BioMed Research International: Biotechnology The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Combinatorial Control of Transgene Expression by Hypoxia-Responsive Promoter and MicroRNA Regulation for Neural Stem Cell-Based Cancer Therapy Thu, 17 Apr 2014 14:11:28 +0000 Owing to their strong migratory capacity, tumor tropism, and tumor inhibitory effect, neural stem cells (NSCs) have recently emerged as one of the most attractive gene delivery vectors for cancer therapy. However, further animal studies found that proportional NSC vectors were distributed to nontarget organs after intravenous injection and the nonspecific transgene expression led to significant cytotoxic effects in these organs. Hence, an expression cassette that controls the transgene expression within NSC vectors in a tumor site-specific manner is desired. Considering hypoxia as a hallmark of tumor microenvironment, we have developed a novel NSC vector platform coupling transcriptional targeting with microRNA (miRNA) regulation for tumor hypoxia targeting. This combinatorial vector employed a hypoxia-responsive promoter and repeated targeting sequences of an miRNA that is enriched in NSCs but downregulated upon hypoxia induction to control the transgene expression. This resulted in significantly improved hypoxic selectivity over the use of a control vector without miRNA regulation. Thus, incorporating miRNA regulation into a transcriptional targeting vector adds an extra layer of security to prevent off-target transgene expression and should be useful for the development of NSC vectors with high targeting specifcity for cancer therapy. Yumei Luo and Detu Zhu Copyright © 2014 Yumei Luo and Detu Zhu. All rights reserved. Coastal Biotechnology: Facing the Global and the Regional Changes Thu, 17 Apr 2014 10:05:48 +0000 Song Qin, Wei Zhang, and Hanzhi Lin Copyright © 2014 Song Qin et al. All rights reserved. Biodegradation and Utilization of Organophosphorus Pesticide Malathion by Cyanobacteria Thu, 17 Apr 2014 08:48:18 +0000 Three strains of filamentous Cyanobacteria were used to study their growth and utilization of organophosphorus pesticide malathion. A sharp decrease in the growth of the algal strains was observed by increasing the concentration of malathion. Amongst them Nostoc muscorum tolerated different concentrations and was recorded as the highest efficient strain for biodegradation (91%) of this compound. Moreover, carbohydrate and protein content of their cells overtopped the other strains especially at higher concentrations. The algal strains were further subjected to grow under P-limitation in absence and presence of malathion. Although, the algal growth under P-limitation recorded a very poor level, a massive enhanced growth and phosphorous content of cells were obtained when the P-limited medium was amended with malathion. This study clarified that N. muscorum with its capability to utilize malathion as a sole phosphorous source is considered as an inexpensive and efficient biotechnology for remediation of organophosphorus pesticide from contaminated wastewater. Wael M. Ibrahim, Mohamed A. Karam, Reda M. El-Shahat, and Asmaa A. Adway Copyright © 2014 Wael M. Ibrahim et al. All rights reserved. Production and Cytotoxicity of Extracellular Insoluble and Droplets of Soluble Melanin by Streptomyces lusitanus DMZ-3 Wed, 16 Apr 2014 13:49:37 +0000 A Streptomyces lusitanus DMZ-3 strain with potential to synthesize both insoluble and soluble melanins was detected. Melanins are quite distinguished based on their solubility for varied biotechnological applications. The present investigation reveals the enhanced production of insoluble and soluble melanins in tyrosine medium by a single culture. Streptomyces lusitanus DMZ-3 was characterized by 16S rRNA gene analysis. An enhanced production of 5.29 g/L insoluble melanin was achieved in a submerged bioprocess following response surface methodology. Combined interactive effect of temperature (50°C), pH (8.5), tyrosine (2.0 g/L), and beef extract (0.5 g/L) were found to be critical variables for enhanced production in central composite design analysis. An optimized indigenous slant culture system was an innovative approach for the successful production (264 mg/L) of pure soluble melanin from the droplets formed on the surface of the culture. Both insoluble and soluble melanins were confirmed and characterized by Chemical, reactions, UV, FTIR, and TLC analysis. First time, cytotoxic study of melanin using brine shrimps was reported. Maximum cytotoxic activity of soluble melanin was Lc50-0.40 µg/mL and insoluble melanin was Lc50-0.80 µg/mL. D. N. Madhusudhan, Bi Bi Zainab Mazhari, Syed G. Dastager, and Dayanand Agsar Copyright © 2014 D. N. Madhusudhan et al. All rights reserved. In Vitro and In Vivo Leishmanicidal Activity of Astronium fraxinifolium (Schott) and Plectranthus amboinicus (Lour.) Spreng against Leishmania (Viannia) braziliensis Wed, 16 Apr 2014 12:23:24 +0000 The aim of the present work was to evaluate antileishmanial activity of Astronium fraxinifolium and Plectranthus amboinicus. For the in vitro tests, essential oil of P. amboinicus (OEPA) and ethanolic extracts from A. fraxinifolium (EEAF) were incubated with 106  promastigotes of L. (Viannia) braziliensis. The OEPA was able to reduce the parasite growth after 48 h; nonetheless, all the EEAFs could totally abolish the parasite growth. For the in vivo studies, BALB/c mice were infected subcutaneously (s.c.) with 107  L. braziliensis promastigotes. Treatment was done by administering OEPA intralesionally (i.l.) for 14 days. No difference was found in lesion thickness when those animals were compared with the untreated animals. Further, golden hamsters were infected s.c. with 106  L. braziliensis promastigotes. The first protocol of treatment consisted of ethanolic leaf extract from A. fraxinifolium (ELEAF) administered i.l. for 4 days and a booster dose at the 7th day. The animals showed a significant reduction of lesion thickness in the 6th week, but it was not comparable to the animals treated with Glucantime. The second protocol consisted of 15 daily intralesional injections. The profiles of lesion thickness were similar to the standard treatment. In conclusion, in vivo studies showed a high efficacy when the infected animals were intralesionally treated with leaf ethanolic extract from A. fraxinifolium. Silvio César Gomes de Lima, Maria Jania Teixeira, José Evaldo Gonçalves Lopes Júnior, Selene Maia de Morais, Alba Fabiola Torres, Milena Aguiar Braga, Raphael Oliveira Rodrigues, Gilvandete Maria Pinheiro Santiago, Alice Costa Martins, and Aparecida Tiemi Nagao-Dias Copyright © 2014 Silvio César Gomes de Lima et al. All rights reserved. Aerobic Sludge Granulation in a Full-Scale Sequencing Batch Reactor Tue, 15 Apr 2014 14:02:21 +0000 Aerobic granulation of activated sludge was successfully achieved in a full-scale sequencing batch reactor (SBR) with 50,000 m3 d−1 for treating a town’s wastewater. After operation for 337 days, in this full-scale SBR, aerobic granules with an average SVI30 of 47.1 mL g−1, diameter of 0.5 mm, and settling velocity of 42 m h−1 were obtained. Compared to an anaerobic/oxic plug flow (A/O) reactor and an oxidation ditch (OD) being operated in this wastewater treatment plant, the sludge from full-scale SBR has more compact structure and excellent settling ability. Denaturing gradient gel electrophoresis (DGGE) analysis indicated that Flavobacterium sp., uncultured beta proteobacterium, uncultured Aquabacterium sp., and uncultured Leptothrix sp. were just dominant in SBR, whereas uncultured bacteroidetes were only found in A/O and OD. Three kinds of sludge had a high content of protein in extracellular polymeric substances (EPS). X-ray fluorescence (XRF) analysis revealed that metal ions and some inorganics from raw wastewater precipitated in sludge acted as core to enhance granulation. Raw wastewater characteristics had a positive effect on the granule formation, but the SBR mode operating with periodic feast-famine, shorter settling time, and no return sludge pump played a crucial role in aerobic sludge granulation. Jun Li, Li-Bin Ding, Ang Cai, Guo-Xian Huang, and Harald Horn Copyright © 2014 Jun Li et al. All rights reserved. Fungal Laccases Degradation of Endocrine Disrupting Compounds Tue, 15 Apr 2014 00:00:00 +0000 Over the past decades, water pollution by trace organic compounds (ng/L) has become one of the key environmental issues in developed countries. This is the case of the emerging contaminants called endocrine disrupting compounds (EDCs). EDCs are a new class of environmental pollutants able to mimic or antagonize the effects of endogenous hormones, and are recently drawing scientific and public attention. Their widespread presence in the environment solicits the need of their removal from the contaminated sites. One promising approach to face this challenge consists in the use of enzymatic systems able to react with these molecules. Among the possible enzymes, oxidative enzymes are attracting increasing attention because of their versatility, the possibility to produce them on large scale, and to modify their properties. In this study five different EDCs were treated with four different fungal laccases, also in the presence of both synthetic and natural mediators. Mediators significantly increased the efficiency of the enzymatic treatment, promoting the degradation of substrates recalcitrant to laccase oxidation. The laccase showing the best performances was chosen to further investigate its oxidative capabilities against micropollutant mixtures. Improvement of enzyme performances in nonylphenol degradation rate was achieved through immobilization on glass beads. Gemma Macellaro, Cinzia Pezzella, Paola Cicatiello, Giovanni Sannia, and Alessandra Piscitelli Copyright © 2014 Gemma Macellaro et al. All rights reserved. Soybean Seeds: A Practical Host for the Production of Functional Subunit Vaccines Mon, 14 Apr 2014 00:00:00 +0000 Soybean seeds possess several inherent qualities that make them an ideal host for the production of biopharmaceuticals when compared with other plant-based and non-plant-based recombinant expression systems (e.g., low cost of production, high protein to biomass ratio, long-term stability of seed proteins under ambient conditions, etc.). To demonstrate the practicality and feasibility of this platform for the production of subunit vaccines, we chose to express and characterize a nontoxic form of S. aureus enterotoxin B (mSEB) as a model vaccine candidate. We show that soy-mSEB was produced at a high vaccine to biomass ratio and represented ~76 theoretical doses of human vaccine per single soybean seed. We localized the model vaccine candidate both intracellularly and extracellularly and found no difference in mSEB protein stability or accumulation relative to subcellular environment. We also show that the model vaccine was biochemically and immunologically similar to native and recombinant forms of the protein produced in a bacterial expression system. Immunization of mice with seed extracts containing mSEB mounted a significant immune response within 14 days of the first injection. Taken together, our results highlight the practicality of soybean seeds as a potential platform for the production of functional subunit vaccines. Laura C. Hudson, Renu Garg, Kenneth L. Bost, and Kenneth J. Piller Copyright © 2014 Laura C. Hudson et al. All rights reserved. Biologic Propensities and Phytochemical Profile of Vangueria madagascariensis J. F. Gmelin (Rubiaceae): An Underutilized Native Medicinal Food Plant from Africa Thu, 10 Apr 2014 14:05:00 +0000 Vangueria madagascariensis (VM), consumed for its sweet-sour fruits, is used as a biomedicine for the management of diabetes and bacterial infections in Africa. The study aims to assess the potential of VM on α-amylase, α-glucosidase, glucose movement, and antimicrobial activity. The antioxidant properties were determined by measuring the FRAP, iron chelating activity, and abilities to scavenge DPPH, HOCl, ∙OH, and NO radicals. Leaf decoction, leaf methanol, and unripe fruit methanol extracts were observed to significantly inhibit α-amylase. Active extracts against α-glucosidase were unripe fruit methanol, unripe fruit decoction, leaf decoction, and ripe fruit methanol, which were significantly lower than acarbose. Kinetic studies revealed a mixed noncompetitive type of inhibition. Leaf methanolic extract was active against S. aureus and E. coli. Total phenolic content showed a strong significant positive correlation () with FRAP. Methanolic leaf extract showed a more efficient NO scavenging potential and was significantly lower than ascorbic acid. Concerning ∙OH-mediated DNA degradation, only the methanol extracts of leaf, unripe fruit, and ripe fruit had IC50 values which were significantly lower than α-tocopherol. Given the dearth of information on the biologic propensities of VM, this study has established valuable primary information which has opened new perspectives for further pharmacological research. Nelvana Ramalingum and M. Fawzi Mahomoodally Copyright © 2014 Nelvana Ramalingum and M. Fawzi Mahomoodally. All rights reserved. Arrabidaea chica Hexanic Extract Induces Mitochondrion Damage and Peptidase Inhibition on Leishmania spp. Wed, 09 Apr 2014 08:54:22 +0000 Currently available leishmaniasis treatments are limited due to severe side effects. Arrabidaea chica is a medicinal plant used in Brazil against several diseases. In this study, we investigated the effects of 5 fractions obtained from the crude hexanic extract of A. chica against Leishmania amazonensis and L. infantum, as well as on the interaction of these parasites with host cells. Promastigotes were treated with several concentrations of the fractions obtained from A. chica for determination of their minimum inhibitory concentration (MIC). In addition, the effect of the most active fraction (B2) on parasite’s ultrastructure was analyzed by transmission electron microscopy. To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases, parasites lysates were treated with the inhibitory and subinhibitory concentrations of the B2 fraction. The minimum inhibitory concentration of B2 fraction was 37.2 and 18.6 μg/mL for L. amazonensis and L. infantum, respectively. Important ultrastructural alterations as mitochondrial swelling with loss of matrix content and the presence of vesicles inside this organelle were observed in treated parasites. Moreover, B2 fraction was able to completely inhibit the peptidase activity of promastigotes at pH 5.5. The results presented here further support the use of A. chica as an interesting source of antileishmanial agents. Igor A. Rodrigues, Mariana M. B. Azevedo, Francisco C. M. Chaves, Celuta S. Alviano, Daniela S. Alviano, and Alane B. Vermelho Copyright © 2014 Igor A. Rodrigues et al. All rights reserved. Chitinase from a Novel Strain of Serratia marcescens JPP1 for Biocontrol of Aflatoxin: Molecular Characterization and Production Optimization Using Response Surface Methodology Wed, 09 Apr 2014 07:50:43 +0000 Chitinase is one of the most important mycolytic enzymes with industrial significance, and produced by a number of organisms. A chitinase producing isolate Serratia marcescens JPP1 was obtained from peanut hulls in Jiangsu Province, China, and exhibited antagonistic activity against aflatoxins. In this study, we describe the optimization of medium composition with increased production of chitinase for the selected bacteria using statistical methods: Plackett-Burman design was applied to find the key ingredients, and central composite design of response surface methodology was used to optimize the levels of key ingredients for the best yield of chitinase. Maximum chitinase production was predicted to be 23.09 U/mL for a 2.1-fold increase in medium containing 12.70 g/L colloidal chitin, 7.34 g/L glucose, 5.00 g/L peptone, 1.32 g/L (NH4)2SO4, 0.7 g/L K2HPO4, and 0.5 g/L MgSO4·7H2O. Polymerase chain reaction (PCR) amplification of the JPP1 chitinase gene was performed and obtained a 1,789 bp nucleotide sequence; its open reading frame encoded a protein of 499 amino acids named as ChiBjp. Kai Wang, Pei-sheng Yan, and Li-xin Cao Copyright © 2014 Kai Wang et al. All rights reserved. Evaluation of Drought Tolerance of the Vietnamese Soybean Cultivars Provides Potential Resources for Soybean Production and Genetic Engineering Mon, 07 Apr 2014 14:02:02 +0000 Drought is one of the greatest constraints to soybean production in many countries, including Vietnam. Although a wide variety of the newly produced cultivars have been produced recently in Vietnam through classical breeding to cope with water shortage, little knowledge of their molecular and physiological responses to drought has been discovered. This study was conducted to quickly evaluate drought tolerance of thirteen local soybean cultivars for selection of the best drought-tolerant cultivars for further field test. Differences in drought tolerance of cultivars were assessed by root and shoot lengths, relative water content, and drought-tolerant index under both normal and drought conditions. Our data demonstrated that DT51 is the strongest drought-tolerant genotype among all the tested cultivars, while the highest drought-sensitive phenotype was observed with MTD720. Thus, DT51 could be subjected to further yield tests in the field prior to suggesting it for use in production. Due to their contrasting drought-tolerant phenotypes, DT51 and MTD720 provide excellent genetic resources for further studies underlying mechanisms regulating drought responses and gene discovery. Our results provide vital information to support the effort of molecular breeding and genetic engineering to improve drought tolerance of soybean. Nguyen Binh Anh Thu, Quang Thien Nguyen, Xuan Lan Thi Hoang, Nguyen Phuong Thao, and Lam-Son Phan Tran Copyright © 2014 Nguyen Binh Anh Thu et al. All rights reserved. Streptomyces flavogriseus HS1: Isolation and Characterization of Extracellular Proteases and Their Compatibility with Laundry Detergents Sun, 06 Apr 2014 14:13:56 +0000 The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5–11) and temperature (25–70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca2+ and Mg2+. EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level. Sofiane Ghorbel, Maher Kammoun, Hala Soltana, Moncef Nasri, and Noomen Hmidet Copyright © 2014 Sofiane Ghorbel et al. All rights reserved. Antioxidant Activity of Essential Oil and Extracts of Valeriana jatamansi Roots Sun, 06 Apr 2014 11:47:37 +0000 Valeriana jatamansi is an indigenous medicinal plant used in the treatment of a number of diseases. In the present study, chemical composition of the essential oil was determined by GC-MS. Seven major components were identified in Valeriana jatamansi essential oil, namely, β-vatirenene, β-patchoulene, dehydroaromadendrene, β-gurjunene, patchoulic alcohol, β-guaiene, and α-muurolene. Methanolic, aqueous, and chloroform extracts of Valeriana jatamansi roots were also prepared and analyzed for their polyphenols and flavonoid content. Antioxidant activity of essential oil and different extracts of Valeriana jatamansi roots was determined by DPPH radical scavenging and chelation power assay. A linear correlation has been obtained by comparing the antioxidant activity and polyphenols and flavonoid content of the extracts. Results indicated that antioxidant activity of methanolic extract could be attributed to the presence of rich amount of polyphenols and flavonoid. Essential oil of Valeriana jatamansi roots showed moderate antioxidant activity. Sakshima Thusoo, Sahil Gupta, Rasleen Sudan, Jaspreet Kour, Sahil Bhagat, Rashid Hussain, and Madhulika Bhagat Copyright © 2014 Sakshima Thusoo et al. All rights reserved. A Plant-Produced Antigen Elicits Potent Immune Responses against West Nile Virus in Mice Thu, 03 Apr 2014 11:49:36 +0000 We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μg DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μg of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability. Junyun He, Li Peng, Huafang Lai, Jonathan Hurtado, Jake Stahnke, and Qiang Chen Copyright © 2014 Junyun He et al. All rights reserved. Optimization of Extraction and Enrichment of Steroidal Alkaloids from Bulbs of Cultivated Fritillaria cirrhosa Tue, 01 Apr 2014 09:03:49 +0000 The bulbs of cultivated Fritillaria cirrhosa (BCFC) are used in China both for food and folk medicine due to its powerful biological activities. The aim of this study is to optimize the extraction and enrichment conditions of alkaloids from BCFC. Firstly, the orthogonal experimental design was used to optimize and evaluate four variables (ethanol concentration, solid-liquid ratio, extraction time, and temperature). Thereafter, resin adsorption was as a means to enrich alkaloids. Among 16 tested resins, H-103 resin presented higher adsorption capacity and desorption ratio. The equilibrium experimental data of the adsorption of total alkaloids, imperialine, and peimisine were well-fitted to the pseudo-first-order kinetics model, Langmuir and Freundlich isotherms models. Finally, in order to optimize the parameters for purifying alkaloids, dynamic adsorption and desorption tests were carried out. After one run treatment with H-103 resin, the contents of total alkaloids, imperialine, and peimisine in the product were 21.40-, 18.31-, and 22.88-fold increased with recovery yields of 94.43%, 90.57%, and 96.16%, respectively. Dongdong Wang, Shu Wang, Qingdan Du, Nanyi Wang, Simei Liu, Xiaoxia Wang, and Jinghui Jiang Copyright © 2014 Dongdong Wang et al. All rights reserved. A Noncellulosomal Mannanase26E Contains a CBM59 in Clostridium cellulovorans Thu, 27 Mar 2014 14:12:20 +0000 A multicomponent enzyme-complex prevents efficient degradation of the plant cell wall for biorefinery. In this study, the method of identifying glycoside hydrolases (GHs) to degrade hemicelluloses was demonstrated. The competence of C. cellulovorans, which changes to be suitable for degradation of each carbon source, was used for the method. C. cellulovorans was cultivated into locust bean gum (LBG) that is composed of galactomannan. The proteins produced by C. cellulovorans were separated into either fractions binding to crystalline cellulose or not. Proteins obtained from each fraction were further separated by SDS-PAGE and were stained with Coomassie Brilliant Blue and were detected for mannanase activity. The proteins having the enzymatic activity for LBG were cut out and were identified by mass spectrometry. As a result, four protein bands were classified into glycosyl hydrolase family 26 (GH26) mannanases. One of the identified mannanases, Man26E, contains a carbohydrate-binding module (CBM) family 59, which binds to xylan, mannan, and Avicel. Although mannose and galactose are the same as a hexose, the expression patterns of the proteins from C. cellulovorans were quite different. More interestingly, zymogram for mannanase activity showed that Man26E was detected in only LBG medium. Kosuke Yamamoto and Yutaka Tamaru Copyright © 2014 Kosuke Yamamoto and Yutaka Tamaru. All rights reserved. Antioxidants, Phytochemicals, and Cytotoxicity Studies on Phaleria macrocarpa (Scheff.) Boerl Seeds Thu, 27 Mar 2014 12:42:01 +0000 In recent years, the utilization of certain medicinal plants as therapeutic agents has drastically increased. Phaleria macrocarpa (Scheff.) Boerl is frequently used in traditional medicine. The present investigation was undertaken with the purpose of developing pharmacopoeial standards for this species. Nutritional values such as ash, fiber, protein, fat, and carbohydrate contents were investigated, and phytochemical screenings with different reagents showed the presence of flavonoids, glycosides, saponin glycosides, phenolic compounds, steroids, tannins, and terpenoids. Our results also revealed that the water fraction had the highest antioxidant activity compared to the methanol extract and other fractions. The methanol and the fractionated extracts (hexane, chloroform, ethyl acetate, and water) of P. macrocarpa seeds were also investigated for their cytotoxic effects on selected human cancer cells lines (MCF-7, HT-29, MDA-MB231, Ca Ski, and SKOV-3) and a normal human fibroblast lung cell line (MRC-5). Information from this study can be applied for future pharmacological and therapeutic evaluations of the species, and may assist in the standardization for quality, purity, and sample identification. To the best of our knowledge, this is the first report on the phytochemical screening and cytotoxic effect of the crude and fractionated extracts of P. macrocarpa seeds on selected cells lines. Ma Ma Lay, Saiful Anuar Karsani, Behrooz Banisalam, Sadegh Mohajer, and Sri Nurestri Abd Malek Copyright © 2014 Ma Ma Lay et al. All rights reserved. Therapeutic Effects of PADRE-BAFF Autovaccine on Rat Adjuvant Arthritis Tue, 25 Mar 2014 07:26:49 +0000 B cell activating factor (BAFF) is a cytokine of tumor necrosis factor family mainly produced by monocytes and dendritic cells. BAFF can regulate the proliferation, differentiation, and survival of B lymphocytes by binding with BAFF-R on B cell membrane. Accumulating evidences showed that BAFF played crucial roles and was overexpressed in various autoimmune diseases such as systemic lupus erythematous (SLE) and rheumatoid arthritis (RA). This suggests that BAFF may be a therapeutic target for these diseases. In the present study, we developed a BAFF therapeutic vaccine by coupling a T helper cell epitope AKFVAAWTLKAA (PADRE) to the N terminus of BAFF extracellular domains (PADRE-BAFF) and expressed this fusion protein in Escherichia coli. The purified vaccine can induce high titer of neutralizing BAFF antibodies and ameliorate the syndrome of complete Freund’s adjuvant (CFA) induced rheumatoid arthritis in rats. Our data indicated that the BAFF autovaccine may be a useful candidate for the treatment of some autoimmune diseases associated with high level of BAFF. Guo-dong Feng, Xiao-chang Xue, Mei-li Gao, Xian-feng Wang, Zhen Shu, Nan Mu, Yuan Gao, Zeng-lu Wang, Qiang Hao, Wei-na Li, Meng Li, Cun Zhang, Wei Zhang, and Ying-qi Zhang Copyright © 2014 Guo-dong Feng et al. All rights reserved. Bacteriostatic Antimicrobial Combination: Antagonistic Interaction between Epsilon-Viniferin and Vancomycin against Methicillin-Resistant Staphylococcus aureus Mon, 24 Mar 2014 16:30:35 +0000 Stilbenoids have been considered as an alternative phytotherapeutic treatment against methicillin-resistant Staphylococcus aureus (MRSA) infection. The combined effect of ε-viniferin and johorenol A with the standard antibiotics, vancomycin and linezolid, was assessed against MRSA ATCC 33591 and HUKM clinical isolate. The minimum inhibitory concentration (MIC) value of the individual tested compounds and the fractional inhibitory concentration index (FICI) value of the combined agents were, respectively, determined using microbroth dilution test and microdilution checkerboard (MDC) method. Only synergistic outcome from checkerboard test will be substantiated for its rate of bacterial killing using time-kill assay. The MIC value of ε-viniferin against ATCC 33591 and johorenol A against both strains was 0.05 mg/mL whereas HUKM strain was susceptible to 0.1 mg/mL of ε-viniferin. MDC study showed that only combination between ε-viniferin and vancomycin was synergistic against ATCC 33591 (FICI 0.25) and HUKM (FICI 0.19). All the other combinations (ε-viniferin-linezolid, johorenol A-vancomycin, and johorenol A-linezolid) were either indifferent or additive against both strains. However, despite the FICI value showing synergistic effect for ε-viniferin-vancomycin, TKA analysis displayed antagonistic interaction with bacteriostatic action against both strains. As conclusion, ε-viniferin can be considered as a bacteriostatic stilbenoid as it antagonized the bactericidal activity of vancomycin. These findings therefore disputed previous report that ε-viniferin acted in synergism with vancomycin but revealed that it targets similar site in close proximity to vancomycin’s action, possibly at the bacterial membrane protein. Hence, this combination has a huge potential to be further studied and developed as an alternative treatment in combating MRSA in future. Dayang Fredalina Basri, Lee Wee Xian, Nur Indah Abdul Shukor, and Jalifah Latip Copyright © 2014 Dayang Fredalina Basri et al. All rights reserved. From Structure to Catalysis: Recent Developments in the Biotechnological Applications of Lipases Mon, 24 Mar 2014 06:51:48 +0000 Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to “order” a “customized” enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design), as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts. Cristiane D. Anobom, Anderson S. Pinheiro, Rafael A. De-Andrade, Erika C. G. Aguieiras, Guilherme C. Andrade, Marcelo V. Moura, Rodrigo V. Almeida, and Denise M. Freire Copyright © 2014 Cristiane D. Anobom et al. All rights reserved. Genetic Analysis of Diversity within a Chinese Local Sugarcane Germplasm Based on Start Codon Targeted Polymorphism Mon, 24 Mar 2014 00:00:00 +0000 In-depth information on sugarcane germplasm is the basis for its conservation and utilization. Data on sugarcane molecular markers are limited for the Chinese sugarcane germplasm collections. In the present study, 20 start codon targeted (SCoT) marker primers were designed to assess the genetic diversity among 107 sugarcane accessions within a local sugarcane germplasm collection. These primers amplified 176 DNA fragments, of which 163 were polymorphic (92.85%). Polymorphic information content (PIC) values ranged from 0.783 to 0.907 with a mean of 0.861. Unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of the SCoT marker data divided the 107 sugarcane accessions into six clusters at 0.674 genetic similarity coefficient level. Relatively abundant genetic diversity was observed among ROC22, ROC16, and ROC10, which occupied about 80% of the total sugarcane acreage in China, indicating their potential breeding value on Mainland China. Principal component analysis (PCA) partitioned the 107 sugarcane accessions into two major groups, the Domestic Group and the Foreign Introduction Group. Each group was further divided based on institutions, where the sugarcane accessions were originally developed. The knowledge of genetic diversity among the local sugarcane germplasm provided foundation data for managing sugarcane germplasm, including construction of a core collection and regional variety distribution and subrogation. Youxiong Que, Yongbao Pan, Yunhai Lu, Cui Yang, Yuting Yang, Ning Huang, and Liping Xu Copyright © 2014 Youxiong Que et al. All rights reserved. Mitogenic Effects of Phosphatidylcholine Nanoparticles on MCF-7 Breast Cancer Cells Thu, 20 Mar 2014 17:38:47 +0000 Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design. Yamila B. Gándola, Sebastián E. Pérez, Pablo E. Irene, Ana I. Sotelo, Johanna G. Miquet, Gerardo R. Corradi, Adriana M. Carlucci, and Lorena Gonzalez Copyright © 2014 Yamila B. Gándola et al. All rights reserved. The Design of a Quantitative Western Blot Experiment Sun, 16 Mar 2014 12:19:16 +0000 Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting. Sean C. Taylor and Anton Posch Copyright © 2014 Sean C. Taylor and Anton Posch. All rights reserved. Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris Thu, 13 Mar 2014 08:28:28 +0000 Heat shock protein 90 (HSP90), a highly conserved molecular chaperone, plays essential roles in folding, keeping structural integrity, and regulating the subset of cytosolic proteins. We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90) by combining homology cloning with rapid amplification of cDNA ends (RACE). Sequence analysis indicated that CvHSP90 is a cytosolic member of the HSP90 family. Quantitative RT-PCR was applied to determine the expression level of messenger RNA (mRNA) in CvHSP90 under different stress conditions. C. vulgaris was kept in different temperatures (5–45°C) for 1 h. The mRNA expression level of CvHSP90 increased with temperature from 5 to 10°C, went further from 35 to 40°C, and reached the maximum at 40°C. On the other hand, for C. vulgaris kept at 35°C for different durations, the mRNA expression level of CvHSP90 increased gradually and reached the peak at 7 h and then declined progressively. In addition, the expression level of CvHSP90 at 40 or 45 in salinity () was almost fourfold of that at 25 in salinity () for 2 h. Therefore, CvHSP90 may be a potential biomarker to monitor environment changes. Zhengyi Liu, Lei Zhang, Yang Pu, Zhaopu Liu, Zhiling Li, Yushan Zhao, and Song Qin Copyright © 2014 Zhengyi Liu et al. All rights reserved. Plant Virus Expression Vector Development: New Perspectives Thu, 13 Mar 2014 00:00:00 +0000 Plant made biologics have elicited much attention over recent years for their potential in assisting those in developing countries who have poor access to modern medicine. Additional applications such as the stockpiling of vaccines against pandemic infectious diseases or potential biological warfare agents are also under investigation. Plant virus expression vectors represent a technology that enables high levels of pharmaceutical proteins to be produced in a very short period of time. Recent advances in research and development have brought about the generation of superior virus expression systems which can be readily delivered to the host plant in a manner that is both efficient and cost effective. This review presents recent innovations in plant virus expression systems and their uses for producing biologics from plants. Kathleen Hefferon Copyright © 2014 Kathleen Hefferon. All rights reserved. Biotechnology and Green Chemistry Wed, 12 Mar 2014 16:40:57 +0000 Bernardo Dias Ribeiro, Isabel Marrucho, Luciana Gonçalves, and Maria Alice Z. Coelho Copyright © 2014 Bernardo Dias Ribeiro et al. All rights reserved. Chemical Constituents from the Fruits of Forsythia suspensa and Their Antimicrobial Activity Wed, 12 Mar 2014 09:36:36 +0000 Lignans and phenylethanoid glycosides purified from Forsythia suspensa were reported to display various bioactivities in the previous literature, including the antimicrobial activity. Therefore, the present research is aimed to purify and identify the chemical constituents of the methanol extracts of fruits of F. suspensa. The methanol extracts of fruits of F. suspensa were fractionated and further purified with the assistance of column chromatography to afford totally thirty-four compounds. Among these isolates, 3β-acetoxy-20α-hydroxyursan-28-oic acid (1) was reported from the natural sources for the first time. Some of the purified principles were subjected to the antimicrobial activity examinations against Escherichia coli to explore new natural lead compounds. Ping-Chung Kuo, Guo-Feng Chen, Mei-Lin Yang, Ya-Hua Lin, and Chi-Chung Peng Copyright © 2014 Ping-Chung Kuo et al. All rights reserved. Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective Wed, 12 Mar 2014 09:01:16 +0000 In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms. Matilde Merlin, Elisa Gecchele, Stefano Capaldi, Mario Pezzotti, and Linda Avesani Copyright © 2014 Matilde Merlin et al. All rights reserved. A Purified Recombinant Lipopeptide as Adjuvant for Cancer Immunotherapy Tue, 11 Mar 2014 13:33:37 +0000 Synthetic lipopeptides have been widely used as vaccine adjuvants to enhance immune responses. The present study demonstrated that the tryptic N-terminal fragment of the lipoprotein rlipo-D1E3 (lipo-Nter) induces superior antitumor effects compared to a synthetic lipopeptide. The lipo-Nter was purified and formulated with protein or peptide vaccines to determine if lipo-Nter could be used as a novel adjuvant and could induce antitumor immunity in a cervical cancer model. Purified lipo-Nter activated the maturation of bone marrow-derived dendritic cells (BM-DCs), leading to the secretion of TNF-α through TLR2/6 but not TLR1/2. A recombinant mutant HPV16 E7 (rE7m) protein was mixed with lipo-Nter to immunize the mice; the anti-E7 antibody titers were increased, and the T helper cells were skewed toward the Th1 fate (increased IL-2 and decreased IL-5 secretion). Single-dose injection of rE7m and lipo-Nter inhibited tumor growth, but the injection of rE7m alone did not. Accordingly, lipo-Nter also enhanced the antitumor immunity of the E7-derived peptide but not the synthetic lipopeptide (Pam3CSK4). We demonstrated that the lipo-Nter of a bacterial-derived recombinant lipoprotein is a novel adjuvant that could be used for the development of a new generation of vaccines. Ying-Chyi Song, Hsueh-Hung Liu, I-Hua Chen, Hsin-Wei Chen, Pele Chong, Chih-Hsiang Leng, and Shih-Jen Liu Copyright © 2014 Ying-Chyi Song et al. All rights reserved. Effect of Age-Related Cartilage Turnover on Serum C-Telopeptide of Collagen Type II and Osteocalcin Levels in Growing Rabbits with and without Surgically Induced Osteoarthritis Wed, 05 Mar 2014 13:23:09 +0000 This study aims to determine the effect of age-related cartilage turnover on the serum C-telopeptide of type II collagen (CTX-II) and osteocalcin (OC) levels in growing rabbits with and without surgically induced osteoarthritis. Twenty-four New Zealand male 3-month-old rabbits were randomized into three operated groups (n = 6 per group, with surgically induced osteroarthritis in the right knee; after blood sampling, the knees were harvested following euthanization at 2, 3, and 6 months after surgery) and a control group (n = 6, blood samples were obtained monthly between 3 and 15 months). Histomorphologically, the medial femoral condyles, particularly the central parts, harbored the most severe osteoarthritic changes among the operated rabbits. The serum levels of CTX-II and OC decreased in the controls from 3 to 11 months and then remained stable. No significant differences in the serum CTX-II and OC levels between the osteoarthritic rabbits and controls were observed. The osteoarthritic-to-normal ratios (ONRs, the ratios of serum CTX-II or OC levels in osteoarthritic rabbits to those of the controls at same ages) enabled an overall assessment of osteoarthritis and age-related cartilage turnover. Elevated CTX-II ONRs were observed in rabbits with mild to advanced osteoarthritis. However, the OC ONRs were unhelpful in assessing osteoarthritic growing rabbits. Chung-Cheng Huang, Chen-Chang Lee, Ching-Jen Wang, Feng-Sheng Wang, Hsuan-Ying Huang, Shu-Hang Ng, Chia-Yi Tseng, and Sheung-Fat Ko Copyright © 2014 Chung-Cheng Huang et al. All rights reserved. Dissolution of Arsenic Minerals Mediated by Dissimilatory Arsenate Reducing Bacteria: Estimation of the Physiological Potential for Arsenic Mobilization Sun, 02 Mar 2014 11:22:27 +0000 The aim of this study was characterization of the isolated dissimilatory arsenate reducing bacteria in the context of their potential for arsenic removal from primary arsenic minerals through reductive dissolution. Four strains, Shewanella sp. OM1, Pseudomonas sp. OM2, Aeromonas sp. OM4, and Serratia sp. OM17, capable of anaerobic growth with As (V) reduction, were isolated from microbial mats from an ancient gold mine. All of the isolated strains: (i) produced siderophores that promote dissolution of minerals, (ii) were resistant to dissolved arsenic compounds, (iii) were able to use the dissolved arsenates as the terminal electron acceptor, and (iii) were able to use copper minerals containing arsenic minerals (e.g., enargite) as a respiratory substrate. Based on the results obtained in this study, we postulate that arsenic can be released from some As-bearing polymetallic minerals (such as copper ore concentrates or middlings) under reductive conditions by dissimilatory arsenate reducers in indirect processes. Drewniak Lukasz, Rajpert Liwia, Mantur Aleksandra, and Sklodowska Aleksandra Copyright © 2014 Drewniak Lukasz et al. All rights reserved. Current Biotechnological Advancements on Sustainable Metal and Nutrient Removal Thu, 27 Feb 2014 13:52:25 +0000 Li-Yuan Chai, Chong-Jian Tang, Qaisar Mahmood, and Xian-Wei Liu Copyright © 2014 Li-Yuan Chai et al. All rights reserved. Antioxidant Activity of Extract and Its Major Constituents from Okra Seed on Rat Hepatocytes Injured by Carbon Tetrachloride Thu, 27 Feb 2014 12:07:59 +0000 The antioxidant activities and protective effects of total phenolic extracts (TPE) and their major components from okra seeds on oxidative stress induced by carbon tetrachloride (CCl4) in rat hepatocyte cell line were investigated. The major phenolic compounds were identified as quercetin 3-O-glucosyl () glucoside (QDG) and quercetin 3-O-glucoside (QG). TPE, QG, and QDG from okra seeds exhibited excellent reducing power and free radical scavenging capabilities including α, α-diphenyl-β-picrylhydrazyl (DPPH), superoxide anions, and hydroxyl radical. Overall, DPPH radical scavenging activity and reducing power of QG and QDG were higher than those of TPE while superoxide and hydroxyl radical scavenging activities of QG and TPE were higher than those of QDG. Furthermore, TPE, QG, and QDG pretreatments significantly alleviated the cytotoxicity of CCl4 on rat hepatocytes, with attenuated lipid peroxidation, increased SOD and CAT activities, and decreased GPT and GOT activities. The protective effects of TPE and QG on rat hepatocytes were stronger than those of QDG. However, the cytotoxicity of CCl4 on rat hepatocytes was not affected by TPE, QG, and QDG posttreatments. It was suggested that the protective effects of TPE, QG, and QDG on rat hepatocyte against oxidative stress were related to the direct antioxidant capabilities and the induced antioxidant enzymes activities. Lianmei Hu, Wenlan Yu, Ying Li, Nagendra Prasad, and Zhaoxin Tang Copyright © 2014 Lianmei Hu et al. All rights reserved. Effect of Acute Gamma Irradiation on Curcuma alismatifolia Varieties and Detection of DNA Polymorphism through SSR Marker Tue, 25 Feb 2014 05:53:55 +0000 The effects of eight different doses (0, 10, 20, 25, 35, 40, 60, and 100 Gy) of acute gamma irradiation on 44 (three varieties of Curcuma alismatifolia: Chiang Mai Red, Sweet Pink, Kimono Pink, and one Curcuma hybrid (Doi Tung 554) individual plants were investigated. Radiation sensitivity tests revealed that the LD50 values of the varieties were achieved at 21 Gy for Chiang Mai Red, 23 Gy for Sweet Pink, 25 Gy for Kimono Pink, and 28 Gy for Doi Tung 554. From the analysis of variance (ANOVA), significant variations were observed for vegetative traits, flowering development, and rhizome characteristics among the four varieties of Curcuma alismatifolia and dose levels as well as the dose × variety interaction. In irradiated plants, the leaf length, leaf width, inflorescence length, the number of true flowers, the number of pink bracts, number of shoots, plant height, rhizome size, number of storage roots, and number of new rhizomes decreased significantly () as the radiation dose increased. The cophenetic correlation coefficient (CCC) between genetic dissimilarity matrix estimated from the morphological characters and the UPGMA clustering method was , showing a proof fit. In terms of genetic variation among the acutely irradiated samples, the number of presumed alleles revealed by simple sequence repeats ranged from two to seven alleles with a mean value of 3.1, 4.5, and 5.3 alleles per locus for radiation doses of 0, 10, and 20 Gy, respectively. The average values of the effective number of alleles, Nei’s gene diversity, and Shannon’s information index were 2.5–3.2, 0.51–0.66, and 0.9–1.3, respectively. The constructed dendrogram grouped the entities into seven clusters. Principal component analysis (PCA) supported the clustering results. Consequently, it was concluded that irradiation with optimum doses of gamma rays efficiently induces mutations in Curcuma alismatifolia varieties. Sima Taheri, Thohirah Lee Abdullah, Zaiton Ahmad, and Nur Ashikin Psyquay Abdullah Copyright © 2014 Sima Taheri et al. All rights reserved. Microwave Assisted Enzymatic Kinetic Resolution of (±)-1-Phenyl-2-propyn-1-ol in Nonaqueous Media Sun, 23 Feb 2014 07:10:45 +0000 Kinetic resolution of 1-phenyl-2-propyn-1-ol, an important chiral synthon, was studied through trans-esterification with acyl acetate to investigate synergism between microwave irradiation and enzyme catalysis. Lipases from different microbial origins were employed for the kinetic resolution of (R/S)-1-phenyl-2-propyn-1-ol, among which Candida antarctica lipase B, immobilized on acrylic resin (Novozym 435), was found to be the best catalyst in n-hexane as solvent. Vinyl acetate was the most effective among different acyl esters studied. The effect of various parameters was studied in a systematic manner. Definite synergism between microwave and enzyme was observed. The initial rate was improved around 1.28 times under microwave irradiation than conventional heating. Under optimum conditions, maximum conversion (48.78%) and high enantiomeric excess (93.25%) were obtained in 2 h. From modeling studies, it is concluded that the reaction follows the Ping-Pong bi-bi mechanism with dead end alcohol inhibition. Kinetic parameters were obtained by using nonlinear regression. This process is green, clean, and easily scalable as compared to the chemical process. Saravanan Devendran and Ganapati D. Yadav Copyright © 2014 Saravanan Devendran and Ganapati D. Yadav. All rights reserved. In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication Thu, 20 Feb 2014 16:38:20 +0000 Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 1014 in the virus-inoculated cells to 109 in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection. Oi Kuan Choong, Parvaneh Mehrbod, Bimo Ario Tejo, and Abdul Rahman Omar Copyright © 2014 Oi Kuan Choong et al. All rights reserved. Production of Biodiesel from Chlorella sp. Enriched with Oyster Shell Extracts Mon, 17 Feb 2014 08:35:56 +0000 This study investigated the cultivation of the marine microalga Chlorella sp. without supplying an inorganic carbon source, but instead with enriching the media with extracts of oyster shells pretreated by a high-pressure homogenization process. The pretreated oyster shells were extracted by a weak acid, acetic acid, that typically has harmful effects on cell growth and also poses environmental issues. The concentration of the residual dissolved carbon dioxide in the medium was sufficient to maintain cell growth at 32 ppm and pH 6.5 by only adding 5% (v/v) of oyster shell extracts. Under this condition, the maximum cell density observed was 2.74 g dry wt./L after 27 days of cultivation. The total lipid content was also measured as 18.1 (%, w/w), and this value was lower than the 23.6 (%, w/w) observed under nitrogen deficient conditions or autotrophic conditions. The fatty acid compositions of the lipids were also measured as 10.9% of C16:1 and 16.4% of C18:1 for the major fatty acids, which indicates that the biodiesel from this culture process should be a suitable biofuel. These results suggest that oyster shells, environmental waste from the food industry, can be used as a nutrient and carbon source with seawater, and this reused material should be important for easily scaling up the process for an outdoor culture system. Cheol Soon Choi, Woon Yong Choi, Do Hyung Kang, and Hyeon Yong Lee Copyright © 2014 Cheol Soon Choi et al. All rights reserved. Impact Assessment of Cadmium Toxicity and Its Bioavailability in Human Cell Lines (Caco-2 and HL-7702) Sun, 16 Feb 2014 13:58:20 +0000 Cadmium (Cd) is a widespread environmental toxic contaminant, which causes serious health-related problems. In this study, human intestinal cell line (Caco-2 cells) and normal human liver cell line (HL-7702 cells) were used to investigate the toxicity and bioavailability of Cd to both cell lines and to validate these cell lines as in vitro models for studying Cd accumulation and toxicity in human intestine and liver. Results showed that Cd uptake by both cell lines increased in a dose-dependent manner and its uptake by Caco-2 cells (720.15 µg mg−1 cell protein) was significantly higher than HL-7702 cells (229.01 µg mg−1 cell protein) at 10 mg L−1. A time- and dose-dependent effect of Cd on cytotoxicity assays (LDH release, MTT assay) was observed in both Cd-treated cell lines. The activities of antioxidant enzymes and differentiation markers (SOD, GPX, and AKP) of the HL-7702 cells were higher than those of Caco-2 cells, although both of them decreased significantly with raising Cd levels. The results from the present study indicate that Cd above a certain level inhibits cellular antioxidant activities and HL-7702 cells are more sensitive to Cd exposure than Caco-2 cells. However, Cd concentrations <0.5 mg L−1 pose no toxic effects on both cell lines. Rukhsanda Aziz, M. T. Rafiq, Jie Yang, Di Liu, Lingli Lu, Zhenli He, M. K. Daud, Tingqiang Li, and Xiaoe Yang Copyright © 2014 Rukhsanda Aziz et al. All rights reserved. Enhancement of Biodiesel Production from Marine Alga, Scenedesmus sp. through In Situ Transesterification Process Associated with Acidic Catalyst Thu, 13 Feb 2014 12:04:43 +0000 The aim of this study was to increase the yield of biodiesel produced by Scenedesmus sp. through in situ transesterification by optimizing various process parameters. Based on the orthogonal matrix analysis for the acidic catalyst, the effects of the factors decreased in the order of reaction temperature (47.5%) > solvent quantity (26.7%) > reaction time (17.5%) > catalyst amount (8.3%). Based on a Taguchi analysis, the effects of the factors decreased in the order of solvent ratio (34.36%) > catalyst (28.62%) > time (19.72%) > temperature (17.32%). The overall biodiesel production appeared to be better using NaOH as an alkaline catalyst rather than using H2SO4 in an acidic process, at 55.07 ± 2.18% (based on lipid weight) versus 48.41 ± 0.21%. However, in considering the purified biodiesel, it was found that the acidic catalyst was approximately 2.5 times more efficient than the alkaline catalyst under the following optimal conditions: temperature of 70°C (level 2), reaction time of 10 hrs (level 2), catalyst amount of 5% (level 3), and biomass to solvent ratio of 1 : 15 (level 2), respectively. These results clearly demonstrated that the acidic solvent, which combined oil extraction with in situ transesterification, was an effective catalyst for the production of high-quantity, high-quality biodiesel from a Scenedesmus sp. Ga Vin Kim, WoonYong Choi, DoHyung Kang, ShinYoung Lee, and HyeonYong Lee Copyright © 2014 Ga Vin Kim et al. All rights reserved. Degradation Potential of Protocatechuate 3,4-Dioxygenase from Crude Extract of Stenotrophomonas maltophilia Strain KB2 Immobilized in Calcium Alginate Hydrogels and on Glyoxyl Agarose Wed, 12 Feb 2014 12:14:44 +0000 Microbial intradiol dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5°C and 10°C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions. Urszula Guzik, Katarzyna Hupert-Kocurek, Marta Krysiak, and Danuta Wojcieszyńska Copyright © 2014 Urszula Guzik et al. All rights reserved. Chemodynamics of Methyl Parathion and Ethyl Parathion: Adsorption Models for Sustainable Agriculture Thu, 06 Feb 2014 07:27:14 +0000 The toxicity of organophosphate insecticides for nontarget organism has been the subject of extensive research for sustainable agriculture. Pakistan has banned the use of methyl/ethyl parathions, but they are still illegally used. The present study is an attempt to estimate the residual concentration and to suggest remedial solution of adsorption by different types of soils collected and characterized for physicochemical parameters. Sorption of pesticides in soil or other porous media is an important process regulating pesticide transport and degradation. The percentage removal of methyl parathion and ethyl parathion was determined through UV-Visible spectrophotometer at 276 nm and 277 nm, respectively. The results indicate that agricultural soil as compared to barren soil is more efficient adsorbent for both insecticides, at optimum batch condition of pH 7. The equilibrium between adsorbate and adsorbent was attained in 12 hours. Methyl parathion is removed more efficiently (by seven orders of magnitude) than ethyl parathion. It may be attributed to more available binding sites and less steric hindrance of methyl parathion. Adsorption kinetics indicates that a good correlation exists between distribution coefficient (Kd) and soil organic carbon. A general increase in Kd is noted with increase in induced concentration due to the formation of bound or aged residue. Noshabah Tabassum, Uzaira Rafique, Khaled S. Balkhair, and Muhammad Aqeel Ashraf Copyright © 2014 Noshabah Tabassum et al. All rights reserved. Use of Synthetic Single-Stranded Oligonucleotides as Artificial Test Soiling for Validation of Surgical Instrument Cleaning Processes Mon, 03 Feb 2014 06:58:16 +0000 Surgical instruments are often strongly contaminated with patients’ blood and tissues, possibly containing pathogens. The reuse of contaminated instruments without adequate cleaning and sterilization can cause postoperative inflammation and the transmission of infectious diseases from one patient to another. Thus, based on the stringent sterility requirements, the development of highly efficient, validated cleaning processes is necessary. Here, we use for the first time synthetic single-stranded DNA (ssDNA_ODN), which does not appear in nature, as a test soiling to evaluate the cleaning efficiency of routine washing processes. Stainless steel test objects were coated with a certain amount of ssDNA_ODN. After cleaning, the amount of residual ssDNA_ODN on the test objects was determined using quantitative real-time PCR. The established method is highly specific and sensitive, with a detection limit of 20 fg, and enables the determination of the cleaning efficiency of medical cleaning processes under different conditions to obtain optimal settings for the effective cleaning and sterilization of instruments. The use of this highly sensitive method for the validation of cleaning processes can prevent, to a significant extent, the insufficient cleaning of surgical instruments and thus the transmission of pathogens to patients. Nadja Wilhelm, Nadja Perle, Robert Simmoteit, Christian Schlensak, Hans P. Wendel, and Meltem Avci-Adali Copyright © 2014 Nadja Wilhelm et al. All rights reserved. Induction of Apoptotic Effects of Antiproliferative Protein from the Seeds of Borreria hispida on Lung Cancer (A549) and Cervical Cancer (HeLa) Cell Lines Wed, 29 Jan 2014 13:33:18 +0000 A 35 KDa protein referred to as F3 was purified from the seeds of Borreria hispida by precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3) on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase of Pyrococcus horikoshii. The protein (F3) exhibited significant cytotoxic activity against lung (A549) and cervical (HeLa) cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds of Borreria hispida with antiproliferative and apoptotic activity in lung (A549) and cervical (HeLa) cancer cells. S. Rupachandra and D. V. L. Sarada Copyright © 2014 S. Rupachandra and D. V. L. Sarada. All rights reserved. Demonstration of Redox Potential of Metschnikowia koreensis for Stereoinversion of Secondary Alcohols/1,2-Diols Mon, 27 Jan 2014 14:07:47 +0000 The present work reports the Metschnikowia koreensis-catalyzed one-pot deracemization of secondary alcohols/1,2-diols and their derivatives with in vivo cofactor regeneration. Reaction is stereoselective and proceeds with sequential oxidation of (R)-secondary alcohols to the corresponding ketones and the reduction of the ketones to (S)-secondary alcohols. Method is applicable to a repertoire of racemic aryl secondary alcohols and 1,2-diols establishing a wide range of substrate specificity of M. koreensis. This ecofriendly method afforded the product in high yield (88%) and excellent optical purity (>98% ee), minimizing the requirement of multistep reaction and expensive cofactor. Vachan Singh Meena, Linga Banoth, and U. C. Banerjee Copyright © 2014 Vachan Singh Meena et al. All rights reserved. Characteristics, Process Parameters, and Inner Components of Anaerobic Bioreactors Thu, 23 Jan 2014 14:02:01 +0000 The anaerobic bioreactor applies the principles of biotechnology and microbiology, and nowadays it has been used widely in the wastewater treatment plants due to their high efficiency, low energy use, and green energy generation. Advantages and disadvantages of anaerobic process were shown, and three main characteristics of anaerobic bioreactor (AB), namely, inhomogeneous system, time instability, and space instability were also discussed in this work. For high efficiency of wastewater treatment, the process parameters of anaerobic digestion, such as temperature, pH, Hydraulic retention time (HRT), Organic Loading Rate (OLR), and sludge retention time (SRT) were introduced to take into account the optimum conditions for living, growth, and multiplication of bacteria. The inner components, which can improve SRT, and even enhance mass transfer, were also explained and have been divided into transverse inner components, longitudinal inner components, and biofilm-packing material. At last, the newly developed special inner components were discussed and found more efficient and productive. Awad Abdelgadir, Xiaoguang Chen, Jianshe Liu, Xuehui Xie, Jian Zhang, Kai Zhang, Heng Wang, and Na Liu Copyright © 2014 Awad Abdelgadir et al. All rights reserved. Microbial Diversity for Biotechnology Thu, 23 Jan 2014 09:12:23 +0000 George Tsiamis, Dimitrios Karpouzas, Ameur Cherif, and Konstantinos Mavrommatis Copyright © 2014 George Tsiamis et al. All rights reserved. Microtexturing of the Conductive PEDOT:PSS Polymer for Superhydrophobic Organic Electrochemical Transistors Wed, 22 Jan 2014 14:01:47 +0000 Superhydrophobic surfaces are bioinspired, nanotechnology artifacts, which feature a reduced friction coefficient, whereby they can be used for a number of very practical applications including, on the medical side, the manipulation of biological solutions. In this work, we integrated superhydrophobic patterns with the conducting polymer PEDOT:PSS, one of the most used polymers in organic electronics because highly sensitive to ionized species in solution. In doing so, we combined geometry and materials science to obtain an advanced device where, on account of the superhydrophobicity of the system, the solutions of interest can be manipulated and, on account of the conductive PEDOT:PSS polymer, the charged molecules dispersed inside can be quantitatively measured. This original substrate preparation allowed to perform electrochemical measurements on ionized species in solution with decreasing concentration down to 10−7 molar. Moreover, it was demonstrated the ability of the device of realizing specific, combined time and space resolved analysis of the sample. Collectively, these results demonstrate how a tight, interweaving integration of different disciplines can provide realistic tools for the detection of pathologies. The scheme here introduced offers breakthrough capabilities that are expected to radically improve both the pace and the productivity of biomedical research, creating an access revolution. Francesco Gentile, Nicola Coppedè, Giuseppe Tarabella, Marco Villani, Davide Calestani, Patrizio Candeloro, Salvatore Iannotta, and Enzo Di Fabrizio Copyright © 2014 Francesco Gentile et al. All rights reserved. Biochemical and Anatomical Changes and Yield Reduction in Rice (Oryza sativa L.) under Varied Salinity Regimes Wed, 22 Jan 2014 08:21:05 +0000 Five Malaysian rice (Oryza sativa L.) varieties, MR33, MR52, MR211, MR219, and MR232, were tested in pot culture under different salinity regimes for biochemical response, physiological activity, and grain yield. Three different levels of salt stresses, namely, 4, 8, and 12 dS m−1, were used in a randomized complete block design with four replications under glass house conditions. The results revealed that the chlorophyll content, proline, sugar content, soluble protein, free amino acid, and yield per plant of all the genotypes were influenced by different salinity levels. The chlorophyll content was observed to decrease with salinity level but the proline increased with salinity levels in all varieties. Reducing sugar and total sugar increased up to 8 dS m−1 and decreased up to 12 dS m−1. Nonreducing sugar decreased with increasing the salinity levels in all varieties. Soluble protein and free amino acid also decreased with increasing salinity levels. Cortical cells of MR211 and MR232 did not show cell collapse up to 8 dS m−1 salinity levels compared to susceptible checks (IR20 and BRRI dhan29). Therefore, considering all parameters, MR211 and MR232 showed better salinity tolerance among the tested varieties. Both cluster and principal component analyses depict the similar results. M. A. Hakim, Abdul Shukor Juraimi, M. M. Hanafi, Mohd Razi Ismail, Ahmad Selamat, M. Y. Rafii, and M. A. Latif Copyright © 2014 M. A. Hakim et al. All rights reserved. Evaluation of Antioxidant Compounds, Antioxidant Activities, and Mineral Composition of 13 Collected Purslane (Portulaca oleracea L.) Accessions Tue, 21 Jan 2014 09:15:19 +0000 The methanolic extracts of 13 accessions of purslane were analyzed for their total phenol content (TPC), total flavonoid contents (TFC), and total carotenoid contents (TCC) and antioxidant activity of extracts was screened using FRAP assay and DPPH radical scavenging methods. The TPC, TFC, and TCC ranged from to  mg GAE/g DW, to  mg RE/g DW, and to  mg (β-carotene equivalent) BCE/g DW, respectively. The DPPH scavenging (IC50) activity varied between  mg/mL and  mg/mL and FRAP ranged from to  μmol TE/g DW. Among all the measured micro- and macrominerals K content was the highest followed by N, Na, Ca, Mg, P, Fe, Zn, and Mn. The overall findings proved that ornamental purslane was richer in antioxidant properties, whereas common purslane possesses more mineral contents than ornamental ones. Md. Amirul Alam, Abdul Shukor Juraimi, M. Y. Rafii, Azizah Abdul Hamid, Farzad Aslani, M. M. Hasan, Mohd Asraf Mohd Zainudin, and Md. Kamal Uddin Copyright © 2014 Md. Amirul Alam et al. All rights reserved. Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of a Plasmodium Region I-Plus Peptide Sun, 19 Jan 2014 13:48:46 +0000 Interferon alpha (IFN α) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFN α is the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFN α may account for its low in vivo biological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFN α2b with a Plasmodium region I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression in E. coli BL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentiality in vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent. Xuemei Lu, Xiaobao Jin, Yanting Huang, Jie Wang, Juan Shen, Fujiang Chu, Hanfang Mei, Yan Ma, and Jiayong Zhu Copyright © 2014 Xuemei Lu et al. All rights reserved. Synergistic Exposure of Rice Seeds to Different Doses of -Ray and Salinity Stress Resulted in Increased Antioxidant Enzyme Activities and Gene-Specific Modulation of TC-NER Pathway Thu, 16 Jan 2014 10:24:21 +0000 Recent reports have underlined the potential of gamma (γ)-rays as tools for seed priming, a process used in seed industry to increase seed vigor and to enhance plant tolerance to biotic/abiotic stresses. However, the impact of γ-rays on key aspects of plant metabolism still needs to be carefully evaluated. In the present study, rice seeds were challenged with different doses of γ-rays and grown in absence/presence of NaCl to assess the impact of these treatments on the early stages of plant life. Enhanced germination efficiency associated with increase in radicle and hypocotyl length was observed, while at later stages no increase in plant tolerance to salinity stress was evident. APX, CAT, and GR were enhanced at transcriptional level and in terms of enzyme activity, indicating the activation of antioxidant defence. The profiles of DNA damage accumulation were obtained using SCGE and the implication of TC-NER pathway in DNA damage sensing and repair mechanisms is discussed. OsXPB2, OsXPD, OsTFIIS, and OsTFIIS-like genes showed differential modulation in seedlings and plantlets in response to γ-irradiation and salinity stress. Altogether, the synergistic exposure to γ-rays and NaCl resulted in enhanced oxidative stress and proper activation of antioxidant mechanisms, thus being compatible with plant survival. Anca Macovei, Bharti Garg, Shailendra Raikwar, Alma Balestrazzi, Daniela Carbonera, Armando Buttafava, Juan Francisco Jiménez Bremont, Sarvajeet Singh Gill, and Narendra Tuteja Copyright © 2014 Anca Macovei et al. All rights reserved. Comparison of Extraction Methods of Chitin from Ganoderma lucidum Mushroom Obtained in Submerged Culture Wed, 15 Jan 2014 16:01:08 +0000 The chitin was isolated from the Ganoderma lucidum submerged cultures mycelium as potential source of chitin under biotechnological processes. The extraction of chitin was carried out through 5 different assays which involved mainly three phases: pulverization of the mushroom, deproteinization of the mycelia with NaOH solution, and a process of decolorization with potassium permanganate and oxalic acid. The chitin contents extracted from 9-day mycelia were 413, 339, 87, 78, and 144 mg/g−1 (milligrams of chitin/grams of dry biomass) for A1, A2, A3, A4, and A5, respectively. Obtained chitin was characterized by X-Ray Diffraction (XRD), by Fourier transform infrared spectroscopy (FTIR), and by thermal analysis (TGA). The results showed that Ganoderma lucidum chitin has similar characteristic of chitin from different fonts. The advantage of the biotechnological processes and the fact that Ganoderma lucidum fungus may be used as a potential raw material for chitin production were demonstrated. Sandra Patricia Ospina Álvarez, David Alexander Ramírez Cadavid, Diana Marcela Escobar Sierra, Claudia Patricia Ossa Orozco, Diego Fernando Rojas Vahos, Paola Zapata Ocampo, and Lucía Atehortúa Copyright © 2014 Sandra Patricia Ospina Álvarez et al. All rights reserved. Production of Fungal Amylases Using Cheap, Readily Available Agriresidues, for Potential Application in Textile Industry Thu, 09 Jan 2014 10:53:52 +0000 The study aimed at isolation and screening of fungal amylase producer, optimization of solid state fermentation conditions for maximum amylase production by the best amylase producer, and characterization of the crude amylases, so produced. Aspergillus fumigatus NTCC1222 showed the highest amylase activity (164.1 U/mL) in secondary screening under SSF conditions and was selected for further studies. The test strain showed maximum amylase production (341.7 U/mL) and supernatant protein concentration (9.7 mg/mL) for incubation period (6 days), temperature (35°C), initial pH (6.0), nutrient salt solution as moistening agent, and beef extract as nitrogen source. Pomegranate peel produced maximum amylase activity, but wheat bran (only slightly lesser amylase activity as compared to that of pomegranate peel) was chosen for further studies, keeping in mind the seasonal availability of pomegranate peel. TLC confirmed the amylase produced to be α-type and 60 kDa was the molecular weight of the partially purified amylase. The enzyme showed maximum enzyme activity at pH 6.0, temperature of 55°C, and incubation time of 60 minutes. UV (616.0 U/mL) and chemical (814.2 U/mL) mutation enhanced amylase activity as compared to wild test strain. The study indicates that Aspergillus fumigatus NTCC1222 can be an