Applied Biochemistry and Biotechnology (v.173, #6)

Here we cloned and expressed two alkaline β-1, 4-endoglucanases of Phaeosphaeria sp. LH21 from deep-sea mud. The two enzymes shared 71 and 63 % of identities with their known β-1, 4-endoglucanases, respectively. According to the primary and spatial structures, the potential active sites of one of the two enzymes could be Asp122 and Asp11, while the other enzyme could be Asp16. The enzymatic properties of their recombinant enzymes from Pichia pastoris GS115 showed that they were optimally active at pH 8 and 60–65 °C, exhibited >90 % residual relative activities at pH 3–10, and obtained relative activities >75 % at pH 5–10.
Keywords: Cloning; Expression; β-1, 4-endoglucanase; Phaeosphaeria sp. LH21; Active sites; Enzymatic properties

Amino Acid Esters Substituted Phosphorylated Emtricitabine and Didanosine Derivatives as Antiviral and Anticancer Agents by Kuruva Chandra Sekhar; Avilala Janardhan; Yellapu Nanda Kumar; Golla Narasimha; Chamarthi Naga Raju; S. K. Ghosh (1303-1318).
Owing to the promising antiviral activity of amino acid ester-substituted phosphorylated nucleosides in the present study, a series of phosphorylated derivatives of emtricitabine and didanosine substituted with bioactive amino acid esters at P-atom were synthesized. Initially, molecular docking studies were screened to predict their molecular interactions with hemagglutinin-neuraminidase protein of Newcastle disease virus and E2 protein of human papillomavirus. The title compounds were screened for their antiviral ability against Newcastle disease virus (NDV) by their in ovo study in embryonated chicken eggs. Compounds 5g and 9c exposed well mode of interactions with HN protein and also exhibited potential growth of NDV inhibition. The remaining compounds exhibited better growth of NDV inhibition than their parent molecules, i.e., emtricitabine (FTC) and didanosine (ddI). In addition, the in vitro anticancer activity of all the title compounds were screenedagainst HeLa cell lines at 10 and 100 μg/mL concentrations. The compounds 5g and 9c showed an effective anticancer activity than that of the remaining title compounds with IC50 values of 40 and 60 μg/mL, respectively. The present in silico and in ovo antiviral and in vitro anticancer results of the title compounds are suggesting that the amino acid ester-substituted phosphorylated FTC and ddI derivatives, especially 5g and 9c, can be used as NDV inhibitors and anticancer agents for the control and management of viral diseases with cancerous condition. ᅟ
Keywords: Phosphorylated derivatives of FTC and ddI; Newcastle disease virus (NDV); Human papillomavirus (HPV); Molecular docking; In ovo antiviral activity; Anticancer activity; HeLa cell lines

Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical conversion of lignocellulosic materials into renewable biofuels and biochemicals. Here, we show that enzymatic hydrolysis of acid-pretreated pine wood with continuous and/or fed-batch enzyme addition improved sugar conversion efficiencies by over sixfold. In addition, specific activity of the hydrolysis enzymes (cellulases, hemicellulases, etc.) increased as a result of continuously washing the residual solids with removal of glucose (avoiding the end product inhibition) and other enzymatic inhibitory compounds (e.g., furfural, hydroxymethyl furfural, organic acids, and phenolics). As part of the continuous hydrolysis, anion exchange resin was tested for its dual application of simultaneous enzyme recovery and removal of potential enzymatic and fermentation inhibitors. Amberlite IRA-96 showed favorable adsorption profiles of inhibitors, especially furfural, hydroxymethyl furfural, and acetic acid with low affinity toward sugars. Affinity of hydrolysis enzymes to adsorb onto the resin allowed for up to 92 % of the enzymatic activity to be recovered using a relatively low-molar NaCl wash solution. Integration of an ion exchange column with enzyme recovery into the proposed fed-batch hydrolysis process can improve the overall biorefinery efficiency and can greatly reduce the production costs of lignocellulosic biorenewable products. Figure A semicontinuous process for the biochemical production of renewable products using detoxification and fed-batch enzyme addition/recycle can increase enzymatic hydrolysis and fermentation efficiencies. Hydrolysis enzymes, inhibitors, sugars, and water can be separated and utilized as high-value steams within the process
Keywords: Biorenewable products; Enzyme recycle; Bioseparations; Enzymatic hydrolysis; Biorefining; High-value coproducts

