bluuebunny/crossref_metadata_2025 huggingface dataset:
A Combined Experimental and Density Functional Theory Study on the Pd‐Mediated Cycloisomerization of o‐Alkynylnitrobenzenes – Synthesis of Isatogens and Their Evaluation as Modulators of ROS‐Mediated Cell Death
Authors: Ramana Chepuri V. , Patel Pitambar , Vanka Kumar , Miao Benchun , Degterev Alexei
Published: August 2010

Highly selective cycloisomerization of o‐alkynylnitrobenzenes, leading to isatogens, has been achieved by employment of a PdII complex. This reaction is very general and functional‐group‐tolerant. The possible mechanism of this reaction was investigated with the help of DFT calculations. Three possible pathways – namely, the addition of the nitro group either in (i) 5‐exo‐dig or (ii) 6‐endo‐dig mode and (iii) halopalladation – and subsequent intramolecular events have been considered and studied in detail. These investigations revealed that pathway (i) is the favored route to isatogen formation. A preliminary screening of the available isatogens reveals the 2‐alkylisatogens to be novel ROS scavengers capable of inhibiting cellular necroptosis.
Electrocatalytic Reduction of Peroxy Disulfate on the Palladized Aluminum Electrode Modified by Prussian Blue: Application to the Analysis of Decolorizing Powders
Authors: Pournaghi‐Azar M. H. , Ahour F.
Published: August 2010

The mechanism of catalytic reduction of peroxydisulfate on the palladized aluminum electrode modified by Prussian blue (PB/Pd‐Al) was studied. The charge transfer‐rate limiting step as well as overall reduction reaction of S2O82− is found to be a one‐electron and two‐electron abstraction respectively. The modified electrode is exploited for the hydrodynamic amperometry of peroxydisulfate. It is found that the calibration graph is linear in the S2O82−concentration range 5×10−6–1.5×10−3 mol L−1. The detection limit of the method was 2.4×10−6 mol L−1 S2O82. The method was successfully used for the determination of S2O82− in decolorizing powders
Silica‐Polyaniline Based Bienzyme Cholesterol Biosensor: Fabrication and Characterization
Authors: Manesh Kalayil Manian , Santhosh Padmanabhan , Gopalan Anantha Iyengar , Lee Kwang‐Pill
Published: August 2010

In the present investigation, silica‐polyaniline based bienzyme cholesterol biosensor is fabricated through a simple one‐step electrochemical method. The one‐step fabrication process involves electrochemical polymerization of N[3‐(trimethoxysilyl)propyl]aniline to result poly(N[3‐(trimethoxysilyl)propyl]aniline) (PTMSPA) and simultaneous immobilization of two enzymes, horseradish peroxidase (HRP) and cholesterol oxidase (ChOx) into PTMSPA matrix. The modified electrode is designated as PTMSPA‐HRP/ChOx‐ME. PTMSPA facilitates direct electron transfer between the electrode surface and the active redox centers of HRP. This enables the operation of a biosensor at a low working potential of about −150 mV (vs. Ag/AgCl) for the detection of hydrogen peroxide. The PTMSPA‐HRP/ChOx‐ME demonstrates excellent analytical performance for the detection of cholesterol between 1 and 25 mM with high sensitivity and selectivity. PTMSPA possesses features suited for the fabrication of third‐generation biosensors.
Fabrication of a microfluidic Ag/AgCl reference electrode and its application for portable and disposable electrochemical microchips
Authors: Zhou Jianhua , Ren Kangning , Zheng Yizhe , Su Jing , Zhao Yihua , Ryan Declan , Wu Hongkai
Published: August 2010

This report describes a convenient method for the fabrication of a miniaturized, reliable Ag/AgCl reference electrode with nanofluidic channels acting as a salt bridge that can be easily integrated into microfluidic chips. The Ag/AgCl reference electrode shows high stability with millivolt variations. We demonstrated the application of this reference electrode in a portable microfluidic chip that is connected to a USB‐port microelectrochemical station and to a computer for data collection and analysis. The low fabrication cost of the chip with the potential for mass production makes it disposable and an excellent candidate for real‐world analysis and measurement. We used the chip to quantitatively analyze the concentrations of heavy metal ions (Cd2+ and Pb2+) in sea water. We believe that the Ag/AgCl reference microelectrode and the portable electrochemical system will be of interest to people in microfluidics, environmental science, clinical diagnostics, and food research.
Polymeric nanopillar arrays for cell traction force measurements
Authors: Kuo Chiung‐Wen , Shiu Jau‐Ye , Chien Fan‐Ching , Tsai Shih‐Min , Chueh Di‐Yan , Chen Peilin
Published: August 2010

This paper reports the development of a novel force measurement device based on polymeric nanopillar arrays. The device was fabricated by a process combining nanosphere lithography, oxygen plasma treatment, deep etching and nano‐molding. Well‐ordered polymeric nanopillar arrays with various diameters and aspect ratios were fabricated and used as cell culture substrates. Cell traction forces were measured by the deflection of the nanopillars. Since the location of the nanopillars can be monitored at all times, this device allows for the measurement of the evolution of adhesion forces over time.
Systematic engineering of microorganisms to improve alcohol tolerance
Authors: Jia Kaizhi , Zhang Yanping , Li Yin
Published: August 2010