important source of amylase and the crude enzyme, hence obtained, can be cost effectively applied in multiple sections of textile wet processing. Shalini Singh, Sanamdeep Singh, Vrinda Bali, Lovleen Sharma, and Jyoti Mangla Copyright © 2014 Shalini Singh et al. All rights reserved. Effect of Temperature on Chinese Rice Wine Brewing with High Concentration Presteamed Whole Sticky Rice Mon, 06 Jan 2014 08:04:27 +0000 Production of high quality Chinese rice wine largely depends on fermentation temperature. However, there is no report on the ethanol, sugars, and acids kinetics in the fermentation mash of Chinese rice wine treated at various temperatures. The effects of fermentation temperatures on Chinese rice wine quality were investigated. The compositions and concentrations of ethanol, sugars, glycerol, and organic acids in the mash of Chinese rice wine samples were determined by HPLC method. The highest ethanol concentration and the highest glycerol concentration both were attained at the fermentation mash treated at 23°C. The highest peak value of maltose (90 g/L) was obtained at 18°C. Lactic acid and acetic acid both achieved maximum values at 33°C. The experimental results indicated that temperature contributed significantly to the ethanol production, acid flavor contents, and sugar contents in the fermentation broth of the Chinese rice wines. Dengfeng Liu, Hong-Tao Zhang, Weili Xiong, Jianhua Hu, Baoguo Xu, Chi-Chung Lin, Ling Xu, and Lihua Jiang Copyright © 2014 Dengfeng Liu et al. All rights reserved. Degradation of Diuron by Phanerochaete chrysosporium: Role of Ligninolytic Enzymes and Cytochrome P450 Tue, 31 Dec 2013 13:31:04 +0000 The white-rot fungus Phanerochaete chrysosporium was investigated for its capacity to degrade the herbicide diuron in liquid stationary cultures. The presence of diuron increased the production of lignin peroxidase in relation to control cultures but only barely affected the production of manganese peroxidase. The herbicide at the concentration of 7 μg/mL did not cause any reduction in the biomass production and it was almost completely removed after 10 days. Concomitantly with the removal of diuron, two metabolites, DCPMU [1-(3,4-dichlorophenyl)-3-methylurea] and DCPU [(3,4-dichlorophenyl)urea], were detected in the culture medium at the concentrations of 0.74 μg/mL and 0.06 μg/mL, respectively. Crude extracellular ligninolytic enzymes were not efficient in the in vitro degradation of diuron. In addition, 1-aminobenzotriazole (ABT), a cytochrome P450 inhibitor, significantly inhibited both diuron degradation and metabolites production. Significant reduction in the toxicity evaluated by the Lactuca sativa L. bioassay was observed in the cultures after 10 days of cultivation. In conclusion, P. chrysosporium can efficiently metabolize diuron without the accumulation of toxic products. Jaqueline da Silva Coelho-Moreira, Adelar Bracht, Aline Cristine da Silva de Souza, Roselene Ferreira Oliveira, Anacharis Babeto de Sá-Nakanishi, Cristina Giatti Marques de Souza, and Rosane Marina Peralta Copyright © 2013 Jaqueline da Silva Coelho-Moreira et al. All rights reserved. Toward Personalized Cell Therapies by Using Stem Cells 2013 Sun, 29 Dec 2013 15:00:07 +0000 Ken-ichi Isobe, Herman S. Cheung, and Ji Wu Copyright © 2013 Ken-ichi Isobe et al. All rights reserved. Coronary CT Angiography in Coronary Artery Disease: Correlation between Virtual Intravascular Endoscopic Appearances and Left Bifurcation Angulation and Coronary Plaques Sat, 28 Dec 2013 08:08:00 +0000 The aim of this study is to investigate the relationship between intraluminal appearances of coronary plaques and left coronary bifurcation angle and plaque components using coronary CT virtual intravascular endoscopy (VIE). Fifty patients suspected of coronary artery disease undergoing coronary CT angiography were included in the study. The left bifurcation angle in patients with diseased left coronary artery which was measured as 94.3° ± 16.5 is significantly larger than that in patients with normal left coronary artery, which was measured as 76.5° ± 15.9 (). Irregular VIE appearances were found in 10 out of 11 patients with mixed plaques in the left anterior descending (LAD) and left circumflex (LCx), while, in 29 patients with calcified plaques in the LAD and LCx, irregular VIE appearances were only noticed in 5 patients. Using 80° as a cut-off value to determine coronary artery disease, smooth VIE appearances were found in 95% of patients (18/19) with left bifurcation angle of less than 80°, while irregular VIE appearances were observed in nearly 50% of patients (15/31) with left bifurcation angle of more than 80°. This preliminary study shows that VIE appearances of the coronary lumen are directly related to the types of plaques. Zhonghua Sun Copyright © 2013 Zhonghua Sun. All rights reserved. Intravenous Transplantation of Mesenchymal Stromal Cells to Enhance Peripheral Nerve Regeneration Thu, 26 Dec 2013 18:13:05 +0000 Peripheral nerve injury is a common and devastating complication after trauma and can cause irreversible impairment or even complete functional loss of the affected limb. While peripheral nerve repair results in some axonal regeneration and functional recovery, the clinical outcome is not optimal and research continues to optimize functional recovery after nerve repair. Cell transplantation approaches are being used experimentally to enhance regeneration. Intravenous infusion of mesenchymal stromal cells (MSCs) into spinal cord injury and stroke was shown to improve functional outcome. However, the repair potential of intravenously transplanted MSCs in peripheral nerve injury has not been addressed yet. Here we describe the impact of intravenously infused MSCs on functional outcome in a peripheral nerve injury model. Rat sciatic nerves were transected followed, by intravenous MSCs transplantation. Footprint analysis was carried out and 21 days after transplantation, the nerves were removed for histology. Labelled MSCs were found in the sciatic nerve lesion site after intravenous injection and regeneration was improved. Intravenously infused MSCs after acute peripheral nerve target the lesion site and survive within the nerve and the MSC treated group showed greater functional improvement. The results of study suggest that nerve repair with cell transplantation could lead to greater functional outcome. Stella M. Matthes, Kerstin Reimers, Insa Janssen, Christina Liebsch, Jeffery D. Kocsis, Peter M. Vogt, and Christine Radtke Copyright © 2013 Stella M. Matthes et al. All rights reserved. Cadmium-Induced Upregulation of Lipid Peroxidation and Reactive Oxygen Species Caused Physiological, Biochemical, and Ultrastructural Changes in Upland Cotton Seedlings Thu, 26 Dec 2013 09:34:31 +0000 Cadmium (Cd) toxicity was investigated in cotton cultivar (ZMS-49) using physiological, ultrastructural, and biochemical parameters. Biomass-based tolerance index decreased, and water contents increased at 500 M Cd. Photosynthetic efficiency determined by chlorophyll fluorescence and photosynthetic pigments declined under Cd stress. Cd contents were more in roots than shoots. A significant decrease in nutrient levels was found in roots and stem. A significant decrease in nutrient levels was found in roots and stems. In response to Cd stress, more MDA and ROS contents were produced in leaves than in other parts of the seedlings. Total soluble proteins were reduced in all parts except in roots at 500 M Cd. Oxidative metabolism was higher in leaves than aerial parts of the plant. There were insignificant alterations in roots and leaves ultrastructures such as a little increase in nucleoli, vacuoles, starch granules, and plastoglobuli in Cd-imposed stressful conditions. Scanning micrographs at 500 M Cd showed a reduced number of stomata as well as near absence of closed stomata. Cd depositions were located in cell wall, vacuoles, and intracellular spaces using TEM-EDX technology. Upregulation of oxidative metabolism, less ultrastructural modification, and Cd deposition in dead parts of cells show that ZMS-49 has genetic potential to resist Cd stress, which need to be explored. Muhammad Daud Khan, Lei Mei, Basharat Ali, Yue Chen, Xin Cheng, and S. J. Zhu Copyright © 2013 Muhammad Daud Khan et al. All rights reserved. Biologic Activity and Biotechnological Development of Natural Products Tue, 24 Dec 2013 08:53:39 +0000 José Carlos Tavares Carvalho, Fabio Ferreira Perazzo, Leandro Machado, and Didier Bereau Copyright © 2013 José Carlos Tavares Carvalho et al. All rights reserved. An Uncommon Plant Growth Regulator, Diethyl Aminoethyl Hexanoate, Is Highly Effective in Tissue Cultures of the Important Medicinal Plant Purple Coneflower (Echinacea purpurea L.) Mon, 23 Dec 2013 18:15:13 +0000 We investigated the effects of various concentrations of diethyl aminoethyl hexanoate (DA-6) on the regeneration and growth of adventitious buds in in vitro purple coneflower cultures. Among the 3 types of explants tested, leaf explants required higher concentrations of DA-6 than petiole and root explants in order to obtain high regeneration rates, while root explants required the lowest concentration of DA-6. Additionally, explants with higher ploidy levels were more sensitive to the addition of DA-6, while explants with lower ploidy levels required higher concentrations of DA-6 to achieve its maximal regeneration rate. Interestingly, the application of a concentration that was conducive to the regeneration of explants with lower ploidy levels was inhibitory to the regeneration of explants with higher ploidy levels. Moreover, during the growth of regenerated buds, DA-6 application significantly improved plant height and weight, root weight, root thickness, root number, primary root length, total root length, and root/top ratio. Differences in the responses of explants to supplementation with DA-6 were also observed among explants with different ploidy levels, with buds having lower ploidy levels responding to lower concentrations of DA-6. Taken together, the results of the present experiments showed that proper application of DA-6 could increase in vitro culture efficiency in purple coneflower. Xiao-Lu Chen, Jun-Jie Zhang, Rong Chen, Qing-Ling Li, Yue-Sheng Yang, and Hong Wu Copyright © 2013 Xiao-Lu Chen et al. All rights reserved. Start-Up Characteristics of a Granule-Based Anammox UASB Reactor Seeded with Anaerobic Granular Sludge Mon, 23 Dec 2013 10:22:50 +0000 The granulation of anammox sludge plays an important role in the high nitrogen removal performance of the anammox reactor. In this study, anaerobic granular sludge was selected as the seeding sludge to start up anammox reactor in order to directly obtain anammox granules. Results showed that the anammox UASB reactor was successfully started up by inoculating anaerobic granular sludge, with substrate capacity of 4435.2 mg/(L·d) and average ammonium and nitrite removal efficiency of 90.36% and 93.29%, respectively. During the start-up course, the granular sludge initially disintegrated and then reaggregated and turned red, suggesting the high anammox performance. Zn-Fe precipitation was observed on the surface of granules during the operation by SEM-EDS, which would impose inhibition to the anammox activity of the granules. Accordingly, it is suggested to relatively reduce the trace metals concentrations, of Fe and Zn in the conventional medium. The findings of this study are expected to be used for a shorter start-up and more stable operation of anammox system. Lei Xiong, Yun-Yan Wang, Chong-Jian Tang, Li-Yuan Chai, Kang-Que Xu, Yu-Xia Song, Mohammad Ali, and Ping Zheng Copyright © 2013 Lei Xiong et al. All rights reserved. Engineered Hypopharynx from Coculture of Epithelial Cells and Fibroblasts Using Poly(ester urethane) as Substratum Sun, 22 Dec 2013 18:11:32 +0000 Porous polymeric scaffolds have been much investigated and applied in the field of tissue engineering research. Poly(ester urethane) (PEU) scaffolds, comprising pores of 1–20 μm in diameter on one surface and ≥200 μm on the opposite surface and in bulk, were fabricated using phase separation method for hypopharyngeal tissue engineering. The scaffolds were grafted with silk fibroin (SF) generated from natural silkworm cocoon to enhance the scaffold’s hydrophilicity and further improve cytocompatibility to both primary epithelial cells (ECs) and fibroblasts of human hypopharynx tissue. Coculture of ECs and fibroblasts was conducted on the SF-grafted PEU scaffold (PEU-SF) to evaluate its in vitro cytocompatibility. After co-culture for 14 days, ECs were lined on the scaffold surface while fibroblasts were distributed in scaffold bulk. The results of in vivo investigation showed that PEU porous scaffold possessed good biocompatibility after it was grafted by silk fibroin. SF grafting improved the cell/tissue infiltration into scaffold bulk. Thus, PEU-SF porous scaffold is expected to be a good candidate to support the hypopharynx regeneration. Zhisen Shen, Jingjing Chen, Cheng Kang, Changfeng Gong, and Yabin Zhu Copyright © 2013 Zhisen Shen et al. All rights reserved. The Uptake Mechanism of Cd(II), Cr(VI), Cu(II), Pb(II), and Zn(II) by Mycelia and Fruiting Bodies of Galerina vittiformis Sun, 22 Dec 2013 18:05:22 +0000 Optimum concentrations of heavy metals like copper, cadmium, lead, chromium, and zinc in soil are essential in carrying out various cellular activities in minimum concentrations and hence help in sustaining all life forms, although higher concentration of these metals is lethal to most of the life forms. Galerina vittiformis, a macrofungus, was found to accumulate these heavy metals into its fleshy fruiting body in the order Pb(II) > Cd(II) > Cu(II) > Zn(II) > Cr(VI) from 50 mg/kg soil. It possesses various ranges of potential cellular mechanisms that may be involved in detoxification of heavy metals and thus increases its tolerance to heavy metal stress, mainly by producing organic acids and phytochelatins (PCs). These components help in repairing stress damaged proteins and compartmentalisation of metals to vacuoles. The stress tolerance mechanism can be deduced by various analytical tools like SEM-EDX, FTIR, and LC-MS. Production of two kinds of phytochelatins was observed in the organism in response to metal stress. Dilna Damodaran, Raj Mohan Balakrishnan, and Vidya K. Shetty Copyright © 2013 Dilna Damodaran et al. All rights reserved. Optimization of Polygalacturonase Production from a Newly Isolated Thalassospira frigidphilosprofundus to Use in Pectin Hydrolysis: Statistical Approach Thu, 19 Dec 2013 11:57:14 +0000 The present study deals with the production of cold active polygalacturonase (PGase) by submerged fermentation using Thalassospira frigidphilosprofundus, a novel species isolated from deep waters of Bay of Bengal. Nonlinear models were applied to optimize the medium components for enhanced production of PGase. Taguchi orthogonal array design was adopted to evaluate the factors influencing the yield of PGase, followed by the central composite design (CCD) of response surface methodology (RSM) to identify the optimum concentrations of the key factors responsible for PGase production. Data obtained from the above mentioned statistical experimental design was used for final optimization study by linking the artificial neural network and genetic algorithm (ANN-GA). Using ANN-GA hybrid model, the maximum PGase activity (32.54 U/mL) was achieved at the optimized concentrations of medium components. In a comparison between the optimal output of RSM and ANN-GA hybrid, the latter favored the production of PGase. In addition, the study also focused on the determination of factors responsible for pectin hydrolysis by crude pectinase extracted from T. frigidphilosprofundus through the central composite design. Results indicated 80% degradation of pectin in banana fiber at 20°C in 120 min, suggesting the scope of cold active PGase usage in the treatment of raw banana fibers. V. P. B. Rekha, Mrinmoy Ghosh, Vijayanand Adapa, Sung-Jong Oh, K. K. Pulicherla, and K. R. S. Sambasiva Rao Copyright © 2013 V. P. B. Rekha et al. All rights reserved. Wound-Healing Potential of Cultured Epidermal Sheets Is Unaltered after Lyophilization: A Preclinical Study in Comparison to Cryopreserved CES Wed, 18 Dec 2013 12:44:44 +0000 Lyophilized Cultured Epidermal Sheets (L-CES) have been reported to be as effective as the cryopreserved CES (F-CES) in treating skin ulcers. However, unlike F-CES, no preclinical study assessing wound-healing effects has been conducted for L-CES. The present study was set out to investigate the microstructure, cytokine profile, and wound-healing effects of L-CES in comparison to those of F-CES. Keratinocytes were cultured to prepare CES, followed by cryopreservation at −70°C and lyophilization. Under microscopic observation, intact cells with apparent intracellular junctions were observed in L-CES. The L-CES, like fresh CES, consisted of three to four well-maintained epidermal layers, as shown by the expression of keratins, involucrin, and p63. There were no differences in the epidermal layer or protein expression between L-CES and F-CES, and both CES were comparable to fresh CES. TGF-, EGF, VEGF, IL-1, and MMPs were detected in L-CES at levels similar to those in F-CES. In a mouse study, wounds treated with L-CES or F-CES completely healed at least 4 days faster than untreated wounds. CES-treated wounds completely healed by day 10, while the untreated wounds did not heal by day 14. Masson’s trichrome staining showed that collagen deposition in the CES-treated wounds was highly increased in the dermis of the wound center compared to that in the control wounds. Thus, this study demonstrates that L-CES is as clinically effective as F-CES for wound treatment. H. Jang, Y. H. Kim, M. K. Kim, K. H. Lee, and S. Jeon Copyright © 2013 H. Jang et al. All rights reserved. Preparative Purification of Recombinant Proteins: Current Status and Future Trends Tue, 17 Dec 2013 13:53:24 +0000 Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications. Mayank Saraswat, Luca Musante, Alessandra Ravidá, Brian Shortt, Barry Byrne, and Harry Holthofer Copyright © 2013 Mayank Saraswat et al. All rights reserved. Combined Industrial Wastewater Treatment in Anaerobic Bioreactor Posttreated in Constructed Wetland Mon, 16 Dec 2013 08:42:27 +0000 Constructed wetland (CW) with monoculture of Arundo donax L. was investigated for the posttreatment of anaerobic bioreactor (ABR) treating combined industrial wastewater. Different dilutions of combined industrial wastewater (20, 40, 60, and 80) and original wastewater were fed into the ABR and then posttreated by the laboratory scale CW. The respective removal efficiencies of COD, BOD, TSS, nitrates, and ammonia were 80%, 78–82%, 91.7%, 88–92%, and 100% for original industrial wastewater treated in ABR. ABR was efficient in the removal of Ni, Pb, and Cd with removal efficiencies in the order of Cd (2.7%) > Ni (79%) > Pb (85%). Posttreatment of the ABR treated effluent was carried out in lab scale CW containing A. donax L. CW was effective in the removal of COD and various heavy metals present in ABR effluents. The posttreatment in CW resulted in reducing the metal concentrations to 1.95 mg/L, 0 mg/L, and 0.004 mg/L for Ni, Pb, and Cd which were within the permissible water quality standards for industrial effluents. The treatment strategy was effective and sustainable for the treatment of combined industrial wastewater. Bibi Saima Zeb, Qaisar Mahmood, Saima Jadoon, Arshid Pervez, Muhammad Irshad, Muhammad Bilal, and Zulfiqar Ahmad Bhatti Copyright © 2013 Bibi Saima Zeb et al. All rights reserved. Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System Sun, 15 Dec 2013 16:36:21 +0000 The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB) is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP) at C-terminus and red fluorescent protein (RFP, DsRed) at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future. Chao-Hsun Yang, Wan-Ting Kuo, Yun-Ting Chuang, Cheng-Yu Chen, and Chih-Chien Lin Copyright © 2013 Chao-Hsun Yang et al. All rights reserved. Fermentative Succinate Production: An Emerging Technology to Replace the Traditional Petrochemical Processes Thu, 12 Dec 2013 18:13:45 +0000 Succinate is a valuable platform chemical for multiple applications. Confronted with the exhaustion of fossil energy resources, fermentative succinate production from renewable biomass to replace the traditional petrochemical process is receiving an increasing amount of attention. During the past few years, the succinate-producing process using microbial fermentation has been made commercially available by the joint efforts of researchers in different fields. In this review, recent attempts and experiences devoted to reduce the production cost of biobased succinate are summarized, including strain improvement, fermentation engineering, and downstream processing. The key limitations and challenges faced in current microbial production systems are also proposed. Yujin Cao, Rubing Zhang, Chao Sun, Tao Cheng, Yuhua Liu, and Mo Xian Copyright © 2013 Yujin Cao et al. All rights reserved. Technological Aptitude and Applications of Leuconostoc mesenteroides Bioactive Strains Isolated from Algerian Raw Camel Milk Wed, 11 Dec 2013 13:45:21 +0000 Two strains (B7 and Z8) of the Leuconostoc mesenteroides subspecies mesenteroides that were isolated from Algerian camel milk from an initial pool of 13 strains and demonstrated a high ability to inhibit the growth of Listeria spp. were selected and characterised at the phenotypic and genotypic levels. Probiotic profiling and inhibition spectra against food borne pathogens in mixed cultures were also investigated. The bacteriocin produced by L. mesenteroides strain B7 was identified as leucocin B by specific PCR. In vitro studies demonstrated that both Leuconostoc mesenteroides strains exhibited a marked probiotic profile, showing high survival at low pH (2-3 and 4) in the presence of 0.5%, 1%, and 2% of bile salts and at pH 3 in the presence of 3 mg/mL pepsin. Susceptibility testing against antimicrobial agents was also performed for both strains. When tested in a mixed culture with Listeria innocua, Listeria ivanovii, or Staphylococcus aureus, strain B7 reduced the numbers of these species by 1.87, 1.78, and 1.38 log units, respectively. Consequently, these two strains were found to possess good probiotic properties in vitro and a high capacity for Listeria spp. inhibition in mixed cultures. Therefore, these strains have a favourable technological aptitude and a potential application as novel probiotic starters. Zineb Benmechernene, Hanane Fatma Chentouf, Bellil Yahia, Ghazi Fatima, Marcos Quintela-Baluja, Pilar Calo-Mata, and Jorge Barros-Velázquez Copyright © 2013 Zineb Benmechernene et al. All rights reserved. Cultivation of Isochrysis galbana in Phototrophic, Heterotrophic, and Mixotrophic Conditions Tue, 10 Dec 2013 14:08:47 +0000 This study compared the growth and biomass production of Isochrysis galbana under hetero-, mixo-, and phototrophic conditions using different organic carbon sources. The growth of I. galbana was inhibited in heterotrophy but was enhanced in mixotrophy compared to that in phototrophy. Subsequently, the influences of organic carbon and environmental factors (light and salinity) on the growth of I. galbana were further investigated. Algal dry weight increased as glycerol concentrations increased from 0 to 200 mmol and the highest algal production occurred at 50 mmol glycerol. At a range of light intensities of 25–200 μmol photons m−2 s−2, the highest algal growth rate occurred at 100 photons μmol m−2 s−2. The growth of I. galbana was significantly affected by photoperiod, and the maximal dry weight was obtained at 12 h light and 12 h dark. In the salinity test, I. galbana could grow in a wide range of salinities from 10 to 65‰, but the 35‰  salinity was optimal. This study suggests that the growth and production of I. galbana can be improved using mixotrophic culture at 50 mmol glycerol in 35‰  salinity. Yousef Alkhamis and Jian G. Qin Copyright © 2013 Yousef Alkhamis and Jian G. Qin. All rights reserved. Genotoxin Induced Mutagenesis in the Model Plant Physcomitrella patens Mon, 09 Dec 2013 17:52:41 +0000 The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype. Marcela Holá, Jaroslav Kozák, Radka Vágnerová, and Karel J. Angelis Copyright © 2013 Marcela Holá et al. All rights reserved. Hexavalent Molybdenum Reduction to Mo-Blue by a Sodium-Dodecyl-Sulfate-Degrading Klebsiella oxytoca Strain DRY14 Mon, 09 Dec 2013 15:45:47 +0000 Bacteria with the ability to tolerate, remove, and/or degrade several xenobiotics simultaneously are urgently needed for remediation of polluted sites. A previously isolated bacterium with sodium dodecyl sulfate- (SDS-) degrading capacity was found to be able to reduce molybdenum to the nontoxic molybdenum blue. The optimal pH, carbon source, molybdate concentration, and temperature supporting molybdate reduction were pH 7.0, glucose at 1.5% (w/v), between 25 and 30 mM, and 25°C, respectively. The optimum phosphate concentration for molybdate reduction was 5 mM. The Mo-blue produced exhibits an absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. None of the respiratory inhibitors tested showed any inhibition to the molybdenum-reducing activity suggesting that the electron transport system of this bacterium is not the site of molybdenum reduction. Chromium, cadmium, silver, copper, mercury, and lead caused approximately 77, 65, 77, 89, 80, and 80% inhibition of the molybdenum-reducing activity, respectively. Ferrous and stannous ions markedly increased the activity of molybdenum-reducing activity in this bacterium. The maximum tolerable concentration of SDS as a cocontaminant was 3 g/L. The characteristics of this bacterium make it a suitable candidate for molybdenum bioremediation of sites cocontaminated with detergent pollutant. M. I. E. Halmi, S. W. Zuhainis, M. T. Yusof, N. A. Shaharuddin, W. Helmi, Y. Shukor, M. A. Syed, and S. A. Ahmad Copyright © 2013 M. I. E. Halmi et al. All rights reserved. Bioleaching of Arsenic-Rich Gold Concentrates by Bacterial Flora before and after Mutation Thu, 05 Dec 2013 15:48:18 +0000 In order to improve the bioleaching efficiency of arsenic-rich gold concentrates, a mixed bacterial flora had been developed, and the mutation breeding method was adopted to conduct the research. The original mixed bacterial flora had been enrichedin acid mine drainage of Dexing copper mine, Jiangxi Province, China. It was induced by UV (ultraviolet), ultrasonic, and microwave, and their combination mutation. The most efficient bacterial flora after mutation was collected for further bioleaching of arsenic-rich gold concentrates. Results indicated that the bacterial flora after mutation by UV 60 s combined with ultrasonic 10 min had the best oxidation rate of ferrous, the biggest density of cells, and the most activity of total protein. During bioleaching of arsenic-rich gold concentrates, the density of the mutant bacterial cells reached to cells/mL at 15 days, more than 10 times compared with that of the original culture. The extraction of iron reached to 95.7% after 15 days, increased by 9.9% compared with that of the original culture. The extraction of arsenic reached to 92.6% after 12 days, which was increased by 46.1%. These results suggested that optimum combined mutation could improve leaching ability of the bacterial flora more significantly. Xuehui Xie, Xuewu Yuan, Na Liu, Xiaoguang Chen, Awad Abdelgadir, and Jianshe Liu Copyright © 2013 Xuehui Xie et al. All rights reserved. The Increasing Interest of ANAMMOX Research in China: Bacteria, Process Development, and Application Thu, 05 Dec 2013 15:14:20 +0000 Nitrogen pollution created severe environmental problems and increasingly has become an important issue in China. Since the first discovery of ANAMMOX in the early 1990s, this related technology has become a promising as well as sustainable bioprocess for treating strong nitrogenous wastewater. Many Chinese research groups have concentrated their efforts on the ANAMMOX research including bacteria, process development, and application during the past 20 years. A series of new and outstanding outcomes including the discovery of new ANAMMOX bacterial species (Brocadia sinica), sulfate-dependent ANAMMOX bacteria (Anammoxoglobus sulfate and Bacillus benzoevorans), and the highest nitrogen removal performance (74.3–76.7 kg-N/m3/d) in lab scale granule-based UASB reactors around the world were achieved. The characteristics, structure, packing pattern and floatation mechanism of the high-rate ANAMMOX granules in ANAMMOX reactors were also carefully illustrated by native researchers. Nowadays, some pilot and full-scale ANAMMOX reactors were constructed to treat different types of ammonium-rich wastewater including monosodium glutamate wastewater, pharmaceutical wastewater, and leachate. The prime objective of the present review is to elucidate the ongoing ANAMMOX research in China from lab scale to full scale applications, comparative analysis, and evaluation of significant findings and to set a design to usher ANAMMOX research in culmination. Mohammad Ali, Li-Yuan Chai, Chong-Jian Tang, Ping Zheng, Xiao-Bo Min, Zhi-Hui Yang, Lei Xiong, and Yu-Xia Song Copyright © 2013 Mohammad Ali et al. All rights reserved. Toxicity of Superparamagnetic Iron Oxide Nanoparticles on Green Alga Chlorella vulgaris Wed, 04 Dec 2013 15:01:16 +0000 Toxicity of superparamagnetic iron oxide nanoparticles (SPION) was investigated on Chlorella vulgaris cells exposed during 72 hours to Fe3O4 (SPION-1), Co0.2Zn0.8Fe2O4 (SPION-2), or Co0.5Zn0.5Fe2O4 (SPION-3) to a range of concentrations from 12.5 to 400 μg mL−1. Under these treatments, toxicity impact was indicated by the deterioration of photochemical activities of photosynthesis, the induction of oxidative stress, and the inhibition of cell division rate. In comparison to SPION-2 and -3, exposure to SPION-1 caused the highest toxic effects on cellular division due to a stronger production of reactive oxygen species and deterioration of photochemical activity of Photosystem II. This study showed the potential source of toxicity for three SPION suspensions, having different chemical compositions, estimated by the change of different biomarkers. In this toxicological investigation, algal model C. vulgaris demonstrated to be a valuable bioindicator of SPION toxicity. Lotfi Barhoumi and David Dewez Copyright © 2013 Lotfi Barhoumi and David Dewez. All rights reserved. Dynamics of Intracellular Polymers in Enhanced Biological Phosphorus Removal Processes under Different Organic Carbon Concentrations Wed, 04 Dec 2013 10:42:55 +0000 Enhanced biological phosphorus removal (EBPR) may deteriorate or fail during low organic carbon loading periods. Polyphosphate accumulating organisms (PAOs) in EBPR were acclimated under both high and low organic carbon conditions, and then dynamics of polymers in typical cycles, anaerobic conditions with excess organic carbons, and endogenous respiration conditions were examined. After long-term acclimation, it was found that organic loading rates did not affect the yield of PAOs and the applied low organic carbon concentrations were advantageous for the enrichment of PAOs. A low influent organic carbon concentration induced a high production of extracellular carbohydrate. During both anaerobic and aerobic endogenous respirations, when glycogen decreased to around 80 ± 10 mg C per gram of volatile suspended solids, PAOs began to utilize polyphosphate significantly. Regressed by the first-order reaction model, glycogen possessed the highest degradation rate and then was followed by polyphosphate, while biomass decay had the lowest degradation rate. Lizhen Xing, Li Ren, Bo Tang, Guangxue Wu, and Yuntao Guan Copyright © 2013 Lizhen Xing et al. All rights reserved. Human Mesenchymal Stem Cells Provide Protection against Radiation-Induced Liver Injury by Antioxidative Process, Vasculature Protection, Hepatocyte Differentiation, and Trophic Effects Thu, 28 Nov 2013 16:50:03 +0000 To evaluate the potential therapeutic effect of the infusion of hMSCs for the correction of liver injuries, we performed total body radiation exposure of NOD/SCID mice. After irradiation, mir-27b level decreases in liver, increasing the directional migration of hMSCs by upregulating SDF1α. A significant increase in plasmatic transaminases levels, apoptosis process in the liver vascular system, and in oxidative stress were observed. hMSC injection induced a decrease in transaminases levels and oxidative stress, a disappearance of apoptotic cells, and an increase in Nrf2, SOD gene expression, which might reduce ROS production in the injured liver. Engrafted hMSCs expressed cytokeratin CK18 and CK19 and AFP genes indicating possible hepatocyte differentiation. The presence of hMSCs expressing VEGF and Ang-1 in the perivascular region, associated with an increased expression of VEGFr1, r2 in the liver, can confer a role of secreting cells to hMSCs in order to maintain the endothelial function. To explain the benefits to the liver of hMSC engraftment, we find that hMSCs secreted NGF, HGF, and anti-inflammatory molecules IL-10, IL1-RA contributing to prevention of apoptosis, increasing cell proliferation in the liver which might correct liver dysfunction. MSCs are potent candidates to repair and protect healthy tissues against radiation damages. Sabine Francois, Moubarak Mouiseddine, Bénédicte Allenet-Lepage, Jan Voswinkel, Luc Douay, Marc Benderitter, and Alain Chapel Copyright © 2013 Sabine Francois et al. All rights reserved. Kinetics of Molybdenum Reduction to Molybdenum Blue by Bacillus sp. Strain A.rzi Thu, 28 Nov 2013 08:50:00 +0000 Molybdenum is very toxic to