bluuebunny/crossref_metadata_2025 huggingface dataset:
Vascular endothelial growth factor C attenuates joint damage in chronic inflammatory arthritis by accelerating local lymphatic drainage in mice
Authors: Zhou Quan , Guo Ruolin , Wood Ronald , Boyce Brendan F. , Liang Qianqian , Wang Yong‐Jun , Schwarz Edward M. , Xing Lianping
Published: May 2011

Objective

To investigate whether the enhancement of joint lymphangiogenesis by injection of vascular endothelial growth factor C (VEGF‐C) adeno‐associated virus (AAV) into the affected joints has therapeutic efficacy in chronic inflammatory arthritis in mice.

Methods

Tumor necrosis factor–transgenic (TNF‐Tg) mice were used as a model of chronic inflammatory arthritis. Human VEGF‐C was cloned into an AAV expression vector to generate AAV‐VEGF‐C. The joints of TNF‐Tg mice were injected with AAV‐VEGF‐C or AAV‐luciferase (AAV‐Luc) as a control. During the 4 months following injection, magnetic resonance imaging of the joints and lymphatic imaging were performed to assess changes in synovial volume and lymph flow from the joint tissues to local draining lymph nodes. Joint inflammation, bone erosion, and cartilage loss were examined by histologic analyses. Lymphatic vessel formation was assessed using immunohistochemistry.

Results

Intraarticular administration of AAV‐VEGF‐C virus significantly attenuated the increase in synovial volume and increased lymphatic vessel number in the joint sections, as compared with that in control AAV‐Luc–injected joints, during the 4‐month period. This was accompanied by a reduction in the area of inflammation, bone erosion, cartilage loss, and osteoclast numbers. Lymph flow from the joints to local draining lymph nodes was slower in TNF‐Tg mice than in wild‐type littermates, and was significantly improved with AAV‐VEGF‐C treatment.

Conclusion

Intraarticular injection of AAV‐VEGF‐C increased lymphangiogenesis and improved lymphatic drainage from the inflamed joints of mice, resulting in attenuation of joint tissue damage. Thus, improvement of joint lymphatic function by local administration of lymphatic growth factors represents a new therapeutic approach for chronic inflammatory arthritis.
Systemic sclerosis–related pulmonary hypertension associated with interstitial lung disease: Impact of pulmonary arterial hypertension therapies
Authors: Le Pavec Jérôme , Girgis Reda E. , Lechtzin Noah , Mathai Stephen C. , Launay David , Hummers Laura K. , Zaiman Ari , Sitbon Olivier , Simonneau Gérald , Humbert Marc , Hassoun Paul M.
Published: May 2011

Objective

Precapillary pulmonary hypertension (PH) is an important cause of death in patients with systemic sclerosis (SSc). It can occur in isolation (pulmonary arterial hypertension [PAH]) or in association with interstitial lung disease (ILD). Importantly, the outcomes and efficacy of PAH therapies in patients with SSc‐related PH complicating ILD (PH‐ILD) remain unknown. This study was undertaken to evaluate our experience with PH‐ILD with regard to the efficacy and safety of PAH therapies in this patient cohort.

Methods

We conducted a retrospective analysis of consecutive SSc patients from 2 large referral centers who had PH‐ILD confirmed by right‐sided heart catheterization and who received targeted PAH therapies. World Health Organization (WHO) functional class, 6‐minute walk distance, and hemodynamic parameters were assessed at baseline and after a mean ± SD of 7.7 ± 6.2 months of treatment for PAH. Kaplan‐Meier and Cox proportional hazards models were used to analyze survival and to identify prognostic factors.

Results

Seventy patients were included in the study. No significant changes were observed in WHO functional class, 6‐minute walk distance, or hemodynamic parameters after therapy. The 1‐, 2‐, and 3‐year survival estimates were 71%, 39%, and 21%, respectively. In the multivariate model, worsening oxygenation during followup and reduced renal function were the only significant risk factors for death.

