bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature dependence of amino acid side chain IR absorptions in the amide I' region
Authors: Anderson Benjamin A. , Literati Alex , Ball Borden , Kubelka Jan
Published: October 2013

Amide I' IR spectra are widely used for studies of structural changes in peptides and proteins as a function of temperature. Temperature dependent absorptions of amino acid side‐chains that overlap the amide I' may significantly complicate the structural analyses. While the side‐chain IR spectra have been investigated previously, thus far their dependence on temperature has not been reported. Here we present the study of the changes in the IR spectra with temperature for side‐chain groups of aspartate, glutamate, asparagine, glutamine, arginine, and tyrosine in the amide I' region (in D2O). Band fitting analysis was employed to extract the temperature dependence of the individual spectral parameters, such as peak frequency, integrated intensity, band width, and shape. As expected, the side‐chain IR bands exhibit significant changes with temperature. The majority of the spectral parameters, particularly the frequency and intensity, show linear dependence on temperature, but the direction and magnitude vary depending on the particular side‐chain group. The exception is arginine, which exhibits a distinctly nonlinear frequency shift with temperature for its asymmetric CN3H5+ bending signal, although a linear fit can account for this change to within ~1/3 cm‐1. The applicability of the determined spectral parameters for estimations of temperature‐dependent side‐chain absorptions in peptides and proteins are discussed. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 536–548, 2014.
Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3‐methyl isoxanthopterin
Authors: Gogichaishvili Shota , Johnson John , Gvarjaladze David , Lomidze Levan , Kankia Besik
Published: October 2013

We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3‐methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI‐based QPA is a useful assay for DNA amplification and detection. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 583–590, 2014.
Cis‐suppression to arrest protein aggregation in mammalian cells
Authors: Gregoire Simpson , Zhang Shaojie , Costanzo Joseph , Wilson Kelly , Fernandez Erik J. , Kwon Inchan
Published: October 2013

Protein misfolding and aggregation are implicated in numerous human diseases and significantly lower production yield of proteins expressed in mammalian cells. Despite the importance of understanding and suppressing protein aggregation in mammalian cells, a protein design and selection strategy to modulate protein misfolding/aggregation in mammalian cells has not yet been reported. In this work, we address the particular challenge presented by mutation‐induced protein aggregation in mammalian cells. We hypothesize that an additional mutation(s) can be introduced in an aggregation‐prone protein variant, spatially near the original mutation, to suppress misfolding and aggregation (cis‐suppression). As a model protein, we chose human copper, zinc superoxide dismutase mutant (SOD1A4V) containing an alanine to valine mutation at residue 4, associated with the familial form of amyotrophic lateral sclerosis. We used the program RosettaDesign to identify Phe20 in SOD1A4V as a key residue responsible for SOD1A4V conformational destabilization. This information was used to rationally develop a pool of candidate mutations at the Phe20 site. After two rounds of mammalian‐cell based screening of the variants, three novel SOD1A4V variants with a significantly reduced aggregation propensity inside cells were selected. The enhanced stability and reduced aggregation propensity of the three novel SOD1A4V variants were verified using cell fractionation and in vitro stability assays. Biotechnol. Bioeng. 2014;111: 462–474. © 2013 Wiley Periodicals, Inc.
Drug susceptibility of matrix‐encapsulated Candida albicans nano‐biofilms
Authors: Srinivasan Anand , Gupta Celia Macias , Agrawal C. Mauli , Leung Kai P. , Lopez‐Ribot Jose L. , Ramasubramanian Anand K.
Published: October 2013

The rise in the use of biomedical devices and implants has seen a concomitant surge in the advent of device‐related nosocomial (hospital‐acquired) infections of bacterial and fungal origins. The most common nosocomial fungal infection is candidiasis caused mainly by Candida albicans biofilms. Candidiasis is associated with an unacceptably high mortality rate, and there is an urgent need for the discovery of new antifungal drugs that prevent or control biofilm formation. To this end, we recently developed an ultra‐high‐throughput microarray platform consisting of nano‐scale biofilms of C. albicans encapsulated in collagen or alginate hydrogel matrices for antifungal drug screening. Here, we report that the choice of matrix influences the apparent susceptibility of C. albicans to the common antifungal drugs, amphotericin B, and caspofungin. While amphotericin B is equally effective against biofilms grown in collagen and alginate matrices, caspofungin is effective only against biofilms grown only in alginate, but not in collagen. We demonstrate differences in the distribution of the drugs in the two matrices may contribute to the susceptibility of C. albicans nano‐biofilms. In a larger context, our results highlight the importance of the choice of matrix as a parameter in 3D cell encapsulation, and suggest a screening strategy to predict drug performance in vivo. Biotechnol. Bioeng. 2014;111: 418–424. © 2013 Wiley Periodicals, Inc.
Combinatorial engineering of mevalonate pathway for improved amorpha‐4,11‐diene production in budding yeast
Authors: Yuan Jifeng , Ching Chi Bun
Published: October 2013

