis. The following extraction conditions were optimized so as to maximize the yield of ovotransferrin from egg white: ethanol % (v/v) and pH and volume (mL) of 25 mM FeCl3/50 mL of egg white. A maximum yield of ~85 ± 2.5% was obtained near the optimum extraction conditions. The yield was calculated based on the theoretical value (934 mg) of ovotransferrin in 100 mL of 1.5x diluted egg white solution. Our results suggest that efficient downstream processing may reduce the cost of overall production process of this promising enzyme, making it a natural and cost-effective alternative to the existing chemically synthesized antimicrobial agents. Eyad M. A. Alshammari, Saif Khan, Arshad Jawed, Mohd Adnan, Mahvish Khan, Gowher Nabi, Mohtashim Lohani, and Shafiul Haque Copyright © 2015 Eyad M. A. Alshammari et al. All rights reserved. Selection of the Strain Lactobacillus acidophilus ATCC 43121 and Its Application to Brewers’ Spent Grain Conversion into Lactic Acid Tue, 10 Nov 2015 06:02:26 +0000 Six Lactobacillus strains were analyzed to select a bacterium for conversion of brewers’ spent grain (BSG) into lactic acid. Among the investigated strains, L. acidophilus ATCC 43121 showed the highest yield of lactic acid production (16.1 g/L after 48 hours) when grown in a synthetic medium. It was then analyzed for its ability to grow on the hydrolysates obtained from BSG after acid-alkaline (AAT) or aqueous ammonia soaking (AAS) pretreatment. The lactic acid production by L. acidophilus ATCC 43121 through fermentation of the hydrolysate from AAS treated BSG was 96% higher than that from the AAT treated one, although similar yields of lactic acid per consumed glucose were achieved due to a higher (46%) glucose consumption by L. acidophilus ATCC 43121 in the AAS BSG hydrolysate. It is worth noting that adding yeast extract to the BSG hydrolysates increased both the yield of lactic acid per substrate consumed and the volumetric productivity. The best results were obtained by fermentation of AAS BSG hydrolysate supplemented by yeast extract, in which the strain produced 22.16 g/L of lactic acid (yield of 0.61 g/g), 27% higher than the value (17.49 g/L) obtained in the absence of a nitrogen source. Rossana Liguori, Carlos Ricardo Soccol, Luciana Porto de Souza Vandenberghe, Adenise Lorenci Woiciechowski, Elena Ionata, Loredana Marcolongo, and Vincenza Faraco Copyright © 2015 Rossana Liguori et al. All rights reserved. Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum Mon, 09 Nov 2015 08:32:04 +0000 Exotoxin A is one of the virulence factors of Pseudomonas aeruginosa, a bacterium that can cause infections resulting in adverse health outcomes and increased burden to health care systems. Current methods of diagnosing P. aeruginosa infections are time consuming and can require significant preparation of patient samples. This study utilized a novel variation of the Systematic Evolution of Ligand by Exponential Enrichment, Decoy-SELEX, to identify an Exotoxin A specific single-stranded DNA (ssDNA) molecular recognition element (MRE). Its emphasis is on increasing stringency in directing binding toward free target of interest and at the same time decreasing binding toward negative targets. A ssDNA MRE with specificity and affinity was identified after fourteen rounds of Decoy-SELEX. Utilizing surface plasmon resonance measurements, the determined equilibrium dissociation constant of the MRE is between 4.2 µM and 4.5 µM, and is highly selective for Exotoxin A over negative targets. A ssDNA MRE modified sandwich enzyme-linked immunosorbent assay (ELISA) has been developed and achieved sensitive detection of Exotoxin A at nanomolar concentrations in human serum. This study has demonstrated the proof-of-principle of using a ssDNA MRE as a clinical diagnostic tool. Ka Lok Hong, Kailey Yancey, Luisa Battistella, Ryan M. Williams, Katherine M. Hickey, Chris D. Bostick, Peter M. Gannett, and Letha J. Sooter Copyright © 2015 Ka Lok Hong et al. All rights reserved. Optimization of Arundo donax Saccharification by (Hemi)cellulolytic Enzymes from Pleurotus ostreatus Thu, 05 Nov 2015 13:14:36 +0000 An enzymatic mixture of cellulases and xylanases was produced by Pleurotus ostreatus using microcrystalline cellulose as inducer, partially characterized and tested in the statistical analysis of Arundo donax bioconversion. The Plackett-Burman screening design was applied to identify the most significant parameters for the enzymatic hydrolysis of pretreated A. donax. As the most significant influence during the enzymatic hydrolysis of A. donax was exercised by the temperature (°C), pH, and time, the combined effect of these factors in the bioconversion by P. ostreatus cellulase and xylanase was analyzed by a 33 factorial experimental design. It is worth noting that the best result of 480.10 mg of sugars/gds, obtained at 45°C, pH 3.5, and 96 hours of incubation, was significant also when compared with the results previously reached by process optimization with commercial enzymes. Rossana Liguori, Elena Ionata, Loredana Marcolongo, Luciana Porto de Souza Vandenberghe, Francesco La Cara, and Vincenza Faraco Copyright © 2015 Rossana Liguori et al. All rights reserved. Facets of Nanotechnology as Seen in Food Processing, Packaging, and Preservation Industry Tue, 03 Nov 2015 12:32:25 +0000 Nanotechnology has proven its competence in almost all possible fields we are aware of. However, today nanotechnology has evolved in true sense by contributing to a very large extent to the food industry. With the growing number of mouths to feed, production of food is not adequate. It has to be preserved in order to reach to the masses on a global scale. Nanotechnology made the idea a reality by increasing the shelf life of different kinds of food materials. It is not an entirely full-proof measure; however it has brought down the extent of wastage of food due to microbial infestation. Not only fresh food but also healthier food is being designed with the help of nano-delivery systems which act as a carrier for the food supplements. There are regulations to follow however as several of them pose serious threats to the wellbeing of the population. In coming days, newer modes of safeguarding food are going to be developed with the help of nanotechnology. In this paper, an overview has been given of the different methods of food processing, packaging, and preservation techniques and the role nanotechnology plays in the food processing, packaging, and preservation industry. Neha Pradhan, Surjit Singh, Nupur Ojha, Anamika Shrivastava, Anil Barla, Vivek Rai, and Sutapa Bose Copyright © 2015 Neha Pradhan et al. All rights reserved. Evaluation of Mediterranean Agricultural Residues as a Potential Feedstock for the Production of Biogas via Anaerobic Fermentation Mon, 02 Nov 2015 13:19:46 +0000 Hydrothermal, dilute acid, and steam explosion pretreatment methods, were evaluated for their efficiency to improve the methane production yield of three Mediterranean agricultural lignocellulosic residues such as olive tree pruning, grapevine pruning, and almond shells. Hydrothermal and dilute acid pretreatments provided low to moderate increase in the digestibility of the biomass samples, whereas steam explosion enabled the highest methane yields to be achieved for almond shells at 232.2 ± 13.0 mL CH4/gVS and olive pruning at 315.4 ± 0.0 mL CH4/gVS. Introduction of an enzymatic prehydrolysis step moderately improved methane yields for hydrothermal and dilute acid pretreated samples but not for the steam exploded ones. Christos Nitsos, Leonidas Matsakas, Kostas Triantafyllidis, Ulrika Rova, and Paul Christakopoulos Copyright © 2015 Christos Nitsos et al. All rights reserved. Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System Thu, 29 Oct 2015 13:26:12 +0000 Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation. Hyuck Joon Kwon and Youngbae Han Copyright © 2015 Hyuck Joon Kwon and Youngbae Han. All rights reserved. Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy Thu, 15 Oct 2015 09:15:33 +0000 Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the production of mAbs for cancer immunotherapy. However, their several limitations have to be resolved for efficient antibody production in plants. Ghislain Moussavou, Kisung Ko, Jeong-Hwan Lee, and Young-Kug Choo Copyright © 2015 Ghislain Moussavou et al. All rights reserved. Current and New Approaches in GMO Detection: Challenges and Solutions Thu, 15 Oct 2015 07:08:43 +0000 In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market and to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owing to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g., loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g., PCR capillary gel electrophoresis, microarray, and Luminex), more accurate quantification of GM targets (e.g., digital PCR), or characterization of partially known (e.g., DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g., NGS) GMO. The benefits and drawbacks of these methods are discussed in this review. Marie-Alice Fraiture, Philippe Herman, Isabel Taverniers, Marc De Loose, Dieter Deforce, and Nancy H. Roosens Copyright © 2015 Marie-Alice Fraiture et al. All rights reserved. Improved Sugar Production by Optimizing Planetary Mill Pretreatment and Enzyme Hydrolysis Process Sun, 11 Oct 2015 12:54:08 +0000 This paper describes an optimization of planetary mill pretreatment and saccharification processes for improving biosugar production. Pitch pine (Pinus rigida) wood sawdust waste was used as biomass feedstock and the process parameters optimized in this study were the buffering media, the milling time, the enzyme quantity, and the incubation time. Glucose yields were improved when acetate buffer was used rather than citrate buffer. Initially, with each process variable tests, the optimal values were 100 minutes of milling, an enzyme concentration of 16 FPU/g-biomass, and a 12-hour enzymatic hydrolysis. Typically, interactions between these experimental conditions and their effects on glucose production were next investigated using RSM. Glucose yields from the Pinus rigida waste exceeded 80% with several of the conditions tested, demonstrating that milling can be used to obtain high levels of glucose bioconversion from woody biomass for biorefinery purposes. Jeong Heo Kwon, Siseon Lee, Jae-Won Lee, Youn-Woo Hong, Jeong Ho Chang, Daekyung Sung, Sung Hyun Kim, Byoung-In Sang, Robert J. Mitchell, and Jin Hyung Lee Copyright © 2015 Jeong Heo Kwon et al. All rights reserved. Biotechnology in Environmental Monitoring and Pollution Abatement 2015 Wed, 07 Oct 2015 09:21:19 +0000 Kannan Pakshirajan, Eldon R. Rene, and Aiyagari Ramesh Copyright © 2015 Kannan Pakshirajan et al. All rights reserved. Enhancing Ecoefficiency in Shrimp Farming through Interconnected Ponds Wed, 07 Oct 2015 07:29:51 +0000 The future development of shrimp farming needs to improve its ecoefficiency. The purpose of this study was to evaluate water quality, flows, and nitrogen balance and production parameters on a farm with interconnected pond design to improve the efficiency of the semi-intensive culture of Litopenaeus vannamei ponds. The study was conducted in 21 commercial culture ponds during 180 days at densities of 30–35 ind m−2 and daily water exchange <2%. Our study provides evidence that by interconnecting ponds nutrient recycling is favored by promoting the growth of primary producers of the pond as chlorophyll a. Based on the mass balance and flow of nutrients this culture system reduces the flow of solid, particulate organic matter, and nitrogen compounds to the environment and significantly increases the efficiency of water (5 to 6.5 m3 kg−1 cycle−1), when compared with traditional culture systems. With this culture system it is possible to recover up to 34% of the total nitrogen entering the system, with production in excess of 4,000 kg ha−1 shrimp. We believe that the production system with interconnected ponds is a technically feasible model to improve ecoefficiency production of shrimp farming. Ramón Héctor Barraza-Guardado, José Alfredo Arreola-Lizárraga, Anselmo Miranda-Baeza, Manuel Juárez-García, Antonio Juvera-Hoyos, and Ramón Casillas-Hernández Copyright © 2015 Ramón Héctor Barraza-Guardado et al. All rights reserved. Fabrication of an Amperometric Flow-Injection Microfluidic Biosensor Based on Laccase for In Situ Determination of Phenolic Compounds Mon, 05 Oct 2015 09:54:11 +0000 We aim to develop an in situ microfluidic biosensor based on laccase from Trametes pubescens with flow-injection and amperometry as the transducer method. The enzyme was directly immobilized by potential step chronoamperometry, and the immobilization was studied using cyclic voltammetry and electrochemical impedance spectroscopy. The electrode response by amperometry was probed using ABTS and syringaldazine. A shift of interfacial electron transfer resistance and the electron transfer rate constant from 18.1 kΩ to 3.9 MΩ and 4.6 × 10−2 cm s−1 to 2.1 × 10−4 cm s−1, respectively, evidenced that laccase was immobilized on the electrode by the proposed method. We established the optimum operating conditions of temperature (55°C), pH (4.5), injection flow rate (200 µL min−1), and applied potential (0.4 V). Finally, the microfluidic biosensor showed better lower limit of detection (0.149 µM) and sensitivity (0.2341 nA µM−1) for ABTS than previous laccase-based biosensors and the in situ operation capacity. Juan C. Gonzalez-Rivera and Johann F. Osma Copyright © 2015 Juan C. Gonzalez-Rivera and Johann F. Osma. All rights reserved. Comparison of Different Strategies for Selection/Adaptation of Mixed Microbial Cultures Able to Ferment Crude Glycerol Derived from Second-Generation Biodiesel Mon, 05 Oct 2015 09:38:56 +0000 Objective of this study was the selection and adaptation of mixed microbial cultures (MMCs), able to ferment crude glycerol generated from animal fat-based biodiesel and produce building-blocks and green chemicals. Various adaptation strategies have been investigated for the enrichment of suitable and stable MMC, trying to overcome inhibition problems and enhance substrate degradation efficiency, as well as generation of soluble fermentation products. Repeated transfers in small batches and fed-batch conditions have been applied, comparing the use of different inoculum, growth media, and Kinetic Control. The adaptation of activated sludge inoculum was performed successfully and continued unhindered for several months. The best results showed a substrate degradation efficiency of almost 100% (about 10 g/L glycerol in 21 h) and different dominant metabolic products were obtained, depending on the selection strategy (mainly 1,3-propanediol, ethanol, or butyrate). On the other hand, anaerobic sludge exhibited inactivation after a few transfers. To circumvent this problem, fed-batch mode was used as an alternative adaptation strategy, which led to effective substrate degradation and high 1,3-propanediol and butyrate production. Changes in microbial composition were monitored by means of Next Generation Sequencing, revealing a dominance of glycerol consuming species, such as Clostridium, Klebsiella, and Escherichia. C. Varrone, T. M. B. Heggeset, S. B. Le, T. Haugen, S. Markussen, I. V. Skiadas, and H. N. Gavala Copyright © 2015 C. Varrone et al. All rights reserved. Perspective for Aquaponic Systems: “Omic” Technologies for Microbial Community Analysis Mon, 05 Oct 2015 08:15:48 +0000 Aquaponics is the combined production of aquaculture and hydroponics, connected by a water recirculation system. In this productive system, the microbial community is responsible for carrying out the nutrient dynamics between the components. The nutrimental transformations mainly consist in the transformation of chemical species from toxic compounds into available nutrients. In this particular field, the microbial research, the “Omic” technologies will allow a broader scope of studies about a current microbial profile inside aquaponics community, even in those species that currently are unculturable. This approach can also be useful to understand complex interactions of living components in the system. Until now, the analog studies were made to set up the microbial characterization on recirculation aquaculture systems (RAS). However, microbial community composition of aquaponics is still unknown. “Omic” technologies like metagenomic can help to reveal taxonomic diversity. The perspectives are also to begin the first attempts to sketch the functional diversity inside aquaponic systems and its ecological relationships. The knowledge of the emergent properties inside the microbial community, as well as the understanding of the biosynthesis pathways, can derive in future biotechnological applications. Thus, the aim of this review is to show potential applications of current “Omic” tools to characterize the microbial community in aquaponic systems. Perla Munguia-Fragozo, Oscar Alatorre-Jacome, Enrique Rico-Garcia, Irineo Torres-Pacheco, Andres Cruz-Hernandez, Rosalia V. Ocampo-Velazquez, Juan F. Garcia-Trejo, and Ramon G. Guevara-Gonzalez Copyright © 2015 Perla Munguia-Fragozo et al. All rights reserved. Magnet-Facilitated Selection of Electrogenic Bacteria from Marine Sediment Sun, 04 Oct 2015 12:18:46 +0000 Some bacteria can carry out anaerobic respiration by depositing electrons on external materials, such as electrodes, thereby creating an electrical current. Into the anode chamber of microbial fuel cells (MFCs) having abiotic air-cathodes we inoculated microorganisms cultured from a magnetic particle-enriched portion of a marine tidal sediment, reasoning that since some external electron acceptors are ferromagnetic, electrogenic bacteria should be found in their vicinity. Two MFCs, one inoculated with a mixed bacterial culture and the other with an axenic culture of a helical bacterium isolated from the magnetic particle enrichment, termed strain HJ, were operated for 65 d. Both MFCs produced power, with production from the mixed culture MFC exceeding that of strain HJ. Strain HJ was identified as a Thalassospira sp. by transmission electron microscopic analysis and 16S rRNA gene comparisons. An MFC inoculated with strain HJ and operated in open circuit produced 47% and 57% of the maximal power produced from MFCs inoculated with the known electrogen Geobacter daltonii and the magnetotactic bacterium Desulfamplus magnetomortis, respectively. Further investigation will be needed to determine whether bacterial populations associated with magnetic particles within marine