Conclusion

This study represents the largest series to date in which the impact of PAH therapies in SSc‐related PH‐ILD was examined. In this cohort, PAH therapies were associated with no clear benefits. Deterioration in oxygenation was an important determinant of long‐term survival. Prospective clinical trials focusing on this group of patients are warranted.
Palladium‐Catalyzed Allyl Cross‐Coupling Reactions with In Situ Generated Organoindium Reagents
Authors: Lee Kooyeon , Kim Hyunseok , Mo Juntae , Lee Phil Ho
Published: May 2011

Inter‐ and intramolecular palladium‐catalyzed allyl cross‐coupling reactions, using allylindium generated in situ from allyl halides and indium, is demonstrated. Allylindium compounds may be effective nucleophilic coupling partners in palladium‐catalyzed cross‐coupling reactions. A variety of allyl halides, such as allyl iodide, allyl bromide, crotyl bromide, prenyl bromide, geranyl bromide, and 3‐bromocyclohexene afforded the allylic cross‐coupling products in good to excellent yields. Stereochemistry of the double bond is retained in the allylic cross‐coupling reactions. Electrophilic cross‐coupling partners, such as aryl and vinyl halides, dibromoolefin, alkynyl iodide, and aryl and vinyl triflates participate in these reactions. The presence of various substituents, such as n‐butyl, ketal, acetyl, ethoxycarbonyl, nitrile, N‐phenylamido, nitro, and chloride groups on the aromatic ring of electrophilic coupling partners showed little effect on the efficiency of the reactions. The present conditions work equally well for not only intermolecular but also intramolecular palladium‐catalyzed cross‐coupling reactions. These methods provide an efficient synthetic method for the introduction of an allyl group, which can be easily further functionalized to afford an sp2‐ and sp‐hybridized carbon. The present method complements existing synthetic methods as a result of advantageous features such as easy preparation and handling, thermal stability, high reactivity and selectivity, operational simplicity, and low toxicity of allylindium reagents.
Cooperative Activation of Alkyne and Thioamide Functionalities; Direct Catalytic Asymmetric Conjugate Addition of Terminal Alkynes to α,β‐Unsaturated Thioamides
Authors: Yazaki Ryo , Kumagai Naoya , Shibasaki Masakatsu
Published: May 2011

A detailed study of the direct catalytic asymmetric conjugate addition of terminal alkynes to α,β‐unsaturated thioamides is described. A soft Lewis acid/hard Brønsted base cooperative catalyst, comprising [Cu(CH3CN)4]PF6, bisphosphine ligand, and Li(OC6H4‐p‐OMe) simultaneously activated both substrates to compensate for the low reactivity of copper alkynylide. A series of control experiments revealed that the intermediate copper‐thioamide enolate functioned as a Brønsted base to generate copper alkynylide from the terminal alkyne, thus driving the catalytic cycle through an efficient proton transfer between substrates. These findings led to the identification of a more convenient catalyst using potassium hexamethyldisilazane (KHMDS) as the Brønsted base, which was particularly effective for the reaction of silylacetylenes. Divergent transformation of the thioamide functionality and a concise enantioselective synthesis of a GPR40 receptor agonist AMG‐837 highlighted the synthetic utility of the present catalysis.
Optimal check up and replacement periods of variable message signs: a case of the Seoul metropolitan transportation center
Authors: Lee Chungwon , Kim Seong W. , Lee Soo‐Beom
Published: May 2011

Variable message signs (VMS) are one kind of traffic management facility installed following the “Guidelines on the Standard of Road Structures and Facilities.” VMS are installed and operated within the Intelligent Transport Systems (ITS). However, existing VMS are operated and managed by designers or operators without any consistent criteria on check up and replacement periods. There has been very little research on check up and replacement periods of VMS. This study was to introduce a new methodology to estimate check up and replacement periods of VMS. This study reflected the actual VMS failure data and VMS and Portable Variable Message Signs luminance test results. Consequently, I hope that this study will be a useful reference for VMS and other ITS facilities. Copyright © 2011 John Wiley & Sons, Ltd.
Productivity changes in consideration of accident costs in Seoul's bus industry
Authors: Oh Miyoung , Shon Euiyoung , Kim Sungsoo
Published: May 2011