Microbial production of alcohol is a promising approach for obtaining important chemicals and fuels from renewable carbon resources. One of the key problems associated with the fermentative production of alcohol is alcohol toxicity and/or growth inhibition, resulting in a low alcohol titer in the fermentation broth. Cost‐effective production of alcohol needs the scientific community to engineer microbes for increased alcohol tolerance. Here, based on the understanding of the mechanisms of alcohol toxicity and alcohol stress resistance, we review the recent progress in engineering microbial alcohol tolerance by rational approaches. In addition, random approaches including traditional mutagenesis and selection, evolution engineering, random knockout and overexpression libraries, genome shuffling, replicational/transcriptional/translational engineering were also included into this review.
Genotoxicity testing: Moving beyond qualitative “screen and bin” approach towards characterization of dose‐response and thresholds
Authors: Pottenger Lynn H. , Gollapudi B. Bhaskar
Published: August 2010

For more than 40+ years, genotoxicity data have been interpreted in a qualitative, binary mode; a chemical is considered either positive or negative for a response in the test system. Although dose–response information is sometimes used in this decision, it is not routine to obtain the amount of information needed to inform risk assessment, for example to determine no‐observed‐genotoxic‐effect‐levels, primarily due to the historical view of genotoxic responses as “linear, no‐threshold.” Only recently have researchers begun to address this issue through robust experimental designs and application of statistical models. A growing body‐of‐evidence supports the existence of response thresholds for a number of mutagenic agents, in vitro and in vivo. Clearly, simple observation of a “hockey‐stick” dose–response curve is not sufficient to establish a threshold. Collection of robust empirical data must be supported with an analysis of biological plausibility for the observed threshold. In this context, a chemical‐specific mode‐of‐action (MOA) approach, which identifies key events responsible for the observed mutagenic effect, is extremely valuable. Biomarkers of key events, providing qualitative and quantitative information, can be integrated in a weight‐of‐evidence‐based assessment of genotoxicity data from multiple test systems and used to identify data gaps to resolve/reduce uncertainties during the risk assessment process. To this end, specific recommendations on study design and data analysis are proposed. As the Environmental Mutagen Society celebrates its 40th anniversary, the field of genetic toxicology is marking a milestone on the path to a new paradigm, using a MOA, data‐driven approach to answer questions about thresholds for genotoxic agents. Environ. Mol. Mutagen., 2010. © 2010 Wiley‐Liss, Inc.
Evaluation of the biotic ligand model to predict long-term toxicity of nickel to Hyalella azteca
Authors: Schroeder J.E. , Borgmann U. , Dixon D.G.
Published: August 2010

Three models were developed and evaluated for their ability to predict long-term bioaccumulation of nickel (Ni) and its toxicity to Hyalella azteca using data from 28-d toxicity tests. One of the models was based on competitive action of Ni with Ca and H (the biotic ligand model; BLM), and the other two models included expressions for the potential noncompetitive action of calcium on the ligand (i.e., acclimation), in addition to, or instead of, its competitive action (not accounted for in the BLM). Each model was able to predict lethal accumulation 50 (accumulation at 50% mortality; LA50s) within a factor of 2 of the corresponding observed LA50. The mean predicted LA50 from all three models was within 13% of the observed mean LA50 of 0.90 µmol/g (dry weight). The median lethal concentrations (LC50s) predicted by the three models were similar and were within a factor of 2 of the observed LC50s for 11 of 13 tests, providing encouragement for further development of a long-term Ni BLM. The similar performance of models based on competitive or noncompetitive action may reflect limitations in the data set or may suggest that effects of calcium on the ligand (LT) were insufficient to hamper the functionality of the competitive model or that the LA50/LT ratio, rather than the LA50 and LT, is constant. Environ. Toxicol. Chem. 2010;29:2498–2504. © 2010 SETAC
Synthesis, characteristic of a novel flame retardant containing phosphorus and its application in poly(ethylene‐co‐vinyl acetate)
Authors: Wang Lichun , Jiang Junqing , Jiang Pingkai , Yu Jinhong
Published: August 2010

A novel flame retardant (SPDH) containing phosphorus was synthesized through the reaction of 10‐(2, 5‐dihydroxyphenyl)‐9, 10‐dihydro‐9‐oxa‐10‐phosphaphenanthrene‐10‐oxide (DOPO‐HQ) and synthesized intermediate product 3, 9‐dichloro‐2, 4, 8, 10‐tetraoxa‐3, 9‐diphosphaspiro(5.5)undecane‐3, 9‐dioxide (SPDPC). The structure and properties of SPDPC and SPDH were characterized by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) spectroscopy and thermogravimetric analysis (TGA). After blending with poly(ethylene‐co‐vinyl acetate) (EVA), the flame‐retardant properties of EVA/SPDH composites were estimated by cone calorimeter, limited oxygen index (LOI) and UL‐94 tests, whereas the thermal stabilities were investigated using TGA. The morphological microstructure of the char formed by EVA/SPDH composite after combustion in cone calorimeter was investigated by scanning electron microscopy (SEM). The results indicate that the flame retardant and thermal stability were improved by incorporation of SPDH. The rich foamy char layers were observed from the residues after combustion in a cone calorimeter, which exactly benefits the improvement of thermal stability and flame retardant property of materials. Copyright © 2010 John Wiley & Sons, Ltd.
A Common Variant in the Patatin-Like Phospholipase 3 Gene ( PNPLA3 ) Is Associated with Fatty Liver Disease in Obese Children and Adolescents
Authors: Santoro Nicola , Kursawe Romy , D'Adamo Ebe , Dykas Daniel J. , Zhang Clarence K. , Bale Allen E. , Calí Anna M. , Narayan Deepak , Shaw Melissa M. , Pierpont Bridget , Savoye Mary , Lartaud Derek , Eldrich Samuel , Cushman Samuel W. , Zhao Hongyu , Shulman Gerald I. , Caprio Sonia
Published: August 2010