Kinetics of Enzymatic Hydrolysis of Olive Oil in Batch and Fed-batch Systems by Paloma Souza Cabral; Arion Zandoná Filho; Fernando Augusto Pedersen Voll; Marcos Lúcio Corazza (1336-1348).
This work reports experimental data, kinetic modeling, and simulations of enzyme-catalyzed hydrolysis of olive oil. This reaction was performed in batch system and an ordered-sequential Bi Bi model was used to model the kinetic mechanism. A fed-batch system was proposed and experimental data were obtained and compared to the simulated values. The kinetic model used was able to correlate the experimental data, in which a satisfactory agreement between the experimental data and modeling results was obtained under different enzyme concentration and initial free water content. Therefore, the modeling allowed a better understanding of the reaction kinetics and affords a fed-batch simulation for this system. From the results obtained, it was observed that the fed-batch approach showed to be more advantageous when compared to the conventional batch system.
Keywords: Acylglycerols; Enzyme-catalyzed; Hydrolysis; Olive oil; Fed-batch system; Mathematical modeling

Chaperones Are Necessary for the Expression of Catalytically Active Potato Apyrases in Prokaryotic Cells by Dorota Porowińska; Joanna Czarnecka; Michał Komoszyński (1349-1359).
NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5′-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.
Keywords: NTPDase; Apyrase; Nucleoside triphosphate diphosphohydrolases; Prokaryotic expression system; Escherichia coli ; Chaperone

Xanthine oxidase is considered as a potential target for treatment of hyperuricemia. Hyperuricemia is predisposing factor for gout, chronic heart failure, atherosclerosis, tissue injury, and ischemia. To date, only two inhibitors of xanthine oxidase viz. allopurinol and febuxostat have been clinically approved for used as drugs. In the process of searching for new xanthine oxidase inhibitors, we screened culture filtrates of 42 endophytic fungi using in vitro qualitative and quantitative XO inhibitory assays. The qualitative assay exhibited potential XO inhibition by culture filtrates of four isolates viz. #1048 AMSTITYEL, #2CCSTITD, #6AMLWLS, and #96 CMSTITNEY. The XO inhibitory activity was present only in the chloroform extract of the culture filtrates. Chloroform extract of culture filtrate #1048 AMSTITYEL exhibited the highest inhibition of XO with an IC50 value of 0.61 μg ml−1 which was better than allopurinol exhibiting an IC50 of 0.937 μg ml−1 while febuxostat exhibited a much lower IC50 of 0.076 μg ml−1. Further, molecular phylogenetic tools and morphological studies were used to identify #1048 AMSTITYEL as Lasiodiplodia pseudotheobromae. This is the first report of an endophytic Lasiodiplodia pseudotheobromae from Aegle marmelos exhibiting potential XO Inhibitory activity.
Keywords: Xanthine oxidase; Endophytic fungi; Allopurinol; Aegle Marmelos ; Lasiodiplodia

An aerobic xylanolytic moderately halophilic and alkali-tolerant bacterium, Gracilibacillus sp. TSCPVG, produces multiple xylanases of unusual halo-acid-alkali-thermo-stable nature. The purification of a major xylanase from TSCPVG culture supernatant was achieved by hydrophobic and gel permeation chromatographic methods followed by electroelution from preparatory PAGE. The molecular mass of the purified xylanase was 42 kDa, as analyzed by SDS-PAGE, with a pI value of 6.1. It exhibited maximal activity in 3.5 % NaCl and retained over 75 % of its activity across the broad salinity range of 0–30 % NaCl, indicating a high halo-tolerance. It showed maximal activity at pH 7.5 and had retained 63 % of its activity at pH 5.0 and 73 % at pH 10.5, signifying the tolerance to broad acid to alkaline conditions. With birchwood xylan as a substrate, K m and specific activity values were 21 mg/ml and 1,667 U/mg, respectively. It is an endoxylanase that degrades xylan to xylose and xylobiose and had no activity on p-nitrophenyl-β-d-xylopyranoside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl acetate, carboxymethylcellulose, and filter paper. Since it showed remarkable stability over different salinities, broad pH, and temperature ranges, it is promising for application in many industries.
Keywords: Gracilibacillus sp. TSCPVG; Enzyme purification; Halo-acid-alkali-thermo-stable β-xylanase; Endoxylanase