agricultural animals. Mo-reducing bacterium can be used to immobilize soluble molybdenum to insoluble forms, reducing its toxicity in the process. In this work the isolation of a novel molybdate-reducing Gram positive bacterium tentatively identified as Bacillus sp. strain A.rzi from a metal-contaminated soil is reported. The cellular reduction of molybdate to molybdenum blue occurred optimally at 4 mM phosphate, using 1% (w/v) glucose, 50 mM molybdate, between 28 and 30°C and at pH 7.3. The spectrum of the Mo-blue product showed a maximum peak at 865 nm and a shoulder at 700 nm. Inhibitors of bacterial electron transport system (ETS) such as rotenone, sodium azide, antimycin A, and potassium cyanide could not inhibit the molybdenum-reducing activity. At 0.1 mM, mercury, copper, cadmium, arsenic, lead, chromium, cobalt, and zinc showed strong inhibition on molybdate reduction by crude enzyme. The best model that fitted the experimental data well was Luong followed by Haldane and Monod. The calculated value for Luong’s constants , , , and was 5.88 μmole Mo-blue hr−1, 70.36 mM, 108.22 mM, and 0.74, respectively. The characteristics of this bacterium make it an ideal tool for bioremediation of molybdenum pollution. A. R. Othman, N. A. Bakar, M. I. E. Halmi, W. L. W. Johari, S. A. Ahmad, H. Jirangon, M. A. Syed, and M. Y. Shukor Copyright © 2013 A. R. Othman et al. All rights reserved. Photosystem II Photochemistry and Phycobiliprotein of the Red Algae Kappaphycus alvarezii and Their Implications for Light Adaptation Wed, 27 Nov 2013 16:02:19 +0000 Photosystem II photochemistry and phycobiliprotein (PBP) genes of red algae Kappaphycus alvarezii, raw material of κ-carrageenan used in food and pharmaceutical industries, were analyzed in this study. Minimum saturating irradiance () of this algal species was less than 115 μmol m−2 s−1. Its actual PSII efficiency (yield II) increased when light intensity enhanced and decreased when light intensity reached 200 μmol m−2 s−1. Under dim light, yield II declined at first and then increased on the fourth day. Under high light, yield II retained a stable value. These results indicate that K. alvarezii is a low-light-adapted species but possesses regulative mechanisms in response to both excessive and deficient light. Based on the PBP gene sequences, K. alvarezii, together with other red algae, assembled faster and showed a closer relationship with LL-Prochlorococcus compared to HL-Prochlorococcus. Many amino acid loci in PBP sequences of K. alvarezii were conserved with those of LL-Prochlorococcus. However, loci conserved with HL-Prochlorococcus but divergent with LL-Prochlorococcus were also found. The diversities of PE and PC are proposed to have played some roles during the algal evolution and divergence of light adaption. Xiangyu Guan, Jinfeng Wang, Jianyi Zhu, Chunyan Yao, Jianguo Liu, Song Qin, and Peng Jiang Copyright © 2013 Xiangyu Guan et al. All rights reserved. Molecular Biomarkers: Tools of Medicine Wed, 27 Nov 2013 13:53:36 +0000 Prabir K. Mandal, Shivani Soni, R. Renee Reams, Tiziano Verri, Anita Mandal, and Sudhish Mishra Copyright © 2013 Prabir K. Mandal et al. All rights reserved. Characterization of an Antihypertensive Angiotensin I-Converting Enzyme Inhibitory Peptide from the Edible Mushroom Hypsizygus marmoreus Wed, 27 Nov 2013 11:17:45 +0000 Hypertension is one of the very serious diseases and, recently, hypertensive patient longevity has been increased significantly. Therefore, the development of new antihypertensive drugs or bioactive compounds is very important to remedy or prevent hypertension. The antihypertensive angiotensin I-converting enzyme (ACE) inhibitor in water extracts from the brown-cultivar-fruiting-body of Hypsizygus marmoreus was purified with ultrafiltration, C18 solid phase extraction chromatography and reverse-phase HPLC, and the purified ACE inhibitor with inhibitory activity of IC50 value of 0.19 mg/mL was obtained. The purified ACE inhibitor was found to be a new oligopeptide with the sequence LSMGSASLSP. Its molecular weight was estimated to be 567.3 Da and the water extracts containing ACE inhibitor from Hypsizygus marmoreus showed a clear antihypertensive action a spontaneously hypertensive rat. Min-Gu Kang, Young-Hun Kim, Zanabaatar Bolormaa, Min-Kyung Kim, Geon-Sik Seo, and Jong-Soo Lee Copyright © 2013 Min-Gu Kang et al. All rights reserved. Generation of Leukemia Inhibitory Factor-Dependent Induced Pluripotent Stem Cells from the Massachusetts General Hospital Miniature Pig Tue, 26 Nov 2013 09:14:30 +0000 The generation and application of porcine induced pluripotent stem cells (iPSCs) may enable the testing for safety and efficacy of therapy in the field of human regenerative medicine. Here, the generation of iPSCs from the Massachusetts General Hospital miniature pig (MGH minipig) established for organ transplantation studies is reported. Fibroblasts were isolated from the skin of the ear of a 10-day-old MGH minipig and transduced with a cocktail of six human factors: POU5F1, NANOG, SOX2, C-MYC, KLF4, and LIN28. Two distinct types of iPSCs were generated that were positive for alkaline phosphatase activity, as well as the classical pluripotency markers: Oct4, Nanog, Sox2, and the surface marker Ssea-1. Only one of two porcine iPSC lines differentiated into three germ layers both in vitro and in vivo. Western blot analysis showed that the porcine iPSCs were dependent on LIF or BMP-4 to sustain self-renewal and pluripotency. In conclusion, the results showed that human pluripotent factors could reprogram porcine ear fibroblasts into the pluripotent state. These cells may provide a useful source of cells that could be used for the treatment of degenerative and genetic diseases and agricultural research and application. Dae-Jin Kwon, Hyelena Jeon, Keon Bong Oh, Sun-A Ock, Gi-Sun Im, Sung-Soo Lee, Seok Ki Im, Jeong-Woong Lee, Sung-Jong Oh, Jin-Ki Park, and Seongsoo Hwang Copyright © 2013 Dae-Jin Kwon et al. All rights reserved. Practical Considerations and Challenges Involved in Surfactant Enhanced Bioremediation of Oil Sun, 24 Nov 2013 14:00:55 +0000 Surfactant enhanced bioremediation (SEB) of oil is an approach adopted to overcome the bioavailability constraints encountered in biotransformation of nonaqueous phase liquid (NAPL) pollutants. Fuel oils contain n-alkanes and other aliphatic hydrocarbons, monoaromatics, and polynuclear aromatic hydrocarbons (PAHs). Although hydrocarbon degrading cultures are abundant in nature, complete biodegradation of oil is rarely achieved even under favorable environmental conditions due to the structural complexity of oil and culture specificities. Moreover, the interaction among cultures in a consortium, substrate interaction effects during the degradation and ability of specific cultures to alter the bioavailability of oil invariably affect the process. Although SEB has the potential to increase the degradation rate of oil and its constituents, there are numerous challenges in the successful application of this technology. Success is dependent on the choice of appropriate surfactant type and dose since the surfactant-hydrocarbon-microorganism interaction may be unique to each scenario. Surfactants not only enhance the uptake of constituents through micellar solubilization and emulsification but can also alter microbial cell surface characteristics. Moreover, hydrocarbons partitioned in micelles may not be readily bioavailable depending on the microorganism-surfactant interactions. Surfactant toxicity and inherent biodegradability of surfactants may pose additional challenges as discussed in this review. Sagarika Mohanty, Jublee Jasmine, and Suparna Mukherji Copyright © 2013 Sagarika Mohanty et al. All rights reserved. Extraction and Separation of Fucoidan from Laminaria japonica with Chitosan as Extractant Sun, 24 Nov 2013 11:40:00 +0000 Herein the extraction method of fucoidan from Laminaria japonica is reported. Firstly, chitosan, chitosan-N-2-hydroxypropyl trimethyl ammonium chloride (HACC), and hexadecyltrimethylammonium bromide (CPAB) were used to extract the fucoidan. The results showed that chitosan was the optimal extractant compared with the other two extractants. After extraction, different aqueous solutions, including NaCl, KCl, and HCl (pH2), were used to separate fucoidan from chitosan-fucoidan complex. The results showed that the separation ability of NaCl was slightly higher than that of KCl. Moreover, the price of NaCl is lower than that of KCl. Given the quality-price rate, NaCl solution was chosen as the separation solution. Thirdly, the concentration and ratio of NaCl solution : sediment influence the separation of fucoidan from chitosan-fucoidan complex. The results showed that the optimal separation conditions include 4 mol/L NaCl solution with the ratio of NaCl solution to sediment at 30 : 1. Fucoidan content was found to be affected by different separation time. Fucoidan content increased with the increase of separation time, and the optimal separation time was 6 h. Compared with traditional alkali extraction method, this method not only reduces the usage of alkali and acid and alleviate environment pollution, but also has the comparable extraction yield of fucoidan. It is a potential method for extraction of fucoidan. Ronge Xing, Song Liu, Huahua Yu, Xiaolin Chen, Yukun Qin, Kecheng Li, and Pengcheng Li Copyright © 2013 Ronge Xing et al. All rights reserved. Pyrolytic and Kinetic Analysis of Two Coastal Plant Species: Artemisia annua and Chenopodium glaucum Tue, 19 Nov 2013 15:33:08 +0000 The large amount of coastal plant species available makes them ideal candidates for energy production. In this study, thermogravimetric analysis was used to evaluate the fuel properties of two coastal plant species, and the distributed activation energy model (DAEM) was employed in kinetic analysis. The major mass loss due to devolatilization started at 154 and 162°C at the heating rate of 10°C min−1 for Artemisia annua and Chenopodium glaucum, respectively. The results showed that the average activation energies of Artemisia annua and Chenopodium glaucum were 169.69 and 170.48 kJ mol−1, respectively. Furthermore, the activation energy changed while the conversion rate increased, and the frequency factor decreased greatly while the activation energy decreased. The results also indicated that the devolatilization of the two coastal plant species underwent a set of first-order reactions and could be expressed by the DAEM. Additionally, a simplified mathematical model was proposed to facilitate the prediction of devolatilization curves. Lili Li, Xiaoning Wang, Jinsheng Sun, Yichen Zhang, and Song Qin Copyright © 2013 Lili Li et al. All rights reserved. From Single Nucleotide Polymorphisms to Constant Immunosuppression: Mesenchymal Stem Cell Therapy for Autoimmune Diseases Tue, 19 Nov 2013 15:13:09 +0000 The regenerative abilities and the immunosuppressive properties of mesenchymal stromal cells (MSCs) make them potentially the ideal cellular product of choice for treatment of autoimmune and other immune mediated disorders. Although the usefulness of MSCs for therapeutic applications is in early phases, their potential clinical use remains of great interest. Current clinical evidence of use of MSCs from both autologous and allogeneic sources to treat autoimmune disorders confers conflicting clinical benefit outcomes. These varied results may possibly be due to MSC use across wide range of autoimmune disorders with clinical heterogeneity or due to variability of the cellular product. In the light of recent genome wide association studies (GWAS), linking predisposition of autoimmune diseases to single nucleotide polymorphisms (SNPs) in the susceptible genetic loci, the clinical relevance of MSCs possessing SNPs in the critical effector molecules of immunosuppression is largely undiscussed. It is of further interest in the allogeneic setting, where SNPs in the target pathway of MSC's intervention may also modulate clinical outcome. In the present review, we have discussed the known critical SNPs predisposing to disease susceptibility in various autoimmune diseases and their significance in the immunomodulatory properties of MSCs. Raghavan Chinnadurai, Edmund K. Waller, Jacques Galipeau, and Ajay K. Nooka Copyright © 2013 Raghavan Chinnadurai et al. All rights reserved. Effects of Seawater Salinity and Temperature on Growth and Pigment Contents in Hypnea cervicornis J. Agardh (Gigartinales, Rhodophyta) Tue, 19 Nov 2013 13:46:54 +0000 This study simulated outdoor environmental living conditions and observed the growth rates and changes of several photosynthetic pigments (Chl a, Car, PE, and PC) in Hypnea cervicornis J. Agardh (Gigartinales, Rhodophyta) by setting up different ranges of salinity (25, 30, 35, 40, 45, and 50) and temperature (15, 20, 25, and 30°C). At conditions of culture, the results are as follows. (1) Changes in salinity and temperature have significant effects on the growth of H. cervicornis. The growth rates first increase then decrease as the temperature increases, while growth tends to decline as salinity increases. The optimum salinity and temperature conditions for growth are 25 and 25°C, respectively. (2) Salinity and temperature have significant or extremely significant effects on photosynthetic pigments (Chl a, Car, PE, and PC) in H. cervicornis. The results of this study are advantageous to ensure propagation and economic development of this species in the southern sea area of China. Lanping Ding, Yuanyuan Ma, Bingxin Huang, and Shanwen Chen Copyright © 2013 Lanping Ding et al. All rights reserved. Microbial Removal of the Pharmaceutical Compounds Ibuprofen and Diclofenac from Wastewater Tue, 19 Nov 2013 13:38:52 +0000 Studies on the occurrence of pharmaceuticals show that the widely used pharmaceuticals ibuprofen and diclofenac are present in relevant concentrations in the environment. A pilot plant treating hospital wastewater with relevant concentrations of these pharmaceuticals was evaluated for its performance to reduce the concentration of the pharmaceuticals. Ibuprofen was completely removed, whereas diclofenac yielded a residual concentration, showing the necessity of posttreatment to remove diclofenac, for example, activated carbon. Successively, detailed laboratory experiments with activated sludge from the same wastewater treatment plant showed bioremediation potential in the treatment plant. The biological degradation pathway was studied and showed a mineralisation of ibuprofen and degradation of diclofenac. The present microbes were further studied in laboratory experiments, and DGGE analyses showed the enrichment and isolation of highly purified cultures that degraded either ibuprofen or diclofenac. This research illuminates the importance of the involved bacteria for the effectiveness of the removal of pharmaceuticals in a wastewater treatment plant. A complete removal of pharmaceuticals from wastewater will stimulate water reuse, addressing the worldwide increasing demand for clean and safe fresh water. Alette Langenhoff, Nadia Inderfurth, Teun Veuskens, Gosse Schraa, Marco Blokland, Katarzyna Kujawa-Roeleveld, and Huub Rijnaarts Copyright © 2013 Alette Langenhoff et al. All rights reserved. Enhancement of Oxygen Mass Transfer and Gas Holdup Using Palm Oil in Stirred Tank Bioreactors with Xanthan Solutions as Simulated Viscous Fermentation Broths Sun, 17 Nov 2013 11:14:37 +0000 Volumetric mass transfer coefficient () is an important parameter in bioreactors handling viscous fermentations such as xanthan gum production, as it affects the reactor performance and productivity. Published literatures showed that adding an organic phase such as hydrocarbons or vegetable oil could increase the . The present study opted for palm oil as the organic phase as it is plentiful in Malaysia. Experiments were carried out to study the effect of viscosity, gas holdup, and on the xanthan solution with different palm oil fractions by varying the agitation rate and aeration rate in a 5 L bench-top bioreactor fitted with twin Rushton turbines. Results showed that 10% (v/v) of palm oil raised the of xanthan solution by 1.5 to 3 folds with the highest value of 84.44 h−1. It was also found that palm oil increased the gas holdup and viscosity of the xanthan solution. The values obtained as a function of power input, superficial gas velocity, and palm oil fraction were validated by two different empirical equations. Similarly, the gas holdup obtained as a function of power input and superficial gas velocity was validated by another empirical equation. All correlations were found to fit well with higher determination coefficients. Suhaila Mohd Sauid, Jagannathan Krishnan, Tan Huey Ling, and Murthy V. P. S. Veluri Copyright © 2013 Suhaila Mohd Sauid et al. All rights reserved. New Trends in Biotechnological Processes to Increase the Environmental Protection Sun, 17 Nov 2013 11:12:06 +0000 Ana Moldes, José Manuel Domínguez González, Ligia Raquel Marona Rodrigues, and Attilio Converti Copyright © 2013 Ana Moldes et al. All rights reserved. Assessing the Detection Capacity of Microarrays as Bio/Nanosensing Platforms Wed, 13 Nov 2013 15:10:37 +0000 Microarray is one of the most powerful detection systems with multiplexing and high throughput capability. It has significant potential as a versatile biosensing platform for environmental monitoring, pathogen detection, medical therapeutics, and drug screening to name a few. To date, however, microarray applications are still limited to preliminary screening of genome-scale transcription profiling or gene ontology analysis. Expanding the utility of microarrays as a detection tool for various biological and biomedical applications requires information about performance such as the limits of detection and quantification, which are considered as an essential information to decide the detection sensitivity of sensing devices. Here we present a calibration design that integrates detection limit theory and linear dynamic range to obtain a performance index of microarray detection platform using oligonucleotide arrays as a model system. Two different types of limits of detection and quantification are proposed by the prediction or tolerance interval for two common cyanine fluorescence dyes, Cy3 and Cy5. Besides oligonucleotide, the proposed method can be generalized to other microarray formats with various biomolecules such as complementary DNA, protein, peptide, carbohydrate, tissue, or other small biomolecules. Also, it can be easily applied to other fluorescence dyes for further dye chemistry improvement. Ju Seok Lee, Joon Jin Song, Russell Deaton, and Jin-Woo Kim Copyright © 2013 Ju Seok Lee et al. All rights reserved. Development of a Novel Reference Plasmid for Accurate Quantification of Genetically Modified Kefeng6 Rice DNA in Food and Feed Samples Wed, 13 Nov 2013 13:42:56 +0000 Reference plasmids are an essential tool for the quantification of genetically modified (GM) events. Quantitative real-time PCR (qPCR) is the most commonly used method to characterize and quantify reference plasmids. However, the precision of this method is often limited by calibration curves, and qPCR data can be affected by matrix differences between the standards and samples. Here, we describe a digital PCR (dPCR) approach that can be used to accurately measure the novel reference plasmid pKefeng6 and quantify the unauthorized variety of GM rice Kefeng6, eliminating the issues associated with matrix effects in calibration curves. The pKefeng6 plasmid was used as a calibrant for the quantification of Kefeng6 rice by determining the copy numbers of event- (77 bp) and taxon-specific (68 bp) fragments, their ratios, and their concentrations. The plasmid was diluted to five different concentrations. The third sample (S3) was optimized for the quantification range of dPCR according to previous reports. The ratio between the two fragments was 1.005, which closely approximated the value certified by sequencing, and the concentration was found to be 792 copies/μL. This method was precise, with an RSD of ~3%. These findings demonstrate the advantages of using the dPCR method to characterize reference materials. Liang Li, Xiujie Zhang, Yusong Wan, and Wujun Jin Copyright © 2013 Liang Li et al. All rights reserved. Biohydrogen Production and Kinetic Modeling Using Sediment Microorganisms of Pichavaram Mangroves, India Mon, 11 Nov 2013 19:02:38 +0000 Mangrove sediments host rich assemblages of microorganisms, predominantly mixed bacterial cultures, which can be efficiently used for biohydrogen production through anaerobic dark fermentation. The influence of process parameters such as effect of initial glucose concentration, initial medium pH, and trace metal (Fe2+) concentration was investigated in this study. A maximum hydrogen yield of 2.34, 2.3, and 2.6 mol H2 mol−1 glucose, respectively, was obtained under the following set of optimal conditions: initial substrate concentration—10,000 mg L−1, initial pH—6.0, and ferrous sulphate concentration—100 mg L−1, respectively. The addition of trace metal to the medium (100 mg L−1 FeSO4·7H2O) enhanced the biohydrogen yield from 2.3 mol H2 mol−1 glucose to 2.6 mol H2 mol−1 glucose. Furthermore, the experimental data was subjected to kinetic analysis and the kinetic constants were estimated with the help of well-known kinetic models available in the literature, namely, Monod model, logistic model and Luedeking-Piret model. The model fitting was found to be in good agreement with the experimental observations, for all the models, with regression coefficient values >0.92. P. Mullai, Eldon R. Rene, and K. Sridevi Copyright © 2013 P. Mullai et al. All rights reserved. Mesenchymal Stem Cell-Like Cells Derived from Mouse Induced Pluripotent Stem Cells Ameliorate Diabetic Polyneuropathy in Mice Mon, 11 Nov 2013 11:03:01 +0000 Background. Although pathological involvements of diabetic polyneuropathy (DPN) have been reported, no dependable treatment of DPN has been achieved. Recent studies have shown that mesenchymal stem cells (MSCs) ameliorate DPN. Here we demonstrate a differentiation of induced pluripotent stem cells (iPSCs) into MSC-like cells and investigate the therapeutic potential of the MSC-like cell transplantation on DPN. Research Design and Methods. For induction into MSC-like cells, GFP-expressing iPSCs were cultured with retinoic acid, followed by adherent culture for 4 months. The MSC-like cells, characterized with flow cytometry and RT-PCR analyses, were transplanted into muscles of streptozotocin-diabetic mice. Three weeks after the transplantation, neurophysiological functions were evaluated. Results. The MSC-like cells expressed MSC markers and angiogenic/neurotrophic factors. The transplanted cells resided in hindlimb muscles and peripheral nerves, and some transplanted cells expressed S100β in the nerves. Impairments of current perception thresholds, nerve conduction velocities, and plantar skin blood flow in the diabetic mice were ameliorated in limbs with the transplanted cells. The capillary number-to-muscle fiber ratios were increased in transplanted hindlimbs of diabetic mice. Conclusions. These results suggest that MSC-like cell transplantation might have therapeutic effects on DPN through secreting angiogenic/neurotrophic factors and differentiation to Schwann cell-like cells. Tatsuhito Himeno, Hideki Kamiya, Keiko Naruse, Zhao Cheng, Sachiko Ito, Masaki Kondo, Tetsuji Okawa, Atsushi Fujiya, Jiro Kato, Hirohiko Suzuki, Tetsutaro Kito, Yoji Hamada, Yutaka Oiso, Kenichi Isobe, and Jiro Nakamura Copyright © 2013 Tatsuhito Himeno et al. All rights reserved. Cultivation-Dependant Assessment, Diversity, and Ecology of Haloalkaliphilic Bacteria in Arid Saline Systems of Southern Tunisia Sun, 10 Nov 2013 13:24:00 +0000 Haloalkaliphiles are polyextremophiles adapted to grow at high salt concentrations and alkaline pH values. In this work, we isolated 122 haloalkaliphilic bacteria upon enrichments of 23 samples from 5 distinct saline systems of southern Tunisia, growing optimally in media with 10% salt and at pH 10. The collection was classified into 44 groups based on the amplification of the 16S–23S rRNA internal transcribed spacers (ITS-PCR). Phylogenetic analysis and sequencing of the 16S rRNA genes allowed the identification of 13 genera and 20 distinct species. Three gram-positive isolates showing between 95 and 96% of 16S rRNA sequence homology with Bacillus saliphilus could represent new species or genus. Beside the difference in bacterial diversity between the studied sites, several species ecological niches correlations were demonstrated such as Oceanobacillus in salt crust, Nesterenkonia in sand, and Salinicoccus in the rhizosphere of the desert plant Salicornia. The collection was further evaluated for the production of extracellular enzymes. Activity tests showed that gram-positive bacteria were mostly active, particularly for protease, lipase, DNase, and amylase production. Our overall results demonstrate the huge phenotypic and phylogenetic diversity of haloalkaliphiles in saline systems of southern Tunisia which represent a valuable source of new lineages and metabolites. Darine El Hidri, Amel Guesmi, Afef Najjari, Hanen Cherif, Besma Ettoumi, Chadlia Hamdi, Abdellatif Boudabous, and Ameur Cherif Copyright © 2013 Darine El Hidri et al. All rights reserved. High Expression and Purification of Amino-Terminal Fragment of Human Amyloid Precursor Protein in Pichia pastoris and Partial Analysis of Its Properties Thu, 07 Nov 2013 17:32:42 +0000 The cleaved amino-terminal fragment of human amyloid precursor protein (N-APP) binds death receptor 6 (DR6) and triggers a caspase-dependent self-destruction process, which was suggested to contribute to Alzheimer’s disease. To investigate the N-APP-DR6-induced degeneration pathway at the molecular level, obtaining abundant and purified N-APP is fundamental and critical. The recombinant N-APP has been produced in mammalian expression system. However, the cost and yield disadvantages of mammalian expression system make it less ideal for protein mass production. Here, we successfully expressed and purified recombinant N-terminal 18-285 amino acid residues of human amyloid precursor protein from the methylotrophic yeast Pichia pastoris with a high yield of 50 mg/L. Flow cytometry indicated the purified N-APP-induced obvious apoptosis of human neuroblastoma SHEP cells. Wei Li, Xiang Gao, Junle Ren, Ting An, and Yan Liu Copyright © 2013 Wei Li et al. All rights reserved. Microbial Enzymes and Their Applications in Industries and Medicine Thu, 07 Nov 2013 16:29:40 +0000 Periasamy Anbu, Subash C. B. Gopinath, Arzu Coleri Cihan, and Bidur Prasad Chaulagain Copyright © 2013 Periasamy Anbu et al. All rights reserved. Adipose-Derived Mesenchymal Cells for Bone Regereneration: State of the Art Thu, 07 Nov 2013 15:28:39 +0000 Adipose tissue represents a hot topic in regenerative medicine because of the tissue source abundance, the relatively easy retrieval, and the inherent biological properties of mesenchymal stem cells residing in its stroma. Adipose-derived mesenchymal stem cells (ASCs) are indeed multipotent somatic stem cells exhibiting growth kinetics and plasticity, proved to induce efficient tissue regeneration in several biomedical applications. A defined consensus for their isolation, classification, and characterization has been very recently achieved. In particular, bone tissue reconstruction and regeneration based on ASCs has emerged as a promising approach to restore structure and function of bone compromised by injury or disease. ASCs have been used in combination with osteoinductive biomaterial and/or osteogenic molecules, in either static or dynamic culture systems, to improve bone regeneration in several animal models. To date, few clinical trials on ASC-based bone reconstruction have been concluded and proved effective. The aim of this review is to dissect the state of the art on ASC use in bone regenerative applications in the attempt to provide a comprehensive coverage of the topics, from the basic laboratory to recent clinical applications. Marta Barba, Claudia Cicione, Camilla Bernardini, Fabrizio Michetti, and Wanda Lattanzi Copyright © 2013 Marta Barba et al. All rights reserved. Back Propagation Neural Network Model for Predicting the Performance of Immobilized Cell Biofilters Handling Gas-Phase Hydrogen Sulphide and Ammonia Thu, 07 Nov 2013 15:01:12 +0000 Lab scale studies were conducted to evaluate the performance of two simultaneously operated immobilized cell biofilters (ICBs) for removing hydrogen sulphide (H2S) and ammonia (NH3) from gas phase. The removal efficiencies (REs) of the biofilter treating H2S varied from 50 to 100% at inlet loading rates (ILRs) varying up to 13 g H2S/m3·h, while the NH3 biofilter showed REs ranging from 60 to 100% at ILRs varying between 0.5 and 5.5 g NH3/m3·h. An application of the back propagation neural network (BPNN) to predict the performance parameter, namely, RE (%) using this experimental data is presented in this paper. The input parameters to the network were unit flow (per min) and inlet concentrations (ppmv), respectively. The accuracy of BPNN-based model predictions were evaluated by providing the trained network topology with a test dataset and also by calculating the regression coefficient (R2) values. The results from this predictive modeling work showed that BPNNs were able to predict the RE of both the ICBs efficiently. Eldon R. Rene, M. Estefanía López, Jung Hoon Kim, and Hung Suck Park Copyright © 2013 Eldon R. Rene et al. All rights reserved. Green and Rapid Synthesis of Anticancerous Silver Nanoparticles by Saccharomyces boulardii and Insight into Mechanism of Nanoparticle Synthesis Tue, 05 Nov 2013 15:12:15 +0000 Rapidly developing field of nanobiotechnology dealing with metallic nanoparticle (MNP) synthesis is primarily lacking control over size, shape, dispersity, yield, and reaction time. Present work describes an ecofriendly method for the synthesis of silver nanoparticles (AgNPs) by cell free extract (CFE) of Saccharomyces boulardii. Parameters such as culture age (stationary phase growth), cell mass concentration (400 mg/mL), temperature (35°C), and reaction time (4 h), have been optimized to exercise a control over the yield of nanoparticles and their properties. Nanoparticle (NP) formation was confirmed by UV-Vis spectroscopy, elemental composition by EDX (energy dispersive X-rays) analysis, and size and shape by transmission electron microscopy. Synthesized nanoparticles had the size range of 3–10 nm with high negative zeta potential (−31 mV) indicating excellent stability. Role of proteins/peptides in NP formation and their stability were also elucidated. Finally, anticancer activity of silver nanoparticles as compared to silver ions was determined on breast cancer cell lines. Abhishek Kaler, Sanyog Jain, and Uttam Chand Banerjee Copyright © 2013 Abhishek Kaler et al. All rights reserved. The Effect of Created Local Hyperosmotic Microenvironment in Microcapsule for the Growth and Metabolism of Osmotolerant Yeast Candida krusei Tue, 05 Nov 2013 10:16:10 +0000 Candida krusei is osmotolerant yeast used for the production of glycerol. Addition of osmolyte such as NaCl into culture medium can increase the production of glycerol from glucose, but osmolytes may burden the glycerol separation. A coencapsulation method was suggested to create local extracellular hyperosmotic stress for glycerol accumulation. Firstly, the influence of osmotic stress induced by the addition of PEG4000 on growth and metabolism of free cell was studied in detail. Glycerol accumulation could be improved by employing PEG4000 as osmoregulator. Secondly, cells and PEG4000 were coentrapped in NaCS/PDMDAAC capsules to create local hyperosmotic stress. The effects of local hyperosmotic microenvironment on the cell growth and metabolism were studied. The coentrapment method increased the glycerol concentration by 25%, and the glycerol concentration attained 50 gL−1 with productivity of 18.8 gL−1Day−1 in shake flask. More importantly, the glycerol could be directly separated from the encapsulated cells. The entrapped cells containing PEG4000 were also cultivated for 15 days in an airlift reactor. The yield and productivity were ca. 35% and 21 gL−1Day−1, respectively. Guo Chen and Shanjing Yao Copyright © 2013 Guo Chen and Shanjing Yao. All rights reserved. A Recombinant Multiepitope Protein for Hepatitis B Diagnosis Tue, 05 Nov 2013 09:05:29 +0000 Hepatitis B is a liver inflammation caused by hepatitis B virus (HBV) and can be diagnosed in clinical stage by hepatitis B core antibody from IgM class (anti-HBcIgM). Hepatitis B core antibody from IgG class (Anti-HBcIgG) appears quickly after IgM, reaching high titers in chronic hepatitis, and remains even after cure. Since hepatitis B core antibody (anti-HBc) is the first antibody identified and sometimes the only marker detected during the course of infection, it can be used both to indicate HBV acute infection (anti-HBc-IgM) and to identify individuals who have come into contact with the virus (anti-HBc-IgG). In this work we propose a recombinant hepatitis B core multiepitope antigen (rMEHB) to be used for diagnosis of hepatitis B. For this purpose, a synthetic gene coding for rMEHB was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal. Time course induction in E. coli showed an induced protein with an apparent molecular mass of ~21 kDa. Protein purification was performed by a single step with affinity chromatography Ni-NTA. Circular dichroism spectroscopy indicated rMEHB as a thermal stable protein at pH 7.0 and 8.0. In these conditions rMEHB was successfully used to perform an enzyme linked immuno sorbent assay (ELISA) with positive and negative sera. Marilen Queiroz