bluuebunny/crossref_metadata_2025_split huggingface dataset:
Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology
Authors: Kennard Malcolm L. , Goosney Danika L. , Monteith Diane , Roe Susanna , Fischer David , Mott John 
Published: May 2009

AbstractIn order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre‐engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO‐S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO‐S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO‐S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO‐S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression. Biotechnol. Bioeng. 2009; 104: 526–539 © 2009 Wiley Periodicals, Inc.
Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices
Authors: Sung Jong Hwan , Choi Jong‐ryul , Kim Donghyun , Shuler Michael L. 
Published: May 2009

AbstractWe describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in MatrigelTM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in MatrigelTM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525 © 2009 Wiley Periodicals, Inc.
Interpreting Experimental Data by Using Molecular Simulation Instead of Model Building
Authors: Gattin Zrinka , Schwartz Julian , Mathad Raveendra I. , Jaun Bernhard , van Gunsteren Wilfred F. 
Published: May 2009

AbstractA proper description of the conformational equilibrium of polypeptides or proteins is essential for a correct description of their function. The conformational ensembles from 16 molecular dynamic simulations of two β‐ heptapeptides were used to interpret the primary NMR data, which were also compared to a set of NMR model structures (see graphic).magnified imageOne of the most used spectroscopic techniques for resolving the structure of a biomolecule, such as a protein or peptide, is NMR spectroscopy. Because only NMR signal intensities and frequencies are measured in the experiment, a conformational interpretation of the primary data, that is, measured data, is not straightforward, especially for flexible molecules. It is hampered by the occurrence of conformational and/or time‐averaging, by insufficient number of experimental data and by insufficient accuracy of experimental data. All three problematic aspects of structure refinement based on NMR nuclear Overhauser effect (NOE) intensities and 3J coupling data are illustrated by using two β‐heptapeptides in methanol as an example. We have performed 16 molecular dynamics (MD) simulations between 20 to 100 ns in length of unrestrained and NOE distance‐restrained cases (instantaneous and time‐averaged) of two β‐heptapeptides with a central β‐HAla(α‐OH) amino acid in methanol at two different temperatures using two different GROMOS force‐field parameter sets, 45 A3 and 53 A6. The created conformational ensembles were used to interpret the primary NMR data on these molecules. They also were compared to a set of NMR model structures derived by single‐structure refinement in vacuo by using standard techniques. It is shown that the conformational interpretation of measured experimental data can be significantly improved by using unrestrained, instantaneous and time‐averaged restrained MD simulations of the peptides by using a thermodynamically calibrated force field and by explicitly including solvent degrees of freedom.
The Steric Nature of the Bite Angle
Authors: van Zeist Willem‐Jan , Visser Ruud , Bickelhaupt F. Matthias 
Published: May 2009

AbstractTwisted! We show, based on DFT analyses, that the well‐known dependence of a catalyst's activity in bond activation on the ligand–metal–ligand angle, or bite angle, has a primarily steric origin, not an electronic one. Thus, by bending or twisting the catalyst a priori, one avoids unfavorable nonbonded interactions with the substrate (see figure).magnified image
A Multicomponent Reaction Towards N‐(Cyanomethyl)amides
Authors: Elders Niels , Ruijter Eelco , de Kanter Frans J. J. , Janssen Elwin , Lutz Martin , Spek Anthony L. , Orru Romano V. A. 
Published: May 2009

AbstractDiversity‐oriented synthesis: The multicomponent reaction of α‐isocyano amides, aldehydes or ketones, and amines affords N‐(cyanomethyl)amides, presenting the fourth class of products from this combination of reagents (see scheme). The scope of the reaction is very broad and various functional groups are tolerated. The outcome of the reaction can also be directed to the formation of 2H‐2‐imidazolines by AgI catalysis.magnified image
Automated quantification of DNA demethylation effects in cells via 3D mapping of nuclear signatures and population homogeneity assessment
Authors: Gertych Arkadiusz , Wawrowsky Kolja A. , Lindsley Erik , Vishnevsky Eugene , Farkas Daniel L. , Tajbakhsh Jian 
Published: May 2009