Combinatorial genome integration of mevalonate pathway genes was performed with the aim of optimizing the metabolic flux for improved production of terpenoids in budding yeast. In the present study, we developed a novel δ‐integration platform to achieve multiple genome integrations through modulating the concentration of antibiotics. By exploiting carotenoid biosynthesis as screening module, we successfully created a library of yeast colonies appeared with various intensities of orange color. As proof‐of‐concept that carotenoid overproducers could serve to boost the titer of other terpenoids, we further tested engineered strains for the production of amorpha‐4,11‐diene, an important precursor for antimalarial drug. However, we experienced some limitations of the carotenoid‐based screening approach as it was only effective in detecting a small range of pathway activity improvement and further increasing mevalonate pathway activity led to a decreased orange color. By far, we were only able to obtain one mutant strain yielded more than 13‐fold amorpha‐4,11‐diene over parental strains, which was approximately 64 mg/L of caryophyllene equivalents. Further qPCR studies confirmed that erg10, erg13, thmg1 and erg12 involved in mevalonate pathway were overexpressed in this mutant strain. We envision the current δ‐integration platform would form the basis of a generalized technique for multiple gene integrations in yeast—a method that would be of significant interest to the metabolic engineering community. Biotechnol. Bioeng. 2014;111: 608–617. © 2013 Wiley Periodicals, Inc.
Simultaneous method for analyzing dimerization and signaling of G‐protein‐coupled receptor in yeast by dual‐color reporter system
Authors: Nakamura Yasuyuki , Takemoto Norika , Ishii Jun , Kondo Akihiko
Published: October 2013

Understanding the role of G‐protein‐coupled receptor (GPCR) dimerization in cellular function has now become a major research focus. The potentially large functional and physiological diversity of dimerization among GPCRs is expected to provide opportunities for novel drug discovery. However, there is currently a lack of cell‐based assays capable of specific profiling for the functional consequences of dimerization linked to ligand‐mediated signaling. Here, we present an advanced method to simultaneously analyze the dimerization and ligand response of GPCRs using two yeast‐based systems for split‐ubiquitin two‐hybrid assay and G‐protein signaling assay. To permit simultaneous detection, we established a two‐color (dual‐color) fluorescence reporter gene assay using enhanced green fluorescent protein (EGFP) and a far‐red derivative of the tetrameric fluorescent protein DsRed‐Express2 (E2‐Crimson). In the present study, we tested our method first by analyzing dimerization and ligand‐mediated signaling by the yeast endogenous pheromone receptor (Ste2p). Second, we showed that the system facilitated mutational analysis of domains involved in dimerization and signaling by Ste2p. Third, we successfully demonstrated that the system could simultaneously monitor homo‐ and hetero‐dimerization and somatostatin‐induced signaling in the test case of the human SSTR5 somatostatin receptor. Our strategy is expected to provide a useful tool for the elucidation of molecular biological functions of GPCR dimers and for the screening of GPCR dimer‐specific agonistic ligands. Biotechnol. Bioeng. 2014;111: 586–596. © 2013 Wiley Periodicals, Inc.
Hydrocarbon production in high density Botryococcus braunii race B continuous culture
Authors: Khatri Waqas , Hendrix Robert , Niehaus Tom , Chappell Joe , Curtis Wayne R.
Published: October 2013

Continuous cultures of Botryococcus braunii race B were maintained at photosynthetic cell densities as high as 20 g dry weight per liter for up to 3 months. Growth associated triterpene hydrocarbon accumulation was nearly constant at 22.5% of dry weight for a range of growth rates maintained by daily replacement of 5–15% of the respective cultures. The ability to achieve high cell concentrations and oil levels of roughly 5 g triterpene oil/L resulted from a combination of high light (∼ 1/4 full sun for 15 h/day) and replenishing stoichiometrically balanced growth medium. Due to light‐limited growth conditions, cell concentration dropped nearly linearly with increased dilution rate. This reduction in cell number resulted in increased productivity per cell at higher dilution rates and was accompanied by a dramatic increase in algae colony size from 0.09 to 0.343 mm at high dilution rate. This change in colony size resulted in an equally dramatic change in optical density (OD) per gram dry weight, which precluded use of simple correlations of OD and cell concentration. A trickle‐film photobioreactor was also demonstrated as a scalable approach to achieving these ultra‐high cell concentrations. Additional media analysis revealed a steady increase in photobioreactor conductivity suggesting an accumulation of ions may be the reason for rapid culture crash and washout observed at all dilution rates after several months of continuous operation. The volumetric productivity of 22.5 mg oil/L/photo‐h reported here is more than an order of magnitude higher than previous reports for B. braunii race B, reflecting the high cell densities used in this work and substantiating a higher metabolic rate for B. braunii race B than previously surmised from its relatively long doubling times. Biotechnol. Bioeng. 2014;111: 493–503. © 2013 Wiley Periodicals, Inc.
Oxygen allows Shewanella oneidensis MR‐1 to overcome mediator washout in a continuously fed bioelectrochemical system
Authors: TerAvest Michaela A. , Rosenbaum Miriam A. , Kotloski Nicholas J. , Gralnick Jeffrey A. , Angenent Largus T.
Published: October 2013