sediments are enriched for electrogens. Larisa Kiseleva, Justina Briliute, Irina V. Khilyas, David J. W. Simpson, Viacheslav Fedorovich, M. Cohen, and Igor Goryanin Copyright © 2015 Larisa Kiseleva et al. All rights reserved. Fungal Antagonism Assessment of Predatory Species and Producers Metabolites and Their Effectiveness on Haemonchus contortus Infective Larvae Sun, 04 Oct 2015 10:36:57 +0000 The objective of this study was to assess antagonism of nematophagous fungi and species producers metabolites and their effectiveness on Haemonchus contortus infective larvae (L3). Assay A assesses the synergistic, additive, or antagonistic effect on the production of spores of fungal isolates of the species Duddingtonia flagrans, Clonostachys rosea, Trichoderma esau, and Arthrobotrys musiformis; Assay B evaluates in vitro the effect of intercropping of these isolates grown in 2% water-agar (2% WA) on L3 of H. contortus. D. flagrans (Assay A) produced 5.3 × 106 spores and associated with T. esau, A. musiformis, or C. rosea reduced its production by 60.37, 45.28, and 49.05%, respectively. T. esau produced 7.9 × 107 conidia and associated with D. flagrans, A. musiformis, or C. rosea reduced its production by 39.24, 82.27, and 96.96%, respectively. A. musiformis produced 7.3 × 109 spores and associated with D. flagrans, T. esau, or C. rosea reduced its production by 99.98, 99.99, and 99.98%, respectively. C. rosea produced 7.3 × 108 conidia and associated with D. flagrans, T. esau, or A. musiformis reduced its production by 95.20, 96.84, and 93.56%, respectively. These results show evidence of antagonism in the production of spores between predators fungi. Manoel Eduardo Silva, Fabio Ribeiro Braga, Pedro Mendoza de Gives, Jair Millán-Orozco, Miguel Angel Mercado Uriostegui, Liliana Aguilar Marcelino, Filippe Elias de Freitas Soares, Andréia Luiza Araújo, Thainá Souza Vargas, Anderson Rocha Aguiar, Thiago Senna, Maria Gorete Rodrigues, Frederico Vieira Froes, and Jackson Victor de Araújo Copyright © 2015 Manoel Eduardo Silva et al. All rights reserved. Efficacy of Clonostachys rosea and Duddingtonia flagrans in Reducing the Haemonchus contortus Infective Larvae Sun, 04 Oct 2015 10:18:53 +0000 The biocontrol is proven effective in reducing in vitro and in situ free-living stages of major gastrointestinal helminths, allowing progress in reducing losses by parasitism, maximizing production, and productivity. This study aimed at evaluating the predatory activity of fungal isolates of Duddingtonia flagrans and Clonostachys rosea species and its association on infective larvae (L3) of H. contortus in microplots formed by grasses and maintained in a protected environment. All groups were added with 10 mL of an aqueous suspension with 618 H. contortus L3 approximately. Group 1 was used as control and only received the infective larvae. Groups 2 and 3 received D. flagrans chlamydospores and C. rosea conidia at doses of 5 × 106. Group 4 received the combination of 5 × 106 D. flagrans chlamydospores + 5 × 106 C. rosea conidia. D. flagrans and C. rosea showed nematicidal effectiveness reducing by 91.5 and 88.9%, respectively, the population of H. contortus L3. However, when used in combination efficiency decreased to 74.5% predation of H. contortus L3. These results demonstrate the need for further studies to determine the existence of additive effects, synergistic or antagonistic, between these species. Manoel Eduardo da Silva, Fabio Ribeiro Braga, Pedro Mendoza de Gives, Miguel Angel Mercado Uriostegui, Manuela Reyes, Filippe Elias de Freitas Soares, Lorendane Millena de Carvalho, Francielle Bosi Rodrigues, and Jackson Victor de Araújo Copyright © 2015 Manoel Eduardo da Silva et al. All rights reserved. Biosorption of Pb(II) Ions by Klebsiella sp. 3S1 Isolated from a Wastewater Treatment Plant: Kinetics and Mechanisms Studies Sun, 04 Oct 2015 08:57:51 +0000 Lead biosorption by Klebsiella sp. 3S1 isolated from a wastewater treatment plant was investigated through a Rotatable Central Composite Experimental Design. The optimisation study indicated the following optimal values of operating variables: 0.4 g/L of biosorbent dosage, pH 5, and 34°C. According to the results of the kinetic studies, the biosorption process can be described by a two-step process, one rapid, almost instantaneous, and one slower, both contributing significantly to the overall biosorption; the model that best fits the experimental results was pseudo-second order. The equilibrium studies showed a maximum lead uptake value of 140.19 mg/g according to the Langmuir model. The mechanism study revealed that lead ions were bioaccumulated into the cytoplasm and adsorbed on the cell surface. The bacterium  Klebsiella sp. 3S1 has a good potential in the bioremoval of lead in an inexpensive and effective process. Antonio Jesús Muñoz, Francisco Espínola, Manuel Moya, and Encarnación Ruiz Copyright © 2015 Antonio Jesús Muñoz et al. All rights reserved. Structure and Antioxidant Activity of Soy Protein Isolate-Dextran Conjugates Obtained by TiO2 Photocatalysis Thu, 01 Oct 2015 11:12:06 +0000 The aim of this study was to investigate the structural characteristics and antioxidant activities of soy protein isolate- (SPI-) dextran conjugates obtained by TiO2 photocatalysis treatment. Results revealed that the UV-vis absorption and the fluorescence intensity increased as the photocatalytic power increased (). Higher photocatalytic power could promote the extent of glycation and the formation of high molecular weight SPI-dextran conjugates, which were evidenced by free amino group content and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The Fourier transform infrared (FT-IR) spectra suggested that the amide I, II, and III bands of SPI were altered by the glycation induced by TiO2 photocatalysis. Moreover, significant changes of secondary structure occurred in SPI-dextran conjugates. The α-helix, β-sheet, β-turns, and random coil were changed from approximately 10.6%, 37.9%, 12.9%, and 38.6% to 3.8%, 10.4%, 17.7%, and 68.8%, respectively, after treatment at photocatalytic power of 1000 W. In addition, SPI-dextran conjugates obtained by TiO2 photocatalysis treatment exhibited high hydroxyl radical scavenging activity and possessed increased reducing power. All data indicated that TiO2 photocatalysis was an efficient method for promoting protein-polysaccharide copolymerisation. Bei Jin, Xiaosong Zhou, Bing Li, Caiyan Chen, Xiaosa Zhang, and Siqiao Chen Copyright © 2015 Bei Jin et al. All rights reserved. The Regulation by Phenolic Compounds of Soil Organic Matter Dynamics under a Changing Environment Thu, 01 Oct 2015 08:26:04 +0000 Phenolics are the most abundant plant metabolites and are believed to decompose slowly in soils compared to other soil organic matter (SOM). Thus, they have often been considered as a slow carbon (C) pool in soil dynamics models. Here, however, we review changes in our concept about the turnover rate of phenolics and quantification of different types of phenolics in soils. Also, we synthesize current research on the degradation of phenolics and their regulatory effects on decomposition. Environmental changes, such as elevated CO2, warming, nitrogen (N) deposition, and drought, could influence the production and form of phenolics, leading to a change in SOM dynamics, and thus we also review the fate of phenolics under environmental disturbances. Finally, we propose the use of phenolics as a tool to control rates of SOM decomposition to stabilize organic carbon in ecosystems. Further studies to clarify the role of phenolics in SOM dynamics should include improving quantification methods, elucidating the relationship between phenolics and soil microorganisms, and determining the interactive effects of combinations of environmental changes on the phenolics production and degradation and subsequent impact on SOM processing. Kyungjin Min, Chris Freeman, Hojeong Kang, and Sung-Uk Choi Copyright © 2015 Kyungjin Min et al. All rights reserved. A Novel Reference Plasmid for the Qualitative Detection of Genetically Modified Rice in Food and Feed Thu, 01 Oct 2015 07:45:58 +0000 Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice. Liang Li, Mei Dong, Na An, Lixia Liang, Yusong Wan, and Wujun Jin Copyright © 2015 Liang Li et al. All rights reserved. Effect of Chitosan Coating with Cinnamon Oil on the Quality and Physiological Attributes of China Jujube Fruits Thu, 01 Oct 2015 07:41:48 +0000 Effects of chitosan coating with cinnamon oil on the physiological attributes and preservation quality of China jujube fruits during storage at 4°C for 60 days were investigated. Results indicated that weight loss and decay of jujube fruits were significantly reduced by chitosan-oil coating during the period of 60-day storage, which also exhibited a quite beneficial effect on maintaining the sensory quality for jujube fruits. Meanwhile, the contents of vitamin C and titratable acid decreased to 3.08 mg·g−1 and 0.342% for the fruits treated by chitosan-oil coating (1.0% + 0.10%), respectively. Polyphenol oxidase, superoxide dismutase, and peroxidase activities were 13.40 U·g−1, 14.53 U·g−1, and 63.6 U·g−1 at the end of storage, respectively. The contents of total soluble phenolics and MDA were 34.51 mg·g−1 and 19.43 