de Souza, Alexsandro Sobreira Galdino, José Carlos dos Santos, Marcus Vinicius Soares, Yanna C. de Nóbrega, Alice da Cunha Morales Álvares, Sonia Maria de Freitas, Fernando Araripe Gonçalves Torres, and Maria Sueli Soares Felipe Copyright © 2013 Marilen Queiroz de Souza et al. All rights reserved. Mass Production of Early-Stage Bone-Marrow-Derived Mesenchymal Stem Cells of Rat Using Gelatin-Coated Matrix Thu, 31 Oct 2013 13:22:08 +0000 Although preparation of early-stage bone-marrow-derived mesenchymal stem cells (BM-MSCs) is critical for successful cell transplantation therapy, no culture system offers a sufficient number of early-stage BM-MSCs for cell transplantation. Accordingly, we developed a culture system capable of producing a large number of early-stage BM-MSCs by using gelatin-coated matrix. The greatest retrieval and proliferation rates of the earliest-stage rat BM-MSCs were detected in bone-marrow-derived cells cultured on 1% (wt/v) gelatin-coated matrix, which showed significantly greater colony forming unit-fibroblast number, diameter, and total cell number. Moreover, continuous culture of the earliest-stage BM-MSCs on 1% (wt/v) gelatin-coated matrix resulted in a maximum of fold increase in the cumulative total number of early-stage BM-MSCs at passage 5. BM-MSCs generated in large quantities due to a reduced doubling time and an increased yield of cell population in S/G2/M phase showed typical fibroblast-like morphology and no significant differences in BM-MSC-related surface marker expression and differentiation potential, except for an increased ratio of differentiation into a neurogenic lineage. The use of gelatin-coated matrix in the retrieval and culture of BM-MSCs contributes greatly to the effective isolation and mass production of early-stage BM-MSCs. Young Hyun Park, Jung Im Yun, Na Rae Han, Hye Jin Park, Ji Yeon Ahn, Choonghyo Kim, Jung Hoon Choi, Eunsong Lee, Jeong Mook Lim, and Seung Tae Lee Copyright © 2013 Young Hyun Park et al. All rights reserved. Human Induced Pluripotent Stem Cells from Basic Research to Potential Clinical Applications in Cancer Mon, 28 Oct 2013 11:52:45 +0000 The human induced pluripotent stem cells (hiPSCs) are derived from a direct reprogramming of human somatic cells to a pluripotent stage through ectopic expression of specific transcription factors. These cells have two important properties, which are the self-renewal capacity and the ability to differentiate into any cell type of the human body. So, the discovery of hiPSCs opens new opportunities in biomedical sciences, since these cells may be useful for understanding the mechanisms of diseases in the production of new diseases models, in drug development/drug toxicity tests, gene therapies, and cell replacement therapies. However, the hiPSCs technology has limitations including the potential for the development of genetic and epigenetic abnormalities leading to tumorigenicity. Nowadays, basic research in the hiPSCs field has made progress in the application of new strategies with the aim to enable an efficient production of high-quality of hiPSCs for safety and efficacy, necessary to the future application for clinical practice. In this review, we show the recent advances in hiPSCs’ basic research and some potential clinical applications focusing on cancer. We also present the importance of the use of statistical methods to evaluate the possible validation for the hiPSCs for future therapeutic use toward personalized cell therapies. Teresa de Souza Fernandez, Cecilia de Souza Fernandez, and André Luiz Mencalha Copyright © 2013 Teresa de Souza Fernandez et al. All rights reserved. Defects in Base Excision Repair Sensitize Cells to Manganese in S. cerevisiae Sun, 27 Oct 2013 14:57:20 +0000 Manganese (Mn) is essential for normal physiologic functioning; therefore, deficiencies and excess intake of manganese can result in disease. In humans, prolonged exposure to manganese causes neurotoxicity characterized by Parkinson-like symptoms. Mn2+ has been shown to mediate DNA damage possibly through the generation of reactive oxygen species. In a recent publication, we showed that Mn induced oxidative DNA damage and caused lesions in thymines. This study further investigates the mechanisms by which cells process Mn2+-mediated DNA damage using the yeast S. cerevisiae. The strains most sensitive to Mn2+ were those defective in base excision repair, glutathione synthesis, and superoxide dismutase mutants. Mn2+ caused a dose-dependent increase in the accumulation of mutations using the CAN1 and lys2-10A mutator assays. The spectrum of CAN1 mutants indicates that exposure to Mn results in accumulation of base substitutions and frameshift mutations. The sensitivity of cells to Mn2+ as well as its mutagenic effect was reduced by N-acetylcysteine, glutathione, and Mg2+. These data suggest that Mn2+ causes oxidative DNA damage that requires base excision repair for processing and that Mn interferes with polymerase fidelity. The status of base excision repair may provide a biomarker for the sensitivity of individuals to manganese. Adrienne P. Stephenson, Tryphon K. Mazu, Jana S. Miles, Miles D. Freeman, R. Renee Reams, and Hernan Flores-Rozas Copyright © 2013 Adrienne P. Stephenson et al. All rights reserved. Microbial Diversity in the Era of Omic Technologies Thu, 24 Oct 2013 11:45:56 +0000 Human life and activity depends on microorganisms, as they are responsible for providing basic elements of life. Although microbes have such a key role in sustaining basic functions for all living organisms, very little is known about their biology since only a small fraction (average 1%) can be cultured under laboratory conditions. This is even more evident when considering that >88% of all bacterial isolates belong to four bacterial phyla, the Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Advanced technologies, developed in the last years, promise to revolutionise the way that we characterize, identify, and study microbial communities. In this review, we present the most advanced tools that microbial ecologists can use for the study of microbial communities. Innovative microbial ecological DNA microarrays such as PhyloChip and GeoChip that have been developed for investigating the composition and function of microbial communities are presented, along with an overview of the next generation sequencing technologies. Finally, the Single Cell Genomics approach, which can be used for obtaining genomes from uncultured phyla, is outlined. This tool enables the amplification and sequencing of DNA from single cells obtained directly from environmental samples and is promising to revolutionise microbiology. Sofia Nikolaki and George Tsiamis Copyright © 2013 Sofia Nikolaki and George Tsiamis. All rights reserved. Evaluation of NK Cell Function by Flowcytometric Measurement and Impedance Based Assay Using Real-Time Cell Electronic Sensing System Wed, 23 Oct 2013 18:35:48 +0000 Although real-time cell electronic sensing (RT-CES) system-based natural killer (NK) cytotoxicity has been introduced, it has not been evaluated using human blood samples. In present study, we measured flowcytometry based assay (FCA) and RT-CES based NK cytotoxicity and analyzed degranulation activity (CD107a) and cytokine production. In 98 healthy individuals, FCA with peripheral blood mononuclear cells (PBMCs) at effector to target (E/T) ratio of 32 revealed 46.5 ± 2.6% cytolysis of K562 cells, and 23.5 ± 1.1% of NK cells showed increased degranulation. In RT-CES system, adherent NIH3T3 target cells were resistant to basal killing by PBMC or NK cells. NK cell activation by adding IL-2 demonstrated real-time dynamic killing activity, and lymphokine-activated PBMC (E/T ratio of 32) from 15 individuals showed 59.1 ± 6.2% cytotoxicity results after 4 hours incubation in RT-CES system. However, there was no significant correlation between FCA and RT-CES cytotoxicity. After K562 target cell stimulation, PBMC produced profound proinflammatory and immunoregulatory cytokines/chemokines including IL-2, IL-8, IL-10, MIP-1αβ, IFN-γ, and TNF-α, and cytokine/chemokine secretion was related to flowcytometry-based NK cytotoxicity. These data suggest that RT-CES and FCA differ in sensitivity, applicability and providing information, and further investigations are necessary in variable clinical conditions. Ki-Hyun Park, Hyesun Park, Myungshin Kim, Yonggoo Kim, Kyungja Han, and Eun-Jee Oh Copyright © 2013 Ki-Hyun Park et al. All rights reserved. Transcriptome Profile Analysis of Sugarcane Responses to Sporisorium scitaminea Infection Using Solexa Sequencing Technology Wed, 23 Oct 2013 13:46:30 +0000 To understand the molecular basis of sugarcane-smut interaction, it is important to identify sugarcane genes that respond to the pathogen attack. High-throughput tag-sequencing (tag-seq) analysis by Solexa technology was performed on sugarcane infected with Sporisorium scitaminea, which should have massively increased the amount of data available for transcriptome profile analysis. After mapping to sugarcane EST databases in NCBI, we obtained 2015 differentially expressed genes, of which 1125 were upregulated and 890 downregulated by infection. Gene ontology (GO) analysis revealed that the differentially expressed genes involve in many cellular processes. Pathway analysis revealed that metabolic pathways and ribosome function are significantly affected, where upregulation of expression dominates over downregulation. Differential expression of three candidate genes involved in MAP kinase signaling pathway, ScBAK1 (GenBank Accession number: KC857629), ScMapkk (GenBank Accession number: KC857627), and ScGloI (GenBank Accession number: KC857628), was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Real-time quantitative PCR (qRT-PCR) analysis concluded that the expression of these genes were all up-regulated after the infection of S. scitaminea and may play a role in pathogen response in sugarcane. The present study provides insights into the molecular mechanism of sugarcane defense to S. scitaminea infection, leading to a more comprehensive understanding of sugarcane-smut interaction. Qibin Wu, Liping Xu, Jinlong Guo, Yachun Su, and Youxiong Que Copyright © 2013 Qibin Wu et al. All rights reserved. Practical Guidance for Implementing Predictive Biomarkers into Early Phase Clinical Studies Tue, 22 Oct 2013 08:46:12 +0000 The recent U.S. Food and Drug Administration (FDA) coapprovals of several therapeutic compounds and their companion diagnostic devices (FDA News Release, 2011, 2013) to identify patients who would benefit from treatment have led to considerable interest in incorporating predictive biomarkers in clinical studies. Yet, the translation of predictive biomarkers poses unique technical, logistic, and regulatory challenges that need to be addressed by a multidisciplinary team including discovery scientists, clinicians, biomarker experts, regulatory personnel, and assay developers. These issues can be placed into four broad categories: sample collection, assay validation, sample analysis, and regulatory requirements. In this paper, we provide a primer for drug development teams who are eager to implement a predictive patient segmentation marker into an early clinical trial in a way that facilitates subsequent development of a companion diagnostic. Using examples of nucleic acid-based assays, we briefly review common issues encountered when translating a biomarker to the clinic but focus primarily on key practical issues that should be considered by clinical teams when planning to use a biomarker to balance arms of a study or to determine eligibility for a clinical study. Matthew J. Marton and Russell Weiner Copyright © 2013 Matthew J. Marton and Russell Weiner. All rights reserved. The World Bacterial Biogeography and Biodiversity through Databases: A Case Study of NCBI Nucleotide Database and GBIF Database Mon, 21 Oct 2013 13:35:13 +0000 Databases are an essential tool and resource within the field of bioinformatics. The primary aim of this study was to generate an overview of global bacterial biodiversity and biogeography using available data from the two largest public online databases, NCBI Nucleotide and GBIF. The secondary aim was to highlight the contribution each geographic area has to each database. The basis for data analysis of this study was the metadata provided by both databases, mainly, the taxonomy and the geographical area origin of isolation of the microorganism (record). These were directly obtained from GBIF through the online interface, while E-utilities and Python were used in combination with a programmatic web service access to obtain data from the NCBI Nucleotide Database. Results indicate that the American continent, and more specifically the USA, is the top contributor, while Africa and Antarctica are less well represented. This highlights the imbalance of exploration within these areas rather than any reduction in biodiversity. This study describes a novel approach to generating global scale patterns of bacterial biodiversity and biogeography and indicates that the Proteobacteria are the most abundant and widely distributed phylum within both databases. Okba Selama, Phillip James, Farida Nateche, Elizabeth M. H. Wellington, and Hocine Hacène Copyright © 2013 Okba Selama et al. All rights reserved. Circulating microRNAs and Kallikreins before and after Radical Prostatectomy: Are They Really Prostate Cancer Markers? Wed, 09 Oct 2013 13:56:00 +0000 The aim of our study was to monitor serum levels of two miRNAs (miR-21 and miR-141) and three KLKs (hK3/PSA, hK11, and hK13) before and 1, 5, and 30 days after radical prostatectomy, in order to characterize their fluctuations after surgery. 38 patients with prostate cancer were included. miR-21 and miR-141 were quantified through real-time PCR, while ELISA assays were used to quantify hK3 (PSA), hK11, and hK13. Both miR-21 and miR-141 showed a significant increase at the 5th postoperative day, after which a gradual return to the preoperative levels was recorded. These findings suggest that miR-21 and miR-141 could be involved in postsurgical inflammatory processes and that radical prostatectomy does not seem to alter their circulating levels. Postoperative serum kallikreins showed a significant decrease, highlighting the potential usefulness of kallikreins apart from PSA as potential prostate cancer markers. Maria Giulia Egidi, Giovanni Cochetti, Maria Rita Serva, Gabriella Guelfi, Danilo Zampini, Luca Mechelli, and Ettore Mearini Copyright © 2013 Maria Giulia Egidi et al. All rights reserved. The Microbiology of Olive Mill Wastes Thu, 03 Oct 2013 15:26:26 +0000 Olive mill wastes (OMWs) are high-strength organic effluents, which upon disposal can degrade soil and water quality, negatively affecting aquatic and terrestrial ecosystems. The main purpose of this review paper is to provide an up-to-date knowledge concerning the microbial communities identified over the past 20 years in olive mill wastes using both culture-dependent and independent approaches. A database survey of 16S rRNA gene sequences (585 records in total) obtained from olive mill waste environments revealed the dominance of members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria. Independent studies confirmed that OMW microbial communities’ structure is cultivar dependant. On the other hand, the detection of fecal bacteria and other potential human pathogens in OMWs is of major concern and deserves further examination. Despite the fact that the degradation and detoxification of the olive mill wastes have been mostly investigated through the application of known bacterial and fungal species originated from other environmental sources, the biotechnological potential of indigenous microbiota should be further exploited in respect to olive mill waste bioremediation and inactivation of plant and human pathogens. The implementation of omic and metagenomic approaches will further elucidate disposal issues of olive mill wastes. Spyridon Ntougias, Kostas Bourtzis, and George Tsiamis Copyright © 2013 Spyridon Ntougias et al. All rights reserved. Microbial Purification of Postfermentation Medium after 1,3-PD Production from Raw Glycerol Wed, 02 Oct 2013 15:55:45 +0000 1,3-Propanediol (1,3-PD) is an important chemical product which can be used to produce polyesters, polyether, and polyurethanes. In the process of conversion of glycerol to 1,3-PD by Clostridium large number of byproducts (butyric, acetic and lactic acid) are generated in the fermentation medium. The aim of this work was to isolate bacteria strains capable of the utilization of these byproducts. Screening of 30 bacterial strains was performed using organic acids as carbon source. Selected isolates were taxonomically characterized and identified as Alcaligenes faecalis and Bacillus licheniformis. The most active strains, Alcaligenes faecalis JP1 and Bacillus licheniformis JP19, were able to utilize organic acids almost totally. Finally, it was find out that by the use of coculture (C. butyricum DSP1 and A. faecalis JP1) increased volumetric productivity of 1,3-PD production (1.07 g/L/h) and the yield equal to 0.53 g/g were obtained in bioreactor fermentation. Moreover, the only by-product present was butyric acid in a concentration below 1 g/L. Daria Szymanowska-Powałowska, Joanna Piątkowska, and Katarzyna Leja Copyright © 2013 Daria Szymanowska-Powałowska et al. All rights reserved. Characteristics of Clinical Shiga Toxin-Producing Escherichia coli Isolated from British Columbia Wed, 02 Oct 2013 09:17:18 +0000 Shiga toxin-producing Escherichia coli (STEC) are significant public health threats. Although STEC O157 are recognized foodborne pathogens, non-O157 STEC are also important causes of human disease. We characterized 10 O157:H7 and 15 non-O157 clinical STEC derived from British Columbia (BC). Eae, hlyA, and were more frequently observed in STEC O157, and 80 and 100% of isolates possessed and , respectively. In contrast, and occurred in 80 and 40% of non-O157 STEC, respectively. Comparative genomic fingerprinting (CGF) revealed three distinct clusters (C). STEC O157 was identified as lineage I (LI; LSPA-6 111111) and clustered as a single group (C1). The cdi gene previously observed only in LII was seen in two LI O157 isolates. CGF C2 strains consisted of diverse non-O157 STEC while C3 included only O103:H25, O118, and O165 serogroup isolates. With the exception of O121 and O165 isolates which were similar in virulence gene complement to STEC O157, C1 O157 STEC produced more Stx2 than non-O157 STEC. Antimicrobial resistance (AMR) screening revealed resistance or reduced sensitivity in all strains, with higher levels occurring in non-O157 STEC. One STEC O157 isolate possessed a mobile gene transferrable across genre via conjugation. Kevin J. Allen, Chad R. Laing, Ana Cancarevic, Yongxiang Zhang, Lili R. Mesak, Hai Xu, Ana Paccagnella, Victor P. J. Gannon, and Linda Hoang Copyright © 2013 Kevin J. Allen et al. All rights reserved. A Novel Differential Predict Model Based on Matrix-Assisted Laser Ionization Time-of-Flight Mass Spectrometry and Serum Ferritin for Acute Graft-versus-Host Disease Tue, 01 Oct 2013 08:56:30 +0000 Clinical diagnosis of acute graft-versus-host disease (aGVHD) mainly depends on clinical manifestation and tissue biopsies, leading to a delayed diagnosis and treatment for aGVHD patients when the early symptom is insignificant. Our objective was to investigate the possibility of prewarning the risk of aGVHD before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT) by serum protein profiling combined with serum ferritin. The difference in polypeptide expression before and after transplantation had been compared by using CLINPROT technology, and serum ferritin levels have been analyzed simultaneously. Through combining serum ferritin and MS spectral data, the diagnosis sensitivity and specificity of our model for prewarning severe aGVHD (III~IV°aGVHD) before transplant all increased to 90.0%, while after transplant, the sensitivity and specificity are 78.3% and 86.4%. Our joint prewarning model could predict the risk of aGVHD, especially severe aGVHD before and after transplant, which also provides a reliable method to the continuous monitoring of the condition of patients. Chun-yan Zhang, Shu-hong Wang, Wen-rong Huang, Guang-hong Guo, Zhu-hong Zhang, Wen-jun Mou, Li Yu, and Ya-Ping Tian Copyright © 2013 Chun-yan Zhang et al. All rights reserved. Improvement of L-Arabinose Fermentation by Modifying the Metabolic Pathway and Transport in Saccharomyces cerevisiae Mon, 30 Sep 2013 11:55:06 +0000 The L-arabinose utilization pathway was established in Saccharomyces cerevisiae, by expressing the codon-optimized araA, araB, and araD genes of Lactobacillus plantarum. After overexpressing the TAL1, TKL1, RPE1, RKI1, and GAL2 genes and adaptive evolution, the L-arabinose utilization of the recombinant strain became efficient. The resulting strain displayed a maximum specific growth rate of 0.075 h−1, a maximum specific L-arabinose consumption rate of 0.61 g h−1 g−1 dry cell weight, and a promising ethanol yield of 0.43 g g−1 from L-arabinose fermentation. Chengqiang Wang, Yu Shen, Yanyan Zhang, Fan Suo, Jin Hou, and Xiaoming Bao Copyright © 2013 Chengqiang Wang et al. All rights reserved. Direct Biodiesel Production from Wet Microalgae Biomass of Chlorella pyrenoidosa through In Situ Transesterification Mon, 30 Sep 2013 08:44:57 +0000 A one-step process was applied to directly converting wet oil-bearing microalgae biomass of Chlorella pyrenoidosa containing about 90% of water into biodiesel. In order to investigate the effects of water content on biodiesel production, distilled water was added to dried microalgae biomass to form wet biomass used to produce biodiesel. The results showed that at lower temperature of 90°C, water had a negative effect on biodiesel production. The biodiesel yield decreased from 91.4% to 10.3% as water content increased from 0% to 90%. Higher temperature could compensate the negative effect. When temperature reached 150°C, there was no negative effect, and biodiesel yield was over 100%. Based on the above research, wet microalgae biomass was directly applied to biodiesel production, and the optimal conditions were investigated. Under the optimal conditions of 100 mg dry weight equivalent wet microalgae biomass, 4 mL methanol, 8 mL n-hexane, 0.5 M H2SO4, 120°C, and 180 min reaction time, the biodiesel yield reached as high as 92.5% and the FAME content was 93.2%. The results suggested that biodiesel could be effectively produced directly from wet microalgae biomass and this effort may offer the benefits of energy requirements for biodiesel production. Hechun Cao, Zhiling Zhang, Xuwen Wu, and Xiaoling Miao Copyright © 2013 Hechun Cao et al. All rights reserved. Production of the Quinone-Methide Triterpene Maytenin by In Vitro Adventitious Roots of Peritassa campestris (Cambess.) A.C.Sm. (Celastraceae) and Rapid Detection and Identification by APCI-IT-MS/MS Wed, 25 Sep 2013 14:47:04 +0000 Establishment of adventitious root cultures of Peritassa campestris (Celastraceae) was achieved from seed cotyledons cultured in semisolid Woody Plant Medium (WPM) supplemented with 2% sucrose, 0.01% PVP, and 4.0 mg L−1 IBA. Culture period on accumulation of biomass and quinone-methide triterpene maytenin in adventitious root were investigated. The accumulation of maytenin in these roots was compared with its accumulation in the roots of seedlings grown in a greenhouse (one year old). A rapid detection and identification of maytenin by direct injection into an atmospheric-pressure chemical ionization ion trap tandem mass spectrometer (APCI-IT-MS/MS) were performed without prior chromatographic separation. In vitro, the greatest accumulation of biomass occurred within 60 days of culture. The highest level of maytenin—972.11 μg·g−1 dry weight—was detected at seven days of cultivation; this value was 5.55-fold higher than that found in the roots of seedlings grown in a greenhouse. Tiago Antunes Paz, Vânia A. F. F. M. dos Santos, Marielle Cascaes Inácio, Edieidia Souza Pina, Ana Maria Soares Pereira, and Maysa Furlan Copyright © 2013 Tiago Antunes Paz et al. All rights reserved. Arsenate Retention by Epipsammic Biofilms Developed on Streambed Sediments: Influence of Phosphate Tue, 24 Sep 2013 17:47:05 +0000 Natural geological conditions together with the impact of human activities could produce environmental problems due to high As concentrations. The aim of this study was to assess the role of epipsammic biofilm-sediment systems onto As (V) sorption and to evaluate the effect of the presence of equimolar P concentrations on As retention. A natural biofilm was grown on sediment samples in the laboratory, using river water as nutrient supplier. Sorption experiments with initial As concentrations 0, 5, 25, 50, 100, 250, and 500 μg L−1 were performed. The average percentage of As sorbed was and % for the sediment and biofilm-sediment systems, respectively. Phosphate decreased by 25% the As sorption capactity in the sediment devoid of biofilm, whereas no significant effect was observed in the systems with biofilm. Freundlich, Sips, and Toth models were the best to describe experimental data. The maximum As sorption capacity of the sediment and biofilm-sediment systems was, respectively, 6.6 and 6.8 μg g−1 and 4.5 and 7.8 μg g−1 in the presence of P. In conclusion, epipsammic biofilms play an important role in the environmental quality of river systems, increasing As retention by the system, especially in environments where both As and P occur simultaneously. D. M. Prieto, R. Devesa-Rey, D. A. Rubinos, F. Díaz-Fierros, and M. T. Barral Copyright © 2013 D. M. Prieto et al. All rights reserved. In Vitro and In Vivo Survival and Colonic Adhesion of Pediococcus acidilactici MTCC5101 in Human Gut Tue, 24 Sep 2013 11:52:42 +0000 The present study aims to investigate the probiotic nature of Pediococcus acidilactici MTCC5101 by an in vitro assay of bacterial adherence to intestinal epithelial cells of human gastrointestinal (GI) tract using Caco-2 cell line. Further to assess the in vivo survival in the GI tract, oral feeding was carried out with the help of 10 healthy volunteers. The effect on wellness was assessed by studying blood biochemical parameters of the volunteers. The survival of the bacteria was assessed using PCR-based detection of P. acidilactici MTCC5101 in fecal samples. The probiotic nature of P. acidilactici MTCC 5101 was strengthened by its adherence to the intestinal epithelial Caco-2 cell line in the in vitro SEM observations. Oral feeding study for assessing the survival of bacteria in GI tract of volunteers showed the strain to be established in the GI tract which survived for about 2 weeks after feeding. Praveen P. Balgir, Baljinder Kaur, Tejinder Kaur, Natisha Daroch, and Gurpreet Kaur Copyright © 2013 Praveen P. Balgir et al. All rights reserved. Skeletal Muscle Regeneration on Protein-Grafted and Microchannel-Patterned Scaffold for Hypopharyngeal Tissue Engineering Mon, 23 Sep 2013 15:10:21 +0000 In the field of tissue engineering, polymeric materials with high biocompatibility like polylactic acid and polyglycolic acid have been widely used for fabricating living constructs. For hypopharynx tissue engineering, skeletal muscle is one important functional part of the whole organ, which assembles the unidirectionally aligned myotubes. In this study, a polyurethane (PU) scaffold with microchannel patterns was used to provide aligning guidance for the seeded human myoblasts. Due to the low hydrophilicity of PU, the scaffold was grafted with silk fibroin (PU-SF) or gelatin (PU-Gel) to improve its cell adhesion properties. Scaffolds were observed to degrade slowly over time, and their mechanical properties and hydrophilicities were improved through the surface grafting. Also, the myoblasts seeded on PU-SF had the higher proliferative rate and better differentiation compared with those on the control or PU-Gel. Our results demonstrate that polyurethane scaffolds seeded with myoblasts hold promise to guide hypopharynx muscle regeneration. Zhisen Shen, Shanshan Guo, Dong Ye, Jingjing Chen, Cheng Kang, Shejie Qiu, Dakai Lu, Qun Li, Kunjie Xu, Jingjing Lv, and Yabin Zhu Copyright © 2013 Zhisen Shen et al. All rights reserved. Constitutive Optimized Production of Streptokinase in Saccharomyces cerevisiae Utilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter of Pichia pastoris Sun, 22 Sep 2013 15:22:25 +0000 A novel expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay and in vitro clot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions. Ravi N. Vellanki, Ravichandra Potumarthi, Kiran K. Doddapaneni, Naveen Anubrolu, and Lakshmi N. Mangamoori Copyright © 2013 Ravi N. Vellanki et al. All rights reserved. Biomedical and Clinical Promises of Human Pluripotent Stem Cells for Neurological Disorders Sun, 22 Sep 2013 15:05:37 +0000 Neurological disorders are characterized by the chronic and progressive loss of neuronal structures and functions. There is a variability of the onsets and causes of clinical manifestations. Cell therapy has brought a new concept to overcome brain diseases, but the advancement of this therapy is limited by the demands of specialized neurons. Human pluripotent stem cells (hPSCs) have been promised as a renewable resource for generating human neurons for both laboratory and clinical purposes. By the modulations of appropriate signalling pathways, desired neuron subtypes can be obtained, and induced pluripotent stem cells (iPSCs) provide genetically matched neurons for treating patients. These hPSC-derived neurons can also be used for disease modeling and drug screening. Since the most urgent problem today in transplantation is the lack of suitable donor organs and tissues, the derivation of neural progenitor cells from hPSCs has opened a new avenue for regenerative medicine. In this review, we summarize the recent reports that show how to generate neural derivatives from hPSCs, and discuss the current evidence of using these cells in animal studies. We also highlight the possibilities and concerns of translating these hPSC-derived neurons for biomedical and clinical uses in order to fight against neurological disorders. Nopporn Jongkamonwiwat and Parinya Noisa Copyright © 2013 Nopporn Jongkamonwiwat and Parinya Noisa. All rights reserved. The use of Multidimensional Data to Identify the Molecular Biomarker for Pancreatic Ductal Adenocarcinoma Sat, 21 Sep 2013 15:01:31 +0000 Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, and the patient has an extremely poor overall survival with a less than 5% 5-year survival rate. Development of potential biomarkers provides a critical foundation for the diagnosis of PDAC. In this project, we have adopted an integrative approach to simultaneously identify biomarker and generate testable hypothesis from multidimensional omics data. We first examine genes for which expression levels are correlated with survival data. The gene list was screened with TF regulation, predicted miRNA targets information, and KEGG pathways. We identified that 273 candidate genes are correlated with patient survival data. 12 TF regulation gene sets, 11 miRNAs targets gene sets, and 15 KEGG pathways are enriched with these survival genes. Notably, CEBPA/miRNA32/PER2 signaling to the clock rhythm qualifies this pathway as a suitable target for therapeutic intervention in PDAC. PER2 expression was highly associated with survival data, thus representing a novel biomarker for earlier detection of PDAC. Liwei Zhuang, Yue Qi, Yun Wu, Nannan Liu, and Yili Fu Copyright © 2013 Liwei Zhuang et al. All rights reserved. Environmental Kuznets Curve Analysis of the Economic Development and Nonpoint Source Pollution in the Ningxia Yellow River Irrigation Districts in China Sat, 21 Sep 2013 15:00:27 +0000 This study applies the environmental Kuznets curve to test the relationship between the regional economic growth and the different types of agricultural nonpoint source pollution loads in the Ningxia Yellow River irrigation area by using the Johnes export coefficient method. Results show that the pollution load generated by crop cultivation and livestock-breeding industries in the Ningxia Yellow River irrigation area shows an inverted U-shaped feature; however, this feature is absent in living-sewage pollution load. Crop pollution has shown a decreasing trend since 1997 because of the increased per capita income of farmers. Livestock-breeding pollution load reached its turning point when the per capita income of farmers reached 8386.74 RMB. Therefore, an increase in the per capita income of farmers corresponds to an increase in the livestock-breeding pollution load in the Ningxia Yellow River irrigation area. Chunlan Mao, Ningning Zhai, Jingchao Yang, Yongzhong Feng, Yanchun Cao, Xinhui Han, Guangxin Ren, Gaihe Yang, and Qing-xiang Meng Copyright © 2013 Chunlan Mao et al. All rights reserved. Clinical Evaluation and Cost-Effectiveness Analysis of Serum Tumor Markers in Lung Cancer Thu, 19 Sep 2013 15:01:10 +0000 The detection of serum tumor markers is valuable for the early diagnosis of lung cancer. Tumor markers are frequently used for the management of cancer patients. However, single markers are less efficient but marker combinations increase the cost, which is troublesome for clinics. To find an optimal serum marker combination panel that benefits the patients and the medical management system as well, four routine lung cancer serum markers (SCCA, NSE, CEA, and CYFRA21-1) were evaluated individually and in combination. Meanwhile, the costs and effects of these markers in clinical practice in China were assessed by cost-effectiveness analysis. As expected, combinations of these tumor markers improved their sensitivity