Many bioelectrochemical systems (BESs) harness the ability of electrode‐active microbes to catalyze reactions between electrodes and chemicals, often to perform useful functions such as wastewater treatment, fuel production, and biosensing. A microbial fuel cell (MFC) is one type of BES, which generates electric power through microbial respiration with an anode as the electron acceptor, and typically with oxygen reduction at the cathode to provide the terminal electron acceptor. Oxygen intrusion into MFCs is typically viewed as detrimental because it competes with anodes for electrons and lowers the coulombic efficiency. However, recent evidence suggests that it does not necessarily lead to lower performances—particularly for the model organism Shewanella oneidensis MR‐1. Because flavin‐mediated electron transfer is important for Shewanella species, which can produce this electron shuttle endogenuously, we investigated the role of flavins in the performance of pure‐culture BESs with S. oneidensis MR‐1 with and without oxygen. We found that oxygen increases current production more than twofold under continuously fed conditions, but only modestly increases current production under batch‐fed conditions. We hypothesized that oxygen is more beneficial under continuously fed conditions because it allows S. oneidensis to grow and produce flavins at a faster rate, and thus lowers flavin washout. Our conclusions were supported by experiments with a flavin‐secretion deficient mutant of S. oneidensis. Biotechnol. Biotechnol. Bioeng. 2014;111: 692–699. © 2013 Wiley Periodicals, Inc.
Enhanced production of coenzyme Q10 by self‐regulating the engineered MEP pathway in Rhodobacter sphaeroides
Authors: Lu Wenqiang , Ye Lidan , Xu Haoming , Xie Wenping , Gu Jiali , Yu Hongwei
Published: October 2013

Fine‐tuning the expression level of an engineered pathway is crucial for the metabolic engineering of a host toward a desired phenotype. However, most engineered hosts suffer from nonfunctional protein expression, metabolic imbalance, cellular burden or toxicity from intermediates when an engineered pathway is first introduced, which can decrease production of the desired product. To circumvent these obstacles, we developed a self‐regulation system utilizing the trc/tac promoter, LacIq protein and ribosomal binding sites (RBS). With the purpose of improving coenzyme Q10 (CoQ10) production by increasing the decaprenyl diphosphate supplement, enzymes DXS, DXR, IDI, and IspD were constitutively overexpressed under the control of the trc promoter in Rhodobacter sphaeroides. Then, a self‐regulation system combining a set of RBSs for adjusting the expression of the LacIq protein was applied to tune the expression of the four genes, resulting in improved CoQ10 production. Finally, another copy of the tac promoter with the UbiG gene (involved in the ubiquinone pathway of CoQ10 biosynthesis) was introduced into the engineered pathway. By optimizing the expression level of both the upstream and downstream pathway, CoQ10 production in the mutants was improved up to 93.34 mg/L (7.16 mg/g DCW), about twofold of the wild‐type (48.25 mg/L, 3.24 mg/g DCW). Biotechnol. Bioeng. 2014;111: 761–769. © 2013 Wiley Periodicals, Inc.
Ultra‐high‐performance liquid chromatography–tandem mass spectrometry for pharmacokinetics of bakuchiol in rats
Authors: Zhang Shuang‐Qing , Fan Yu‐Ming
Published: October 2013

Psoralea Corylifolia L. is a traditional Chinese medicine with many beneficial effects in medical therapies. Bakuchiol was the main active ingredient of Psoralea Corylifolia L., used for the treatment of various diseases and also as a natural food additive. A specific and reliable ultra‐high performance liquid chromatography–tandem mass spectrometry has been developed and fully validated for the quantification of bakuchiol in rat plasma. Chromatographic separation of bakuchiol and an internal standard, daidzein, was achieved on a Hypersil Gold C18 column with gradient elution that consisted of methanol and water at a flow rate of 0.2 mL/min. The compounds were detected at negative ionization mode using mass transition m/z 255.2 → 172.0 and 252.9 → 132.0 for bakuchiol and daidzein, respectively. Good linearity was obtained over the range of 2–1000 ng/mL and the lower limit of quantification was 2 ng/mL. The intra‐ and inter‐day accuracies ranged from 91.1 to 105.7% and precisions (relative standard deviations) were within 9.3%. Bakuchiol was found to be stable under three freeze–thaw cycles, short‐term temperature, post‐preparative and long‐term temperature conditions. The method was applied to a pharmacokinetic study of bakuchiol intravenously administered to rats at a dose of 5 mg/kg. Copyright © 2013 John Wiley & Sons, Ltd.
Studies on excretion kinetics of ten constituents in rat urine after oral administration of Shensong Yangxin Capsule by UPLC‐MS/MS
Authors: Liu Minyan , Li Song , Zhao Shaohua , Wang Hongtao , Wang Hairong , Tu Pengfei
Published: October 2013