μmol·g−1 for the combined coating treated samples and control fruits, respectively. These results suggested that the chitosan-oil coating might be recognized as one efficiency technology on the preservation quality of jujube fruits during the storage time. Yage Xing, Hongbin Lin, Dong Cao, Qinglian Xu, Wenfeng Han, Ranran Wang, Zhenming Che, and Xihong Li Copyright © 2015 Yage Xing et al. All rights reserved. Integrated Evaluation of Urban Water Bodies for Pollution Abatement Based on Fuzzy Multicriteria Decision Approach Thu, 01 Oct 2015 07:21:58 +0000 Today’s ecology is erected with miscellaneous framework. However, numerous sources deteriorate it, such as urban rivers that directly cause the environmental pollution. For chemical pollution abatement from urban water bodies, many techniques were introduced to rehabilitate the water quality of these water bodies. In this research, Bacterial Technology (BT) was applied to urban rivers escalating the necessity to control the water pollution in different places (Xuxi River (XXU); Gankeng River (GKS); Xia Zhang River (XZY); Fenghu and Song Yang Rivers (FSR); Jiu Haogang River (JHH)) in China. For data analysis, the physiochemical parameters such as temperature, chemical oxygen demand (COD), dissolved oxygen (DO), total phosphorus (TP), and ammonia nitrogen (NH3N) were determined before and after the treatment. Multicriteria Decision Making (MCDM) method was used for relative significance of different water quality on each station, based on fuzzy analytical hierarchy process (FAHP). The overall results revealed that the pollution is exceeding at “JHH” due to the limit of “COD” as critical water quality parameter and after treatment, an abrupt recovery of the rivers compared with the average improved efficiency of nutrients was 79%, 74%, 68%, and 70% of COD, DO, TP, and NH3N, respectively. The color of the river’s water changed to its original form and aquatic living organism appeared with clear effluents from them. Sarfraz Hashim, Xie Yuebo, Muhammad Saifullah, Ramila Nabi Jan, and Adila Muhetaer Copyright © 2015 Sarfraz Hashim et al. All rights reserved. Vulnerability Assessment and Application of Bacterial Technology on Urban Rivers for Pollution Eradication Thu, 01 Oct 2015 06:57:41 +0000 To protect against the environmental pollution, the present research was undertaken to enumerate the Bacterial Technologies (BTs) on the restoration of polluted urban rivers, that is, Fenghu-Song Yang River (FSR) and Xuxi River (XXR). Experimental research accounted for the physiochemical parameters (pH; temperature; dissolved oxygen (DO); chemical oxygen demand (COD); total phosphorus (TP); total nitrogen (TN); and ammonia nitrogen (NH3N)) before and after the BT operation. The results declared that the BT is efficient to restore the polluted rivers up to reliable condition. These results were analyzed by using multivariate statistical techniques (principal component analysis (PCA) and cluster analysis (CA)). These techniques interpreted the complex data sets and expressed the point source information about the water quality of these rivers at SA5, SA6, and SB3 under highly polluted regions. For better understanding, water quality index (WQI) was applied to compute the single numeric value. WQI results are evidence of the above results which prove the water quality of both rivers faced under outrageous condition (below 50 WQI scores) before the BT treatment, but, after the treatment, the rivers were restored from fair to good level (above 50 WQI scores) and overall output of these scores was quite similar to detect the point source of pollution. These results described an abrupt recovery of the urban rivers up to reliable condition for aquatic organism and clear effluents from the rivers. Sarfraz Hashim, Xie Yuebo, Fiaz Ahmad, Chaudhry Arslan, and Muhammad Saifullah Copyright © 2015 Sarfraz Hashim et al. All rights reserved. Evaluation of Natural Materials as Exogenous Carbon Sources for Biological Treatment of Low Carbon-to-Nitrogen Wastewater Thu, 01 Oct 2015 06:54:10 +0000 In the bacterial processes involved in the mitigation of nitrogen pollution, an adequately high carbon-to-nitrogen (C : N) ratio is key to sustain denitrification. We evaluated three natural materials (woodchips, barley grains, and peanut shells) as carbon sources for low C : N wastewater. The amount of organic matter released from these materials to aqueous media was evaluated, as well as their pollution swapping potential by measuring the release of total Kjeldahl nitrogen, N-, , and , and total phosphorous. Barley grains yielded the highest amount of organic matter, which also showed to be the most easily biodegradable. Woodchips and peanut shells released carbon rather steadily and so they would not require frequent replenishment from biological reactors. These materials produced eluates with lower concentrations of nutrients than the leachates from barley grains. However, as woodchips yielded lower amounts of suspended solids, they constitute an adequate exogenous source for the biological treatment of carbon-deficient effluents. Juan Ramírez-Godínez, Icela Beltrán-Hernández, Alejandro Álvarez-Hernández, Claudia Coronel-Olivares, Elizabeth Contreras-López, Maribel Quezada-Cruz, and Gabriela Vázquez-Rodríguez Copyright © 2015 Juan Ramírez-Godínez et al. All rights reserved. Decolorization of Distillery Spent Wash Using Biopolymer Synthesized by Pseudomonas aeruginosa Isolated from Tannery Effluent Thu, 01 Oct 2015 06:31:44 +0000 A bacterial strain was isolated from tannery effluent which can tolerate high concentrations of potassium dichromate up to 1000 ppm. The isolated microorganism was identified as Pseudomonas aeruginosa by performing biochemical tests and molecular characterization. In the presence of excess of carbohydrate source, which is a physiological stress, this strain produces Polyhydroxybutyrate (PHB). This intracellular polymer, which is synthesized, is primarily a product of carbon assimilation and is employed by microorganisms as an energy storage molecule to be metabolized when other common energy sources are limitedly available. Efforts were taken to check whether the PHB has any positive effect on spent wash decolorization. When a combination of PHB and the isolated bacterial culture was added to spent wash, a maximum color removal of 92.77% was found which was comparatively higher than the color removed when the spent wash was treated individually with the PHB and Pseudomonas aeruginosa. PHB behaved as a support material for the bacteria to bind to it and thus develops biofilm, which is one of the natural physiological growth forms of microorganisms. The bacterial growth in the biofilm and the polymer together acted in synergy, adsorbing and coagulating the pollutants in the form of color pigments. Charles David, M. Arivazhagan, M. N. Balamurali, and Dhivya Shanmugarajan Copyright © 2015 Charles David et al. All rights reserved. Antioxidant Potential in Different Parts and Callus of Gynura procumbens and Different Parts of Gynura bicolor Wed, 30 Sep 2015 14:31:43 +0000 Plants from Gynura family was used in this study, namely, Gynura procumbens and Gynura bicolor. Gynura procumbens is well known for its various medicinal properties such as antihyperglycaemic, antihyperlipidaemic, and antiulcerogenic; meanwhile, G. bicolor remains unexploited. Several nonenzymatic antioxidants methods were utilized to study the antioxidant capacity, which include ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, total flavonoid content, total phenolic content, and ascorbic acid content determination. DPPH assay reveals G. procumbens shoot as the lowest (66.885%) and G. procumbens root as the highest (93.499%) DPPH radical inhibitor. In FRAP assay, reducing power was not detected in G. procumbens leaf callus (0.000 TEAC mg/g FW) whereby G. procumbens root exhibits the highest (1.103 TEAC mg/g FW) ferric reducing power. Total phenolic content and total flavonoid content exhibited similar trend for both the intact plants analysed. In all antioxidant assays, G. procumbens callus culture exhibits very low antioxidant activity. However, G. procumbens root exhibited highest phenolic content, flavonoid content, and ascorbic acid content with 4.957 TEAC mg/g FW, 543.529 QE µg/g FW, and 54.723 µg/g FW, respectively. This study reveals that G. procumbens root extract is a good source of natural antioxidant. Vijendren Krishnan, Syahida Ahmad, and Maziah Mahmood Copyright © 2015 Vijendren Krishnan et al. All rights reserved. Characteristics of Biological Nitrogen Removal in a Multiple Anoxic and Aerobic Biological Nutrient Removal Process Wed, 30 Sep 2015 14:09:04 +0000 Two sequencing batch reactors, one with the conventional anoxic and aerobic (AO) process and the other with the multiple AO process, were operated to examine characteristics of biological nitrogen removal, especially of the multiple AO process. The long-term operation showed that the total nitrogen removal percentage of the multiple AO reactor was 38.7% higher than that of the AO reactor. In the multiple AO reactor, at the initial SBR cycle stage, due to the occurrence of simultaneous nitrification and denitrification, no nitrite and/or nitrate were accumulated. In the multiple AO reactor, activities