The genetic factors associated with susceptibility to nonalcoholic fatty liver disease (NAFLD) in pediatric obesity remain largely unknown. Recently, a nonsynonymous single-nucleotide polymorphism (rs738409), in the patatin-like phospholipase 3 gene (

PNPLA3

) has been associated with hepatic steatosis in adults. In a multiethnic group of 85 obese youths, we genotyped the

PNLPA3

single-nucleotide polymorphism, measured hepatic fat content by magnetic resonance imaging and insulin sensitivity by the insulin clamp. Because

PNPLA3

might affect adipogenesis/lipogenesis, we explored the putative association with the distribution of adipose cell size and the expression of some adipogenic/lipogenic genes in a subset of subjects who underwent a subcutaneous fat biopsy. Steatosis was present in 41% of Caucasians, 23% of African Americans, and 66% of Hispanics. The frequency of

PNPLA3

(rs738409) G allele was 0.324 in Caucasians, 0.183 in African Americans, and 0.483 in Hispanics. The prevalence of the G allele was higher in subjects showing hepatic steatosis. Surprisingly, subjects carrying the G allele showed comparable hepatic glucose production rates, peripheral glucose disposal rate, and glycerol turnover as the CC homozygotes. Carriers of the G allele showed smaller adipocytes than those with CC genotype (

P

= 0.005). Although the expression of

PNPLA3, PNPLA2, PPARγ2

(peroxisome proliferator-activated receptor gamma 2),

SREBP1c

(sterol regulatory element binding protein 1c), and

ACACA

(acetyl coenzyme A carboxylase) was not different between genotypes, carriers of the G allele showed lower leptin (

LEP

)(

P

= 0.03) and sirtuin 1 (

SIRT1

) expression (

P

= 0.04).

Conclusion:

A common variant of the

PNPLA3

gene confers susceptibility to hepatic steatosis in obese youths without increasing the level of hepatic and peripheral insulin resistance. The rs738409

PNPLA3

G allele is associated with morphological changes in adipocyte cell size. (Hepatology 2010.)
Scenario and multiple criteria decision analysis for energy and environmental security of military and industrial installations
Authors: Karvetski Christopher W , Lambert James H , Linkov Igor
Published: August 2010

Military and industrial facilities need secure and reliable power generation. Grid outages can result in cascading infrastructure failures as well as security breaches and should be avoided. Adding redundancy and increasing reliability can require additional environmental, financial, logistical, and other considerations and resources. Uncertain scenarios consisting of emergent environmental conditions, regulatory changes, growth of regional energy demands, and other concerns result in further complications. Decisions on selecting energy alternatives are made on an ad hoc basis. The present work integrates scenario analysis and multiple criteria decision analysis (MCDA) to identify combinations of impactful emergent conditions and to perform a preliminary benefits analysis of energy and environmental security investments for industrial and military installations. Application of a traditional MCDA approach would require significant stakeholder elicitations under multiple uncertain scenarios. The approach proposed in this study develops and iteratively adjusts a scoring function for investment alternatives to find the scenarios with the most significant impacts on installation security. A robust prioritization of investment alternatives can be achieved by integrating stakeholder preferences and focusing modeling and decision-analytical tools on a few key emergent conditions and scenarios. The approach is described and demonstrated for a campus of several dozen interconnected industrial buildings within a major installation. Integr Environ Assess Manag 2011;7:228–236. © 2010 SETAC
PKA knockdown enhances cell killing in response to radiation and androgen deprivation
Authors: Hensley Harvey H. , Hannoun‐Levi Jean‐Michel , Hachem Paul , Mu Zhaomei , Stoyanova Radka , Khor Li‐Yan , Agrawal Sudhir , Pollack Alan
Published: August 2010

The therapeutic efficacy of Gem®231, a second generation antisense molecule targeted to the RIα subunit of PKARIα (AS‐PKA), administered in combination with androgen deprivation (AD) and radiation therapy (RT), was examined in androgen sensitive (LNCaP) and insensitive (PC3) cell lines. Apoptosis was assayed by Caspase 3 + 7 activity and Annexin V binding. AS‐PKA significantly increased apoptosis in vitro from RT (both lines), with further increases in LNCaP cells grown in AD medium. In LNCaP cells, AD increased phosphorylated mitogen activated protein‐kinase (pMAPK), which was reduced by AS‐PKA relative to the mismatch (MM) controls. AS‐PKA also reduced pMAPK levels in PC3 cells. Cell death was measured by clonogenic survival assays. In vivo, LNCaP cells were grown orthotopically in nude mice. Tumor kinetics were measured by magnetic resonance imaging and serum prostate‐specific antigen. PC3 cells were grown subcutaneously and tumor volume assessed by caliper measurements. In PC3 xenografts, AS‐PKA caused a significant increase in tumor doubling time relative to MM controls as a monotherapy or in combination with RT. In orthotopic LNCaP tumors, AS‐PKA was ineffective as a monotherapy; however, it caused a statistically significant increase in tumor doubling time relative to MM controls when used in combination with AD, with or without RT. PKARIα levels in tumors were quantified via immunohistochemical (IHC) staining and image analysis. IHC measurements in LNCaP cells exhibited that AS‐PKA reduced PKARIα levels in vivo. We demonstrate for the first time that AS‐PKA enhances cell killing androgen sensitive prostate cancer cells to AD ± RT and androgen insensitive cells to RT.
Cervical nitric oxide release and persistence of high‐risk human papillomavirus in women
Authors: Rahkola Päivi , Väisänen‐Tommiska Mervi , Tuomikoski Pauliina , Ylikorkala Olavi , Mikkola Tomi S.
Published: August 2010