for lung cancer and different combination panels had their own usefulness. NSE + CEA + CYFRA21-1 was the optimal combination panel with highest Youden’s index (0.64), higher sensitivity (75.76%), and specificity (88.57%), which can aid the clinical diagnosis of lung cancer. Nevertheless, the most cost-effective combination was SCCA + CEA, which can be used to screen the high-risk group. Rong Wang, Guoqing Wang, Nan Zhang, Xue Li, and Yunde Liu Copyright © 2013 Rong Wang et al. All rights reserved. Biological Pretreatment of Rubberwood with Ceriporiopsis subvermispora for Enzymatic Hydrolysis and Bioethanol Production Thu, 19 Sep 2013 11:46:44 +0000 Rubberwood (Hevea brasiliensis), a potential raw material for bioethanol production due to its high cellulose content, was used as a novel feedstock for enzymatic hydrolysis and bioethanol production using biological pretreatment. To improve ethanol production, rubberwood was pretreated with white rot fungus Ceriporiopsis subvermispora to increase fermentation efficiency. The effects of particle size of rubberwood (1 mm, 0.5 mm, and 0.25 mm) and pretreatment time on the biological pretreatment were first determined by chemical analysis and X-ray diffraction and their best condition obtained with 1 mm particle size and 90 days pretreatment. Further morphological study on rubberwood with 1 mm particle size pretreated by fungus was performed by FT-IR spectra analysis and SEM observation and the result indicated the ability of this fungus for pretreatment. A study on enzymatic hydrolysis resulted in an increased sugar yield of 27.67% as compared with untreated rubberwood (2.88%). The maximum ethanol concentration and yield were 17.9 g/L and 53% yield, respectively, after 120 hours. The results obtained demonstrate that rubberwood pretreated by C. subvermispora can be used as an alternative material for the enzymatic hydrolysis and bioethanol production. Forough Nazarpour, Dzulkefly Kuang Abdullah, Norhafizah Abdullah, Nazila Motedayen, and Reza Zamiri Copyright © 2013 Forough Nazarpour et al. All rights reserved. Early Continence Recovery after Preservation of Maximal Urethral Length until the Level of Verumontanum during Radical Prostatectomy: Primary Oncological and Functional Outcomes after 1 Year of Follow-Up Thu, 19 Sep 2013 11:43:17 +0000 Purpose. To investigate the effect of preventing maximal urethral length until the level of verumontanum during radical prostatectomy on both oncologic and functional outcomes. Patients and Methods. We recruited 329 patients, and they underwent an open radical prostatectomy by a single surgeon. The study cohort was randomized in 2 groups. A standard radical prostatectomy was performed in group A patients, while in group B the urethra was preserved until the level of verumontanum. Results. There was no statistically significant difference between the study groups in terms of positive surgical margins or biochemical relapse. Regarding the functional results, the incidence of incontinence, urgency, and nocturia at 1st month, statistically significant higher rates were seen in group A. In addition, there was a statistically significant difference in the number of pads/day in favor of group B at the 1st, 3rd, and 6th months after surgery. However, this difference was eliminated at 12 months postoperatively. Similar results were seen with the scores of the ICIQ-SF and IIQ-SF questionnaires. Conclusions. Without compromising the oncological outcome, our surgical modificated technique showed earlier recovery of continence in the first 6 months, having though the same rates of continence at 12 months. Stavros Sfoungaristos, Stavros Kontogiannis, and Petros Perimenis Copyright © 2013 Stavros Sfoungaristos et al. All rights reserved. Immune Parameters in The Prognosis and Therapy Monitoring of Cutaneous Melanoma Patients: Experience, Role, and Limitations Thu, 19 Sep 2013 10:28:58 +0000 Cutaneous melanoma is an immune-dependent aggressive tumour. Up to our knowledge, there are no reports regarding immune parameters monitoring in longitudinal followup of melanoma patients. We report a followup for 36 months of the immune parameters of patients diagnosed in stages I–IV. The circulatory immune parameters comprised presurgery and postsurgery immune circulating peripheral cells and circulating intercommunicating cytokines. Based on our analysis, the prototype of the intratumor inflammatory infiltrate in a melanoma with good prognosis is composed of numerous T cells CD3+, few or even absent B cells CD20+, few or absent plasma cells CD138+, and present Langerhans cells CD1a+ or langerin+. Regarding circulatory immune cells, a marker that correlates with stage is CD4+/CD8+ ratio, and its decrease clearly indicates a worse prognosis of the disease. Moreover, even in advanced stages, patients that have an increased overall survival rate prove the increase of this ratio. The decrease in the circulating B lymphocytes with stage is balanced by an increase in circulating NK cells, a phenomenon observed in stage III. Out of all the tested cytokines in the followup, IL-6 level correlated with the patient’s survival, while in our study, IL-8, IL-10, and IL-12 did not correlate statistically in a significant way with overall survival, or relapse-free survival. Monica Neagu, Carolina Constantin, and Sabina Zurac Copyright © 2013 Monica Neagu et al. All rights reserved. Stem Cell Therapy in Bladder Dysfunction: Where Are We? And Where Do We Have to Go? Mon, 16 Sep 2013 18:45:10 +0000 To date, stem cell therapy for the bladder has been conducted mainly on an experimental basis in the areas of bladder dysfunction. The therapeutic efficacy of stem cells was originally thought to be derived from their ability to differentiate into various cell types. Studies about stem cell therapy for bladder dysfunction have been limited to an experimental basis and have been less focused than bladder regeneration. Bladder dysfunction was listed in MESH as “urinary bladder neck obstruction”, “urinary bladder, overactive”, and “urinary bladder, neurogenic”. Using those keywords, several articles were searched and studied. The bladder dysfunction model includes bladder outlet obstruction, cryoinjured, diabetes, ischemia, and spinal cord injury. Adipose derived stem cells (ADSCs), bone marrow stem cells (BMSCs), and skeletal muscle derived stem cells (SkMSCs) are used for transplantation to treat bladder dysfunction. The main mechanisms of stem cells to reconstitute or restore bladder dysfunction are migration, differentiation, and paracrine effects. The aim of this study is to review the stem cell therapy for bladder dysfunction and to provide the status of stem cell therapy for bladder dysfunction. Jae Heon Kim, Sang-Rae Lee, Yun Seob Song, and Hong Jun Lee Copyright © 2013 Jae Heon Kim et al. All rights reserved. Nanobiotechnology Mon, 16 Sep 2013 14:17:23 +0000 Susana N. Diniz, Alejandro Sosnik, Huiling Mu, and Claudete J. Valduga Copyright © 2013 Susana N. Diniz et al. All rights reserved. Extending the Serum Half-Life of G-CSF via Fusion with the Domain III of Human Serum Albumin Sun, 15 Sep 2013 13:39:43 +0000 Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF), the domain III of human serum albumin (3DHSA) was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned into pPICZαA along with the open reading frame of the α-factor signal under the control of the AOX1 promoter. The recombinant expression vector was transformed into Pichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC) counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF. Shuqiang Zhao, Yu Zhang, Hong Tian, Xiaofei Chen, Di Cai, Wenbing Yao, and Xiangdong Gao Copyright © 2013 Shuqiang Zhao et al. All rights reserved. Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase from Aspergillus niger ATCC 20611 Sun, 15 Sep 2013 11:38:10 +0000 The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO3, Zn+2, and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination () more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO3, 1.5 mM (v/v) Zn+2, and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry. Mojdeh Dinarvand, Malahat Rezaee, Malihe Masomian, Seyed Davoud Jazayeri, Mohsen Zareian, Sahar Abbasi, and Arbakariya B. Ariff Copyright © 2013 Mojdeh Dinarvand et al. All rights reserved. Diversity and Antimicrobial Properties of Lactic Acid Bacteria Isolated from Rhizosphere of Olive Trees and Desert Truffles of Tunisia Sat, 14 Sep 2013 09:03:10 +0000 A total of 119 lactic acid bacteria (LAB) were isolated, by culture-dependant method, from rhizosphere samples of olive trees and desert truffles and evaluated for different biotechnological properties. Using the variability of the intergenic spacer 16S-23S and 16S rRNA gene sequences, the isolates were identified as the genera Lactococcus, Pediococcus, Lactobacillus, Weissella, and Enterococcus. All the strains showed proteolytic activity with variable rates 42% were EPS producers, while only 10% showed the ability to grow in 9% NaCl. In addition, a low rate of antibiotic resistance was detected among rhizospheric enterococci. Furthermore, a strong antibacterial activity against plant and/or pathogenic bacteria of Stenotrophomonas maltophilia, Pantoea agglomerans, Pseudomonas savastanoi, the food-borne Staphylococcus aureus, and Listeria monocytogenes was recorded. Antifungal activity evaluation showed that Botrytis cinerea was the most inhibited fungus followed by Penicillium expansum, Verticillium dahliae, and Aspergillus niger. Most of the active strains belonged to the genera Enterococcus and Weissella. This study led to suggest that environmental-derived LAB strains could be selected for technological application to control pathogenic bacteria and to protect food safety from postharvest deleterious microbiota. Imene Fhoula, Afef Najjari, Yousra Turki, Sana Jaballah, Abdelatif Boudabous, and Hadda Ouzari Copyright © 2013 Imene Fhoula et al. All rights reserved. A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device Sat, 14 Sep 2013 08:47:29 +0000 Existing chemotaxis assays do not generate stable chemotactic gradients and thus—over time—functionally measure only nonspecific random motion (chemokinesis). In comparison, microfluidic technology has the capacity to generate a tightly controlled microenvironment that can be stably maintained for extended periods of time and is, therefore, amenable to adaptation for assaying chemotaxis. We describe here a novel microfluidic device for sensitive assay of cellular migration and show its application for evaluating the chemotaxis of smooth muscle cells in a chemokine gradient. Chen Zhang, Sunyoung Jang, Ovid C. Amadi, Koichi Shimizu, Richard T. Lee, and Richard N. Mitchell Copyright © 2013 Chen Zhang et al. All rights reserved. Comparative Gene Expression Profiling in Human Cumulus Cells according to Ovarian Gonadotropin Treatments Thu, 12 Sep 2013 15:16:40 +0000 In in vitro fertilization cycles, both HP-hMG and rFSH gonadotropin treatments are widely used to control human follicle development. The objectives of this study are (i) to characterize and compare gene expression profiles in cumulus cells (CCs) of periovulatory follicles obtained from patients stimulated with HP-hMG or rFSH in a GnRH antagonist cycle and (ii) to examine their relationship with in vitro embryo development, using Human Genome U133 Plus 2.0 microarrays. Genes that were upregulated in HP-hMG-treated CCs are involved in lipid metabolism (GM2A) and cell-to-cell interactions (GJA5). Conversely, genes upregulated in rFSH-treated CCs are implicated in cell assembly and organization (COL1A1 and COL3A1). Interestingly, some genes specific to each gonadotropin treatment (NPY1R and GM2A for HP-hMG; GREM1 and OSBPL6 for rFSH) were associated with day 3 embryo quality and blastocyst grade at day 5, while others (STC2 and PTX3) were related to in vitro embryo quality in both gonadotropin treatments. These genes may prove valuable as biomarkers of in vitro embryo quality. Said Assou, Delphine Haouzi, Hervé Dechaud, Anna Gala, Alice Ferrières, and Samir Hamamah Copyright © 2013 Said Assou et al. All rights reserved. Arsenic, Zinc, and Aluminium Removal from Gold Mine Wastewater Effluents and Accumulation by Submerged Aquatic Plants (Cabomba piauhyensis, Egeria densa, and Hydrilla verticillata) Thu, 12 Sep 2013 15:04:07 +0000 The potential of three submerged aquatic plant species (Cabomba piauhyensis, Egeria densa, and Hydrilla verticillata) to be used for As, Al, and Zn phytoremediation was tested. The plants were exposed for 14 days under hydroponic conditions to mine waste water effluents in order to assess the suitability of the aquatic plants to remediate elevated multi-metals concentrations in mine waste water. The results show that the E. densa and H. verticillata are able to accumulate high amount of arsenic (95.2%) and zinc (93.7%) and resulted in a decrease of arsenic and zinc in the ambient water. On the other hand, C. piauhyensis shows remarkable aluminium accumulation in plant biomass (83.8%) compared to the other tested plants. The ability of these plants to accumulate the studied metals and survive throughout the experiment demonstrates the potential of these plants to remediate metal enriched water especially for mine drainage effluent. Among the three tested aquatic plants, H. verticillata was found to be the most applicable (84.5%) and suitable plant species to phytoremediate elevated metals and metalloid in mine related waste water. Ahmad Farid Abu Bakar, Ismail Yusoff, Ng Tham Fatt, Faridah Othman, and Muhammad Aqeel Ashraf Copyright © 2013 Ahmad Farid Abu Bakar et al. All rights reserved. Discoloration of Indigo Carmine Using Aqueous Extracts from Vegetables and Vegetable Residues as Enzyme Sources Thu, 12 Sep 2013 12:15:21 +0000 Several vegetables and vegetable residues were used as sources of enzymes capable to discolor indigo carmine (IC), completely or partially. Complete discoloration was achieved with aqueous extracts of green pea seeds and peels of green pea, cucumber, and kohlrabi, as well as spring onion leaves. The source of polyphenol oxidase (PPO), pH, time, and aeration is fundamental for the discoloration process catalyzed by PPO. The PPO present in the aqueous extract of green pea seeds was able to degrade 3,000 ppm of IC at a pH of 7.6 and magnetic stirring at 1,800 rpm in about 36 h. In addition, at 1,800 rpm and a pH of 7.6, this extract discolored 300 ppm of IC in 1:40 h; in the presence of 10% NaCl, the discoloration was complete in 5:50 h, whereas it was completed in 4:30 h with 5% NaCl and 2% laundry soap. A. Solís, F. Perea, M. Solís, N. Manjarrez, H. I. Pérez, and J. Cassani Copyright © 2013 A. Solís et al. All rights reserved. Improved Properties of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Produced by Comamonas sp. EB172 Utilizing Volatile Fatty Acids by Regulating the Nitrogen Source Wed, 11 Sep 2013 10:32:51 +0000 This study presents the effect of carbon to nitrogen ratio (C/N) (mol/mol) on the cell growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) accumulation by Comamonas sp. EB172 in 2 L fermenters using volatile fatty acids (VFA) as the carbon source. This VFA was supplemented with ammonium sulphate and yeast extract in the feeding solution to achieve C/N (mol/mol) 5, 15, 25, and 34.4, respectively. By extrapolating the C/N and the source of nitrogen, the properties of the polymers can be regulated. The number average molecular weight () of P(3HB-co-3HV) copolymer reached the highest at 838 × 103 Da with polydispersity index (PDI) value of 1.8, when the culture broth was supplemented with yeast extract (C/N 34.4). Tensile strength and Young’s modulus of the copolymer containing 6–8 mol% 3HV were in the ranges of 13–14.4 MPa and 0.26–0.34 GPa, respectively, comparable to those of polyethylene (PE). Thus, Comamonas sp. EB172 has shown promising bacterial isolates producing polyhydroxyalkanoates from renewable carbon materials. Mohd Rafein Zakaria, Hidayah Ariffin, Suraini Abd-Aziz, Mohd Ali Hassan, and Yoshihito Shirai Copyright © 2013 Mohd Rafein Zakaria et al. All rights reserved. Recovery of Stored Aerobic Granular Sludge and Its Contaminants Removal Efficiency under Different Operation Conditions Wed, 11 Sep 2013 08:49:20 +0000 The quick recovery process of contaminants removal of aerobic granular sludge (AGS) is complex, and the influencing factors are still not clear. The effects of dissolved oxygen (DO, air intensive aeration rate), organic loading rate (OLR), and C/N on contaminants removal characteristics of AGS and subsequently long-term operation of AGS bioreactor were investigated in this study. DO had a major impact on the recovery of AGS. The granules reactivated at air intensive aeration rate of 100 L/h achieved better settling property and contaminants removal efficiency. Moreover, protein content in extracellular polymeric substance (EPS) was almost unchanged, which demonstrated that an aeration rate of 100 L/h was more suitable for maintaining the biomass and the structure of AGS. Higher OLR caused polysaccharides content increase in EPS, and unstable C/N resulted in the overgrowth of filamentous bacteria, which presented worse NH4+-N and PO43−-P removal. Correspondingly, quick recovery of contaminants removal was accomplished in 12 days at the optimized operation conditions of aeration rate 100 L/h, OLR 4 g/L·d, and C/N 100 : 10, with COD, NH4+-N, and PO43−-P removal efficiencies of 87.2%, 86.9%, and 86.5%, respectively. The renovation of AGS could be successfully utilized as the seed sludge for the rapid start-up of AGS bioreactor. Zhiwei Zhao, Shuo Wang, Wenxin Shi, and Ji Li Copyright © 2013 Zhiwei Zhao et al. All rights reserved. A Broader View: Microbial Enzymes and Their Relevance in Industries, Medicine, and Beyond Wed, 11 Sep 2013 08:10:45 +0000 Enzymes are the large biomolecules that are required for the numerous chemical interconversions that sustain life. They accelerate all the metabolic processes in the body and carry out a specific task. Enzymes are highly efficient, which can increase reaction rates by 100 million to 10 billion times faster than any normal chemical reaction. Due to development in recombinant technology and protein engineering, enzymes have evolved as an important molecule that has been widely used in different industrial and therapeutical purposes. Microbial enzymes are currently acquiring much attention with rapid development of enzyme technology. Microbial enzymes are preferred due to their economic feasibility, high yields, consistency, ease of product modification and optimization, regular supply due to absence of seasonal fluctuations, rapid growth of microbes on inexpensive media, stability, and greater catalytic activity. Microbial enzymes play a major role in the diagnosis, treatment, biochemical investigation, and monitoring of various dreaded diseases. Amylase and lipase are two very important enzymes that have been vastly studied and have great importance in different industries and therapeutic industry. In this review, an approach has been made to highlight the importance of different enzymes with special emphasis on amylase and lipase in the different industrial and medical fields. Neelam Gurung, Sumanta Ray, Sutapa Bose, and Vivek Rai Copyright © 2013 Neelam Gurung et al. All rights reserved. Improvement of Daptomycin Production in Streptomyces roseosporus through the Acquisition of Pleuromutilin Resistance Tue, 10 Sep 2013 14:21:17 +0000 Daptomycin, a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, displays potent activity against a variety of gram-positive pathogens. There is a demand for generating high-producing strains for industrial production of this valuable antibiotic. Ribosome engineering is a powerful strategy to enhance the yield of secondary metabolites. In this study, the effect of a diterpenoid antibiotic pleuromutilin resistance mutation on daptomycin production was assessed. Spontaneous pleuromutilin-resistant derivatives of S. roseosporus were isolated. Sequencing of rplC locus (encoding the ribosomal protein L3) showed a point mutation at nt 455, resulting in the substitution of glycine with valine. G152V mutants showed increased production of daptomycin by approximately 30% in comparison with the wild-type strain. Its effect on daptomycin production was due to enhanced gene transcription of the daptomycin biosynthetic genes. In conclusion, pleuromutilin could be used as a novel ribosome engineering agent to improve the production of desired secondary metabolites. Linli Li, Tingmei Ma, Qing Liu, Yuqi Huang, Changhua Hu, and Guojian Liao Copyright © 2013 Linli Li et al. All rights reserved. Application of Universal Stress Proteins in Probing the Dynamics of Potent Degraders in Complex Terephthalate Metagenome Tue, 10 Sep 2013 11:28:01 +0000 The culture-independent strategies to study microbial diversity and function have led to a revolution in environmental genomics, enabling fundamental questions about the distribution of microbes and their influence on bioremediation to be addressed. In this research we used the expression of universal stress proteins as a probe to determine the changes in degrading microbial population from a highly toxic terephthalate wastewater to a less toxic activated sludge bioreactor. The impact of relative toxicities was significantly elaborated at the levels of genus and species. The results indicated that 23 similar prokaryotic phyla were represented in both metagenomes irrespective of their relative abundance. Furthermore, the following bacteria taxa Micromonosporaceae, Streptomyces, Cyanothece sp. PCC 7822, Alicyclobacillus acidocaldarius, Bacillus halodurans, Leuconostoc mesenteroides, Lactococcus garvieae, Brucellaceae, Ralstonia solanacearum, Verminephrobacter eiseniae, Azoarcus, Acidithiobacillus ferrooxidans, Francisella tularensis, Methanothermus fervidus, and Methanocorpusculum labreanum were represented only in the activated sludge bioreactor. These highly dynamic microbes could serve as taxonomic biomarkers for toxic thresholds related to terephthalate and its derivatives. This paper, highlights the application of universal stress proteins in metagenomics analysis. Dynamics of microbial consortium of this nature can have future in biotechnological applications in bioremediation of toxic chemicals and radionuclides. Andreas N. Mbah and Raphael D. Isokpehi Copyright © 2013 Andreas N. Mbah and Raphael D. Isokpehi. All rights reserved. Evaluation of a New Lipase from Staphylococcus sp. for Detergent Additive Capability Tue, 10 Sep 2013 11:17:17 +0000 Lipases are the enzymes of choice for laundry detergent industries owing to their triglyceride removing ability from the soiled fabric which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In the present study, a partially purified bacterial lipase from Staphylococcus arlettae JPBW-1 isolated from the rock salt mine has been assessed for its triglyceride removing ability by developing a presoak solution so as to use lipase as an additive in laundry detergent formulations. The effects of selected surfactants, commercial detergents, and oxidizing agents on lipase stability were studied in a preliminary evaluation for its further usage in the industrial environment. Partially purified lipase has shown good stability in presence of surfactants, commercial detergents, and oxidizing agents. Washing efficiency has been found to be enhanced while using lipase with 0.5% nonionic detergent than the anioinic detergent. The wash performance using 0.5% wheel with 40 U lipase at 40°C in 45 min results in maximum oil removal (62%) from the soiled cotton fabric. Hence, the present study opens the new era in enzyme-based detergent sector for formulation of chemical-free detergent using alkaline bacterial lipase. Mamta Chauhan, Rajinder Singh Chauhan, and Vijay Kumar Garlapati Copyright © 2013 Mamta Chauhan et al. All rights reserved. A Biosurfactant-Sophorolipid Acts in Synergy with Antibiotics to Enhance Their Efficiency Mon, 09 Sep 2013 15:00:59 +0000 Sophorolipids (SLs), biosurfactants with antimicrobial properties, have been tried to address the problem of antibiotic resistance. The synergistic action of SL and antibiotics was checked using standard microdilution and spread plate methods. With Staphylococcus aureus, SL-tetracycline combination achieved total inhibition before 4 h of exposure while tetracycline alone couldnot achieve total inhibition till the end of 6 h. The inhibition caused by exposure of bacterium to SL-tetracycline mixture was ~25% more as compared to SL alone. In spite of known robustness of gram-negative bacteria, SL-cefaclor mixture proved to be efficient against Escherichia coli which showed ~48% more inhibition within 2 h of exposure as compared to cefaclor alone. Scanning electron microscopy of the cells treated with mixture revealed bacterial cell membrane damage and pore formation. Moreover, SLs being a type of asymmetric bola, they are expected to form self-assemblies with unique functionality. This led to the speculation that SLs being amphiphilic in nature can span through the structurally alike cell membrane and facilitate the entry of drug molecules. Kasturi Joshi-Navare and Asmita Prabhune Copyright © 2013 Kasturi Joshi-Navare and Asmita Prabhune. All rights reserved. Lignocellulosic Fermentation of Wild Grass Employing Recombinant Hydrolytic Enzymes and Fermentative Microbes with Effective Bioethanol Recovery Mon, 09 Sep 2013 10:02:48 +0000 Simultaneous saccharification and fermentation (SSF) studies of steam exploded and alkali pretreated different leafy biomass were accomplished by recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for bioethanol production. The recombinant C. thermocellum GH5 cellulase and GH43 hemicellulase genes expressed in Escherichia coli cells were grown in repetitive batch mode, with the aim of enhancing the cell biomass production and enzyme activity. In batch mode, the cell biomass () of E. coli cells and enzyme activities of GH5 cellulase and GH43 hemicellulase were 1.4 and 1.6 with 2.8 and 2.2 U·mg−1, which were augmented to 2.8 and 2.9 with 5.6 and 3.8 U·mg−1 in repetitive batch mode, respectively. Steam exploded wild grass (Achnatherum hymenoides) provided the best ethanol titres as compared to other biomasses. Mixed enzyme (GH5 cellulase, GH43 hemicellulase) mixed culture (Saccharomyces cerevisiae, Candida shehatae) system gave 2-fold higher ethanol titre than single enzyme (GH5 cellulase) single culture (Saccharomyces cerevisiae) system employing 1% (w/v) pretreated substrate. 5% (w/v) substrate gave 11.2 g·L−1 of ethanol at shake flask level which on scaling up to 2 L bioreactor resulted in 23 g·L−1 ethanol. 91.6% (v/v) ethanol was recovered by rotary evaporator with 21.2% purification efficiency. Saprativ P. Das, Arabinda Ghosh, Ashutosh Gupta, Arun Goyal, and Debasish Das Copyright © 2013 Saprativ P. Das et al. All rights reserved. Copper Enhanced Monooxygenase Activity and FT-IR Spectroscopic Characterisation of Biotransformation Products in Trichloroethylene Degrading Bacterium: Stenotrophomonas maltophilia PM102 Sun, 08 Sep 2013 15:28:35 +0000 Stenotrophomonas maltophilia PM102 (NCBI GenBank Acc. no. JQ797560) is capable of growth on trichloroethylene as the sole carbon source. In this paper, we report the purification and characterisation of oxygenase present in the PM102 isolate. Enzyme activity was found to be induced 10.3-fold in presence of 0.7 mM copper with a further increment to 14.96-fold in presence of 0.05 mM NADH. Optimum temperature for oxygenase activity was recorded at . The reported enzyme was found to have enhanced activity at pH 5 and pH 8, indicating presence of two isoforms. Maximum activity was seen on incubation with benzene compared to other substrates like TCE, chloroform, toluene, hexane, and petroleum benzene. and for benzene were 3.8 mM and 340 U/mg/min and those for TCE were 2.1 mM and 170 U/mg/min. The crude enzyme was partially purified by ammonium sulphate precipitation followed by dialysis. Zymogram analysis revealed two isoforms in the 70% purified enzyme fraction. The activity stain was more prominent when the native gel was incubated in benzene as substrate in comparison to TCE. Crude enzyme and purified enzyme fractions were assayed for TCE degradation by the Fujiwara test. TCE biotransformation products were analysed by FT-IR spectroscopy. Piyali Mukherjee and Pranab Roy Copyright © 2013 Piyali Mukherjee and Pranab Roy. All rights reserved. Green Synthesis of Silver Nanoparticles Using Pinus eldarica Bark Extract Sun, 08 Sep 2013 11:22:44 +0000 Recently, development of reliable experimental protocols for synthesis of metal nanoparticles with desired morphologies and sizes has become a major focus of researchers. Green synthesis of metal nanoparticles using organisms has emerged as a nontoxic and ecofriendly method for synthesis of metal nanoparticles. The objectives of this study were production of silver nanoparticles using Pinus eldarica bark extract and optimization of the biosynthesis process. The effects of quantity of extract, substrate concentration, temperature, and pH on the formation of silver nanoparticles are studied. TEM images showed that biosynthesized silver nanoparticles (approximately in the range of 10–40 nm) were predominantly spherical in shape. The preparation of nano-structured silver particles using P. eldarica bark extract provides an environmentally friendly option, as compared to currently available chemical and/or physical methods. Siavash Iravani and Behzad Zolfaghari Copyright © 2013 Siavash Iravani and Behzad Zolfaghari. All rights reserved. In Vitro Cytotoxic, Antioxidant, and Antimicrobial Activities of Mesua beccariana (Baill.) Kosterm., Mesua ferrea Linn., and Mesua congestiflora Extracts Sun, 08 Sep 2013 10:07:48 +0000 The in vitro cytotoxicity tests on the extracts of Mesua beccariana, M. ferrea, and M. congestiflora against Raji, SNU-1, HeLa, LS-174T, NCI-H23, SK-MEL-28, Hep-G2, IMR-32, and K562 were achieved using MTT assay. The methanol extracts of Mesua beccariana showed its potency towards the proliferation of B-lymphoma cell (Raji). In addition, only the nonpolar to semipolar extracts (hexane to ethyl acetate) of the three Mesua species indicated cytotoxic effects on the tested panel of human cancer cell lines. Antioxidant assays were evaluated using DPPH scavenging radical assay and Folin-Ciocalteu method. The methanol extracts of M. beccariana and M. ferrea showed high antioxidant activities with low EC50 values of 12.70 and 9.77 μg/mL, respectively, which are comparable to that of ascorbic acid (EC50 = 5.62 μg/mL). Antibacterial tests were carried out using four Gram positive and four Gram negative bacteria on Mesua beccariana extracts. All the extracts showed negative results in the inhibition of Gram negative bacteria. Nevertheless, methanol extracts showed some activities against Gram positive bacteria which are Bacillus cereus, methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus aureus (MRSA), while the hexane extract also contributed some activities towards Bacillus cereus. Soek Sin Teh, Gwendoline Cheng Lian Ee, Siau Hui Mah, Yoke Keong Yong, Yang Mooi Lim, Mawardi Rahmani, and Zuraini Ahmad Copyright © 2013 Soek Sin Teh et al. All rights reserved. Human Body Fluid Sun, 08 Sep 2013 09:38:40 +0000 Shih-Bin Su, Terence Chuen Wai Poon, and Visith Thongboonkerd Copyright © 2013 Shih-Bin Su et al. All rights reserved. Aptamers: Novel Molecules as Diagnostic Markers in Bacterial and Viral Infections? Thu, 05 Sep 2013 13:31:25 +0000 Worldwide the entire human population is at risk of infectious diseases of which a high degree is caused by pathogenic protozoans, worms, bacteria, and virus infections. Moreover the current medications against pathogenic agents are losing their efficacy due to increasing and even further spreading drug resistance. Therefore, there is an urgent need to discover novel diagnostic as well as therapeutic tools against infectious agents. In view of that, the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) represents a powerful technology to target selectively pathogenic factors as well as entire bacteria or viruses. SELEX uses a large combinatorial oligonucleic acid library (DNA or RNA) which is processed a by high-flux in vitro screen of iterative cycles. The selected ligands, termed aptamers, are characterized by high specificity and affinity to their target molecule, which are already exploited in diagnostic and therapeutic applications. In this minireview we will discuss the current status of the SELEX technique applied on bacterial and viral pathogens. Flávia M. Zimbres, Attila Tárnok, Henning Ulrich, and Carsten Wrenger Copyright © 2013 Flávia M. Zimbres et al. All rights reserved. An Insight into the “-Omics” Based Engineering of Streptomycetes for Secondary Metabolite Overproduction Mon, 02 Sep 2013 13:26:03 +0000 Microorganisms produce a range of chemical substances representing a vast diversity of fascinating molecular architectures not available in any other system. Among them, Streptomyces are frequently used to produce useful enzymes and a wide variety of secondary metabolites with potential biological activities. Streptomyces are preferred over other microorganisms for producing more than half of the clinically useful naturally originating pharmaceuticals. However, these compounds are usually produced in very low amounts (or not at all) under typical laboratory conditions. Despite the superiority of Streptomyces, they still lack well documented genetic information and a large number of in-depth molecular biological tools for strain improvement. Previous attempts to produce high yielding strains required selection of the genetic material through classical mutagenesis for commercial production