AbstractToday's advanced microscopic imaging applies to the preclinical stages of drug discovery that employ high‐throughput and high‐content three‐dimensional (3D) analysis of cells to more efficiently screen candidate compounds. Drug efficacy can be assessed by measuring response homogeneity to treatment within a cell population. In this study, topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti‐cancer agents: 5‐azacytidine (5‐AZA) and octreotide (OCT). Mouse pituitary folliculostellate TtT‐GF cells treated with 5‐AZA and OCT for 48 hours, and untreated populations, were studied by immunofluorescence with a specific antibody against 5‐methylcytosine (MeC), and 4,6‐diamidino‐2‐phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei (n = 163). Cell images were processed utilizing an automated 3D analysis software that we developed by combining seeded watershed segmentation to extract nuclear shells with measurements of Kullback‐Leibler's (K‐L) divergence to analyze cell population homogeneity in the relative nuclear distribution patterns of MeC versus DAPI stained sites. Each cell was assigned to one of the four classes:similar,likely similar,unlikely similar, anddissimilar. Evaluation of the different cell groups revealed a significantly higher number of cells with similar or likely similar MeC/DAPI patterns among untreated cells (approximately 100%), 5‐AZA‐treated cells (90%), and a lower degree of same type of cells (64%) in the OCT‐treated population. The latter group contained (28%) of unlikely similar or dissimilar (7%) cells. Our approach was successful in the assessment of cellular behavior relevant to the biological impact of the applied drugs, i.e., the reorganization of MeC/DAPI distribution by demethylation. In a comparison with other metrics, K‐L divergence has proven to be a more valuable and robust tool for categorization of individual cells within a population, with potential applications in epigenetic drug screening. © 2009 International Society for Advancement of Cytometry
BMI, body composition, and the energy requirement for body weight gain in patients with anorexia nervosa
Authors: Yamashita Sakino , Kawai Keisuke , Yamanaka Takeharu , Inoo Takehiro , Yokoyama Hiroaki , Morita Chihiro , Takii Masato , Kubo Chiharu 
Published: May 2009

AbstractObjectiveTheories abound about the energy requirements for body weight gain in anorexia nervosa (AN). We hypothesized that malnutrition status affects the energy requirements.MethodOn admission, 97 AN patients were measured for body composition by dual‐energy X‐ray absorptiometry. The relationship between body mass index (BMI) and body composition was investigated. In addition, 21 patients who completed our treatment program were tested for energy intake and body weight.ResultsThe relationship between BMI and both fat‐free mass and fat mass (FM) on admission was curvilinear. The weight gain per excess energy was greater in the group of patients with FM < 4 kg or BMI < 14 kg/m2 than in the group with FM ≥ 4 kg or BMI ≥ 14 kg/m2 (p = .037, p = .055, respectively).DiscussionThe energy requirements for weight gain in AN patients are related to the initial FM and BMI. © 2009 by Wiley Periodicals, Inc. Int J Eat Disord 2010
Palladium‐Catalysed Cross‐Coupling and Related Processes: Some Interesting Observations That Have Been Exploited in Synthetic Chemistry
Authors: McGlacken Gerard P. , Fairlamb Ian J. S. 
Published: May 2009

AbstractPalladium‐catalysed cross‐coupling reactions are routinely used in the synthesis of important organic and organometallic compounds, including advanced materials and natural products. Industry and academia depend heavily on these ubiquitous transformations. Their success is quite remarkable, but not without their share of problematic and sometimes unusual observations, which continue to probe our fundamental understanding of these reactions. Through an identification of side‐reactions, our train of thought of a well‐known reaction can be challenged. In this review we identify exemplary processes and show how they have been exploited in synthetic chemistry. A deeper insight into well‐established processes is provided, which alerts us to new synthetic transformations. On one hand, it is of significant interest to understand how to minimise unwanted reactions and their side‐products, but on the other, one can seek to exploit such reaction pathways and develop new synthetic methodologies. Either way, synthetic chemists only stand to benefit from such findings, which, more often than not, stimulate further research into this ever‐expanding field. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)
Value of fine‐needle aspiration biopsy of salivary gland lesions
Authors: Christensen Rikke Kølby , Bjørndal Kristine , Godballe Christian , Krogdahl Annelise 
Published: May 2009