Nitric oxide may serve as one cofactor for human papillomavirus (HPV)‐induced development of cervical cancer. Therefore, we first assessed the levels of cervical fluid nitric oxide metabolite (NOx) in 283 women with and without high‐risk (hr) HPV. The NOx level in women with hr HPV (48.4 μmol/L [95% CI: 39.4–56.6], n = 199) was higher (p < 0.001) than that in women without hr HPV (24.6 μmol/L [95% CI: 19.1–38.7], n = 84). Second, we evaluated if cervical fluid NOx levels could predict the persistence of hr HPV. Therefore, we followed up 113 women with detectable hr HPV without any treatment for 12 mo and repeated hr HPV test. High‐risk HPV persisted in 72 women (64%) and disappeared in 41 women (36%). The median basal levels of NOx were higher (p = 0.02) in women with persistent hr HPV (56.9 μmol/L [95% CI: 48.7–81.0]) compared to those with eradicated hr HPV (37.7 μmol/L [95% CI: 27.0–58.0]). The NOx level higher than the 75th percentile (>87.0 μmol/L) predicted hr HPV persistence (OR = 4.1 [95% CI: 1.3–13.1]). This cutoff level of NOx showed 33% sensitivity and 90% specificity in predicting the persistence of hr HPV, but it failed to predict cytological progression or regression in 12 mo. In conclusion, high cervical fluid NOx appears to be connected to the persistence of hr HPV, but the low predictive capacity of NOx prevents its clinical use at this phase.
Correlation between tissue oxygenation and erythrocytes elasticity
Authors: Wu Yu‐Tsung , Chiou Arthur , Sun Chia‐Wei
Published: August 2010

In this paper, near‐infrared spectroscopy (NIRS) and jumping optical tweezers were used to measure the tissue oxygenation and the elasticity of erythrocytes, respectively. The correlation between tissue oxygenation induced by arterial occlusion test (AOT) and the mechanical properties of individual erythrocytes from a blood sample obtained after AOT was studied. The experimental results show a linear correlation between the oxygenation signal caused by AOT and the elasticity of erythrocytes. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
CB2 cannabinoid receptor targets mitogenic Gi protein–cyclin D1 axis in osteoblasts
Authors: Ofek Orr , Attar-Namdar Malka , Kram Vardit , Dvir-Ginzberg Mona , Mechoulam Raphael , Zimmer Andreas , Frenkel Baruch , Shohami Esther , Bab Itai
Published: August 2010

CB2 is a Gi protein–coupled receptor activated by endo- and phytocannabinoids, thus inhibiting stimulated adenylyl cyclase activity. CB2 is expressed in bone cells and Cb2 null mice show a marked age-related bone loss. CB2-specific agonists both attenuate and rescue ovariectomy-induced bone loss. Activation of CB2 stimulates osteoblast proliferation and bone marrow derived colony-forming units osteoblastic. Here we show that selective and nonselective CB2 agonists are mitogenic in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures. The CB2 mitogenic signaling depends critically on the stimulation of Erk1/2 phosphorylation and de novo synthesis of MAP kinase–activated protein kinase 2 (Mapkapk2) mRNA and protein. Further downstream, CB2 activation enhances CREB transcriptional activity and cyclin D1 mRNA expression. The CB2-induced stimulation of CREB and cyclin D1 is inhibitable by pertussis toxin, the MEK-Erk1/2 inhibitors PD098059 and U0126, and Mapkapk2 siRNA. These data demonstrate that in osteoblasts CB2 targets a Gi protein–cyclin D1 mitogenic axis. Erk1/2 phosphorylation and Mapkapk2 protein synthesis are critical intermediates in this axis. © 2011 American Society for Bone and Mineral Research.
Glypican 3 binds to GLUT1 and decreases glucose transport activity in hepatocellular carcinoma cells
Authors: Cho Hye‐Sim , Ahn Jung‐Mo , Han Ho‐Jae , Cho Je‐Yoel
Published: August 2010

Glypican 3 (GPC3), a member of heparin sulfate proteoglycans, is attached to the cell surface by a glycosylphosphatidylinositol anchor and is reported to be overexpressed in liver cancers. In order to identify GPC3 binding proteins on the cell surface, we constructed a cDNA containing the C‐terminal cell surface‐attached form of GPC3 (GPC3c) in a baculoviral vector. The GPC3c bait protein was produced by expressing the construct in Sf21 insect cells and double purified using a His column and Flag immunoprecipitation. Purified GPC3c was used to uncover GPC3c‐interacting proteins. Using an LC–MS/MS proteomics strategy, we identified glucose transporter 1 (GLUT1) as a novel GPC3 interacting protein from the HepG2 hepatoma cell lysates. The interaction was confirmed by immunoprecipitation (IP)–WB analysis and surface plasmon resonance (SPR). SPR result showed the interaction of GLUT1 to GPC3c with equilibrium dissociation constants (KD) of 1.61 nM. Moreover, both incubation with GPC3c protein and transfection of Gpc3c cDNA into HepG2 cells resulted in reduced glucose uptake activity. Our results indicate that GPC3 plays a role in glucose transport by interacting with GLUT1. J. Cell. Biochem. 111: 1252–1259, 2010. © 2010 Wiley‐Liss, Inc.
Nitric oxide involved in the IL‐1β‐induced inhibition of fructose intestinal transport
Authors: García‐Barrios Alberto , Guillén Natalia , Gascón Sonia , Osada Jesús , Vazquez Carmen Mª , Miguel‐Carrasco José Luis , Rodríguez‐Yoldi Mª Jesús
Published: August 2010