A rapid and sensitive ultra‐high performance liquid chromatography–mass spectrometry (UPLC‐MS/MS) method was developed and validated for the quantification of 10 major active constituents in rat urine after oral administration of Shensong Yangxin Capsule (SSYX) using diazepam as an internal standard (IS). The urine samples were pretreated and extracted by solid‐phase extraction prior to UPLC. Chromatographic separation was achieved on a Waters C18 (2.1 × 50 mm, 1.7 µm) column using a gradient elution program with 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.4 mL/min. Detection and quantitation were accomplished by a hybrid quadrupole mass spectrometer using electrospray ionization source and multiple reaction monitoring in the positive ionization mode. The mass transition ion‐pairs (m/z) for quantitation were all optimized and the total run time was 4.50 min. The specificity, linearity, accuracy, precision, recovery, matrix effect and stabilities were all validated for the analytes in urine samples. The validation results indicated that this method was simple, rapid, specific and reliable. The proposed method was successfully applied to investigate the urinary excretion kinetics of 10 compounds in rat after oral administration of SSYX. Copyright © 2013 John Wiley & Sons, Ltd.
Out of the Cold – the Rising Importance of Environmental Management in the Corporate Governance of Russian Oil and Gas Producers
Authors: Thurner Thomas , Proskuryakova Liliana Nikolaevna
Published: October 2013

Oil and gas producers play a pivotal role in the Russian economy. The industry itself is very energy consuming and capable of producing major environmental damage. For a long time, however, innovative ways of production were low on their priority list, leading to heavily outdated equipment and hardware. Recently, Russian oil‐ and gas producers have kicked off major programs to catch up with their western counterparts. Until now, however, insights into the importance of environmental management of oil and gas producers are very limited. We studied the annual reports and environmental reports of the six most prominent industry actors between 2008 and 2010 and analyze their changing approaches to green management. Most companies start in 2009 to address their environmental activities in the statement of the CEO and chairmen. In 2010, the environmental activities have made it up to the list of top priorities in the companies. On an industry‐level, we find that management's own initiatives drive companies' adoption of greener production technologies and are much more influential than government regulation. In fact, we see that leading industry actors inform government regulation and thereby lift up the greening of production also in late followers. Green innovation either stems from international cooperation for industry leaders or out of in‐house knowledge generated through own R&D institutes. Copyright © 2013 John Wiley & Sons, Ltd and ERP Environment.
The Integration of Quality, Environmental and Health and Safety Management by Car Manufacturers – a Long‐Term Empirical Study
Authors: von Ahsen Anette
Published: October 2013

The diversity of stakeholder requirements is currently a pivotal challenge for companies. Stakeholders expect companies to increasingly consider environmental and social aspects in their decisions. Thus, corporate goal systems are including not only qualitative and financial goals, but also environmental and social ones. Management systems for ensuring quality, environmental and occupational safety play an important role in achieving these objectives. Considering the interdependency between the different systems constitutes a central challenge. Much of the literature and the results from empirical studies suggest that the spatial application of integrated management systems is state of the art. Integrated management systems in particular are regarded as more effective and more efficient than separate and distinct management systems. However, the present long‐term study demonstrates that for car manufacturers in Germany these integration efforts are implemented in very different ways, and that some integration approaches are partially abandoned after a period of unsatisfactory implementation. Copyright © 2013 John Wiley & Sons, Ltd and ERP Environment
Using Social Cognitive Theory to Investigate Green Consumer Behavior
Authors: Lin Hsiu‐Yi , Hsu Meng‐Hsiang
Published: October 2013

Green consumer behavior can induce industries to develop green production methods and convert wasteful patterns of consumption into green consumption patterns. This study demonstrates that the practice of green consumption relies on the self‐sanction concept in individuals. The objective of this study was to develop a research model of green consumer behavior based on social cognitive theory. The results show that personal self‐concepts (the most significant of which is green consumption self‐efficacy), personal outcome expectation and social sanction all have a significant influence. On the other hand, the influences of the expected outcome of green consumer behavior, climate change and the mass media were not significant. Copyright © 2015 John Wiley & Sons, Ltd and ERP Environment.
A Comparison of Insufficient Effort Rates, Neuropsychological Functioning, and Neuropsychiatric Symptom Reporting in Military Veterans and Civilians with Chronic Traumatic Brain Injury
Authors: Gfeller Jeffrey D. , Roskos P. Tyler
Published: October 2013