of secondary metabolites, optimizing culture conditions, and random selection. However, a profound effect on the strategy for strain development has occurred with the recent advancement of whole-genome sequencing, systems biology, and genetic engineering. In this review, we demonstrate a few of the major issues related to the potential of “-omics” technology (genomics, transcriptomics, proteomics, and metabolomics) for improving streptomycetes as an intelligent chemical factory for enhancing the production of useful bioactive compounds. Amit Kumar Chaudhary, Dipesh Dhakal, and Jae Kyung Sohng Copyright © 2013 Amit Kumar Chaudhary et al. All rights reserved. Genetic and Biochemical Diversity of Paenibacillus larvae Isolated from Tunisian Infected Honey Bee Broods Mon, 02 Sep 2013 10:26:56 +0000 Paenibacillus larvae is the causative agent of American foulbrood (AFB), a virulent disease of honeybee (Apis mellifera) larvae. In Tunisia, AFB has been detected in many beekeeping areas, where it causes important economic losses, but nothing is known about the diversity of the causing agent. Seventy-five isolates of P. larvae, identified by biochemical tests and 16S rRNA gene sequencing, were obtained from fifteen contaminated broods showing typical AFB symptoms, collected in different locations in the northern part of the country. Using BOX-PCR, a distinct profile of P. larvae with respect to related Paenibacillus species was detected which may be useful for its identification. Some P. larvae-specific bands represented novel potential molecular markers for the species. BOX-PCR fingerprints indicated a relatively high intraspecific diversity among the isolates not described previously with several molecular polymorphisms identifying six genotypes on polyacrylamide gel. Polymorphisms were also detected in several biochemical characters (indol production, nitrate reduction, and methyl red and oxidase tests). Contrary to the relatively high intraspecies molecular and phenotypic diversity, the in vivo virulence of three selected P. larvae genotypes did not differ significantly, suggesting that the genotypic/phenotypic differences are neutral or related to ecological aspects other than virulence. Chadlia Hamdi, Jihène Essanaa, Luigi Sansonno, Elena Crotti, Khaoula Abdi, Naima Barbouche, Annalisa Balloi, Elena Gonella, Alberto Alma, Daniele Daffonchio, Abdellatif Boudabous, and Ameur Cherif Copyright © 2013 Chadlia Hamdi et al. All rights reserved. Preventative and Therapeutic Probiotic Use in Allergic Skin Conditions: Experimental and Clinical Findings Sun, 01 Sep 2013 13:34:53 +0000 Probiotics are ingested live microbes that can modify intestinal microbial populations in a way that benefits the host. The interest in probiotic preventative/therapeutic potential in allergic diseases stemmed from the fact that probiotics have been shown to improve intestinal dysbiosis and permeability and to reduce inflammatory cytokines in human and murine experimental models. Enhanced presence of probiotic bacteria in the intestinal microbiota is found to correlate with protection against allergy. Therefore, many studies have been recently designed to examine the efficacy of probiotics, but the literature on the allergic skin disorders is still very scarce. Here, our objective is to summarize and evaluate the available knowledge from randomized or nonrandomized controlled trials of probiotic use in allergic skin conditions. Clinical improvement especially in IgE-sensitized eczema and experimental models such as atopic dermatitis-like lesions (trinitrochlorobenzene and picryl chloride sensitizations) and allergic contact dermatitis (dinitrofluorobenzene sensitization) has been reported. Although there is a very promising evidence to recommend the addition of probiotics into foods, probiotics do not have a proven role in the prevention or the therapy of allergic skin disorders. Thus, being aware of possible measures, such as probiotics use, to prevent/heal atopic diseases is essential for the practicing allergy specialist. Öner Özdemir and Azize Yasemin Göksu Erol Copyright © 2013 Öner Özdemir and Azize Yasemin Göksu Erol. All rights reserved. Performance Study of Chromium (VI) Removal in Presence of Phenol in a Continuous Packed Bed Reactor by Escherichia coli Isolated from East Calcutta Wetlands Thu, 29 Aug 2013 11:33:30 +0000 Organic pollutants, like phenol, along with heavy metals, like chromium, are present in various industrial effluents that pose serious health hazard to humans. The present study looked at removal of chromium (VI) in presence of phenol in a counter-current continuous packed bed reactor packed with E. coli cells immobilized on clay chips. The cells removed 85% of 500 mg/L of chromium (VI) from MS media containing glucose. Glucose was then replaced by 500 mg/L phenol. Temperature and pH of the MS media prior to addition of phenol were 30°C and 7, respectively. Hydraulic retention times of phenol- and chromium (VI)-containing synthetic media and air flow rates were varied to study the removal efficiency of the reactor system. Then temperature conditions of the reactor system were varied from 10°C to 50°C, the optimum being 30°C. The pH of the media was varied from pH 1 to pH 12, and the optimum pH was found to be 7. The maximum removal efficiency of 77.7% was achieved for synthetic media containing phenol and chromium (VI) in the continuous reactor system at optimized conditions, namely, hydraulic retention time at 4.44 hr, air flow rate at 2.5 lpm, temperature at 30°C, and pH at 7. Bhaswati Chakraborty, Suvendu Indra, Ditipriya Hazra, Rupal Betai, Lalitagauri Ray, and Srabanti Basu Copyright © 2013 Bhaswati Chakraborty et al. All rights reserved. Phenolic Biotransformations during Conversion of Ferulic Acid to Vanillin by Lactic Acid Bacteria Wed, 28 Aug 2013 14:24:15 +0000 Vanillin is widely used as food additive and as a masking agent in various pharmaceutical formulations. Ferulic acid is an important precursor of vanillin that is available in abundance in cell walls of cereals like wheat, corn, and rice. Phenolic biotransformations can occur during growth of lactic acid bacteria (LAB), and their production can be made feasible using specialized LAB strains that have been reported to produce ferulic acid esterases. The present study aimed at screening a panel of LAB isolates for their ability to release phenolics from agrowaste materials like rice bran and their biotransformation to industrially important compounds such as ferulic acid, 4-ethyl phenol, vanillic acid, vanillin, and vanillyl alcohol. Bacterial isolates were evaluated using ferulic acid esterase, ferulic acid decarboxylase, and vanillin dehydrogenase assays. This work highlights the importance of lactic acid bacteria in phenolic biotransformations for the development of food grade flavours and additives. Baljinder Kaur, Debkumar Chakraborty, and Balvir Kumar Copyright © 2013 Baljinder Kaur et al. All rights reserved. Class A -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine Wed, 28 Aug 2013 14:12:01 +0000 Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications from basic research to medical applications. This review focuses on the use of class A β-lactamases as versatile scaffolds to design hybrid enzymes (referred to as β-lactamase hybrid proteins, BHPs) in which an exogenous peptide, protein or fragment thereof is inserted at various permissive positions. We discuss how BHPs can be specifically designed to create bifunctional proteins, to produce and to characterize proteins that are otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to better understand the structure/function relationship of proteins. Céline Huynen, Patrice Filée, André Matagne, Moreno Galleni, and Mireille Dumoulin Copyright © 2013 Céline Huynen et al. All rights reserved. Sympathetic Innervation Induced in Engrafted Engineered Cardiomyocyte Sheets by Glial Cell Line Derived Neurotrophic Factor In Vivo Wed, 28 Aug 2013 08:52:24 +0000 The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue. However, this strategy has been hampered by lack of functional integration of grafts with native myocardium. Autonomic innervation may be crucial for grafts to function properly with host myocardium. In this study, we explored the feasibility of in vivo induction of autonomic innervation to engineered myocardial tissue using genetic modulation by adenovirus encoding glial cell line derived neurotrophic factor (GDNF). GFP-transgene (control group) or GDNF overexpressing (GDNF group) engineered cardiomyocyte sheets were transplanted on cryoinjured hearts in rats. Nerve fibers in the grafts were examined by immunohistochemistry at 1, 2, and 4 weeks postoperatively. Growth associated protein-43 positive growing nerves and tyrosine hydroxylase positive sympathetic nerves were first detected in the grafts at 2 weeks postoperatively in control group and 1 week in GDNF group. The densities of growing nerve and sympathetic nerve in grafts were significantly increased in GDNF group. No choline acetyltransferase immunopositive parasympathetic nerves were observed in grafts. In conclusion, sympathetic innervation could be effectively induced into engrafted engineered cardiomyocyte sheets using GDNF. Xian-ming Fu, Jong-Kook Lee, Keiko Miwa, Tatsuya Shimizu, Yoshiko Takagishi, Masumi Hirabayashi, Kazuhiko Watabe, Akihiko Usui, Itsuo Kodama, and Yuichi Ueda Copyright © 2013 Xian-ming Fu et al. All rights reserved. Combined Treatment of Hydroxytyrosol with Carbon Monoxide-Releasing Molecule-2 Prevents TNFα-Induced Vascular Endothelial Cell Dysfunction through NO Production with Subsequent NFκB Inactivation Tue, 27 Aug 2013 15:44:15 +0000 This study investigated the atheroprotective properties of olive oil polyphenol, hydroxytyrosol (HT), in combination with carbon monoxide-releasing molecule-2 (CORM-2) that acts as a carbon monoxide donor using vascular endothelial cells (VECs). Our results showed that CORM-2 could strengthen the cytoprotective and anti-apoptotic effects of HT against TNFα-induced cellular damage by enhancing cell survival and the suppression of caspase-3 activation. While HT alone attenuated NFκBp65 phosphorylation and IκBα degradation triggered by TNFα in a dose-dependent manner, combined treatment of HT with CORM-2 but not iCORM-2 nearly completely blocked these TNFα effects. Furthermore, combined action of both compounds results in the inhibition of NFκB nuclear translocation. Results also indicate that both compounds time-dependently increased eNOS phosphorylation levels and the combination of HT with CORM-2 was more effective in enhancing eNOS activation and NO production in VECs. The NOS inhibitor, L-NMMA, significantly suppressed the combined effects of HT and CORM-2 on TNFα-triggered NFκBp65 and IκBα phosphorylation as well as decreased cell viability. Together, these data suggest that carbon monoxide-dependent regulation of NO production by the combination of HT with CORM-2 may provide a therapeutic benefit in the treatment of endothelial dysfunction and atherosclerosis. Houda Zrelli, Che Wei Wu, Nahla Zghonda, Hidehisa Shimizu, and Hitoshi Miyazaki Copyright © 2013 Houda Zrelli et al. All rights reserved. Characterization of the Bacterial Community Associated with Larvae and Adults of Anoplophora chinensis Collected in Italy by Culture and Culture-Independent Methods Tue, 27 Aug 2013 14:59:19 +0000 The wood-boring beetle Anoplophora chinensis Forster, native to China, has recently spread to North America and Europe causing serious damage to ornamental and forest trees. The gut microbial community associated with these xylophagous beetles is of interest for potential biotechnological applications in lignocellulose degradation and development of pest-control measures. In this study the gut bacterial community of larvae and adults of A. chinensis, collected from different host trees in North Italy, was investigated by both culture and culture-independent methods. Larvae and adults harboured a moderately diverse bacterial community, dominated by Proteobacteria, Actinobacteria, and Firmicutes. The gammaproteobacterial family Enterobacteriaceae (genera Gibbsiella, Enterobacter, Raoultella, and Klebsiella) was the best represented. The abundance of such bacteria in the insect gut is likely due to the various metabolic abilities of Enterobacteriaceae, including fermentation of carbohydrates derived from lignocellulose degradation and contribution to nitrogen intake by nitrogen-fixing activity. In addition, bacteria previously shown to have some lignocellulose-degrading activity were detected at a relatively low level in the gut. These bacteria possibly act synergistically with endogenous and fungal enzymes in lignocellulose breakdown. The detection of actinobacterial symbionts could be explained by a possible role in the detoxification of secondary plant metabolites and/or protection against pathogens. Aurora Rizzi, Elena Crotti, Luigimaria Borruso, Costanza Jucker, Daniela Lupi, Mario Colombo, and Daniele Daffonchio Copyright © 2013 Aurora Rizzi et al. All rights reserved. Recent Developments in Production and Biotechnological Applications of C-Phycocyanin Mon, 26 Aug 2013 17:33:55 +0000 An extensive range of pigments including phycobiliproteins are present in algae. C-phycocyanin (C-PC), a phycobiliprotein, is one of the key pigments of Spirulina, a microalgae used in many countries as a dietary supplement. Algal pigments have massive commercial value as natural colorants in nutraceutical, cosmetics, and pharmaceutical industries, besides their health benefits. At present, increasing awareness of harmful effects of synthetic compounds and inclination of community towards the usage of natural products have led to the exploitation of microalgae as a source of natural pigments/colors. This review describes recent findings about the sources and production of C-PC, with emphasis on specific techniques for extraction and purification, along with potential industrial applications in diagnostics, foods, cosmetics, and pharmaceutical industries. M. Kuddus, P. Singh, G. Thomas, and Awdah Al-Hazimi Copyright © 2013 M. Kuddus et al. All rights reserved. Population Abundance of Potentially Pathogenic Organisms in Intestinal Microbiome of Jungle Crow (Corvus macrorhynchos) Shown with 16S rRNA Gene-Based Microbial Community Analysis Sat, 24 Aug 2013 09:29:18 +0000 Jungle Crows (Corvus macrorhynchos) prefer human habitats because of their versatility in feeding accompanied with human food consumption. Therefore, it is important from a public health viewpoint to characterize their intestinal microbiota. However, no studies have been involved in molecular characterization of the microbiota based on huge and reliable number of data acquisition. In this study, 16S rRNA gene-based microbial community analysis coupled with the next-generation DNA sequencing techniques was applied to the taxonomic classification of intestinal microbiome for three jungle crows. Clustering of the reads into 130 operational taxonomic units showed that at least 70% of analyzed sequences for each crow were highly homologous to Eimeria sp., which belongs to the protozoan phylum Apicomplexa. The microbiotas of three crows also contained potentially pathogenic bacteria with significant percentages, such as the genera Campylobacter and Brachyspira. Thus, the profiling of a large number of 16S rRNA gene sequences in crow intestinal microbiomes revealed the high-frequency existence or vestige of potentially pathogenic microorganisms. Isamu Maeda, Mohammad Shohel Rana Siddiki, Tsutomu Nozawa-Takeda, Naoki Tsukahara, Yuri Tani, Taki Naito, and Shoei Sugita Copyright © 2013 Isamu Maeda et al. All rights reserved. Poly β-Hydroxybutyrate Production by Bacillus subtilis NG220 Using Sugar Industry Waste Water Wed, 21 Aug 2013 14:00:46 +0000 The production of poly β-hydroxybutyrate (PHB) by Bacillus subtilis NG220 was observed utilizing the sugar industry waste water supplemented with various carbon and nitrogen sources. At a growth rate of 0.14 g h−1 L−1, using sugar industry waste water was supplemented with maltose (1% w/v) and ammonium sulphate (1% w/v); the isolate produced 5.297 g/L of poly β-hydroxybutyrate accumulating 51.8% (w/w) of biomass. The chemical nature of the polymer was confirmed with nuclear magnetic resonance, Fourier transform infrared, and GC-MS spectroscopy whereas thermal properties were monitored with differential scanning calorimetry. In biodegradability study, when PHB film of the polymer (made by traditional solvent casting technique) was subjected to degradation in various natural habitats like soil, compost, and industrial sludge, it was completely degraded after 30 days in the compost having 25% (w/w) moisture. So, the present study gives insight into dual benefits of conversion of a waste material into value added product, PHB, and waste management. Gulab Singh, Anish Kumari, Arpana Mittal, Anita Yadav, and Neeraj K. Aggarwal Copyright © 2013 Gulab Singh et al. All rights reserved. Treatment of Slaughter House Wastewater in a Sequencing Batch Reactor: Performance Evaluation and Biodegradation Kinetics Tue, 20 Aug 2013 10:32:43 +0000 Slaughterhouse wastewater contains diluted blood, protein, fat, and suspended solids, as a result the organic and nutrient concentration in this wastewater is vary high and the residues are partially solubilized, leading to a highly contaminating effect in riverbeds and other water bodies if the same is let off untreated. The performance of a laboratory-scale Sequencing Batch Reactor (SBR) has been investigated in aerobic-anoxic sequential mode for simultaneous removal of organic carbon and nitrogen from slaughterhouse wastewater. The reactor was operated under three different variations of aerobic-anoxic sequence, namely, (4+4), (5+3), and (3+5) hr. of total react period with two different sets of influent soluble COD (SCOD) and ammonia nitrogen (-N) level  mg/L, and  mg/L,  mg/L and  mg/L, respectively. It was observed that from 86 to 95% of SCOD removal is accomplished at the end of 8.0 hr of total react period. In case of (4+4) aerobic-anoxic operating cycle, a reasonable degree of nitrification 90.12 and 74.75% corresponding to initial -N value of 96.58 and 176.85 mg/L, respectively, were achieved. The biokinetic coefficients (, , , ) were also determined for performance evaluation of SBR for scaling full-scale reactor in future operation. Pradyut Kundu, Anupam Debsarkar, and Somnath Mukherjee Copyright © 2013 Pradyut Kundu et al. All rights reserved. In Vitro Characterization of a Multifunctional Staphylokinase Variant with Reduced Reocclusion, Produced from Salt Inducible E. coli GJ1158 Tue, 13 Aug 2013 08:23:08 +0000 The thrombolytic therapy with clinically approved drugs often ensues with recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, a staphylokinase (SAK)-based bacterial friendly multifunctional recombinant protein SRH (staphylokinase (SAK) linked with tripeptide RGD and dodecapeptide Hirulog (SRH)) was constructed to have Hirulog as an antithrombin agent and RGD (Arg-Gly-Asp) as an antiplatelet agent in the present study. This multifunctional fusion protein SRH was expressed in osmotically inducible E. coli GJ1158 as soluble form and purified with a yield of 0.27 g/L and functionally characterized in vitro. SRH retained the fibrinolytic activity and plasminogen activation rate comparable to the parental counterpart SAK. The antithrombin activity of SRH was significantly higher than SAK. The platelet rich clot lysis assay indicated that SRH had enhanced platelet binding activity and and C50 of SRH were significantly lower than that of SAK. Furthermore, SRH inhibited the ADP-induced platelet aggregation in dose-dependent manner while SAK had no significant effect on platelet aggregation. Thus, the current study suggests that the SAK variant produced from osmotically inducible GJ1158 is more potent thrombolytic agent with antithrombin and antiplatelet aggregation activities for reduction of reocclusion in thrombolytic therapy. K. K. Pulicherla, Anmol Kumar, G. S. Gadupudi, Seetha Ram Kotra, and K. R. S. Sambasiva Rao Copyright © 2013 K. K. Pulicherla et al. All rights reserved. Promotion of Hepatic Differentiation of Bone Marrow Mesenchymal Stem Cells on Decellularized Cell-Deposited Extracellular Matrix Wed, 07 Aug 2013 13:43:40 +0000 Interactions between stem cells and extracellular matrix (ECM) are requisite for inducing lineage-specific differentiation and maintaining biological functions of mesenchymal stem cells by providing a composite set of chemical and structural signals. Here we investigated if cell-deposited ECM mimicked in vivo liver's stem cell microenvironment and facilitated hepatogenic maturation. Decellularization process preserved the fibrillar microstructure and a mix of matrix proteins in cell-deposited ECM, such as type I collagen, type III collagen, fibronectin, and laminin that were identical to those found in native liver. Compared with the cells on tissue culture polystyrene (TCPS), bone marrow mesenchymal stem cells (BM-MSCs) cultured on cell-deposited ECM showed a spindle-like shape, a robust proliferative capacity, and a suppressed level of intracellular reactive oxygen species, accompanied with upregulation of two superoxide dismutases. Hepatocyte-like cells differentiated from BM-MSCs on ECM were determined with a more intensive staining of glycogen storage, an elevated level of urea biosynthesis, and higher expressions of hepatocyte-specific genes in contrast to those on TCPS. These results demonstrate that cell-deposited ECM can be an effective method to facilitate hepatic maturation of BM-MSCs and promote stem-cell-based liver regenerative medicine. Hongliang He, Xiaozhen Liu, Liang Peng, Zhiliang Gao, Yun Ye, Yujie Su, Qiyi Zhao, Ke Wang, Yihong Gong, and Fan He Copyright © 2013 Hongliang He et al. All rights reserved. Production, Characterization and Antioxidant Potential of Protease from Streptomyces sp. MAB18 Using Poultry Wastes Wed, 07 Aug 2013 09:11:49 +0000 Poultry waste is an abundant renewable source for the recovery of several value-added metabolites with potential industrial applications. This study describes the production of protease on poultry waste, with the subsequent use of the same poultry waste for the extraction of antioxidants. An extracellular protease-producing strain was isolated from Cuddalore coast, India, and identified as Streptomyces sp. MAB18. Its protease was purified 17.13-fold with 21.62% yield with a specific activity of 2398.36 U/mg and the molecular weight was estimated as 43 kDa. The enzyme was optimally active at pH 8–10 and temperature 50–60°C and it was most stable up to pH 12 and 6–12% of NaCl concentration. The enzyme activity was reduced when treated with Hg2+, Pb2+, and SDS and stimulated by Fe2+, Mg2+, Triton X-100, DMSO (dimethyl sulfoxide), sodium sulphite, and β-mercaptoethanol. Furthermore, the antioxidant activities of protease were evaluated using in vitro antioxidant assays, such as DPPH radical-scavenging activity, O2 scavenging activity, NO scavenging activity, Fe2+ chelating activity, and reducing power. The enzyme showed important antioxidant potential with an IC50 value of  mg/mL. Results of the present study indicate that the poultry waste-derived protease may be useful as supplementary protein and antioxidant in the animal feed formulations. Panchanathan Manivasagan, Jayachandran Venkatesan, Kannan Sivakumar, and Se-Kwon Kim Copyright © 2013 Panchanathan Manivasagan et al. All rights reserved. Microbial Adhesion and Biofilm Formation on Microfiltration Membranes: A Detailed Characterization Using Model Organisms with Increasing Complexity Tue, 06 Aug 2013 13:11:48 +0000 Since many years, membrane biofouling has been described as the Achilles heel of membrane fouling. In the present study, an ecological assay was performed using model systems with increasing complexity: a monospecies assay using Pseudomonas aeruginosa or Escherichia coli separately, a duospecies assay using both microorganisms, and a multispecies assay using activated sludge with or without spiked P. aeruginosa. The microbial adhesion and biofilm formation were evaluated in terms of bacterial cell densities, species richness, and bacterial community composition on polyvinyldifluoride, polyethylene, and polysulfone membranes. The data show that biofouling formation was strongly influenced by the kind of microorganism, the interactions between the organisms, and the changes in environmental conditions whereas the membrane effect was less important. The findings obtained in this study suggest that more knowledge in species composition and microbial interactions is needed in order to understand the complex biofouling process. This is the first report describing the microbial interactions with a membrane during the biofouling development. L. Vanysacker, C. Denis, P. Declerck, A. Piasecka, and I. F. J. Vankelecom Copyright © 2013 L. Vanysacker et al. All rights reserved. Distribution of ABO Blood Group and Major Cardiovascular Risk Factors with Coronary Heart Disease Mon, 05 Aug 2013 08:35:50 +0000 The purpose of this study is to establish whether ABO blood group is related to coronary heart disease in an individual in Asian Indian Bengali population of eastern part of India. Two hundred and fifty (250) CHD patients and two hundred and fifty (250) age and sex matched healthy subjects were enrolled in the study. ABO blood group distribution in patients was compared with control group. Frequency of major cardiac risk factors was determined to find any correlation between blood groups and cardiovascular risk factors. The distribution of ABO blood groups in patients versus control group was A in 24.00 versus 21.60%, B in 30.80 versus 32.40%, O in 38.40 versus 21.60%, and AB in 6.80 versus 24.40%. The analysis showed significant difference in frequency of O (OR = 1.857, 95%CI = 1.112–3.100, ) and AB (OR = 0.447, 95%CI = 0.227–0.882, ) blood group between healthy controls and CHD individuals. Our results may suggest that the AB blood group decreases the risk of CHD in healthy controls, and it might be due to the higher concentration of high density lipoprotein cholesterol (HDL-c), while the O blood group increases the risk of CHD due to lower HDL-c levels in Bengali population of eastern part of India. Santanu Biswas, Pradip K. Ghoshal, Bhubaneswar Halder, and Nripendranath Mandal Copyright © 2013 Santanu Biswas et al. All rights reserved. Enhancement of Phosphate Absorption by Garden Plants by Genetic Engineering: A New Tool for Phytoremediation Sun, 04 Aug 2013 14:34:33 +0000 Although phosphorus is an essential factor for proper plant growth in natural environments, an excess of phosphate in water sources causes serious pollution. In this paper we describe transgenic plants which hyperaccumulate inorganic phosphate (Pi) and which may be used to reduce environmental water pollution by phytoremediation. AtPHR1, a transcription factor for a key regulator of the Pi starvation response in Arabidopsis thaliana, was overexpressed in the ornamental garden plants Torenia, Petunia, and Verbena. The transgenic plants showed hyperaccumulation of Pi in leaves and accelerated Pi absorption rates from hydroponic solutions. Large-scale hydroponic experiments indicated that the enhanced ability to absorb Pi in transgenic torenia (AtPHR1) was comparable to water hyacinth a plant that though is used for phytoremediation causes overgrowth problems. Keisuke Matsui, Junichi Togami, John G. Mason, Stephen F. Chandler, and Yoshikazu Tanaka Copyright © 2013 Keisuke Matsui et al. All rights reserved. Isolation and Characterization of Chicken Dermis-Derived Mesenchymal Stem/Progenitor Cells Sun, 04 Aug 2013 10:37:02 +0000 Dermis-derived mesenchymal stem/progenitor cells (DMS/PCs) were isolated from the skin tissue of 16-day-old chick embryos and then characterized by immunofluorescence and RT-PCR. We found that primary DMS/PCs could be expanded for 15 passages. Expression of β-integrin, CD44, CD71, and CD73 was observed by immunofluorescence and RT-PCR. Passage 3 DMS/PCs were successfully induced to differentiate into osteoblasts, adipocytes, and neurocytes. The results indicate the potential for multilineage differentiation of DMS/PCs that may represent an ideal candidate for cellular transplantation therapy. Yuhua Gao, Chunyu Bai, Hui Xiong, Qian Li, Zhiqiang Shan, Linsheng Huang, Yuehui Ma, and Weijun Guan Copyright © 2013 Yuhua Gao et al. All rights reserved. Evaluation of Human Body Fluids for the Diagnosis of Fungal Infections Thu, 01 Aug 2013 13:03:57 +0000 Invasive fungal infections are a major cause of morbidity and mortality in immunocompromised patients. Because the etiologic agents of these infections are abundant in nature, their isolation from biopsy material or sterile body fluids is needed to document infection. This review evaluates and discusses different human body fluids used to diagnose fungal infections. Parisa Badiee Copyright © 2013 Parisa Badiee. All rights reserved. β-Glucosidases from the Fungus Trichoderma: An Efficient Cellulase Machinery in Biotechnological Applications Thu, 01 Aug 2013 09:42:26 +0000 β-glucosidases catalyze the selective cleavage of glucosidic linkages and are an important class of enzymes having significant prospects in industrial biotechnology. These are classified in family 1 and family 3 of glycosyl hydrolase family. β-glucosidases, particularly from the fungus Trichoderma, are widely recognized and used for the saccharification of cellulosic biomass for biofuel production. With the rising trends in energy crisis and depletion of fossil fuels, alternative strategies for renewable energy sources need to be developed. However, the major limitation accounts for low production of β-glucosidases by the hyper secretory strains of Trichoderma. In accordance with the increasing significance of β-glucosidases in commercial applications, the present review provides a detailed insight of the enzyme family, their classification, structural parameters, properties, and studies at the genomics and proteomics levels. Furthermore, the paper discusses the enhancement strategies employed for their utilization in biofuel generation. Therefore, β-glucosidases are prospective toolbox in bioethanol production, and in the near future, it might be successful in meeting the requirements of alternative renewable sources of energy. Pragya Tiwari, B. N. Misra, and Neelam S. Sangwan Copyright © 2013 Pragya Tiwari et al. All rights reserved. Biomedical Applications of Fermenticin HV6b Isolated from Lactobacillus fermentum HV6b MTCC10770 Mon, 29 Jul 2013 14:59:19 +0000 Fermenticin HV6b is a class IIa antimicrobial peptide produced by Lactobacillus fermentum HV6b MTCC 10770 isolated from human vaginal ecosystem. It shows growth inhibition of a wide range of opportunistic pathogens of humans, for example, Bacteroides, Gardnerella vaginalis, Mobiluncus, Staphylococci, and Streptococci, associated with bacterial vaginosis in humans. It does possess an impressive sperm immobilization and spermicidal activity tested against human sperms which makes it an attractive proposition for formulating antibacterial vaginosis and contraceptive products. Apart from this, in vitro studies conducted against four different tissue models have indicated its potential to be used as a component of anticancerous drug therapy as it is reported to induce apoptosis in cancerous cells. This information could be integrated in future studies focusing on in vivo assessment of anticancerous activity of lactic acid bacterial toxins or bacteriocins. Baljinder Kaur, Praveen P. Balgir, Bharti Mittu, Balvir Kumar, and Neena Garg Copyright © 2013 Baljinder Kaur et al. All rights reserved. Pesticide Residue Screening Using a Novel Artificial Neural Network Combined with a Bioelectric Cellular Biosensor Sun, 28 Jul 2013 09:41:40 +0000 We developed a novel artificial neural network (ANN) system able to detect and classify pesticide residues. The novel ANN is coupled, in a customized way, to a cellular biosensor operation based on the bioelectric recognition assay (BERA) and able to simultaneously assay eight samples in three minutes. The novel system was developed using the data (time series) of the electrophysiological responses of three different cultured cell lines against three different pesticide groups (carbamates, pyrethroids, and organophosphates). Using the novel system, we were able to classify correctly the presence of the investigated pesticide groups with an overall success rate of 83.6%. Considering that only 70,000–80,000 samples are annually tested in Europe with current conventional technologies (an extremely minor fraction of the actual screening needs), the system reported in the present study could contribute to a screening system milestone for the future landscape in food safety control. Konstantinos P. Ferentinos, Costas P. Yialouris, Petros Blouchos, Georgia Moschopoulou, and Spyridon Kintzios Copyright © 2013 Konstantinos P. Ferentinos et al. All rights reserved. Antigenicity and Immunogenicity of Rotavirus VP6 Protein Expressed on the Surface of Lactococcus lactis Wed, 24 Jul 2013 11:15:25 +0000 Group A rotaviruses are the major etiologic agents of acute gastroenteritis worldwide in children and young animals. Among its structural proteins, VP6 is the most immunogenic and is highly conserved within this group. Lactococcus lactis is a food-grade, Gram-positive, and nonpathogenic lactic acid bacteria that has already been explored as a mucosal delivery system of heterologous antigens. In this work, the nisin-controlled expression system was used to display the VP6 protein at the cell surface of L. lactis. Conditions for optimal gene expression were established by testing different nisin concentrations, cell density