Optimization of Anaerobic Co-digestion of Strawberry and Fish Waste by Antonio Serrano; José A. Siles; M. Carmen Gutiérrez; M. Ángeles Martín (1391-1404).
Anaerobic co-digestion of agri-food waste is a promising management alternative. Its implementation, however, requires evaluating the proportion in which waste should be mixed to optimize their centralized treatment. The combined treatment of strawberry extrudate and fish waste, which are widely generated in Mediterranean areas, was optimized. Strawberry extrudate and fish waste were mixed and treated at different proportions (88:12, 94:6, and 97:3, respectively; wet basis). The proportions selected for the mixture allow the different flows to be absorbed simultaneously. The highest methane production was observed for the ratio 94:6 (0.205 m3 STP CH4/kg volatile solid) (VS) (STP; 0 °C, 1 atm), with a methane production rate in the range of 5 · 10−3–9 · 10−3 m3 STP/kg VS · d, while the highest organic loading rate was observed for the mixture at a proportion 88:12 (1.9 ± 0.1 kg VS/m3 · d). Biodegradability was found to be similar for the 88:12 and 94:6 proportions, with values around 90 % in VS. Nevertheless, the 97:3 ratio was not viable due to a low methane production. An inhibition phenomenon occurred at increasing loads due to the effect of some compounds contained in the fish waste such as chloride or nitrogen.
Keywords: Strawberry extrudate; Fish waste; Anaerobic co-digestion; Optimization; Chloride; Free ammonia

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0–4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.
Keywords: Ni-Eupergit C; Immobilization; Penicillium occitanis ; Xylanase; Corncob xylan; Xylooligosaccharides

Gene Cloning, Expression, and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9 by Jian Gao; You-Yong Xu; Hong-Mei Yang; Hong Xu; Feng Xue; Sha Li; Xiao-Hai Feng (1419-1430).
An inulinase-producing strain, Paenibacillus polymyxa ZJ-9, was isolated from natural sources to produce R,R-2,3-butanediol via one-step fermentation of raw inulin extracted from Jerusalem artichoke tubers. The inulinase gene from P. polymyxa ZJ-9 was cloned and overexpressed in Escherichia coli BL21 (DE3), and the purified recombinant inulinase was estimated to be approximately 56 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and gel filtration chromatography. This result suggests that the active form of the inulinase is probably a monomer. Terminal hydrolysis fructose units from the inulin indicate that enzymes are exo-inulinase. The purified recombinant enzyme showed maximum activity at 25 °C and pH 6.0, which indicate its extreme suitability for industrial applications. Zn2+, Fe2+, and Mg2+ stimulated the activity of the purified enzyme, whereas Co2+, Cu2+, and Ni2+ inhibited enzyme activity. The K m and V max values for inulin hydrolysis were 1.72 mM and 21.69 μmol min−1 mg−1 protein, respectively. The same parameters toward sucrose were 41.09 mM and 78.7 μmol min−1 mg−1 protein, respectively. Considering its substrate specificity and other enzymatic characteristics, we believe that this inulinase gene from P. polymyxa ZJ-9 could be transformed into other special bacterial strains to allow inulin conversion to other biochemicals and bioenergy through one-step fermentation.
Keywords: Characterization; Cloning; Exo-inulinase; Paenibacillus polymyxa ZJ-9; R,R-2,3-butanediol