Neuropsychological evaluation of persons with chronic traumatic brain injury (TBI) symptoms is complicated by multiple factors. The authors explored the impact of mechanism of injury, effort testing performance, and neuropsychiatric status in a sample of military veterans (V‐TBI) and civilians (C‐TBI) with chronic TBI. V‐TBI (n = 74), C‐TBI (n = 67), and healthy civilian control (C‐HC) participants (n = 66), completed a battery of neuropsychological, effort, and self‐report neuropsychiatric measures. Results indicated that C‐HC and C‐TBI participants exhibited comparably low failure rates on effort tests (6% and 3%, respectively). V‐TBI participants exhibited significantly higher rates of failure (18%). Subgroups (n = 20) of effort‐screened participants matched for demographics and disability level were compared regarding neuropsychological performance and neuropsychiatric self‐report. Both TBI groups exhibited limited neuropsychological impairment, relative to the C‐HC participants. The V‐TBI group exhibited pronounced neuropsychiatric symptomology compared with the other participant groups. The implications of these findings are discussed for evaluation in the context of disability and litigation. Copyright © 2013 John Wiley & Sons, Ltd.
Urea enhances cell growth and pyruvate production in Torulopsis glabrata
Authors: Yang Songxin , Chen Xiulai , Xu Nan , Liu Liming , Chen Jian
Published: October 2013

Torulopsis glabrata is a strain of yeast that is used for the industrial production of pyruvate. Determination of the optimal nutrient environment is vital for obtaining the most efficient production system. In this study, the fermentation parameters, gene transcription levels, activities of key enzymes and metabolites levels were analyzed when either urea or ammonium chloride was used as the sole source of nitrogen. Urea caused an increase in the dry cell weight (18%) and pyruvate productivity was significantly increased (14%). The transcription levels of CAGL0M05533g (DUR1,2), CAGL0J07612g (ZWF1), and CAGL0I02200g (SOL3) were upregulated, but CAGL0G05698g (GDH2) and CAGL0L01089g (GLT1) were down‐regulated. The activities of urea amidolyase, NADPH dependent glutamate dehydrogenase and glucose‐6‐phosphate dehydrogenase were increased by 380, 430, and 140%, respectively. The activities of arginase and glutamate synthase were decreased by 40 and 35%, respectively. The NADPH content was increased by 33%, whilst ATP content was decreased by 37%. This changed the intracellular levels of organic acids and amino acids. The results expand the understanding of the physiological characteristics of yeast species grown with different sources of nitrogen. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:19–27, 2014
Design of experiments applications in bioprocessing: Concepts and approach
Authors: Kumar Vijesh , Bhalla Akriti , Rathore Anurag S.
Published: October 2013

Most biotechnology unit operations are complex in nature with numerous process variables, feed material attributes, and raw material attributes that can have significant impact on the performance of the process. Design of experiments (DOE)‐based approach offers a solution to this conundrum and allows for an efficient estimation of the main effects and the interactions with minimal number of experiments. Numerous publications illustrate application of DOE towards development of different bioprocessing unit operations. However, a systematic approach for evaluation of the different DOE designs and for choosing the optimal design for a given application has not been published yet. Through this work we have compared the I‐optimal and D‐optimal designs to the commonly used central composite and Box–Behnken designs for bioprocess applications. A systematic methodology is proposed for construction of the model and for precise prediction of the responses for the three case studies involving some of the commonly used unit operations in downstream processing. Use of Akaike information criterion for model selection has been examined and found to be suitable for the applications under consideration. © 2013 American Institute of Chemical EngineersBiotechnol. Prog., 30:86–99, 2014
Optimization of five environmental factors to increase beta‐propeller phytase production in Pichia pastoris and impact on the physiological response of the host
Authors: Viader‐Salvadó José M. , Castillo‐Galván Miguel , Fuentes‐Garibay José A. , Iracheta‐Cárdenas María M. , Guerrero‐Olazarán Martha
Published: October 2013

Recently, we engineered Pichia pastoris Muts strains to produce several beta‐propeller phytases, one from Bacillus subtilis and the others designed by a structure‐guided consensus approach. Furthermore, we demonstrated the ability of P. pastoris to produce and secrete these phytases in an active form in shake‐flask cultures. In the present work, we used a design of experiments strategy (Simplex optimization method) to optimize five environmental factors that define the culture conditions in the induction step to increase beta‐propeller phytase production in P. pastoris bioreactor cultures. With the optimization process, up to 347,682 U (82,814 U/L or 6.4 g/L culture medium) of phytase at 68 h of induction was achieved. In addition, the impact of the optimization process on the physiological response of the host was evaluated. The results indicate that the increase in extracellular phytase production through the optimization process was correlated with an increase in metabolic activity of P. pastoris, shown by an increase in oxygen demand and methanol consumption, that increase the specific growth rate. The increase in extracellular phytase production also occurred with a decrease in extracellular protease activity. Moreover, the optimized culture conditions increased the recombinant protein secretion by up to 88%, along with the extracellular phytase production efficiency per cell. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1377–1385, 2013
Production of cellulase‐free xylanase by the recombinant Bacillus subtilis and its applicability in paper pulp bleaching
Authors: Verma Digvijay , Satyanarayana T.
Published: October 2013