of nitrite oxidizing bacteria were inhibited due to the multiple AO operating mode applied, resulting in the partial nitrification. Denitrifiers in the multiple AO reactor mainly utilized internal organic carbon for denitrification, and their activities were lower than those of denitrifiers in the AO reactor utilizing external organic carbon. Huoqing Wang, Yuntao Guan, Li Li, and Guangxue Wu Copyright © 2015 Huoqing Wang et al. All rights reserved. Antiviral Potential of Algae Polysaccharides Isolated from Marine Sources: A Review Mon, 21 Sep 2015 11:45:49 +0000 From food to fertilizer, algal derived products are largely employed in assorted industries, including agricultural, biomedical, food, and pharmaceutical industries. Among different chemical compositions isolated from algae, polysaccharides are the most well-established compounds, which were subjected to a variety of studies due to extensive bioactivities. Over the past few decades, the promising results for antiviral potential of algae-derived polysaccharides have advocated them as inordinate candidates for pharmaceutical research. Numerous studies have isolated various algal polysaccharides possessing antiviral activities, including carrageenan, alginate, fucan, laminaran, and naviculan. In addition, different mechanisms of action have been reported for these polysaccharides, such as inhibiting the binding or internalization of virus into the host cells or suppressing DNA replication and protein synthesis. This review strives for compiling previous antiviral studies of algae-derived polysaccharides and their mechanism of action towards their development as natural antiviral agents for future investigations. Azin Ahmadi, Soheil Zorofchian Moghadamtousi, Sazaly Abubakar, and Keivan Zandi Copyright © 2015 Azin Ahmadi et al. All rights reserved. Two-Dimensional Crystallization Procedure, from Protein Expression to Sample Preparation Thu, 27 Aug 2015 14:07:58 +0000 Membrane proteins play important roles for living cells. Structural studies of membrane proteins provide deeper understanding of their mechanisms and further aid in drug design. As compared to other methods, electron microscopy is uniquely suitable for analysis of a broad range of specimens, from small proteins to large complexes. Of various electron microscopic methods, electron crystallography is particularly well-suited to study membrane proteins which are reconstituted into two-dimensional crystals in lipid environments. In this review, we discuss the steps and parameters for obtaining large and well-ordered two-dimensional crystals. A general description of the principle in each step is provided since this information can also be applied to other biochemical and biophysical methods. The examples are taken from our own studies and published results with related proteins. Our purpose is to give readers a more general idea of electron crystallography and to share our experiences in obtaining suitable crystals for data collection. Qie Kuang, Pasi Purhonen, and Hans Hebert Copyright © 2015 Qie Kuang et al. All rights reserved. Biological Activities of Tetrodotoxin-Producing Enterococcus faecium AD1 Isolated from Puffer Fishes Mon, 24 Aug 2015 12:24:09 +0000 Puffer fishes were collected from the central sea in Vietnam from spring to summer season. The eggs were incubated in MRS broth that was used to test the toxicity in mice and isolate the lactic acid bacteria community that could produce tetrodotoxin (TTX). Thin layer chromatography (TLC) and high performance lipid chromatography (HPLC) were used to detect and quantify TTX. As a result, Enterococcus faecium AD1 which was identified by biochemical test and 16S rRNA analysis could produce TTX 0.3 mg/mL when cultured in MRS broth. The bacterium was optimized for TTX production and gave 0.18 mg/mL, 0.07 mg/mL, and 0.15 mg/mL in media prepared from the meat-washing water of freshwater fishes (Pangasius bocourti, Oreochromis sp.) and sea fish (Auxis thazard), respectively, that are also hopeful to answer some poisoning cases related to eating fishes. Enterococcus faecium also showed the wide antimicrobial activities on yeast, Gram-negative and -positive bacteria. Extracted exopolysaccharide (EPS) that reacted with 2,2-diphenyl-1-picrylhydrazyl to give IC50 at 5 mg/mL equaled 11 mg/mL ascorbic acid which could show effects on Hela-6 and Hep G2 using sulforhodamine B test. Enterococcus faecium can be claimed as a promising source in tetrodotoxin and biological compounds. Tu Hoang Khue Nguyen, Huu Ngoc Nguyen, Dat Van Nghe, and Kim Hoang Nguyen Copyright © 2015 Tu Hoang Khue Nguyen et al. All rights reserved. Optimization of Fermentation Medium for Extracellular Lipase Production from Aspergillus niger Using Response Surface Methodology Thu, 20 Aug 2015 16:16:13 +0000 Lipase produced by Aspergillus niger is widely used in various industries. In this study, extracellular lipase production from an industrial producing strain of A. niger was improved by medium optimization. The secondary carbon source, nitrogen source, and lipid were found to be the three most influential factors for lipase production by single-factor experiments. According to the statistical approach, the optimum values of three most influential parameters were determined: 10.5 g/L corn starch, 35.4 g/L soybean meal, and 10.9 g/L soybean oil. Using this optimum medium, the best lipase activity was obtained at 2,171 U/mL, which was 16.4% higher than using the initial medium. All these results confirmed the validity of the model. Furthermore, results of the Box-Behnken Design and quadratic models analysis indicated that the carbon to nitrogen (C/N) ratio significantly influenced the enzyme production, which also suggested that more attention should be paid to the C/N ratio for the optimization of enzyme production. Jia Jia, Xiaofeng Yang, Zhiliang Wu, Qian Zhang, Zhi Lin, Hongtao Guo, Carol Sze Ki Lin, Jianying Wang, and Yunshan Wang Copyright © 2015 Jia Jia et al. All rights reserved. Hydrothermal Pretreatment of Date Palm (Phoenix dactylifera L.) Leaflets and Rachis to Enhance Enzymatic Digestibility and Bioethanol Potential Wed, 12 Aug 2015 14:27:47 +0000 Date palm residues are one of the most promising lignocellulosic biomass for bioethanol production in the Middle East. In this study, leaflets and rachis were subjected to hydrothermal pretreatment to overcome the recalcitrance of the biomass for enzymatic conversion. Evident morphological, structural, and chemical changes were observed by scanning electron microscopy, X-ray diffraction, and infrared spectroscopy after pretreatment. High glucan (>90% for both leaflets and rachis) and xylan (>75% for leaflets and >79% for rachis) recovery were achieved. Under the optimal condition of hydrothermal pretreatment (210°C/10 min) highly digestible (glucan convertibility, 100% to leaflets, 78% to rachis) and fermentable (ethanol yield, 96% to leaflets, 80% to rachis) solid fractions were obtained. Fermentability test of the liquid fractions proved that no considerable inhibitors to Saccharomyces cerevisiae were produced in hydrothermal pretreatment. Given the high sugar recovery, enzymatic digestibility, and ethanol yield, production of bioethanol by hydrothermal pretreatment could be a promising way of valorization of date palm residues in this region. Chuanji Fang, Jens Ejbye Schmidt, Iwona Cybulska, Grzegorz P. Brudecki, Christian Grundahl Frankær, and Mette Hedegaard Thomsen Copyright © 2015 Chuanji Fang et al. All rights reserved. Specialized Bioactive Microbial Metabolites: From Gene to Product Wed, 05 Aug 2015 12:07:51 +0000 Flavia Marinelli, Olga Genilloud, Victor Fedorenko, and Eliora Z. Ron Copyright © 2015 Flavia Marinelli et al. All rights reserved. A Novel Microbisporicin Producer Identified by Early Dereplication during Lantibiotic Screening Tue, 04 Aug 2015 11:50:53 +0000 With the increasing need of effective antibiotics against multi-drug resistant pathogens, lantibiotics are an attractive option of a new class of molecules. They are ribosomally synthetized and posttranslationally modified peptides possessing potent antimicrobial activity against aerobic and anaerobic Gram-positive pathogens, including those increasingly resistant to β-lactams and glycopeptides. Some of them (actagardine, mersacidin, planosporicin, and microbisporicin) inhibit cell wall biosynthesis in pathogens and their effect is not antagonized by vancomycin. Hereby, we apply an efficient strategy for lantibiotic screening to 240 members of a newly described genus of filamentous actinomycetes, named Actinoallomurus, that is considered a yet-poorly-exploited promising source for novel bioactive metabolites. By combining antimicrobial differential assay against Staphylococcus aureus and its L-form (also in the presence of a β-lactamase cocktail or Ac-Lys-D-alanyl-D-alanine tripeptide), with LC-UV-MS dereplication coupled with bioautography, a novel producer of the potent microbisporicin complex was rapidly identified. Under the commercial name of NAI-107, it is currently in late preclinical phase for the treatment of multi-drug resistant Gram-positive pathogens. To our knowledge, this is the first report on a lantibiotic produced by an Actinoallomurus sp. and on a microbisporicin producer not