Interleukin‐1β (IL‐1β) is a pleiotropic cytokine produced by cells of the immune system and a large variety of other cell types including endothelial cells. It is released during inflammatory and infectious diseases, and possesses a wide spectrum of autocrine, paracrine and endocrine activities. The aim of this work was to examine the IL‐1β effect on D‐fructose transport across rabbit jejunum and try to identify the mediators implicated in this process. A sepsis condition was induced for 90 min after intravenous (iv) administration of IL‐1β and body temperature was recorded. Studies on cellular intestinal integrity have not shown modifications of the epithelium and the basement membrane. D‐fructose intestinal transport was studied in rabbit jejunum from control and treated animals and it was reduced in the latter ones. This cytokine decreased both the mucosal to serosal transepithelial flux and the transport across brush‐border membrane vesicles of D‐fructose. The inhibition was reversed by L‐NAME (nitric oxide [NO] synthase inhibitor), but not by indomethacin (cyclooxygenase 1 and 2 inhibitor). Both inhibitors were administered iv 15 min before the IL‐1β. The protein levels of GLUT5 were not changed in all animal groups and those of mRNA were even increased. In summary, these findings indicate that IL‐1β, at the time assayed, induced a significant reduction in the relative intrinsic activity of GLUT5 and in this decrease are involved NO signalling pathways. In this way, blockage of D‐fructose intestinal uptake by IL‐1β may be playing an essential role in the pathophysiology of septic shock. J. Cell. Biochem. 111: 1321–1329, 2010. © 2010 Wiley‐Liss, Inc.
Prenatal diagnosis of fetal hypothyroidism after maternal radioactive iodine exposure during pregnancy
Authors: Basbug Mustafa , Ozgun Mahmut T. , Murat Nurcan , Batukan Cem , Ozcelik Bulent , Kurtoglu Selim
Published: August 2010

A 33‐year‐old woman with a history of surgically treated papillary thyroid carcinoma was inadvertently given radioactive iodine when she was 16 weeks pregnant. Sonographic examination revealed fetal thyroid hypoplasia, and cordocentesis confirmed fetal hypothyroidism at 22 weeks. The pregnancy was terminated at 24 weeks. We report the first case of fetal thyroid hypoplasia diagnosed by ultrasound and cordocentesis. © 2010 Wiley Periodicals, Inc. J Clin Ultrasound, 2010
Critical design criteria for minimal antibiotic‐free plasmid vectors necessary to combine robust RNA Pol II and Pol III‐mediated eukaryotic expression with high bacterial production yields
Authors: Carnes Aaron E. , Luke Jeremy M. , Vincent Justin M. , Anderson Sheryl , Schukar Angela , Hodgson Clague P. , Williams James A.
Published: August 2010

Background

For safety considerations, regulatory agencies recommend the elimination of antibiotic resistance markers and non‐essential sequences from plasmid DNA‐based gene medicines. In the present study, we analyzed antibiotic‐free (AF) vector design criteria impacting upon bacterial production and mammalian transgene expression.

Methods

Both CMV‐HTLV‐I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed‐batch fermentation. RNA Pol II‐directed enhanced green fluorescent protein and RNA Pol III‐directed RNA expression were quantified by fluorometry and quantitative real‐time polymerase chain reaction, respectively, after transfection of human HEK293 cells.

Results

Sucrose‐selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA‐based AF selection with highly productive fermentation manufacturing (>1000 mg/l plasmid DNA) and high‐level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin‐resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1740 mg/l was developed.

Conclusions

We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly‐identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors. Copyright © 2010 John Wiley & Sons, Ltd.
Synthesis and reactivity of 6‐methyl‐4H‐furo[3,2c]pyran‐3,4‐dione
Authors: Hikem‐Oukacha Djamila , Rachedi Yahïa , Hamdi Maamar , Silva Artur M.S.
Published: August 2010

An efficient synthesis of 3‐bromoacetyl‐4‐hydroxy‐6‐methyl‐2H‐pyran‐2‐one by bromination of dehydroacetic acid in glacial acetic acid is described. Novel 4‐hydroxy‐6‐methyl‐3‐(2‐substituted‐thiazol‐4‐yl)‐2H‐pyran‐2‐ones have been prepared from the reaction of 3‐bromoacetyl‐4‐hydroxy‐6‐methyl‐2H‐pyran‐2‐one with thioamides, thiourea, and diphenylthiocarbazone. The condensation reaction of 6‐methyl‐4H‐furo[3,2c]pyran‐3,4‐dione, obtained from the reaction of 3‐bromoacetyl‐4‐hydroxy‐6‐methyl‐2H‐pyran‐2‐one with aliphatic amines, with benzaldehydes and acetophenones led to novel 2‐arylidene‐6‐methyl‐2H‐furo[3,2‐c]pyran‐3,4‐diones and 6‐(2‐arylprop‐1‐enyl)‐2H‐furo[3,2‐c]pyran‐3,4‐diones. The structure of all compounds was established by elemental analysis, IR, NMR, and mass spectra. J. Heterocyclic Chem., 2011.
E‐1,2‐diarylsulfonylethene—Source for new class of heterocycles
Authors: Padmaja Adivireddy , Payani Thalari , Muralikrishna Akkarapalli , Mahesh Konda
Published: August 2010