AbstractBackground.The aim of this study was to assess the utility of fine‐needle aspiration biopsy (FNAB) in the diagnosis and treatment planning of the lesions of the salivary gland.Methods.Eight hundred seventy‐nine aspiration biopsies of the lesion of the salivary gland over a 10‐year period, from 1997 to 2006, were reviewed with special reference to its value in the clinical treatment of patients.Results.Cytologic as well as histologic diagnoses of 382 patients were available. In these diagnoses, the sensitivity of malignancy was 83% and specificity was 99%. The positive predictive value was 98%, and the negative predictive value was 97%. The overall accuracy was 93%. The correct subtyping of the benign lesions was 97%, and the exact type‐specific concordance of the malignant lesions was 71%.Conclusion.Considerable benefit to the patient may result from the cautious use of FNAB of lesions of the salivary gland. The close co‐operation between pathologist and surgeon can improve individual treatment. © 2009 Wiley Periodicals, Inc. Head Neck, 2010
Capital gains: expatriate adjustment and the psychological contract in international careers
Authors: Haslberger Arno , Brewster Chris 
Published: May 2009

AbstractThis paper argues that the notion of adjustment to careers involving international assignments needs to be developed further than the current literature reflects. An expatriate assignment is an expatriate's opportunity to build career capital and a company's opportunity to generate social and intellectual capital. The extent of the capital gains will depend considerably on the expatriate's adjustment during and after the assignment, which is influenced by the psychological contract. We argue that our understanding of the career impact of expatriation will be enhanced by a more refined picture of the adjustment that expatriates experience during the assignment and during repatriation. In particular, we examine adjustment as process rather than as event. We propose a broad conception of expatriate adjustment and its link to careers. © 2009 Wiley Periodicals, Inc.
Dealing with rough times: a capabilities development approach to surviving and thriving
Authors: Mohrman Susan Albers , Worley Christopher G. 
Published: May 2009

AbstractLeaders during a deep and prolonged downturn should introduce organizational practices that build capability in the organization, not only to withstand the uncertainties of rough times better, but also to emerge stronger for the future. We present a set of organization design changes that can create closer connections to the marketplace and better use knowledge in the organization to introduce new ways to deliver value while consuming fewer resources. During rapid change and extreme threats, steering the ship is not sufficient. Leaders must introduce approaches that tap employees' energies throughout the organization. © 2009 Wiley Periodicals, Inc.
Downregulation of p21‐activated kinase‐1 inhibits the growth of gastric cancer cells involving cyclin B1
Authors: Liu Funan , Li Xiaodong , Wang Chunyu , Cai Xinze , Du Zhongmin , Xu Huimian , Li Feng 
Published: May 2009

Abstractp21‐activated kinase 1 (Pak1), a serine/threonine kinase, has been implicated in cytoskeletal remodelling, cell motility, apoptosis and transformation. However, the role of Pak1 in gastric cancer remains unclear. In this study, we detected Pak1 expression in gastric cancer tissues from 40 patients by western blot. Overexpression of Pak1 was associated with progression, metastasis and prognosis of gastric cancer. In addition, we found that knockdown of Pak1 expression significantly inhibited anchorage‐dependent and anchorage‐independent growth in gastric cancer cells, and markedly inhibited gastric cancer cell xenograft tumor growth. In further study, data showed that Pak1 could regulate the expression of cyclin B1 at the mRNA and protein levels, and impact the subcellular distribution and the promoter activity of cyclin B1. Results from deletion and mutant analysis supplied a new NF‐κB binding sites at position ‐321 of cyclin B1 promoter, and indicated that Pak1 regulated the transcription of cyclin B1 in gastric cancer through NF‐κB. In conclusion, Pak1 may be a potential prognostic marker and therapeutic target in gastric cancer. © 2009 UICC
Quantitative expression and immunogenicity of MAGE‐3 and ‐6 in upper aerodigestive tract cancer
Authors: Filho Pedro A. Andrade , López‐Albaitero Andrés , Xi Liqiang , Gooding William , Godfrey Tony , Ferris Robert L. 
Published: May 2009