The Seoul Metropolitan Government has introduced a quasi‐public operational system since the public transport systems were reformed in July 2004. Thus, there has been controversy on moral hazards. A question addressed by researchers of transportation is the extent to which the public transport reforms have led to improvements in efficiency and productivity growth. The directional distance function and the Malmquist Luenberger Index, respectively, were used to investigate the impacts of public transport reforms on efficiency and productivity growth both with and without accident costs in Seoul's bus industry. The data cover 2003 and 2005; 2003 is the pre‐reform year and 2005 is the post‐reform year. It is found that the overall productivity increased a little regardless of the consideration of accident costs; it increased a little bit more in the presence of accident costs. This means that the overall productivity at least did not decrease and the level of service in terms of safety was improved following the public transport reforms. Copyright © 2011 John Wiley & Sons, Ltd.
Stochastic modeling of the equilibrium speed–density relationship
Authors: Wang Haizhong , Ni Daiheng , Chen Qian‐Yong , Li Jia
Published: May 2011

As the graphical and mathematical representation of relationships among traffic flow, speed, and density, the fundamental diagram has been the foundation of traffic flow theory and transportation engineering. Underlying the fundamental diagram is the speed–density relationship which was originally documented in Greenshields' seminal work and followed by a variety of equilibrium models over the past 75 years. Observed in these efforts was their deterministic nature striving to pursue two seemingly competing goals: mathematical elegance and empirical accuracy, the former of which is attractive to mathematical modeling of traffic dynamics, and the latter is required if such modeling is meant to be realistic. As a continued effort of such a pursuit, this paper presents a stochastic speed–density model. The motivation is twofold: first, it is desirable to have a model which achieves both goals reasonably and second, the stochastic model can potentially lead to probabilistic traffic flow modeling and prediction which is typically not offered by a macroscopic approach. Copyright © 2011 John Wiley & Sons, Ltd.
Diabetic embryopathy: A role for the epigenome?
Authors: Salbaum J. Michael , Kappen Claudia
Published: May 2011

Embryonic development under adverse conditions, such as maternal diabetes or obesity during pregnancy, constitutes a major risk factor for birth defects, as well as for long‐term health consequences and disease susceptibility in the offspring. While contributions from epigenetic changes have been invoked previously to explain the long‐term changes in terms of developmental programming, we here review how maternal metabolism may directly affect the embryonic epigenome in relationship to teratogenic processes. We consider four epigenetic modalities – DNA methylation, non‐coding RNA, transcription factors, and histone modifications – and their contribution to epigenetic memory, and discuss how epigenomic changes may mediate the altered control of embryonic gene expression brought about by maternal diabetes. In combination, the epigenomic modalities serve to define transcription‐permissive domains of the genome, resulting in distinct epigenomic landscapes in different developmental cell types. We evaluate experimental approaches to characterize the epigenome in adverse pregnancy conditions, highlighting the role of next‐generation sequencing on the technological side, while emphasizing the necessity to study defined cell populations in terms of biologic impact. Finally, we outline the challenges in moving from findings that correlate epigenomics to developmental phenotypes to scenarios that establish teratogenic causality. Birth Defects Research (Part A), 2011. © 2011 Wiley‐Liss, Inc.
Chemischer Angriff auf verpresste Anker und Pfähle
Authors: Rigo Ellen , Unterderweide Klaus
Published: May 2011