A metagenomic xylanase gene (Mxyl) was successfully cloned into shuttle vector pWH1520 and expressed in Bacillus subtilis extracellularly. On induction with xylose, recombinant xylanase secretion commenced after 6 h. Identifying critical variables for recombinant xylanase production by one‐variable‐at‐time approach followed by optimization of the selected variables (xylose, inoculum density, incubation density) by response surface methodology (RSM) led to three‐fold enhancement in extracellular xylanase production (119 U mL−1). When the pulp was treated with recombinant xylanase at 80°C and pH 9.0, kappa number of the pulp was reduced with concomitant increase in brightness and 24% reduction in chlorine consumption. This is the first report on the expression of metagenomic xylanase gene in Bacillus subtilis extracellularly and its utility in developing an environment‐friendly pulp bleaching process. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1441–1447, 2013
Kinetic characterization of galacto‐oligosaccharide (GOS) synthesis by three commercially important β‐galactosidases
Authors: Warmerdam Anja , Zisopoulos Filippos K. , Boom Remko M. , Janssen Anja E. M.
Published: October 2013

Many β‐galactosidases show large differences in galacto‐oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various β‐galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the β‐galactosidase of Bacillus circulans than the β‐galactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the β‐galactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why β‐galactosidase of B. circulans gives higher yields in GOS production than other β‐galactosidases. Many of the reaction rate constants for the β‐galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform β‐gal‐A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:38–47, 2014
MALDI‐TOF characterization of hGH1 produced by hairy root cultures of Brassica oleracea var. italica grown in an airlift with mesh bioreactor
Authors: López Edgar García , Ramírez Emma Gloria Ramos , Gúzman Octavio Gómez , Calva Graciano Calva , Ariza‐Castolo Armando , Pérez‐Vargas Josefina , Rodríguez Herminia Guadalupe Martínez
Published: October 2013

Expression systems based on plant cells, tissue, and organ cultures have been investigated as an alternative for production of human therapeutic proteins in bioreactors. In this work, hairy root cultures of Brassica oleracea var. italica (broccoli) were established in an airlift with mesh bioreactor to produce isoform 1 of the human growth hormone (hGH1) as a model therapeutic protein. The hGH1 cDNA was cloned into the pCAMBIA1105.1 binary vector to induce hairy roots in hypocotyls of broccoli plantlets via Agrobacterium rhizogenes. Most of the infected plantlets (90%) developed hairy roots when inoculated before the appearance of true leaves, and keeping the emerging roots attached to hypocotyl explants during transfer to solid Schenk and Hildebrandt medium. The incorporation of the cDNA into the hairy root genome was confirmed by PCR amplification from genomic DNA. The expression and structure of the transgenic hGH1 was assessed by ELISA, western blot, and MALDITOF‐MS analysis of the purified protein extracted from the biomass of hairy roots cultivated in bioreactor for 24 days. Production of hGH1 was 5.1 ± 0.42 µg/g dry weight (DW) for flask cultures, and 7.8 ± 0.3 µg/g DW for bioreactor, with productivity of 0.68 ± 0.05 and 1.5 ± 0.06 µg/g DW*days, respectively, indicating that the production of hGH1 was not affected by the growth rate, but might be affected by the culture system. These results demonstrate that hairy root cultures of broccoli have potential as an alternative expression system for production of hGH1, and might also be useful for production of other therapeutic proteins. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:161–171, 2014
Changes of oral microcirculation in chemotherapy patients: A possible correlation with mucositis?
Authors: Scardina Giuseppe A. , Cacioppo Antonino , Messina Pietro
Published: October 2013

The aim of this investigation is to appraise labial oral microcirculation in chemotherapy patients to clarify the effects of cytotoxic agents on oral microvessels. Twenty‐five patients with diagnosis of head and neck tumors were recruited in the study. All the patients were submitted to chemotherapy. Labial oral microcirculation was evaluated on labial mucosa using oral videocapillaroscopy. The statistical significance was checked with the Mann–Whitney U‐test (P < 0.05). The analysis of videocapillaroscopic patterns showed statistically significant variations relative to the diameter of the incoming loop; the diameter of the outgoing loop; and loop tortuosity. This study shows that capillary alterations to patients who receive chemotherapy occur in labial oral microcirculation. Clin. Anat. 27:417–422, 2014. © 2013 Wiley Periodicals, Inc.
Software engineering education for a graduate course: A web‐based tool for conducting process improvement initiatives with local industry collaboration
Authors: Garcia I. A. , Calvo‐Manzano J. A. , Pacheco C. L. , Perez C. A.
Published: October 2013