belonging to the Microbispora genus. Lucia Carrano, Monica Abbondi, Paola Turconi, Gianpaolo Candiani, and Flavia Marinelli Copyright © 2015 Lucia Carrano et al. All rights reserved. Toxicity of Environmental Contaminants Tue, 04 Aug 2015 11:40:21 +0000 Sunil Kumar, Patrick Hettiaratchi, Nanjappa Ashwath, and Petros Gikas Copyright © 2015 Sunil Kumar et al. All rights reserved. Improved Production of Sublancin 168 Biosynthesized by Bacillus subtilis 168 Using Chemometric Methodology and Statistical Experimental Designs Mon, 03 Aug 2015 12:05:52 +0000 Sublancin 168, as a distinct S-linked antimicrobial glycopeptide produced by Bacillus subtilis 168, is effective in killing specific microorganisms. However, the reported yield of sublancin 168 is at a low level of no more than 60 mg from 1 L fermentation culture of B. subtilis 168 by using the method in the literature. Thus optimization of fermentation condition for efficiently producing sublancin 168 is required. Here, Box-Behnken design was used to determine the optimal combination of three fermentation parameters, namely, corn powder, soybean meal, and temperature that were identified previously by Plackett-Burman design and the steepest ascent experiment. Subsequently, based on the response surface methodology, the quadratic regression model for optimally producing sublancin 168 was developed, and the optimal combination of culture parameters for maximum sublancin 168 production of 129.72 mg/L was determined as corn powder 28.49 g/L, soybean meal 22.99 g/L, and incubation temperature 30.8°C. The results showed that sublancin 168 production obtained experimentally was coincident with predicted value of 125.88 mg/L, and the developed model was proved to be adequate, and the aim of efficiently producing sublancin 168 was achieved. Shengyue Ji, Weili Li, Haiyun Xin, Shan Wang, and Binyun Cao Copyright © 2015 Shengyue Ji et al. All rights reserved. Bioconcentration and Acute Intoxication of Brazilian Freshwater Fishes by the Methyl Parathion Organophosphate Pesticide Mon, 03 Aug 2015 11:49:36 +0000 Three species of freshwater Brazilian fishes (pacu, Piaractus mesopotamicus; piavussu, Leporinus macrocephalus, and curimbatá, Prochilodus lineatus) were exposed to an acute dose of 5 ppm methyl parathion organophosphate pesticide. Three to five individuals per species were exposed, one at a time, to 40 liters tap water spiked with Folidol 600. Pesticide concentrations and cholinesterase (ChE) activities were evaluated in serum, liver, brain, heart, and muscle. The bioconcentration of methyl parathion was similar for all studied fishes. Brain tissue showed the highest pesticide concentration, reaching 80 ppm after exposure for 30 min to methyl parathion. Three to 5 hours of 5 ppm methyl parathion exposure provoked the death of all P. lineatus at 92% brain AChE inhibition, whereas fish from the other two species survived for up to 78 hours with less than 80% brain AChE inhibition. Our results indicate that acute toxic effects of methyl parathion to fish are correlated with brain AChE sensitivity to methyl paraoxon. João Bosco de Salles, Renato Matos Lopes, Cristiane M. C. de Salles, Vicente P. F. Cassano, Manildo Marcião de Oliveira, Vera L. F. Cunha Bastos, and Jayme Cunha Bastos Copyright © 2015 João Bosco de Salles et al. All rights reserved. Considerable Variation of Antibacterial Activity of Cu Nanoparticles Suspensions Depending on the Storage Time, Dispersive Medium, and Particle Sizes Mon, 03 Aug 2015 11:46:27 +0000 Suspensions of Cu nanoparticles are promising for creating the new class of alternative antimicrobial products. In this study we examined copper nanoparticles of various sizes obtained by the method of wire electric explosion: nanopowder average size 50 nm (Cu 50) and 100 nm (Cu 100). The paper presents the complex study of the influence of physicochemical properties such as particle size and concentration of the freshly prepared and 24-hour suspensions of Cu nanoparticles in distilled water and physiological solution upon their toxicity to bacteria E. coli M-17. Ionic solution of Cu2+ and sodium dichloroisocyanurate was used for comparison study. It has been shown that decrease in the nanoparticle size leads to changes in the correlation between toxicity and concentration as toxicity peaks are observed at low concentrations (0.0001⋯0.01 mg/L). It has been observed that antibacterial properties of Cu 50 nanoparticle suspensions are ceased after 24-hour storage, while for Cu 100 suspensions no correlation between antibacterial properties and storage time has been noted. Cu 100 nanoparticle suspensions at 10 mg/L concentration display higher toxicity at substituting physiological solution for water than Cu 50 suspensions. Dependence of the toxicity on the mean particle aggregates size in suspension was not revealed. Olga V. Zakharova, Anna Yu. Godymchuk, Alexander A. Gusev, Svyatoslav I. Gulchenko, Inna A. Vasyukova, and Denis V. Kuznetsov Copyright © 2015 Olga V. Zakharova et al. All rights reserved. Antibacterial Discovery and Development: From Gene to Product and Back Mon, 03 Aug 2015 11:35:01 +0000 Concern over the reports of antibiotic-resistant bacterial infections in hospitals and in the community has been publicized in the media, accompanied by comments on the risk that we may soon run out of antibiotics as a way to control infectious disease. Infections caused by Enterococcus faecium, Staphylococcus aureus, Klebsiella species, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and other Enterobacteriaceae species represent a major public health burden. Despite the pharmaceutical sector’s lack of interest in the topic in the last decade, microbial natural products continue to represent one of the most interesting sources for discovering and developing novel antibacterials. Research in microbial natural product screening and development is currently benefiting from progress that has been made in other related fields (microbial ecology, analytical chemistry, genomics, molecular biology, and synthetic biology). In this paper, we review how novel and classical approaches can be integrated in the current processes for microbial product screening, fermentation, and strain improvement. Victor Fedorenko, Olga Genilloud, Liliya Horbal, Giorgia Letizia Marcone, Flavia Marinelli, Yossi Paitan, and Eliora Z. Ron Copyright © 2015 Victor Fedorenko et al. All rights reserved. Biologically Active Metabolites Synthesized by Microalgae Mon, 03 Aug 2015 11:25:43 +0000 Microalgae are microorganisms that have different morphological, physiological, and genetic traits that confer the ability to produce different biologically active metabolites. Microalgal biotechnology has become a subject of study for various fields, due to the varied bioproducts that can be obtained from these microorganisms. When microalgal cultivation processes are better understood, microalgae can become an environmentally friendly and economically viable source of compounds of interest, because production can be optimized in a controlled culture. The bioactive compounds derived from microalgae have anti-inflammatory, antimicrobial, and antioxidant activities, among others. Furthermore, these microorganisms have the ability to promote health and reduce the risk of the development of degenerative diseases. In this context, the aim of this review is to discuss bioactive metabolites produced by microalgae for possible applications in the life sciences. Michele Greque de Morais, Bruna da Silva Vaz, Etiele Greque de Morais, and Jorge Alberto Vieira Costa Copyright © 2015 Michele Greque de Morais et al. All rights reserved. Violacein: Properties and Production of a Versatile Bacterial Pigment Mon, 03 Aug 2015 11:20:01 +0000 Violacein-producing bacteria, with their striking purple hues, have undoubtedly piqued the curiosity of scientists since their first discovery. The bisindole violacein is formed by the condensation of two tryptophan molecules through the action of five proteins. The genes required for its production, vioABCDE, and the regulatory mechanisms employed have been studied within a small number of violacein-producing strains. As a compound, violacein is known to have diverse biological activities, including being an anticancer agent and being an antibiotic against Staphylococcus aureus and other Gram-positive pathogens. Identifying the biological roles of this pigmented molecule is of particular interest, and understanding violacein’s function and mechanism of action has relevance to those unmasking any of its commercial or therapeutic benefits. Unfortunately, the production of violacein and its related derivatives is not easy and so various groups are also seeking to improve the fermentative yields of violacein through genetic engineering and synthetic biology. This review discusses the recent trends in the research and production of violacein by both natural and genetically modified bacterial strains. Seong Yeol Choi, Kyoung-hye Yoon, Jin Il Lee, and Robert J. Mitchell Copyright © 2015 Seong Yeol Choi et al. All rights reserved. Comparison of Simultaneous Nitrification and Denitrification for Three Different Reactors Mon, 03 Aug 2015 11:18:36 +0000 Discharge of high NH4-N containing wastewater into water bodies has become a critical and