A new class of pyrazolidinediones, isoxazolidinediones, pyrimidinetriones, and thioxopyrimidinediones were synthesized by the reaction of Michael adduct, dimethyl 2‐(1′,2′‐diarylsulfonyl)ethylmalonate with different nucleophiles, hydrazine hydrate, hydroxylamine hydrochloride, and urea derivatives. J. Heterocyclic Chem., 2011.
A novel, facile, simple and convenient oxidative aromatization of Hantzsch 1,4‐dihydropyridines to pyridines using polymeric iodosobenzene with KBr
Authors: Kumar Parvin
Published: August 2010

magnified image

An easy, safe, effective and handy method for oxidative aromatization of Hantzsch 1,4‐dihydropyridines catalyzed by hypervalent iodine (iodosobenzene) and potassium bromide to corresponding pyridine derivatives in high‐yields and within short span of time was described. Dealkylation in case of 4‐n‐alkyl substituted 1,4‐dihydropyridines was not obtained. J. Heterocyclic Chem., (2010).
Analysis and design of enhanced directivity microstrip antenna at terahertz frequency by using electromagnetic bandgap material
Authors: Jha Kumud Ranjan , Singh G.
Published: August 2010

In this paper, an electromagnetic bandgap (EBG) material in the range of 0.5–0.8 THz has been simulated with Arlon AR 600 (ε=6.0) as host material and air‐cylinders as the foreign embedded material. The effective dielectric permittivity of this material has been analyzed at the frequency range of 0.590–0.620 THz. The reduced effective dielectric permittivity substrate of thickness 50 µm has been used to design the microstrip antenna at 600 GHz with a novel approach. This proposed antenna shows an enhanced directivity of 15.1 dBi at 600 GHz frequency. To compare the analysis, the operating frequency has been scaled down by a factor of 100 and various results have been compared by using two different commercially available simulators CST Microwave Studio based on the finite integral technique and Ansoft HFSS based on the finite element method. Copyright © 2010 John Wiley & Sons, Ltd.
Ih “run‐up” in rat neocortical neurons and transiently rat or human HCN1‐expressing HEK293 cells
Authors: Battefeld Arne , Bierwirth Claudia , Li Ying Chuan , Barthel Lennart , Velmans Tanja , Strauss Ulf
Published: August 2010

Hyperpolarization‐activated cyclic nucleotide‐gated ion channels (HCN) are key determinants of CNS functions. Here we describe an increase in hyperpolarization‐activated current (Ih) at the beginning of whole‐cell recordings in rat layer 5 cortical neurons. For a closer investigation of this Ih increase, we overexpressed the predominant layer 5 rat subunit HCN1 in HEK293 cells. We characterized the resulting Ih in the cell‐attached and whole‐cell configurations. Breaking into whole‐cell configuration led to about a 30% enhancement of rat HCN1‐mediated Ih accompanied by a depolarizing shift in voltage dependence and an accelerated time course of activation. This current enhancement is not species specific; for human HCN1, the current similarly increases in amount and kinetics. Although the changes were bound to cytosolic solution exchange, they were independent of cAMP, ATP, GTP, and the phosphate group donor phosphocreatine. Together, these data provide a characterization of heterologous expression of rat HCN1 and suggest that cytosolic contents suppress Ih. Such a mechanism might constitute a reserve in h‐channel function in vivo. © 2010 Wiley‐Liss, Inc.
Prokaryotic diversity of a non‐sulfide, low‐salt cold spring sediment of Shawan County, China
Authors: Zeng Jun , Yang Hong‐mei , Lou Kai
Published: August 2010

The prokaryotic diversity of a non‐sulfide, low‐salt cold spring sediment was investigated by constructing bacterial and archaeal clone libraries of the 16S rRNA gene. 241 bacterial clones were screened, which could be grouped into 86 ribotypes, based on restriction fragment length polymorphism (RFLP) analysis. These were divided into 11 phyla (Actinobacteria, Acidobacteria, Bacteroidetes, Chlorobi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Proteobacteria, Planctomycetes and Verrucomicrobia). Of these, Acidobacteria and Proteobacteria were the most dominant, representing 48% and 25% of the total bacteria clone library, respectively. For the archaeal clone library, 121 positive clones were screened and 22 ribotypes were determined. BLAST analysis indicated that all ribotypes were affiliated with the phylum Crenarchaeota. Phylogenetic analysis classified them into three subgroups (Groups I–III). Groups I and III, belonging to the Soil–Freshwater‐subsurface group and Marine group I, respectively, were the dominant groups, representing 50% and 47% of the library, respectively. Of them, 20% of ribotypes were related to the cold‐loving Crenarchaeota. These findings show that bacteria in spring sediments are more diverse than are archaea; in addition, the spring harbors a large number of novel bacterial and archaeal species and maybe exist novel lineages. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Applying target region amplification polymorphism markers for analyzing genetic diversity of Lentinula edodes in China
Authors: Xiao Yang , Liu Wei , Lu Ying‐Ying , Gong Wen‐Bing , Bian Yin‐Bing
Published: August 2010

The target region amplification polymorphism (TRAP) technique was utilized for assessing the genetic diversity of 55 wild strains and one cultivated strain of Lentinula edodes in China. From these strains, 932 DNA fragments were amplified using 12 primer combinations, 929 fragments (99.68%) of which were polymorphic between two or more strains. The average coefficient of pairwise genetic similarity was 0.696, within a range from 0.503 to 0.947. Cluster analysis and principal coordinate analysis separated the tested strains of L. edodes into two major groups. Group A was further divided into seven subgroups. In most cases, the strains from the same or adjoining regions could be preferentially clustered into small groups. The results from the average genetic similarity and the weighted average value of Shannon's Information Index among the tested strains of L. edodes from the same region revealed a vast genetic diversity in the natural germplasm found in China. Compared with the L. edodes strains from other regions, those found on the Yunnan Plateau, in the Hengduanshan Mountains, in Taiwan, South China, and Northeast China showed greater genetic diversity. The results of the present study indicated that the wild strains of L. edodes in China possessed abundant genetic variation, and the genetic relationships among them were highly associated with the geographic distribution. This is the first report demonstrating that TRAP markers were powerful for analyzing the genetic diversity of L. edodes, and the study lays the foundation for a further application of this remarkable technique to other fungi. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Optimization of a genome‐walking method to suit GC‐rich template DNA from biotechnological relevant Actinobacteria
Authors: Gröning Janosch A. D. , Tischler Dirk , Kaschabek Stefan R. , Schlömann Michael
Published: August 2010