In recent years, there has been an on‐going demand for better services and functionality in software products; as a consequence many models, techniques, and tools have been developed such as CMMI‐DEV v1.2, TSP, or Scrum. However, software products still suffer from excessive costs, delays in delivery, and low quality. Furthermore, there is a lack of educational material providing high levels of interaction between students and the software industry to learn about how enterprises adopt these models, techniques, and tools into their daily work. This article describes a web‐based Tool (EduSysProVAL) to support a graduate course in collaboration with the local software industry. The main goal of this research is to demonstrate that a Software Engineering course may use the EduSysProVAL tool to improve students' practical and professional skills, thus increasing their participation and effort in improvement initiatives, in comparison to traditional educational approaches which are only based on theory classes. This research uses a four‐category questionnaire and follow‐up interviews to evaluate a satisfactory level of tool effectiveness with undergraduate students and summarizes the industry's positive perceptions about its contribution to the course. © 2013 Wiley Periodicals, Inc. Comput Appl Eng Educ 23:117–136, 2015; View this article online at wileyonlinelibrary.com/journal/cae; DOI 10.1002/cae.21584
The Use of the Child Abuse Potential Inventory in the Assessment of Parents involved in Care Proceedings
Authors: Laulik Sarah , Allam Jayne , Browne Kevin
Published: October 2013

Assessing for potential physical abuse is a fundamental task for those professionals undertaking assessments of parents involved in care proceedings. One tool developed to help assist in this endeavour is the Child Abuse Potential (CAP) Inventory (Milner, , ). The CAP Inventory provides an estimate of parental risk of child abuse and is one of the most widely used psychometric tools by psychologists in child protection settings. This review evaluates the psychometric properties of this measure and provides an overview of its potential uses, as well as its limitations. It concludes that there is evidence to support the validity and reliability of the CAP Inventory for use in care proceedings. Specifically, the review indicates that the CAP Inventory has good levels of reliability and validity (construct, content and concurrent). The only aspect of validity for which there is a lack of evidence is that of predictive validity and this is due to a dearth of prospective studies. Given the above, the practical, clinical and ethical implications for the use of the CAP Inventory in the assessment of parents involved in care proceedings are discussed. Copyright © 2013 John Wiley & Sons, Ltd.

‘There is evidence to support the validity and reliability of the CAP Inventory for use in care proceedings’
Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation
Authors: Roloff Alexander , Seitz Oliver
Published: October 2013

Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA‐triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid‐programmed ligation reactions. A DNA‐triggered ligation–cyclization sequence (“cycligation”) of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA‐thioesters and isocysteinyl‐PNA‐cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product‐template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur‐halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2–3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25–0.01 equiv). We believe that cyclization of products from DNA‐templated reactions could ultimately afford systems that completely overcome product inhibition.
Fibronectin promotes migration and invasion of ovarian cancer cells through up‐regulation of FAK–PI3K/Akt pathway
Authors: Yousif Nasser Ghaly
Published: October 2013

Ovarian cancer is the leading cause of death from gynecological malignancy, and the fourth most common cause of cancer death among American women. This study investigates the mechanism of fibronectin (FN) in stimulating ovarian cancer cell migration and invasion through up‐regulation of focal adhesion kinase (FAK) pathway. Human ovarian cancer cells (OVCAR‐3, A2780/CP70) were cultured and treated with fibronectin (10 µg/mL). Trans‐well plates were used to conduct the migration assay, real‐time RT‐PCR for FAK mRNA expression, and FAK siRNA for blocking FAK expression. Western blots were used for P‐FAK, P‐PI3K, and P‐Akt analysis. Fibronectin‐treated OVCAR‐3, A2780/CP70 cells have increased ability to migrate and invade. It significantly promoted this behavior through the phosphorylation of FAK. The cell displayed significantly increased signaling regulation of the FAK pathway (p‐PI3K/P‐Akt). Furthermore, siRNA FAK‐treated cells had reduced the levels of p‐PI3K/P‐Akt after induced by fibronectin. Our results indicate that FAK inhibition can suppress ovarian cancer cells migration and invasion through inhibiting downstream signaling (PI3K/AKT), which might be a therapeutic target or biomarker for ovarian cancer.
Comparison of different methods for the isolation of mesenchymal stem cells from umbilical cord matrix: Proliferation and multilineage differentiation as compared to mesenchymal stem cells from umbilical cord blood and bone marrow
Authors: Hua Jie , Gong Jian , Meng Hongbo , Xu Bin , Yao Le , Qian Mingping , He Zhigang , Zou Shaowu , Zhou Bo , Song Zhenshun
Published: October 2013

We have identified the most appropriate method of isolating human umbilical cord matrix‐derived mesenchymal stem cells (UCM‐MSCs) and compared morphological, phenotypic, proliferative and differentiation characteristics of UCM‐MSCs with bone marrow‐derived MSCs (BM‐MSCs) and umbilical cord blood‐derived MSCs (UCB‐MSCs). Three explant culture methods and three enzymatic methods were compared with regards to time for primary culture, cell number, cell morphology, immune phenotype and differentiation potential. Morphological, phenotypic, proliferative and differentiation characteristics of UCM‐MSCs, BM‐MSCs and UCB‐MSCs were also compared. UCM‐MSCs isolated using the 10 mm size tissue explant method led to shorter primary culture time, higher numbers of isolated cells and higher proliferation rates compared with other isolation methods. Immune phenotype and multilineage differentiation capacity did not differ significantly among six groups. UCM‐MSCs had similar characteristics as BM‐MSCs and UCB‐MSCs, including fibroblastic morphology, typical immunophenotypic markers and multilineage differentiation capacity. In comparison with UCB‐MSCs and BM‐MSCs, UCM‐MSCs have higher proliferative capacity, higher rate of chondrogenic differentiation, and higher expression of CD 146. The results suggest that the 10 mm size tissue culture method is the optimal protocol for the isolation of UCM‐MSCs. Given the distinct advantages of UC, such as accessibility, painless acquisition and abundance of cells obtained, we propose that UC be considered an alternative to BM and UCB as a source of MSCs for cell therapy.
Retrograde aortic valve crossing of the corevalve prosthesis using the buddy balloon technique
Authors: Noble Stephane , Roffi Marco
Published: October 2013