Die für die Herstellung und Ausführung von verpressten Pfählen und Ankern relevanten Regelwerke enthalten für die chemischen Angriffe durch Ammonium‐ und Chloridionen keine konkreten Angaben zur Sicherstellung der Dauerhaftigkeit. Dies führt zu großen Unsicherheiten beim Planer. Mit Hilfe numerischer Simulation wurde unter Berücksichtigung hoher Ammonium‐ und Chloridionenkonzentrationen (150 mg/L NH4+, 2500 mg/L Cl–) die Dauerhaftigkeit von mit Zementleimsuspensionen verpressten Pfählen und Ankern berechnet. Dazu wurde das Programm Transreac genutzt, welches den Transport in porösen Materialien mit der Berechnung der chemischen Reaktionen kombiniert. Es konnte gezeigt werden, dass innerhalb von 100 Jahren eine sehr geringe Schädigung (< 1 mm) hinsichtlich des lösenden Angriffes durch Ammonium zu erwarten ist. Für den Stahlkörper der Pfähle ergibt die Prognose weder durch die pH‐Wert‐Absenkung infolge des lösenden Ammoniumangriffes noch hinsichtlich eines kritischen Chloridgehaltes eine bedeutende Korrosion.

Chemical Attack on Grouted Piles and Anchors A Numerical Prognosis of Durability

With the aid of numerical simulation, the degradation of grouted piles and anchors in presence of high amounts of ammoniumions (up to 150 mg/L NH4+) and chloride‐ions (up to 2500 mg/L Cl–) was calculated. Given these environmental conditions it can be assumed, that the grout (cement paste with high w/b) is dissolved by the ammonium‐ions, and the steel of the piles and anchors is corroded because of the reduction of the pH‐value and the attack of the chloride‐ions. Until now there is not one technical rule or a code that deals with the durability of piles and anchors that suffer from chemical attack due to ammonium or chloride or both combined. The designers are not sure how to establish the durability in that case. Due to this fact a numerical calculation of combined chemical attack of ammonium‐ions and chloride‐ions is performed for real environmental conditions. For that purpose the numerical model Transreac is used. This model combines the calculation of transport processes in porous materials and the calculation of chemical reactions. The numerical prognosis shows that the grout is not likely to degenerate within 100 years by reason of the attack of ammonium‐ions. Furthermore, there will not be a considerable consumption of steel in the anchor within the given conditions, neither from the decrease in the pH‐value nor from the chloride.
Ein Beitrag zur Berechnung des kriechbedingten Abbaues von Zwangschnittgrößen aus Setzungen
Authors: Strauß Peter
Published: May 2011

Es wird ein Rechenverfahren vorgestellt, mit dem der kriechbedingte Abbau von Zwangschnittgrößen für beliebige stetig zunehmende Zeit‐Setzungsverläufe berechnet werden kann. Die primäre Zielsetzung des Verfahrens ist die Bestimmung des quantitativen Kriecheinflusses auf die Setzungsschnittgrößen bei der Nachrechnung bestehender Bauwerke mit bekannten Setzungsverläufen. An einem Beispiel wird die Anwendung des Rechenverfahrens auf einen konkreten Nachweisfall gezeigt.

About the Calculation of Creep‐related Relief of Restraint Forces out of Settlements

A method is presented here with which it is possible to calculate the creep‐related relief of restraint forces in sections for any settlement processes that constantly increase over time. The chief aim of this method is to determine the quantitative influence of creep on the settlement forces in sections when analysing existing structures with known settlement patterns. The application of the method of calculation is demonstrated in an example based on a specific case.
Online naphazoline quality control by micellar‐enhanced spectrofluorimetry
Authors: Peralta Cecilia Mariana , Silva Raúl Alejandro , Fernández Liliana Patricia , Masi Adriana Noemí
Published: May 2011

The aim of this study was to develop a method for online spectrofluorimetric quality control of naphazoline (NPZ) in pharmaceuticals and raw drugs. A combination of a flow‐injection analysis (FIA) system with micellar‐enhanced fluorescence detection is presented as a powerful alternative for the rapid and sensitive analysis of naphazoline. Since NPZ shows low native fluorescence, the use of an anionic surfactant, such as sodium dodecyl sulphate (SDS), provides a considerable enhancement of fluorescence intensity and the nature of the technique allows a possible and easy adaptation to a FIA system. Using λexc = 280 nm and λem = 326 nm, a good linear relationship (LOL) was obtained in the range 0.003–10 µg mL−1 with a detection limit (LOD) of 3 × 10−4 µg mL−1 (s/n = 3). Parameters related to the nature of the analytical signal and to the FIA manifold were optimized. Satisfactory recoveries were obtained in the analysis of commercial pharmaceutical formulations. The proposed method is simple, accurate and allows for high‐speed sampling and considerably shorter analysis times. In addition, it requires inexpensive equipment and reagents and has easy operational conditions and no side effects, thus avoiding environmental pollution through toxic waste. Copyright © 2011 John Wiley & Sons, Ltd.
Study of the interaction between ofloxacin and human serum albumin by spectroscopic methods
Authors: Varlan Aurica , Ionescu Sorana , Hillebrand Mihaela
Published: May 2011