at induction, and incubation times after induction. Cytoplasmic and cell wall protein extracts were analyzed by Western blot and surface expression was confirmed by flow cytometry. Both analysis provided evidence that VP6 was efficiently expressed and displayed on the cell surface of L. lactis. Furthermore, the humoral response of mice immunized with recombinant L. lactis was evaluated and the displayed recombinant VP6 protein proved to be immunogenic. In conclusion, this is the first report of displaying VP6 protein on the surface of L. lactis to induce a specific immune response against rotavirus. These results provide the basis for further evaluation of this VP6-displaying L. lactis as a mucosal delivery vector in a mouse model of rotavirus infection. L. E. Esteban, C. F. Temprana, M. H. Argüelles, G. Glikmann, and A. A. Castello Copyright © 2013 L. E. Esteban et al. All rights reserved. Advances in the Applications of Polyhydroxyalkanoate Nanoparticles for Novel Drug Delivery System Wed, 24 Jul 2013 10:24:28 +0000 Drug delivery technology is emerging as an interdisciplinary science aimed at improving human health. The controlled delivery of pharmacologically active agents to the specific site of action at the therapeutically optimal rate and dose regimen has been a major goal in designing drug delivery systems. Over the past few decades, there has been considerable interest in developing biodegradable drug carriers as effective drug delivery systems. Polymeric materials from natural sources play an important role in controlled release of drug at a particular site. Polyhydroxyalkanoates, due to their origin from natural sources, are given attention as candidates for drug delivery materials. Biodegradable and biocompatible polyhydroxyalkanoates are linear polyesters produced by microorganisms under unbalanced growth conditions, which have emerged as potential polymers for use as biomedical materials for drug delivery due to their unique physiochemical and mechanical properties. This review summarizes many of the key findings in the applications of polyhydroxyalkanoates and polyhydroxyalkanoate nanoparticles for drug delivery system. Anupama Shrivastav, Hae-Yeong Kim, and Young-Rok Kim Copyright © 2013 Anupama Shrivastav et al. All rights reserved. Immunomodulatory Effect of Continuous Venovenous Hemofiltration during Sepsis: Preliminary Data Tue, 23 Jul 2013 09:02:31 +0000 Introduction. Severe sepsis and septic shock are the primary causes of multiple organ dysfunction syndrome (MODS), which is the most frequent cause of death in intensive care unit patients. Many pro- and anti-inflammatory mediators, such as interleukin-6 (IL-6), play a strategic role in septic syndrome. Continuous renal replacement therapy (CRRT) removes in a nonselective way pro- and anti-inflammatory mediators. Objective. To investigate the effects of continuous venovenous hemofiltration (CVVH) as an immunomodulatory treatment of sepsis in a prospective clinical study. Methods. High flux hemofiltration (Qf = 60 ml/Kg/hr) was performed for 72 hr in thirteen critically ill patients suffering from severe sepsis or septic shock with acute renal failure (ARF). IL-6 gene expression was measured by real-time PCR analysis on RNA extracted from peripheral blood mononuclear cell before beginning of treatment (T0) and after 12, 24, 48, and 72 hours (T1–4). Results. Real-time PCR analysis demonstrated in twelve patients IL-6 mRNA reduction after 12 hours of treatment and a progressive increase after 24, 48, and 72 hours. Conclusions. We suggest that an immunomodulatory effect might exist during CVVH performed in critically ill patients with severe sepsis and septic shock. Our data show that the transcriptional activity of IL-6 increases during CVVH. Giuseppe Servillo, Maria Vargas, Antonio Pastore, Alfredo Procino, Michele Iannuzzi, Alfredo Capuano, Andrea Memoli, Eleonora Riccio, and Bruno Memoli Copyright © 2013 Giuseppe Servillo et al. All rights reserved. Improvement of Medium Chain Fatty Acid Content and Antimicrobial Activity of Coconut Oil via Solid-State Fermentation Using a Malaysian Geotrichum candidum Thu, 18 Jul 2013 10:20:34 +0000 Coconut oil is a rich source of beneficial medium chain fatty acids (MCFAs) particularly lauric acid. In this study, the oil was modified into a value-added product using direct modification of substrate through fermentation (DIMOSFER) method. A coconut-based and coconut-oil-added solid-state cultivation using a Malaysian lipolytic Geotrichum candidum was used to convert the coconut oil into MCFAs-rich oil. Chemical characteristics of the modified coconut oils (MCOs) considering total medium chain glyceride esters were compared to those of the normal coconut oil using ELSD-RP-HPLC. Optimum amount of coconut oil hydrolysis was achieved at 29% moisture content and 10.14% oil content after 9 days of incubation, where the quantitative amounts of the modified coconut oil and MCFA were 0.330 mL/g of solid media (76.5% bioconversion) and 0.175 mL/g of solid media (53% of the MCO), respectively. MCOs demonstrated improved antibacterial activity mostly due to the presence of free lauric acid. The highest MCFAs-rich coconut oil revealed as much as 90% and 80% antibacterial activities against Staphylococcus aureus and Escherichia coli, respectively. The results of the study showed that DIMOSFER by a local lipolytic G. candidum can be used to produce MCFAs as natural, effective, and safe antimicrobial agent. The produced MCOs and MCFAs could be further applied in food and pharmaceutical industries. Anahita Khoramnia, Afshin Ebrahimpour, Raheleh Ghanbari, Zahra Ajdari, and Oi-Ming Lai Copyright © 2013 Anahita Khoramnia et al. All rights reserved. Anaerobic Ammonium Oxidation: From Laboratory to Full-Scale Application Wed, 17 Jul 2013 09:23:53 +0000 From discovery in the early 1990s to completion of full-scale anammox reactor, it took almost two decades to uncover the secret veil of anammox bacteria. There were three milestones during the commercialization of anammox: the development of the first enrichment culture medium, the completion of the first commercial anammox reactor, and the fast start-up of full-scale anammox plant. Till now, the culture of anammox bacteria experienced a big progress through two general strategies: (a) to start up a reactor from scratch and (b) to seed the reactor with enriched anammox sludge. The first full-scale anammox reactor took 3.5 years to realize full operation using the first approach due to several reasons besides the lack of anammox sludge. On the other hand, the first Asian anammox reactor started up in two months, thanks to the availability of anammox seed. Along with the implementation of anammox plants, anammox eventually becomes the priority choice for ammonium wastewater treatment. Shou-Qing Ni and Jian Zhang Copyright © 2013 Shou-Qing Ni and Jian Zhang. All rights reserved. Uptake of Gold Nanoparticles in Several Rat Organs after Intraperitoneal Administration In Vivo: A Fluorescence Study Wed, 17 Jul 2013 08:33:28 +0000 Background. The gold nanoparticles (GNPs) have potential applications in cancer diagnosis and therapy. In an attempt to characterise the potential toxicity or hazards of GNPs as a therapeutic or diagnostic tool, the fluorescence spectra in several rat organs in vivo were measured after intraperitoneal administration of GNPs. Methods. The experimental rats were divided into control and six groups (G1A, G1B, G2A, G2B, G3A, and G3B; G1: 20 nm; G2: 10 nm; G3: 50 nm; A: infusion of GNPs for 3 days; B: infusion of GNPs for 7 days). The fluorescence measurements were investigated in the liver, kidney, heart, and lung organs of rats after intraperitoneal administration of GNPs for periods of 3 and 7 days in vivo. Results. The 10 and 20 nm GNPs exhibited spherical morphology shape, while the 50 nm GNPs exhibited hexagonal shape. A sharp decrease in the fluorescence intensity induced with the larger 50 nm GNPs in the liver, kidney, heart, and lung organs of rats at the exposure duration of 3 and 7 days in vivo compared with the smaller 10 and 20 nm GNPs was observed. Conclusions. The decrease in fluorescence intensity may be attributed to occurrence of strong quenching, decrease in number and surface area of GNPs, and high clearance of GNPs via urine and bile. Moreover, decreasing size may lead to an exponential increase in surface area relative to volume, thus making GNPs surface more reactive on aggregation and to its surrounding biological components. The size, shape, surface area, number, and clearance of GNPs play a key role in toxicity and accumulation in the different rat organs. This study demonstrates that fluorescence peak intensity is particle size and exposure duration dependent. This study suggests that fluorescence intensity can be used as a useful tool for pointing to bioaccumulation and toxicity induced by GNPs in the different rat organs. Mohamed Anwar K. Abdelhalim Copyright © 2013 Mohamed Anwar K. Abdelhalim. All rights reserved. Hydroxycinnamic Acid Antioxidants: An Electrochemical Overview Tue, 16 Jul 2013 11:43:11 +0000 Hydroxycinnamic acids (such as ferulic, caffeic, sinapic, and p-coumaric acids) are a group of compounds highly abundant in food that may account for about one-third of the phenolic compounds in our diet. Hydroxycinnamic acids have gained an increasing interest in health because they are known to be potent antioxidants. These compounds have been described as chain-breaking antioxidants acting through radical scavenging activity, that is related to their hydrogen or electron donating capacity and to the ability to delocalize/stabilize the resulting phenoxyl radical within their structure. The free radical scavenger ability of antioxidants can be predicted from standard one-electron potentials. Thus, voltammetric methods have often been applied to characterize a diversity of natural and synthetic antioxidants essentially to get an insight into their mechanism and also as an important tool for the rational design of new and potent antioxidants. The structure-property-activity relationships (SPARs) correlations already established for this type of compounds suggest that redox potentials could be considered a good measure of antioxidant activity and an accurate guideline on the drug discovery and development process. Due to its magnitude in the antioxidant field, the electrochemistry of hydroxycinnamic acid-based antioxidants is reviewed highlighting the structure-property-activity relationships (SPARs) obtained so far. José Teixeira, Alexandra Gaspar, E. Manuela Garrido, Jorge Garrido, and Fernanda Borges Copyright © 2013 José Teixeira et al. All rights reserved. Microbial Inoculants and Their Impact on Soil Microbial Communities: A Review Thu, 11 Jul 2013 14:07:49 +0000 The knowledge of the survival of inoculated fungal and bacterial strains in field and the effects of their release on the indigenous microbial communities has been of great interest since the practical use of selected natural or genetically modified microorganisms has been developed. Soil inoculation or seed bacterization may lead to changes in the structure of the indigenous microbial communities, which is important with regard to the safety of introduction of microbes into the environment. Many reports indicate that application of microbial inoculants can influence, at least temporarily, the resident microbial communities. However, the major concern remains regarding how the impact on taxonomic groups can be related to effects on functional capabilities of the soil microbial communities. These changes could be the result of direct effects resulting from trophic competitions and antagonistic/synergic interactions with the resident microbial populations, or indirect effects mediated by enhanced root growth and exudation. Combination of inoculants will not necessarily produce an additive or synergic effect, but rather a competitive process. The extent of the inoculation impact on the subsequent crops in relation to the buffering capacity of the plant-soil-biota is still not well documented and should be the focus of future research. Darine Trabelsi and Ridha Mhamdi Copyright © 2013 Darine Trabelsi and Ridha Mhamdi. All rights reserved. Production and Characterization of Keratinolytic Protease from New Wool-Degrading Bacillus Species Isolated from Egyptian Ecosystem Thu, 11 Jul 2013 13:38:46 +0000 Novel keratin-degrading bacteria were isolated from sand soil samples collected from Minia Governorate, Egypt. In this study, the isolates were identified as Bacillus amyloliquefaciens MA20 and Bacillus subtilis MA21 based on morphological and biochemical characteristics as well as 16S rRNA gene sequencing. B. amyloliquefaciens MA20 and B. subtilis MA21 produced alkaline keratinolytic serine protease when cultivated in mineral medium containing 1% of wool straight off sheep as sole carbon and nitrogen source. The two strains were observed to degrade wool completely to powder at pH 7 and 37°C within 5 days. Under these conditions the maximum activity of proteases produced by B. amyloliquefaciens MA20 and B. subtilis MA21 was 922 and 814 U/ml, respectively. The proteases exhibited optimum temperature and pH at 60°C and 9, respectively. However, the keratinolytic proteases were stable in broad range of temperature and pH values towards casein Hammerstein. Furthermore the protease inhibitor studies indicated that the produced proteases belong to serine protease because of their sensitivity to PMSF while they were inhibited partially in presence of EDTA. The two proteases are stable in most of the used organic solvents and enhanced by metals suggesting their potential use in biotechnological applications such as wool industry. Mohamed A. Hassan, Bakry M. Haroun, Amro A. Amara, and Ehab A. Serour Copyright © 2013 Mohamed A. Hassan et al. All rights reserved. Acetylcholinesterase as a Biomarker in Environmental and Occupational Medicine: New Insights and Future Perspectives Thu, 11 Jul 2013 09:58:06 +0000 Acetylcholinesterase (AChE) is a key enzyme in the nervous system. It terminates nerve impulses by catalysing the hydrolysis of neurotransmitter acetylcholine. As a specific molecular target of organophosphate and carbamate pesticides, acetylcholinesterase activity and its inhibition has been early recognized to be a human biological marker of pesticide poisoning. Measurement of AChE inhibition has been increasingly used in the last two decades as a biomarker of effect on nervous system following exposure to organophosphate and carbamate pesticides in occupational and environmental medicine. The success of this biomarker arises from the fact that it meets a number of characteristics necessary for the successful application of a biological response as biomarker in human biomonitoring: the response is easy to measure, it shows a dose-dependent behavior to pollutant exposure, it is sensitive, and it exhibits a link to health adverse effects. The aim of this work is to review and discuss the recent findings about acetylcholinesterase, including its sensitivity to other pollutants and the expression of different splice variants. These insights open new perspective for the future use of this biomarker in environmental and occupational human health monitoring. Maria Giulia Lionetto, Roberto Caricato, Antonio Calisi, Maria Elena Giordano, and Trifone Schettino Copyright © 2013 Maria Giulia Lionetto et al. All rights reserved. Biosynthesis, Antimicrobial and Cytotoxic Effect of Silver Nanoparticles Using a Novel Nocardiopsis sp. MBRC-1 Thu, 11 Jul 2013 09:25:38 +0000 The biosynthesis of nanoparticles has been proposed as a cost effective environmental friendly alternative to chemical and physical methods. Microbial synthesis of nanoparticles is under exploration due to wide biomedical applications, research interest in nanotechnology and microbial biotechnology. In the present study, an ecofriendly process for the synthesis of nanoparticles using a novel Nocardiopsis sp. MBRC-1 has been attempted. We used culture supernatant of Nocardiopsis sp. MBRC-1 for the simple and cost effective green synthesis of silver nanoparticles. The reduction of silver ions occurred when silver nitrate solution was treated with the Nocardiopsis sp. MBRC-1 culture supernatant at room temperature. The nanoparticles were characterized by UV-visible, TEM, FE-SEM, EDX, FTIR, and XRD spectroscopy. The nanoparticles exhibited an absorption peak around 420 nm, a characteristic surface plasmon resonance band of silver nanoparticles. They were spherical in shape with an average particle size of  nm. The EDX analysis showed the presence of elemental silver signal in the synthesized nanoparticles. The FTIR analysis revealed that the protein component in the form of enzyme nitrate reductase produced by the isolate in the culture supernatant may be responsible for reduction and as capping agents. The XRD spectrum showed the characteristic Bragg peaks of 1 2 3, 2 0 4, 0 4 3, 1 4 4, and 3 1 1 facets of the face centered cubic silver nanoparticles and confirms that these nanoparticles are crystalline in nature. The prepared silver nanoparticles exhibited strong antimicrobial activity against bacteria and fungi. Cytotoxicity of biosynthesized AgNPs against in vitro human cervical cancer cell line (HeLa) showed a dose-response activity. IC50 value was found to be 200 μg/mL of AgNPs against HeLa cancer cells. Further studies are needed to elucidate the toxicity and the mechanism involved with antimicrobial and anticancer activity of the synthesized AgNPs as nanomedicine. Panchanathan Manivasagan, Jayachandran Venkatesan, Kalimuthu Senthilkumar, Kannan Sivakumar, and Se-Kwon Kim Copyright © 2013 Panchanathan Manivasagan et al. All rights reserved. Analytical Variables Affecting Analysis of F2-Isoprostanes and F4-Neuroprostanes in Human Cerebrospinal Fluid by Gas Chromatography/Mass Spectrometry Wed, 10 Jul 2013 10:14:05 +0000 F2-isoprostanes (F2-IsoPs) are a gold marker of lipid peroxidation in vivo, whereas F4-neuroprostanes (F4-NPs) measured in cerebrospinal fluid (CSF) or brain tissue selectively indicate neuronal oxidative damage. Gas chromatography/negative-ion chemical-ionization mass spectrometry (GC/NICI-MS) is the most sensitive and robust method for quantifying these compounds, which is essential for CSF samples because abundance of these compounds in CSF is very low. The present study revealed potential interferences on the analysis of F2-IsoPs and F4-NPs in CSF by GC/NICI-MS due to the use of improper analytical methods that have been employed in the literature. First, simultaneous quantification of F2-IsoPs and F4-NPs in CSF samples processed for F4-NPs analysis could cause poor chromatographic separation and falsely higher F2-IsoPs values for CSF samples with high levels of F2-IsoPs and F4-NPs. Second, retention of unknown substances in GC columns from CSF samples during F4-NPs analysis and from plasma samples during F2-IsoPs analysis might interfere with F4-NPs analysis of subsequent runs, which could be solved by holding columns at a high temperature for a period of time after data acquisition. Therefore, these special issues should be taken into consideration when performing analysis of F2-IsoPs and F4-NPs in CSF to avoid misleading results. Hsiu-Chuan Yen, Hsing-Ju Wei, and Ting-Wei Chen Copyright © 2013 Hsiu-Chuan Yen et al. All rights reserved. Effect of NaCl on Heat Resistance, Antibiotic Susceptibility, and Caco-2 Cell Invasion of Salmonella Mon, 08 Jul 2013 15:45:07 +0000 This study evaluated the effects of NaCl on heat resistance, antibiotic susceptibility, and Caco-2 cell invasion of Salmonella. Salmonella typhimurium NCCP10812 and Salmonella enteritidis NCCP12243 were exposed to 0, 2, and 4% NaCl and to sequential increase of NaCl concentrations from 0 to 4% NaCl for 24 h at 35°C. The strains were then investigated for heat resistance (60°C), antibiotic susceptibility to eight antibiotics, and Caco-2 cell invasion efficiency. S. typhimurium NCCP10812 showed increased thermal resistance () after exposure to single NaCl concentrations. A sequential increase of NaCl concentration decreased () the antibiotic sensitivities of S. typhimurium NCCP10812 to chloramphenicol, gentamicin, and oxytetracycline. NaCl exposure also increased () Caco-2 cell invasion efficiency of S. enteritidis NCCP12243. These results indicate that NaCl in food may cause increased thermal resistance, cell invasion efficiency, and antibiotic resistance of Salmonella. Hyunjoo Yoon, Beom-Young Park, Mi-Hwa Oh, Kyoung-Hee Choi, and Yohan Yoon Copyright © 2013 Hyunjoo Yoon et al. All rights reserved. High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli Tue, 02 Jul 2013 11:18:11 +0000 OG2 is a modified antimicrobial peptide, that is, derived from the frog peptide Palustrin-OG1. It has high antimicrobial activity and low cytotoxicity, and it is therefore promising as a therapeutic agent. Both prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) production host systems were used to produce OG2 in our previous study; however, it was difficult to achieve high expression yields and efficient purification. In this study, we achieved high-yield OG2 expression using the intein fusion system. The optimized OG2 gene was cloned into the pTWIN1 vector to generate pTWIN-OG2-intein2 (C-terminal fusion vector) and pTWIN-intein1-OG2 (N-terminal fusion vector). Nearly 70% of the expressed OG2-intein2 was soluble after the IPTG concentration and induction temperature were decreased, whereas only 42% of the expressed of intein1-OG2 was soluble. Up to 75 mg of OG2-intein2 was obtained from a 1 l culture, and 85% of the protein was cleaved by 100 mM DTT. Intein1-OG2 was less amenable to cleavage due to the inhibition of cleavage by the N-terminal amino acid of OG2. The purified OG2 exhibited strong antimicrobial activity against E. coli K88. The intein system is the best currently available system for the cost-effective production of OG2. Yong-Gang Xie, Fei-Fei Han, Chao Luan, Hai-Wen Zhang, Jie Feng, Young-Jun Choi, Denis Groleau, and Yi-Zhen Wang Copyright © 2013 Yong-Gang Xie et al. All rights reserved. Rhizobium pongamiae sp. nov. from Root Nodules of Pongamia pinnata Tue, 02 Jul 2013 09:47:44 +0000 Pongamia pinnata has an added advantage of N2-fixing ability and tolerance to stress conditions as compared with other biodiesel crops. It harbours “rhizobia” as an endophytic bacterial community on its root nodules. A gram-negative, nonmotile, fast-growing, rod-shaped, bacterial strain VKLR-01T was isolated from root nodules of Pongamia that grew optimal at 28°C, pH 7.0 in presence of 2% NaCl. Isolate VKLR-01 exhibits higher tolerance to the prevailing adverse conditions, for example, salt stress, elevated temperatures and alkalinity. Strain VKLR-01T has the major cellular fatty acid as C18:1  ω7c (65.92%). Strain VKLR-01T was found to be a nitrogen fixer using the acetylene reduction assay and PCR detection of a nifH gene. On the basis of phenotypic, phylogenetic distinctiveness and molecular data (16S rRNA, recA, and atpD gene sequences, G + C content, DNA-DNA hybridization etc.), strain VKLR-01T = (MTCC 10513T = MSCL 1015T) is considered to represent a novel species of the genus Rhizobium for which the name Rhizobium pongamiae sp. nov. is proposed. Rhizobium pongamiae may possess specific traits that can be transferred to other rhizobia through biotechnological tools and can be directly used as inoculants for reclamation of wasteland; hence, they are very important from both economic and environmental prospects. Vigya Kesari, Aadi Moolam Ramesh, and Latha Rangan Copyright © 2013 Vigya Kesari et al. All rights reserved. Evaluation of an Adsorbent Based on Agricultural Waste (Corn Cobs) for Removal of Tyrosine and Phenylalanine from Aqueous Solutions Tue, 02 Jul 2013 08:14:14 +0000 Adsorption of phenolic amino acids, such as phenylalanine and tyrosine, is quite relevant for the production of protein hydrolysates used as dietary formulations for patients suffering from congenital disorders of amino acid metabolism, such as phenylketonuria. In this study, an adsorbent prepared from corn cobs was evaluated for the removal of tyrosine (Tyr) from both a single component solution and a binary aqueous solution with phenylalanine (Phe). The adsorption behavior of tyrosine was similar to that of phenylalanine in single component solutions, however, with a much lower adsorption capacity (14 mg g−1 for Tyr compared to 109 mg g−1 for Phe). Tyr adsorption kinetics was satisfactorily described by a pseudosecond-order model as it was for Phe. In adsorption equilibrium studies for binary mixtures, the presence of Tyr in Phe solutions favored Phe faster adsorption whereas the opposite behavior was observed for the presence of Phe in Tyr solutions. Such results indicate that, in binary systems, Phe will be adsorbed preferably to Tyr, and this is a welcome feature when employing the prepared adsorbent for the removal of Phe from protein hydrolysates to be used in dietary formulations for phenylketonuria treatment. Cibele C. O. Alves, Adriana S. Franca, and Leandro S. Oliveira Copyright © 2013 Cibele C. O. Alves et al. All rights reserved. Polyisoprenylated Methylated Protein Methyl Esterase Is Both Sensitive to Curcumin and Overexpressed in Colorectal Cancer: Implications for Chemoprevention and Treatment Mon, 01 Jul 2013 15:47:27 +0000 Inhibition of PMPMEase, a key enzyme in the polyisoprenylation pathway, induces cancer cell death. In this study, purified PMPMEase was inhibited by the chemopreventive agent, curcumin, with a of 0.3 μM (IC50 = 12.4 μM). Preincubation of PMPMEase with 1 mM curcumin followed by gel-filtration chromatography resulted in recovery of the enzyme activity, indicative of reversible inhibition. Kinetics analysis with N-para-nitrobenzoyl-S-trans,trans-farnesylcysteine methyl ester substrate yielded values of 23.6 ± 2.7 and 85.3 ± 15.3 μM in the absence or presence of 20 μM curcumin, respectively. Treatment of colorectal cancer (Caco2) cells with curcumin resulted in concentration-dependent cell death with an EC50 of 22.0 μg/mL. PMPMEase activity in the curcumin-treated cell lysate followed a similar concentration-dependent profile with IC50 of 22.6 μg/mL. In colorectal cancer tissue microarray studies, PMPMEase immunoreactivity was significantly higher in 88.6% of cases compared to normal colon tissues . The mean scores ± SEM were 91.7 ± 11.4 (normal), 75.0 ± 14.4 (normal adjacent), 294.8 ± 7.8 (adenocarcinoma), and 310.0 ± 22.6 (mucinous adenocarcinoma), respectively. PMPMEase overexpression in colorectal cancer and cancer cell death stemming from its inhibition is an indication of its possible role in cancer progression and a target for chemopreventive agents. Felix Amissah, Randolph Duverna, Byron J. Aguilar, Rosemary A. Poku, and Nazarius S. Lamango Copyright © 2013 Felix Amissah et al. All rights reserved. Plant Growth Promotion Potential Is Equally Represented in Diverse Grapevine Root-Associated Bacterial Communities from Different Biopedoclimatic Environments Wed, 26 Jun 2013 18:01:52 +0000 Plant-associated bacteria provide important services to host plants. Environmental factors such as cultivar type and pedoclimatic conditions contribute to shape their diversity. However, whether these environmental factors may influence the plant growth promoting (PGP) potential of the root-associated bacteria is not widely understood. To address this issue, the diversity and PGP potential of the bacterial assemblage associated with the grapevine root system of different cultivars in three Mediterranean environments along a macrotransect identifying an aridity gradient were assessed by culture-dependent and independent approaches. According to 16S rRNA gene PCR-DGGE, the structure of endosphere and rhizosphere bacterial communities was highly diverse () and was associated with a cultivar/latitudinal/climatic effect. Despite being diverse, the bacterial communities associated with Egyptian grapevines shared a higher similarity with the Tunisian grapevines than those cultivated in North Italy. A similar distribution, according to the cultivar/latitude/aridity gradients, was observed for the cultivable bacteria. Many isolates (23%) presented in vitro multiple stress resistance capabilities and PGP activities, the most frequent being auxin synthesis (82%), insoluble phosphate solubilisation (61%), and ammonia production (70%). The comparable numbers and types of potential PGP traits among the three different environmental settings indicate a strong functional homeostasis of beneficial bacteria associated with grape root. Ramona Marasco, Eleonora Rolli, Marco Fusi, Ameur Cherif, Ayman Abou-Hadid, Usama El-Bahairy, Sara Borin, Claudia Sorlini, and Daniele Daffonchio Copyright © 2013 Ramona Marasco et al. All rights reserved. Natural Treatment Systems as Sustainable Ecotechnologies for the Developing Countries Wed, 26 Jun 2013 17:48:13 +0000 The purpose of natural treatment systems is the re-establishment of disturbed ecosystems and their sustainability for benefits to human and nature. The working of natural treatment systems on ecological principles and their sustainability in terms of low cost, low energy consumption, and low mechanical technology is highly desirable. The current review presents pros and cons of the natural treatment systems, their performance, and recent developments to use them in the treatment of various types of wastewaters. Fast population growth and economic pressure in some developing countries compel the implementation of principles of natural treatment to protect natural environment. The employment of these principles for waste treatment not only helps in environmental cleanup but also conserves biological communities. The systems particularly suit developing countries of the world. We reviewed information on constructed wetlands, vermicomposting, role of mangroves, land treatment systems, soil-aquifer treatment, and finally aquatic systems for waste treatment. Economic cost and energy requirements to operate various kinds of natural treatment systems were also reviewed. Qaisar Mahmood, Arshid Pervez, Bibi Saima Zeb, Habiba Zaffar, Hajra Yaqoob, Muhammad Waseem, Zahidullah, and Sumera Afsheen Copyright © 2013 Qaisar Mahmood et al. All rights reserved. Persistent Organic Pollutants Induced Protein Expression and Immunocrossreactivity by Stenotrophomonas maltophilia PM102: A Prospective Bioremediating Candidate Wed, 26 Jun 2013 11:31:42 +0000 A novel bacterium capable of growth on trichloroethylene as the sole carbon source was identified as Stenotrophomonas maltophilia PM102 by 16S rDNA sequencing (accession number of NCBI GenBank: JQ797560). In this paper, we report the growth pattern, TCE degradation, and total proteome of this bacterium in presence of various other carbon sources: toluene, phenol, glucose, chloroform, and benzene. TCE degradation was comparatively enhanced in presence of benzene. Densitometric analysis of the intracellular protein profile revealed four proteins of 78.6, 35.14, 26.2, and 20.47 kDa while the extracellular protein profile revealed two distinct bands at 14 kDa and 11 kDa that were induced by TCE, benzene, toluene, and chloroform but absent in the glucose lane. A rabbit was immunised with the total protein extracted from the bacteria grown in 0.2% TCE + 0.2% peptone. Antibody preadsorbed on proteins from peptone grown PM102 cells reacted with a single protein of 35.14 kDa (analysed by MALDI-TOF-mass-spectrometry) from TCE, benzene, toluene, or chloroform grown cells. No reaction was seen for proteins of PM102 grown with glucose. The PM102 strain was immobilised in calcium alginate beads, and TCE degradation by immobilised cells was almost double of that by free cells. The beads could be reused 8 times. Piyali Mukherjee and Pranab Roy Copyright © 2013 Piyali Mukherjee and Pranab Roy. All rights reserved. Antimicrobial Resistance Mechanisms among Campylobacter Mon, 24 Jun 2013 15:41:32 +0000 Campylobacter jejuni and Campylobacter coli are recognized as the most common causative agents of bacterial gastroenteritis in the world. Humans most often become infected by ingesting contaminated food, especially undercooked chicken, but also other sources of bacteria have been described. Campylobacteriosis is normally a self-limiting disease. Antimicrobial treatment is needed only in patients with more severe disease and in those who are immunologically compromised. The most common antimicrobial agents used in the treatment of Campylobacter infections are macrolides, such as erythromycin, and fluoroquinolones, such as ciprofloxacin. Tetracyclines have been suggested as an alternative choice in the treatment of clinical campylobacteriosis but in practice are not often used. However, during the past few decades an increasing number of resistant Campylobacter isolates have developed resistance to fluoroquinolones and other antimicrobials such as macrolides, aminoglycosides, and beta-lactams. Trends in antimicrobial resistance have shown a clear correlation between use of antibiotics in the veterinary medicine and animal production and resistant isolates of Campylobacter in humans. In this review, the patterns of emerging resistance to the antimicrobial agents useful in treatment of the disease are presented and the mechanisms of resistance to these drugs in Campylobacter are discussed. Kinga Wieczorek and Jacek Osek Copyright © 2013 Kinga Wieczorek and Jacek Osek. All rights reserved. Strategies to Characterize Fungal Lipases for Applications in Medicine and Dairy Industry Mon, 24 Jun 2013 08:57:45 +0000 Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications. Subash C. B. Gopinath, Periasamy Anbu, Thangavel Lakshmipriya, and Azariah Hilda Copyright © 2013 Subash C. B. Gopinath et al. All rights reserved.