In rare cases, retrograde aortic valve crossing during transcatheter aortic valve implantation may be challenging despite improvements in delivery catheter profile, size, and steerability compared with the first generation devices. Herein, we report a case of challenging transfemoral Medtronic CoreValve placement that was possible, thanks to the buddy balloon technique, using a peripheral 6‐F compatible balloon. © 2013 Wiley Periodicals, Inc.
UK multicenter experience using the gore septal occluder (GSOTM) for atrial septal defect closure in children and adults
Authors: Smith Ben , Thomson John , Crossland David , Spence Mark S. , Morgan Gareth J.
Published: October 2013

Background

Percutaneous closure of atrial defects (ASD) has evolved as the treatment of choice for the majority of defects and patent oval foramens. The Gore Septal Occluder (GSO) is an innovative device consisting mostly of a folded thin GoreTex tube for use in the closure of septal defects.

Methods

Reviewed is the multicenter UK experience of the first 22 ASD occlusions with the GSOTM device. All implantations were performed by consultant operators experienced in ASD device closure. The inclusion criterion was the presence of a hemodynamically significant secundum ASD with a diameter of <18 mm. Procedural data and acute and mid‐term closure rates were retrospectively matched to a cohort of patients having defect closure using the Amplatzer Septal OccluderTM (ASOTM).

Results

Acute and 3‐month follow‐up closure rates for the GSOTM were 100% and 100% vs. 100% and 100% closure with the ASOTM implants. The difference in paired procedure times was not statistically significant (56 min: GSOTM; 42 min: ASOTM device P = ns), nor was the paired difference in fluoroscopic screening times (12 min: GSOTM vs. 8.4 min: ASOTM, P = ns). One GSO device embolized immediately after deployment and was successfully retrieved at the same procedure. There were no other significant complications in either group.

Conclusions

The GSOTM implant can achieve comparable closure rates to the ASOTM in small to moderate atrial septal defects after 3 months. Longer fluoroscopy and procedure times are a drawback; however these should improve with familiarity with the implant and deployment system. The larger sheath size was not associated with increased complications in our cohort. © 2013 Wiley Periodicals, Inc.
Lower extremity mass
Authors: Saurabh Shireesh , Singh Namrata
Published: October 2013

Key Clinical Message

Soft tissue sarcomas are rare tumors accounting for less than 1% of all malignancies. Multidisciplinary approach is used in the management of these patients.
Type II Flavin‐Containing Monooxygenases: A New Class of Biocatalysts that Harbors Baeyer–Villiger Monooxygenases with a Relaxed Coenzyme Specificity
Authors: Riebel Anette , Fink Michael J. , Mihovilovic Marko D. , Fraaije Marco W.
Published: October 2013

Within a newly identified set of flavin‐containing monooxygenases (FMOs) from Rhodococcus jostii RHA1, we have identified three monooxygenases (FMO‐E, FMO‐F, and FMO‐G) that are effective in catalyzing Baeyer–Villiger oxidations. These type II FMOs display relaxed coenzyme specificity by accepting both NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) and NADH (reduced form of nicotinamide adenine dinucleotide), as a coenzyme, which is a novel and attractive feature among biocatalysts capable of conducting Baeyer–Villiger oxidations. We purified FMO‐E and determined that the Michaelis constants for both coenzymes were in the micromolar range, whereas the activity was highest for NADH. By using the stopped‐flow technique, formation of a peroxyflavin–enzyme intermediate was observed, which indicated that type II FMOs follow a catalytic mechanism similar to that of other class B flavoprotein monooxygenases. A set of cyclobutanones and cyclohexanones were used to probe the regio‐ and enantioselectivity of all three recombinant monooxygenases. The biocatalysts readily accepted small cyclic ketones, which enabled the conversion of previously poorly accepted substrates by other monooxygenases (especially norcamphor), and exhibited excellent and unique regio‐ and enantioselectivities. Sequence analysis revealed that type II FMOs that act as Baeyer–Villiger monooxygenases contain a unique N‐terminal domain. Sequence conservation in this protein domain can be used to identify new NADH‐dependent Baeyer–Villiger monooxygenases, which would facilitate future biocatalyst discovery efforts.