Evaluation of Internal Control for Gene Expression in Phalaenopsis by Quantitative Real-Time PCR by Xiu-Yun Yuan; Su-Hua Jiang; Mo-Fei Wang; Jie Ma; Xian-Yun Zhang; Bo Cui (1431-1445).
The selection of appropriate reference genes is one of the most important steps to obtain reliable results for normalizing quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) of MADS-box gene in Phalaenopsis. In this study, we cloned 12 candidate reference genes including 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), cytoskeletal structural protein actin (ACT1, ACT2, ACT3, ACT4, ACT5), ubiquitin protein (UBQ1 and UBQ2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the cytoskeletal structural proteins α-tubulin (TUA) and β-tubulin (TUB) in Phalaenopsis and evaluated their expression reliability. The expression of these candidate reference genes was analyzed using geNorm and normFinder software packages; the results showed that ACT2 and ACT4 were the highest stability reference genes for all experiment sets based on normFinder, followed by ACT1 or ACT3, while ACT3 and ACT4 were the highest stability reference genes for most experiment sets based on geNorm, then TUB or others. Taken together, Actin genes were the higher stability reference genes for all tissues at total developmental stages, and similar results came from analysis by normFinder. According to geNorm analysis, ACT3 and ACT4 were the most stable reference genes for all tissues tested and tissues at reproductive stages; TUB and ACT5 or ACT4 were the most stable reference genes for vegetative tissues or roots. The most stable reference genes for all vegetative tissues and only leaves were ACT4 and ACT5, ACT2 and ACT3, respectively; ACT1 and ACT3 were the most stable genes and sufficient for reliable normalization of flower tissues. While EF1α, UBQ1, UBQ2, and GAPDH were found to be unsuitable as a reference gene in our analysis for flower tissues, total tissues, and reproductive stages; UBQ2 and 18S were identified as the least stable reference genes for vegetative tissues at different stages, different tissues at vegetative stages; TUA and 18S were the least reliable reference genes for the samples from roots at all developmental stages. This is the first systematic report on the selection of reference genes in Phalaenopsis, and these data will facilitate future work on gene expression in orchid.
Keywords: Phalaenopsis ; Quantitative real-time PCR; Reference genes; Gene expression

Pangasianodon hypophthalmus is one of the fast-growing catfish of freshwater origin, and its growth is attributed by the action of growth hormone (GH). In this study, the growth hormone gene (PhGH) of 3.0 kb was characterized, and it is composed of five exons and four introns and having characteristics of an upstream region that contains TATA, CAAT boxes, and binding sites of important transcription factors like Pit-1a, CRE, CREB, CREBP, Ap-1, SP1, and TBP. The full-length cDNA sequence of 1,069 bp was isolated using RACE technique, and it is composed of untranslated regions of 60 and 403 bp at 5′ and 3′, respectively, with an open reading frame of 603 bp that encodes a putative polypeptide of 200 amino acids with an estimated molecular mass of 22.57 kDa. The precursor of PhGH is composed of 22 amino acid signal peptides and 178 amino acid mature peptides. Five conserved Cys residues (Cys71, Cys135, Cys173, Cys190, and Cys198) and two possible sites of N-glycosylation (145th and 197th) were detected on GH polypeptide. The PhGH gene showed more than 90 % sequence similarity with other catfishes, and the phylogeny constructed revealed the close proximity of Siluriformes fishes with Cypriniformes fishes. The PhGH gene was observed to be expressed predominantly in pituitary tissues while weekly expressed in extrapituitary tissues. Further, the recombinant PhGH was expressed in Escherichia coli using His-tag expression vector pET 32(a), and the recombinant protein of ~23 kDa was confirmed by western blotting. Our findings suggest that the identified functional GH gene would provide basic information in transgenic studies aiming for faster growth rate. This recombinant growth hormone (GH) may be produced in large scale to exploit its growth-promoting function in other cultured fishes.
Keywords: Growth enhancement; Aquaculture; Transgenic fish; Recombinant growth hormone; Tissue-specific expression