The binding of ofloxacin (OFLX) to human serum albumin (HSA) was investigated by fluorescence and circular dichroism (CD) techniques. The binding parameters have been evaluated by a fluorescence quenching method. Competitive binding measurements were performed in the presence of warfarin and ibuprofen and suggest binding to the warfarin site I of HSA. The distance r between donor (HSA) and acceptor (OFLX) was estimated according to the Forster's theory of non‐radiatiative energy transfer. CD spectra revealed that the binding of OFLX to HSA induced conformational changes in HSA. Molecular docking was performed and shows that for the lowest energy complex OFLX is located in site I of HSA, which correlate to the competitive binding experiments. Copyright © 2011 John Wiley & Sons, Ltd.
Characterization of secondary amide peptide bond isomerization: Thermodynamics and kinetics from 2D NMR spectroscopy
Authors: Zhang Jin , Germann Markus W.
Published: May 2011

Secondary amide cis peptide bonds are of even lower abundance than the cis tertiary amide bonds of prolines, yet they are of biochemical importance. Using 2D NMR exchange spectroscopy (EXSY) we investigated the formation of cis peptide bonds in several oligopeptides: Ac‐G‐G‐G‐NH2, Ac‐I‐G‐G‐NH2, Ac‐I‐G‐G‐N‐NH2 and its cyclic form: I‐G‐G‐N in dimethylsulfoxide (DMSO). From the NMR studies, using the amide protons as monitors, an occurrence of 0.13–0.23% of cis bonds was obtained at 296 K. The rate constants for the trans to cis conversion determined from 2D EXSY spectroscopy were 4–9 × 10−3 s−1. Multiple minor conformations were detected for most peptide bonds. From their thermodynamic and kinetic properties the cis isomers are distinguished from minor trans isomers that appear because of an adjacent cis peptide bond. Solvent and sequence effects were investigated utilizing N‐methylacetamide (NMA) and various peptides, which revealed a unique enthalpy profile in DMSO. The cyclization of a tetrapeptide resulted in greatly lowered cis populations and slower isomerization rates compared to its linear counterpart, further highlighting the impact of structural constraints. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 755‐762, 2011.
An evaluation of Poisson–Boltzmann electrostatic free energy calculations through comparison with experimental mutagenesis data
Authors: Gorham Ronald D. , Kieslich Chris A. , Nichols Aaron , Sausman Noriko U. , Foronda Marisse , Morikis Dimitrios
Published: May 2011

For systems involving highly and oppositely charged proteins, electrostatic forces dominate association and contribute to biomolecular complex stability. Using experimental or theoretical alanine‐scanning mutagenesis, it is possible to elucidate the contribution of individual ionizable amino acids to protein association. We evaluated our electrostatic free energy calculations by comparing calculated and experimental data for alanine mutants of five protein complexes. We calculated Poisson–Boltzmann electrostatic free energies based on a thermodynamic cycle, which incorporates association in a reference (Coulombic) and solvated (solution) state, as well as solvation effects. We observe that Coulombic and solvation free energy values correlate with experimental data in highly and oppositely charged systems, but not in systems comprised of similarly charged proteins. We also observe that correlation between solution and experimental free energies is dependent on dielectric coefficient selection for the protein interior. Free energy correlations improve as protein dielectric coefficient increases, suggesting that the protein interior experiences moderate dielectric screening, despite being shielded from solvent. We propose that higher dielectric coefficients may be necessary to more accurately predict protein–protein association. Additionally, our data suggest that Coulombic potential calculations alone may be sufficient to predict relative binding of protein mutants. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 746‐754, 2011.
Spatiotemporal modeling and analysis of transient gene delivery
Authors: Jandt Uwe , Shao Shi , Wirth Manfred , Zeng An‐Ping
Published: May 2011