AbstractThe MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aerodigestive tract (UADT) tumor cells and its association with T‐cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE‐specific immunotherapy. Using quantitative RT‐PCR (QRT‐PCR), we evaluated the expression of MAGE‐3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA‐A*0201+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using Western blot. HLA‐A*0201:MAGE‐3‐ (271–279) specific cytotoxic T lymphocytes (MAGE‐CTL) from SCCHN patients and healthy donors showed that MAGE‐3/6 expression was highly associated with CTL recognition in vitro. On the basis of the MAGE‐3/6 expression, we could identify 31 (47%) of the 65 UADT tumors, which appeared to express MAGE‐3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE‐3 expression was responsible for CTL recognition, 2 MAGE‐3/6 mRNAhigh SCCHN cell lines, PCI‐13 and PCI‐30, were subjected to MAGE‐3/6‐specific knockdown. RNAi‐transfected cells showed that MAGE expression and MAGE‐CTL recognition were significantly reduced. Furthermore, treatment of cells expressing low MAGE‐3/6 mRNA with a demethylating agent, 5‐aza‐2′‐deoxycytidine (DAC), increased the expression of MAGE‐3/6 and CTL recognition. Thus, using QRT‐PCR UADT cancers frequently express MAGE‐3/6 at levels sufficient for CTL recognition, supporting the use of a QRT‐PCR‐based assay for the selection of candidates likely to respond to MAGE‐3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials. © 2009 UICC
Artificial reaction coordinate “tunneling” in free‐energy calculations: The catalytic reaction of RNase H
Authors: Rosta Edina , Woodcock H. Lee , Brooks Bernard R. , Hummer Gerhard 
Published: May 2009

AbstractWe describe a method for the systematic improvement of reaction coordinates in quantum mechanical/molecular mechanical (QM/MM) calculations of reaction free‐energy profiles. In umbrella‐sampling free‐energy calculations, a biasing potential acting on a chosen reaction coordinate is used to sample the system in reactant, product, and transition states. Sharp, nearly discontinuous changes along the resulting reaction path are used to identify coordinates that are relevant for the reaction but not properly sampled. These degrees of freedom are then included in an extended reaction coordinate. The general formalism is illustrated for the catalytic cleavage of the RNA backbone of an RNA/DNA hybrid duplex by the RNase H enzyme ofBacillus halodurans. We find that in the initial attack of the phosphate diester by water, the oxygen‐phosphorus distances alone are not sufficient as reaction coordinates, resulting in substantial hysteresis in the proton degrees of freedom and a barrier that is too low (∼10 kcal/mol). If the proton degrees of freedom are included in an extended reaction coordinate, we obtain a barrier of 21.6 kcal/mol consistent with the experimental rates. As the barrier is approached, the attacking water molecule transfers one of its protons to the O1P oxygen of the phosphate group. At the barrier top, the resulting hydroxide ion forms a penta‐coordinated phosphate intermediate. The method used to identify important degrees of freedom, and the procedure to optimize the reaction coordinate are general and should be useful both in classical and in QM/MM free‐energy calculations. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009
Simultaneous inhibition of anti‐coagulation and inflammation: crystal structure of phospholipase A2 complexed with indomethacin at 1.4 Å resolution reveals the presence of the new common ligand‐binding site
Authors: Singh Nagendra , Kumar Ramasamy Prem , Kumar Sanjit , Sharma Sujata , Mir Rafia , Kaur Punit , Srinivasan Alagiri , Singh Tej P. 
Published: May 2009

AbstractA novel ligand‐binding site with functional implications has been identified in phospholipase A2 (PLA2). The binding of non‐steroidal anti‐inflammatory agent indomethacin at this site blocks both catalytic and anti‐coagulant actions of PLA2. A group IIA PLA2 has been isolated from Daboia russelli pulchella (Russell's viper) which is enzymatically active as well as induces a strong anti‐coagulant action. The binding studies have shown that indomethacin reduces the effects of both anti‐coagulant and pro‐inflammatory actions of PLA2. A group IIA PLA2 was co‐crystallized with indomethacin and the structure of the complex has been determined at 1.4 Å resolution. The structure determination has revealed the presence of an indomethacin molecule in the structure of PLA2 at a site which is distinct from the conventional substrate‐binding site. One of the carboxylic group oxygen atoms of indomethacin interacts with Asp 49 and His 48 through the catalytically important water molecule OW 18 while the second carboxylic oxygen atom forms an ionic interaction with the side chain of Lys 69. It is well known that the residues, His 48 and Asp 49 are essential for catalysis while Lys 69 is a part of the anti‐coagulant loop (residues, 54–77). Indomethacin binds in such a manner that it blocks the access to both, it works as a dual inhibitor for catalytic and anti‐coagulant actions of PLA2. This new binding site in PLA2 has been observed for the first time and indomethacin is the first compound that has been shown to bind at this novel site resulting in the prevention of anti‐coagulation and inflammation. Copyright © 2009 John Wiley & Sons, Ltd.