serious issue due to its negative impact on water and environmental quality. In this research, the performance of three different reactors was assessed and compared with regard to the removal of NH4-N from wastewater. The highest nitrogen removal efficiency of 98.3% was found when the entrapped sludge reactor (ESR), in which the sludge was entrapped in polyethylene glycol polymer, was used. Under intermittent aeration, nitrification and denitrification occurred simultaneously in the aerobic and anaerobic periods. Moreover, internal carbon was consumed efficiently for denitrification. On the other hand, internal carbon consumption was not found to occur in the suspended sludge reactor (SSR) and the mixed sludge reactor (MSR) and this resulted in nitrogen removal efficiencies of SSR and MSR being 64.7 and 45.1%, respectively. Nitrification and denitrification were the main nitrogen removal processes in the aerobic and anaerobic periods, respectively. However, due to the absence of sufficient organic carbon, denitrification was uncompleted resulting in high NO3-N contents in the effluent. W. Khanitchaidecha, A. Nakaruk, P. Koshy, and K. Futaba Copyright © 2015 W. Khanitchaidecha et al. All rights reserved. Isolation and Characterization of Hydrocarbon-Degrading Yeast Strains from Petroleum Contaminated Industrial Wastewater Mon, 03 Aug 2015 10:49:23 +0000 Two yeast strains are enriched and isolated from industrial refinery wastewater. These strains were observed for their ability to utilize several classes of petroleum hydrocarbons substrates, such as n-alkanes and aromatic hydrocarbons as a sole carbon source. Phylogenetic analysis based on the D1/D2 variable domain and the ITS-region sequences indicated that strains HC1 and HC4 were members of the genera Candida and Trichosporon, respectively. The mechanism of hydrocarbon uptaking by yeast, Candida, and Trichosporon has been studied by means of the kinetic analysis of hydrocarbons-degrading yeasts growth and substrate assimilation. Biodegradation capacity and biomass quantity were daily measured during twelve days by gravimetric analysis and gas chromatography coupled with mass spectrometry techniques. Removal of n-alkanes indicated a strong ability of hydrocarbon biodegradation by the isolated yeast strains. These two strains grew on long-chain n-alkane, diesel oil, and crude oil but failed to grow on short-chain n-alkane and aromatic hydrocarbons. Growth measurement attributes of the isolates, using n-hexadecane, diesel oil, and crude oil as substrates, showed that strain HC1 had better degradation for hydrocarbon substrates than strain HC4. In conclusion, these yeast strains can be useful for the bioremediation process and decreasing petroleum pollution in wastewater contaminated with petroleum hydrocarbons. Boutheina Gargouri, Najla Mhiri, Fatma Karray, Fathi Aloui, and Sami Sayadi Copyright © 2015 Boutheina Gargouri et al. All rights reserved. Prenatal Exposure to Sodium Arsenite Alters Placental Glucose 1, 3, and 4 Transporters in Balb/c Mice Mon, 03 Aug 2015 09:36:50 +0000 Inorganic arsenic (iAs) exposure induces a decrease in glucose type 4 transporter (GLUT4) expression on the adipocyte membrane, which may be related to premature births and low birth weight infants in women exposed to iAs at reproductive age. The aim of this study was to analyze the effect of sodium arsenite (NaAsO2) exposure on GLUT1, GLUT3, and GLUT4 protein expression and on placental morphology. Female Balb/c mice () were exposed to 0, 12, and 20 ppm of NaAsO2 in drinking water from 8th to 18th day of gestation. Morphological changes and GLUT1, GLUT3, and GLUT4 expression were evaluated in placentas by immunohistochemical and image analysis and correlated with iAs and arsenical species concentration, which were quantified by atomic absorption spectroscopy. NaAsO2 exposure induced a significant decrease in fetal and placental weight () and increases in infarctions and vascular congestion. Whereas GLUT1 expression was unchanged in placentas from exposed group, GLUT3 expression was found increased. In contrast, GLUT4 expression was significantly lower () in placentas from females exposed to 12 ppm. The decrease in placental GLUT4 expression might affect the provision of adequate fetal nutrition and explain the low fetal weight observed in the exposed groups. Daniela Sarahí Gutiérrez-Torres, Carmen González-Horta, Luz María Del Razo, Rocío Infante-Ramírez, Ernesto Ramos-Martínez, Margarita Levario-Carrillo, and Blanca Sánchez-Ramírez Copyright © 2015 Daniela Sarahí Gutiérrez-Torres et al. All rights reserved. Gestational Hypertension and Organophosphorus Pesticide Exposure: A Cross-Sectional Study Mon, 03 Aug 2015 09:34:12 +0000 Hypertension is the most common medical problem encountered during pregnancy, complicating 2-3% of pregnancies. High blood pressure (BP) with diastolic BP ≥ 90 mm Hg and/or systolic BP ≥ 140 mm Hg arising after week 22 of pregnancy and resolving after delivery is defined as gestational hypertension (GHY). The aim of this cross-sectional study was to investigate whether occupational and/or environmental exposure to organophosphorus (OP) pesticide affects GHY. Women at approximately 22 weeks of gestation were recruited. OP pesticide exposure in the first trimester of pregnancy was classified into four categories: no exposure, indirect exposure, domestic exposure, and occupational exposure. Application of the exclusion criteria left 2203 participants (mean age 30.4 ± 11.6 years). Data analysis showed that in women with indirect OP pesticide exposure the incidence of GHY was slightly higher than that in the world population, whereas domestic exposure involved a 7% increase and occupational exposure a 12% increase. Analysis of the pesticides used by participants highlighted a possible role for malathion and diazinon (adjusted OR 1.09 and 1.14, resp.). Further investigation of exposed workers and the general population is clearly warranted given the broad diffusion of OP pesticides and their possible public health impact, maybe by including a wider range of health outcomes. Caterina Ledda, Maria Fiore, Lory Santarelli, Massimo Bracci, Giuseppe Mascali, Maria Grazia D’Agati, Alfredo Busà, Margherita Ferrante, and Venerando Rapisarda Copyright © 2015 Caterina Ledda et al. All rights reserved. ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in LTEP-a-2 Cells Mon, 03 Aug 2015 09:02:32 +0000 Owing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥0.01 μg/mL) significantly inhibited proliferation () and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30–40% corresponding to significant depletion (approximately 70–80%) in GSH content in LTEP-a-2 cells (), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment. Caixia Wang, Xiaoke Hu, Yan Gao, and Yinglu Ji Copyright © 2015 Caixia Wang et al. All rights reserved. Study of Cytotoxic Effects of Benzonitrile Pesticides Mon, 03 Aug 2015 06:23:52 +0000 The benzonitrile herbicides bromoxynil, chloroxynil, dichlobenil, and ioxynil have been used actively worldwide to control weeds in agriculture since 1970s. Even though dichlobenil is prohibited in EU since 2008, studies addressing the fate of benzonitrile herbicides in the environment show that some metabolites of these herbicides are very persistent. We tested the cytotoxic effects of benzonitrile herbicides and their microbial metabolites using two human cell lines, Hep G2 and HEK293T, representing liver and kidneys as potential target organs in humans. The cell viability and proliferation were determined by MTT test and RTCA DP Analyzer system, respectively. The latter allows real-time monitoring of the effect of added substances. As the cytotoxic compounds could compromise cell membrane integrity, the lactate dehydrogenase test was performed as well. We observed high toxic effects of bromoxynil, chloroxynil, and ioxynil on both tested cell lines. In contrast, we determined only low inhibition of cell growth in presence of dichlobenil and microbial metabolites originating from the tested herbicides. Petra Lovecka, Marketa Thimova, Petra Grznarova, Jan Lipov, Zdenek Knejzlik, Hana Stiborova, Tjokorda Gde Tirta Nindhia, Katerina Demnerova, and Tomas Ruml Copyright © 2015 Petra Lovecka et al. All rights reserved. Subtoxic Doses of Cadmium Modulate Inflammatory Properties of Murine RAW 264.7 Macrophages Mon, 03 Aug 2015 06:21:31 +0000 Cadmium (Cd) is a toxic heavy metal that exhibits various adverse effects in the human and animal organism. Its resemblance to essential metals such as calcium, iron, and zinc leads to an unintended uptake in cells after intake through inhalation and ingestion. In this study we investigated the toxicity and the immunomodulatory potential of Cd in nonactivated and activated murine macrophages (i.e., cell line RAW 264.7). Cadmium alone caused a dose-dependent decreased viability of exposed cells. Subtoxic Cd concentrations delayed cell death in macrophages, resulting from cytotoxic storm, producing reactive oxygen species (ROS) and nitric oxide (NO), in response to their stimulation by bacterial antigens via pattern-recognition receptors (PRRs). In addition, production of selected pro- and anti-inflammatory cytokines, the chemokine CXCL1 (KC), and NO was determined. We observed that