SiteFinding‐PCR has been recently reported to be a useful technique in order to identify unknown DNA fragments located adjacent to available sequences. However, this method has so far only been applied to few DNA sources including plants, samples from bioleaching communities, and a Pseudomonas strain. In order to complete the sequence information of two gene clusters in Gram‐positive rhodococci the original protocol was applied yielding amplicons of insufficient size. The binding site of the previously published SiteFinder‐2 oligo proved to be unsuitable for Rhodococcus and other members of the Actinobacteria since the binding motif occurred too frequently. Available genome sequences of different Actinobacteria were analysed and the binding site of the SiteFinder oligo modified. Moreover, PCR conditions were adapted to the high GC content of the template DNA allowing the successful adaptation of this method to two members of the Actinobacteria. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Purification and characterization of the ncgl2923 ‐encoded 3‐hydroxybenzoate 6‐hydroxylase from Corynebacterium glutamicum
Authors: Yang Yi‐Fan , Zhang Jun‐Jie , Wang Song‐He , Zhou Ning‐Yi
Published: August 2010

Corynebacterium glutamicum ATCC 13032 metabolizes 3‐hydroxybenzoate via gentisate. We have now characterized the ncgl2923 ‐encoded 3‐hydroxybenzoate 6‐hydroxylase involved in the initial step of 3‐hydroxybenzoate catabolism by this strain, a first 3‐hydroxybenzoate 6‐hydroxylase molecularly and biochemically characterized from a Gram‐positive strain. The ncg12923 gene from Corynebacterium glutamicum ATCC 13032 was shown to encode 3‐hydroxybenzoate 6‐hydroxylase, the enzyme that catalyzes the NADH‐dependent conversion of 3‐hydroxybenzoate to gentisate. Ncgl2923 was expressed with an N‐terminal six‐His tag and purified to apparent homogeneity by Ni2+‐nitrilotriacetic acid affinity chromatography. The purified H6‐Ncgl2923 showed a single band at apparent molecular mass of 49 kDa on a sodium dodecyl sulfate polyacrylamide gel electrophoresis and was found to be most likely a trimer as determined by gel filtration chromatography. It had a specific activity of 6.92 ± 0.39 U mg–1 against 3‐hydroxybenzoate and with a Km value of 53.4 ± 4.7 μM using NADH as a cofactor. The product formed from the 3‐hydroxybenzoate hydroxylation catalyzed by H6‐Ncgl2923 was identified by high‐performance liquid chromatography as gentisate, a ring‐cleavage substrate in the microbial aromatic degradation. The enzyme exhibited a maximum activity at pH 7.5 in phosphate buffer, and adding flavin adenine dinucleotide to a final concentration of 15 μM would enhance the activity by three‐fold. Although this enzyme shares no more than 33% identity with any of reported 3‐hydroxybenzoate 6‐hydroxylases from Gram‐negative bacterial strains, there is little difference in subunit sizes and biochemical characteristics between them. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Molecular diversity of native rhizobia trapped by five field pea genotypes in Indian soils
Authors: Wadhwa K. , Dudeja S. S. , Yadav R. K.
Published: August 2010

Five pea cultivars; HFP 4, HVP 3–5, HFP 9426, Jayanti and Hariyal, being grown in CCS Haryana Agricultural University farm were used to isolate native rhizobia. Selected 54 rhizobia, from all cultivars, were authenticated as rhizobia by plant infectivity test. Along with nodulation, symbiotic effectiveness in terms of symbiotic ratios showed wide range of effectiveness of pea rhizobia from 1.11 to 5.0. DNA of all the 54 rhizobia was extracted and amplified by PCR, using ERIC and 16S rDNA primers. Dendrogram based on ERIC profiles of these 54 rhizobia showed the formation of 13 subclusters at 80% level of similarity. Dendrogram based on RFLP of 16S rDNA by three restriction endonucleases; Msp I, Csp 6I and Rsa I; also formed 13 subclusters at 80% level of similarity. However, positioning of subclusters was different from that of ERIC based dendrogram. Majority of the isolates i.e. 64.8% by ERIC profiles and 44.4% by RFLP of 16S rDNA formed one cluster. Isolates from same nodule were not 100% similar. Considering each cluster representing a rhizobial genotype, both techniques used to assess molecular diversity indicated the presence of 13 genotypes of field pea rhizobia in CCS Haryana Agricultural University farm soil. Two pea rhizobial genotypes were able to nodulate all the five pea cultivars. Furthermore, high strain richness index (0.43–0.5) of field pea rhizobia was observed by both the techniques. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Validation of nested PCR and a selective biochemical method as alternatives for mycoplasma detection
Authors: Cheong Kyung Ah , Agrawal Santosh Rani , Lee Ai‐Young
Published: August 2010