A Novel Low Molecular Weight Endo-xylanase from Streptomyces sp. CS628 Cultivated in Wheat Bran by Md. Arifur Rahman; Yun Hee Choi; Pradeep G. C.; Yoon Seok Choi; Eun Joo Choi; Seung Sik Cho; Jin Cheol Yoo (1469-1480).
An extracellular low molecular weight xylanase (Xyn628) from Streptomyces sp. CS628 was isolated from Korean soil sample, produced in wheat bran medium, purified, and biochemically characterized. Xyn628 was purified 4.8-fold with a 33.78 % yield using Sepharose CL-6B column chromatography. The purified xylanase was ~18.1 kDa estimated by SDS-PAGE and xylan zymography. N-terminal amino acid sequences of Xyn628 were AYIKEVVSRAYM. The enzyme was found to be stable in a broad range of pH (5.0–13.0) and up to 60 °C and have optimal pH and temperature of pH 11.0 and 60 °C, respectively. Xyn628 activities were remarkable affected by various detergents, chelators, modulators, and metal ions. The xylanase produced xylobiose and xylotriose as principal hydrolyzed end products from the xylan. It was found to degrade agro-waste materials like corn cob and wheat bran by Xyn628 (20 U/g) as shown by electron microscopy. As being simple in purification, low molecular weight, alkaline, thermostable, and ability to produce xylooligosaccharides show that Xyn628 has potential applications in bioindustries as a biobleaching agent or/and xylooligosaccharides production with an appropriate utilization of agro-waste.
Keywords: Streptomyces ; Low molecular weight; Xylooligosaccharides; Agro-waste; Wheat bran

In Vitro Propagation, Encapsulation, and Genetic Fidelity Analysis of Terminalia arjuna: a Cardioprotective Medicinal Tree by Amit K. Gupta; Harish; Manoj K. Rai; Mahendra Phulwaria; Tanvi Agarwal; N. S. Shekhawat (1481-1494).
The present study described an improved and reproducible in vitro regeneration system for Terminalia arjuna using nodal segment explants obtained from a mature plant. Shoot tips excised from in vitro proliferated shoots were encapsulated in 3 % sodium alginate and 100 mM CaCl2⋅2H2O for the development of synthetic seeds which may be applicable in short-term storage and germplasm exchange of elite genotype. Shoot multiplication was significantly influenced by a number of factors, namely types and concentrations of plant growth regulators, medium composition, repeated transfer of mother explants, subculturing of in vitro regenerated shoot clumps, agar concentrations, and temperature. Maximum numbers of shoots (16.50 ± 3.67) were observed on modified Murashige and Skoog (MMS) medium containing 0.5 mg l−1 of benzylaminopurine (BAP) and 0.1 mg l−1 of naphthalene acetic acid (NAA). To shortening the regeneration pathway, rooting of micropropagated shoots under in vitro condition was excluded and an experiment on ex vitro rooting was conducted and it was observed that the highest percentage of shoots rooted ex vitro when treated with indole-3-butyric acid (IBA, 250 mg l−1) + 2-naphthoxy acetic acid (NOA, 250 mg l−1) for 5 min. The well-developed ex vitro rooted shoots were acclimatized successfully in soilrite under greenhouse conditions with 80 % survival of plants. Randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants were genetically identical to the mother plant, suggesting the absence of detectable genetic variation in the regenerated plantlets. To the best of our knowledge, this is the first report on synthetic seed production as well as ex vitro rooting and genetic fidelity assessment of micropropagated shoots of T. arjuna.
Keywords: Ex vitro rooting; Micropropagation; Molecular marker; Somaclonal variation; Synthetic seed