proinflammatory IL-1β and also CXCL1 were highly upregulated whereas anti-inflammatory or regulatory cytokines IL-6 and IL-10 were suppressed by 10 µM Cd. Also production of antibacterial NO was significantly reduced through exposure to 10 µM Cd, maybe explaining better survival of macrophages. Additionally, we could show by analysis via ICP-MS that different effects of Cd in nonactivated and activated macrophages definitely did not result from different Cd uptake rates. Sina Riemschneider, Martin Herzberg, and Jörg Lehmann Copyright © 2015 Sina Riemschneider et al. All rights reserved. Pollution Problem in River Kabul: Accumulation Estimates of Heavy Metals in Native Fish Species Mon, 03 Aug 2015 06:20:28 +0000 The contamination of aquatic systems with heavy metals is affecting the fish population and hence results in a decline of productivity rate. River Kabul is a transcountry river originating at Paghman province in Afghanistan and inters in Khyber Pakhtunkhwa province of Pakistan and it is the major source of irrigation and more than 54 fish species have been reported in the river. Present study aimed at the estimation of heavy metals load in the fish living in River Kabul. Heavy metals including chromium, nickel, copper, zinc, cadmium, and lead were determined through atomic absorption spectrophotometer after tissue digestion by adopting standard procedures. Concentrations of these metals were recorded in muscles and liver of five native fish species, namely, Wallago attu, Aorichthys seenghala, Cyprinus carpio, Labeo dyocheilus, and Ompok bimaculatus. The concentrations of chromium, nickel, copper, zinc, and lead were higher in both of the tissues, whereas the concentration of cadmium was comparatively low. However, the concentration of metals was exceeding the RDA (Recommended Dietary Allowance of USA) limits. Hence, continuous fish consumption may create health problems for the consumers. The results of the present study are alarming and suggest implementing environmental laws and initiation of a biomonitoring program of the river. Habib Ahmad, Ali Muhammad Yousafzai, Muhammad Siraj, Rashid Ahmad, Israr Ahmad, Muhammad Shahid Nadeem, Waqar Ahmad, Nazia Akbar, and Khushi Muhammad Copyright © 2015 Habib Ahmad et al. All rights reserved. Green Tea Increases the Concentration of Total Mercury in the Blood of Rats following an Oral Fish Tissue Bolus Sun, 02 Aug 2015 13:40:25 +0000 Fish has many health benefits but is also the most common source of methylmercury. The bioavailability of methylmercury in fish may be affected by other meal components. In this study, the effect of green tea on the bioavailability of methylmercury from an oral bolus of fish muscle tissue was studied in rats and compared to a water treated control group and a group treated with meso-2,3-dimercaptosuccinic acid (DMSA), a compound used medically to chelate mercury. Rats were given a single oral dose of fish tissue via gavage and one of the treatments. Rats were given access to food for 3 h at 12 h intervals. They were dosed with each of the treatments with each meal. Blood samples were collected for 95 hours. Green tea significantly increased the concentration of total mercury in blood relative to the control, whereas DMSA significantly decreased it. In addition, feeding caused a slight increase in blood mercury for several meals following the initial dose. Elsa M. Janle, Helene Freiser, Christopher Manganais, Tzu-Ying Chen, Bruce A. Craig, and Charles R. Santerre Copyright © 2015 Elsa M. Janle et al. All rights reserved. Evaluation of Ricinus communis L. for the Phytoremediation of Polluted Soil with Organochlorine Pesticides Sun, 02 Aug 2015 13:38:40 +0000 Phytoremediation is an attractive alternative to conventional treatments of soil due to advantages such as low cost, large application areas, and the possibility of in situ treatment. This study presents the assessment of phytoremediation processes conducted under controlled experimental conditions to evaluate the ability of Ricinus communis L., tropical plant species, to promote the degradation of 15 persistent organic pollutants (POPs), in a 66-day period. The contaminants tested were hexachlorocyclohexane (HCH), DDT, heptachlor, aldrin, and others. Measurements made in rhizosphere soil indicate that the roots of the studied species reduce the concentration of pesticides. Results obtained during this study indicated that the higher the hydrophobicity of the organic compound and its molecular interaction with soil or root matrix the greater its tendency to concentrate in root tissues and the research showed the following trend: HCHs < diclofop-methyl < chlorpyrifos < methoxychlor < heptachlor epoxide < endrin < o,p′-DDE < heptachlor < dieldrin < aldrin < o,p′-DDT < p,p′-DDT by increasing order of log values. The experimental results confirm the importance of vegetation in removing pollutants, obtaining remediation from 25% to 70%, and demonstrated that Ricinus communis L. can be used for the phytoremediation of such compounds. Sandra Regina Rissato, Mário Sergio Galhiane, João Roberto Fernandes, Marli Gerenutti, Homero Marques Gomes, Renata Ribeiro, and Marcos Vinícius de Almeida Copyright © 2015 Sandra Regina Rissato et al. All rights reserved. Dichloroacetate Decreases Cell Health and Activates Oxidative Stress Defense Pathways in Rat Alveolar Type II Pneumocytes Sun, 02 Aug 2015 13:31:30 +0000 Dichloroacetate (DCA) is a water purification byproduct that is known to be hepatotoxic and hepatocarcinogenic and to induce peripheral neuropathy and damage macrophages. This study characterizes the effects of the haloacetate on lung cells by exposing rat alveolar type II (L2) cells to 0–24 mM DCA for 6–24 hours. Increasing DCA concentration and the combination of increasing DCA concentration plus longer exposures decrease measures of cellular health. Length of exposure has no effect on oxidative stress biomarkers, glutathione, SOD, or CAT. Increasing DCA concentration alone does not affect total glutathione or its redox ratio but does increase activity in the SOD/CAT oxidative stress defense pathway. These data suggest that alveolar type II cells rely on SOD and CAT more than glutathione to combat DCA-induced stress. Alexis Valauri-Orton, Frizzi Bschorer, and Karen K. Bernd Copyright © 2015 Alexis Valauri-Orton et al. All rights reserved. Air Pollution by Hydrothermal Volcanism and Human Pulmonary Function Sun, 02 Aug 2015 12:59:13 +0000 The aim of this study was to assess whether chronic exposure to volcanogenic air pollution by hydrothermal soil diffuse degassing is associated with respiratory defects in humans. This study was carried in the archipelago of the Azores, an area with active volcanism located in the Atlantic Ocean where Eurasian, African, and American lithospheric plates meet. A cross-sectional study was performed on a study group of 146 individuals inhabiting an area where volcanic activity is marked by active fumarolic fields and soil degassing (hydrothermal area) and a reference group of 359 individuals inhabiting an area without these secondary manifestations of volcanism (nonhydrothermal area). Odds ratio (OR) and 95% confidence intervals (CIs) were adjusted for age, gender, fatigue, asthma, and smoking. The OR for restrictive defects and for exacerbation of obstructive defects (COPD) in the hydrothermal area was 4.4 (95% CI 1.78–10.69) and 3.2 (95% CI 1.82–5.58), respectively. Increased prevalence of restrictions and all COPD severity ranks (mild, moderate, and severe) was observed in the population from the hydrothermal area. These findings may assist health officials in advising and keeping up with these populations to prevent and minimize the risk of respiratory diseases. Diana Linhares, Patrícia Ventura Garcia, Fátima Viveiros, Teresa Ferreira, and Armindo dos Santos Rodrigues Copyright © 2015 Diana Linhares et al. All rights reserved. Three-Dimensional Modelling inside a Differential Pressure Laminar Flow Bioreactor Filled with Porous Media Sun, 02 Aug 2015 11:15:22 +0000 A three-dimensional computational fluid dynamics- (CFD-) model based on a differential pressure laminar flow bioreactor prototype was developed to further examine performance under changing culture conditions. Cell growth inside scaffolds was simulated by decreasing intrinsic permeability values and led to pressure build-up in the upper culture chamber. Pressure release by an integrated bypass system allowed continuation of culture. The specific shape of the bioreactor culture vessel supported a homogenous flow profile and mass flux at the scaffold level at various scaffold permeabilities. Experimental data showed an increase in oxygen concentration measured inside a collagen scaffold seeded with human mesenchymal stem cells when cultured in the perfusion bioreactor after 24 h compared to static culture in a Petri dish (dynamic: 11% O2 versus static: 3% O2). Computational fluid simulation can support design of bioreactor systems for tissue engineering application. Birgit Weyand, Meir Israelowitz, James Kramer, Christian Bodmer, Mariel Noehre, Sarah Strauss, Elmar Schmälzlin, Christoph Gille, Herbert P. von Schroeder, Kerstin Reimers, and Peter M. Vogt Copyright © 2015 Birgit Weyand et al. 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.