Direct culture is the most common way to reliably detect mycoplasma, but it is not practical for the qualitative control of cell therapeutics because of the elaborate culture medium, the prolonged incubation time, and the large sample volumes. Here, we chose two alternative methods using commercial detection kits, the PCR mycoplasma detection kit with nested PCR and the selective biochemical method, MycoAlert®, and validated them with the direct culture method as a reference. We tested eight mycoplasma species and five validation parameters: specificity, detection limit, robustness, repeatability, and ruggedness, based on the regulatory guidelines in the US Pharmacopoeia. All experiments were performed using fibroblasts spiked with mycoplasma. Specificity tests for both methods included all mycoplasma species, except Mycoplasma pneumonia and M. genitalium for the nested PCR and Ureaplasma urealyticum for the MycoAlert® assay. Regarding the detection limit, the nested PCR proved to be as sensitive as the direct culture method and more sensitive than the MycoAlert® assay. The predicted median for probit = 0.9 was 54 (44–76) CFU/ml for M. hyorhinis and 16 (13–23) CFU/ml for M. hominis by the nested PCR, but 431 (346–593) CFU/ml and 105 (87–142) CFU/ml, respectively, with MycoAlert®. Changes in the concentration of reagents, reagent lot, or individual analysts did not influence the results of the examined methods. The results of this study support nested PCR as a valuable alternative for mycoplasma detection. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Titanium particles activate toll‐like receptor 4 independently of lipid rafts in RAW264.7 murine macrophages
Authors: Islam Andrew S. , Beidelschies Michelle A. , Huml Anne , Greenfield Edward M.
Published: August 2010

Adherent pathogen‐associated molecular patterns (PAMPs) act through toll‐like receptor 2 (TLR2) and TLR4 to increase the biological activity of orthopedic wear particles in cell culture and animal models of implant loosening. This study tested whether this is dependent on TLR association with lipid rafts as reported for the response to soluble TLR ligands. For this purpose, RAW264.7 murine macrophages were activated by exposure to titanium particles with adherent PAMPs, soluble lipopolysaccharide (LPS), soluble lipotecichoic acid (LTA), or heat‐killed bacteria that had been extensively washed to remove soluble PAMPs. Lipid rafts were isolated by two independent methods and the location of TLR4 and TLR2 was analyzed by Western blotting. The cognate TLRs associated with lipid rafts when the macrophages were activated with soluble LPS and LTA but not after stimulation with either titanium particles with adherent PAMPs or heat‐killed bacteria. The lipid raft disruptor, methyl‐β‐cyclodextrin, dose‐dependently inhibited TNF‐α release in response to LPS but had no affect on TNF‐α release in response to titanium particles with adherent PAMPs. We conclude, therefore, that titanium particles with adherent PAMPs and heat‐killed bacteria activate TLR2 and TLR4 in macrophages without inducing either TLR to associate with lipid rafts. These results have important implications for the mechanisms of orthopedic implant loosening as well the mechanisms for TLR activation in other inflammatory situations. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:211–217, 2011
Role of mitogen‐activated protein kinase in osteoblast differentiation
Authors: Lin Fu‐Hsiung , Chang Jessica B. , Brigman Brian E.
Published: August 2010

Local control of osteoblast differentiation and bone formation is not well understood. We have previously seen biphasic effects on cell differentiation in response to the short‐ and long‐term exposure to IL‐1β in rat calvarial osteoblasts. To characterize the signaling pathway mechanisms regulating IL‐1β biphasic effects, we examined the contribution of mitogen‐activated protein kinase (MAPK) family. Cells were pretreated with specific inhibitors to extracellular signal‐regulated kinase (ERK, PD98059), p38 (SB203580), and c‐JUN N‐terminal kinase (JNK, SP600125), then co‐cultured with IL‐1β for 2, 4, and 6 days. Cell differentiation was determined by measuring bone nodules after 10 days of culture. These inhibitors did not alter biphasic effects of IL‐1β on cell differentiation. However, PD98059 and U2016, another inhibitor of ERK activation robustly increased osteoblast differentiation compared to vehicle‐treated control in a time‐ and dose‐dependent manner. PD98059 appears to stimulate alkaline phosphatase (ALP) activity to promote cell differentiation, where IL‐1β appears to suppress it. Interestingly, continuous ERK inhibition with PD98059, after 2 and 4 days of IL‐1β treatment, enhanced the IL‐1β anabolic effect by increasing bone nodules formed. These observations provide a potential mechanism involving ERK pathway in osteoblasts differentiation and suggest that MAPK family may not directly regulate IL‐1β biphasic effects. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:204–210, 2011
Surgical synovectomy decreases density of sensory nerve fibers in synovial tissue of non‐inflamed controls and rheumatoid arthritis patients
Authors: Ossyssek B. , Anders S. , Grifka J. , Straub R.H.
Published: August 2010

Surgical synovectomy is a technique to treat synovitis and pain in patients with rheumatoid arthritis (RA) resistant to DMARDs or therapy with biologics. Indication to synovectomy is subject to tight cooperation of orthopaedic surgeons and rheumatologists. It was thought that synovectomy leads to a reduction of sensory nerve fibers, called sensory denervation. Since sensory denervation after synovectomy has never been histologically tested, we aimed to investigate sensory and sympathetic innervation in synovial tissue before and after synovectomy. Eight non‐inflamed control subjects and eight patients with RA were included in this study with a two‐stage synovectomy approach (interval 40–50 days). Nerve fibers and cells in synovial tissue were detected and counted using immunofluorescence. Density of sympathetic nerve fibers did not change after synovectomy, whereas density of sensory nerve fibers decreased in all control subjects and seven of eight patients with RA. In parallel, the density of synovial cells increased after synovectomy in all control subjects and six of eight RA patients, which is indicative of a wound healing response. In one female RA patient, density of sensory nerve fibers increased and a very marked rise of cellular density was observed, too. This indicates that probably not all patients profit from surgical synovectomy. The majority of patients (94%) demonstrated sensory denervation after surgical synovectomy accompanied by a wound healing cell response. This study can help to explain the positive effects of surgical synovectomy which usually leads to pain reduction and improved mobility. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:297–302, 2011