The aim of this study is to optimize the lipid accumulation in microalgae by using two agricultural residues of pineapple peels and sugarcane bagasse as low-cost organic carbon sources. Green microalgae Scenedesmus acutus was isolated and selected for cultivation. Effects of three initial sugar concentrations and the stage for adding sugar during cultivation on biomass and lipid production were investigated. The results clearly showed that two-stage cultivation is more suitable than one-stage. The maximum biomass concentration and productivity were obtained at 3.85 g/L and 160.42 mg/L/day when sugarcane bagasse was used. The highest lipid content and lipid yield was reached at 28.05 % and 0.93 g/L when pineapple peels were used, while in the case of sugarcane bagasse, 40.89 % and 1.24 g/L lipid content and yield were obtained. Lipid content was found in normal condition (autotrophic) at 17.71 % which was approximately 2.13-fold lower than when sugarcane bagasse was used (40.89 %). Biodiesel production via in situ transesterification was also investigated; the main fatty acids of palmitic acid and oleic acid were found. This work indicates that using agricultural residues as organic carbon sources could be able to increase lipid content and reduce the cost of biofuel production.
Keywords: Microalgae; Scenedesmus acutus ; Agricultural residues; Pineapple peels; Sugarcane bagasse; Lipid; Biodiesel

Carbon nanotube/nanoparticle hybrid materials have been proven to exhibit high electrocatalytic activity suggesting broad potential applications in the field of electroanalysis. For the first time, modification of Ta electrode with aligned multi-walled carbon nanotubes/Au nanoparticles introduced for the sensitive determination of the antibiotic drug, cefazolin (CFZ). The electrochemical response characteristics of the modified electrode toward CFZ were investigated by means of cyclic and linear sweep voltammetry. The modified electrode showed an efficient catalytic activity for the reduction of CFZ, leading to a remarkable decrease in reduction overpotential and a significant increase of peak current. Under optimum conditions, the highly sensitive modified electrode showed a wide linear range from 50 pM to 50 μM with a sufficiently low detection limit of 1 ± 0.01 pM (S/N = 3). The results indicated that the prepared electrode presents suitable characteristics in terms of sensitivity (458.2 ± 2.6 μAcm−2/μM), accuracy, repeatability (RSD of 1.8 %), reproducibility (RSD of 2.9 %), stability (14 days), and good catalytic activity in physiological conditions. The method was successfully applied for accurate determination of trace amounts of CFZ in pharmaceutical and clinical preparations without the necessity for samples pretreatment or any time-consuming extraction or evaporation steps prior to the analysis.
Keywords: Multi-walled carbon nanotube; Au nanoparticle; Voltammetry; Electrochemical sensor; Cefazolin; Electrochemistry

Lentivirus-Mediated Knockdown of CUGBP1 Suppresses Gastric Cancer Cell Proliferation In Vitro by Xudong Wang; Haizhu Wang; Fujian Ji; Shutao Zhao; Xuedong Fang (1529-1536).
Gastric cancer is the second most common cause of cancer-related death worldwide. This study was designed to examine the role of CUGBP1 in cell growth via an RNA interference (RNAi) lentivirus system in gastric cancer cells in vitro. The expression of CUGBP1 was much stronger in gastric cancer tissues than that in adjacent normal tissues. The lentivirus-mediated knockdown of CUGBP1 resulted in a significant reduction of CUGBP1 expression in MGC-803 gastric cancer cells. The cell viability was remarkably decreased by 50 % after 5 days of infection, as determined by MTT assay. Moreover, the size and the number of colonies formed in MGC-803 cells were markedly reduced in the absence of CUGBP1. Furthermore, the silencing of CUGBP1 downregulated the expression levels of cyclin B1 and cyclin D1, which are involved in cell cycle control. These results clearly indicated that CUGBP1 is essential for the growth of gastric cancer cells. Therefore, silencing of CUGBP1 by RNAi could be developed as a promising therapeutic approach for gastric cancer.
Keywords: Gastric cancer; MGC-803; CUGBP1; Lentivirus; Growth

Dry Anaerobic Co-digestion of Cow Dung with Pig Manure for Methane Production by Jianzheng Li; Ajay Kumar Jha; Tri Ratna Bajracharya (1537-1552).
The performance of dry anaerobic digestions of cow dung, pig manure, and their mixtures into different ratios were evaluated at 35 ± 1 °C in single-stage batch reactors for 63 days. The specific methane yields were 0.33, 0.37, 0.40, 0.38, 0.36, and 0.35 LCH4/gVSr for cow dung to pig manure ratios of 1:0, 4:1, 3:2, 2:3, 1:4, and 0:1, respectively, while volatile solid (VS) and chemical oxygen demand (COD) removal efficiencies were 48.59, 50.79, 53.20, 47.73, 46.10, and 44.88 % and 55.44, 57.96, 60.32, 56.96, 53.32, and 50.86 %, respectively. The experimental results demonstrated that the co-digestions resulted in 5.10–18.01 % higher methane yields, 2.03–12.95 % greater VS removals, 2.98–12.52 % greater COD degradation and so had positive synergism. The various mixtures of pig manure with cow dung might persuade a better nutrient balance and dilution of high ammonia concentration in pig manure and therefore enhanced digester performance efficiency and higher biogas yields. The dry co-digestion of 60 % cow dung and 40 % pig manure achieved the highest methane yield and the greatest organic materials removal efficiency than other mixtures and controls.
Keywords: Dry anaerobic digestion; Co-digestion; Manures; Biogas; Organic materials removal

Genome Shuffling and Ribosome Engineering of Streptomyces actuosus for High-Yield Nosiheptide Production by Qingling Wang; Dong Zhang; Yudong Li; Fuming Zhang; Cao Wang; Xinle Liang (1553-1563).
Nosiheptide is one of the EU-approved sulfur-containing peptides in feed industry to inhibit the growth of the majority of Gram-positive bacteria. The main purpose of this study is directed to breed the high nosiheptide-producers by genome shuffling and ribosome engineering in Streptomyces actuosus AW7. The starting population for shuffling was generated by combining 60Coγ-irradiation with LiCl mutagenesis treatments on the spores. After four rounds of protoplast fusion exposed to streptomycin as adaptive pressure, a high-yield recombinant strain D92 was obtained. In a 10-L fermenter, nosiheptide production reached 1.54 g/L which was 9.20-fold compared to that of the parental strain. Hyphae development, metabolic process, and ribosomal protein S12 sequence were investigated to characterize the differentiation among the recombinants. Several mutations in S12 were believed to be responsible to streptomycin resistance in the tested strain. The results demonstrated that the combination of genome shuffling and ribosome engineering is an efficient approach to breed high-yield industrial strains.
Keywords: Nosiheptide; Genome shuffling; Ribosome engineering; Sequence difference; Streptomyces actuosus

Effects of Pipe Materials on Chlorine-resistant Biofilm Formation Under Long-term High Chlorine Level by Zebing Zhu; Chenguang Wu; Dan Zhong; Yixing Yuan; Lili Shan; Jie Zhang (1564-1578).
Drinking water distribution systems are composed of various pipe materials and may harbor biofilms even in the continuous presence of disinfectants. Biofilms formation on five pipe materials (copper (Cu), polyethylene (PE), stainless steel (STS), cast iron (CI), and concrete-coated polycarbonate (CP)) within drinking water containing 1.20 mg/L free chlorine, was investigated by flow cytometry, heterotrophic plate counts, and denaturing gradient gel electrophoresis analysis. Results showed that the biofilms formation varied in pipe materials. The biofilm formed on CP initially emerged the highest biomass in 12 days, but CI presented the significantly highest biomass after 28 days, and Cu showed the lowest bacterial numbers before 120 days, while STS expressed the lowest bacterial numbers after 159 days. In the biofilm community structure, Moraxella osloensis and Sphingomonas sp. were observed in all the pipe materials while Bacillus sp. was detected except in the CP pipe and Stenotrophomonas maltophila was found from three pipe materials (Cu, PE, and STS). Other bacteria were only found from one or two pipe materials. It is noteworthy that there are 11 opportunistic pathogens in the 17 classified bacterial strains. This research has afforded crucial information regarding the influence of pipe materials on chlorine-resistant biofilm formation.
Keywords: Drinking water distribution systems; Biofilm; Pipe material; Chlorine-resistant bacteria; Pathogenic bacteria

Erratum to: Effect of Cortisol on Calpains in the C2C12 and 3 T3-L1 Cells by Pandurangan Muthuraman; Sambandam Ravikumar; Veerappan Muthuviveganandavel; Jongpil Kim (1579-1579).