A quantitative and mechanistic understanding of intracellular transport processes in eukaroytic cells during transient transfection is an important prerequisite for the systematic and specific optimization of transient gene expression procedures for pharmaceutic and industrial protein production. There is evidence that intracellular transport processes during gene delivery and their regulation may have significant influence on the transfection efficiency. This contribution describes a compartmented, spatiotemporally resolved and stochastic modeling approach that describes intracellular transport processes responsible for gene delivery during transient transfection. It enables a detailed prediction and analysis and identification of potential bottlenecks. This model is currently being adapted to a model cell line, HEK293s. The simulated results are compared with experimental quantitative polymerase chain reaction (qPCR) data and confocal imaging data obtained with transfected and stained HEK293 cells. Global parameter estimation is performed to qPCR data based on two different novel plasmid constructs in order to identify candidates for plasmid‐specific transport parameter variations. The influence of the specific property of HEK293 cells to grow in clusters is investigated and the impact of active microtubule transport depending on cell morphology and clustering is examined. A general sensitivity analysis allows for the identification of the sensitive parameters. Biotechnol. Bioeng. 2011;108:2205–2217. © 2011 Wiley Periodicals, Inc.
A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies
Authors: Kim Minsoo , O'Callaghan Peter M. , Droms Kurt A. , James David C.
Published: May 2011

One of the most significant problems in industrial bioprocessing of recombinant proteins using engineered mammalian cells is the phenomenon of cell line instability, where a production cell line suffers a loss of specific productivity (qP). This phenomenon occurs with unpredictable kinetics and has been widely observed in Chinese hamster ovary (CHO) cell lines and with all commonly used gene expression systems. The underlying causes (both genetic and physiological) and the precise molecular mechanisms underpinning cell line instability have yet to be fully elucidated, although recombinant gene silencing and loss of recombinant gene copies have been shown to cause qP loss. In this work we have investigated the molecular mechanisms underpinning qP instability over long‐term sub‐culture in CHO cell lines producing recombinant IgG1 and IgG2 monoclonal antibodies (Mab's). We demonstrate that production instability derives from two primary mechanisms: (i) epigenetic—methylation‐induced transcriptional silencing of the CMV promoter driving Mab gene transcription and (ii) genetic—progressive loss of recombinant Mab gene copies in a proliferating CHO cell population. We suggest that qP decline resulting from loss of recombinant genes is a consequence of the inherent genetic instability of recombinant CHO cell lines. Biotechnol. Bioeng. 2011;108: 2434–2446. © 2011 Wiley Periodicals, Inc.
Microprinted feeder cells guide embryonic stem cell fate
Authors: Tavana Hossein , Mosadegh Bobak , Zamankhan Parsa , Grotberg James B. , Takayama Shuichi
Published: May 2011

We introduce a non‐contact approach to microprint multiple types of feeder cells in a microarray format using immiscible aqueous solutions of two biopolymers. Droplets of cell suspension in the denser aqueous phase are printed on a substrate residing within a bath of the immersion aqueous phase. Due to their affinity to the denser phase, cells remain localized within the drops and adhere to regions of the substrate underneath the drops. We show the utility of this technology for creating duplex heterocellular stem cell niches by printing two different support cell types on a gel surface and overlaying them with mouse embryonic stem cells (mESCs). As desired, the type of printed support cell spatially direct the fate of overlaid mESCs. Interestingly, we found that interspaced mESCs colonies on differentiation‐inducing feeder cells show enhanced neuronal differentiation and give rise to dense networks of neurons. This cell printing technology provides unprecedented capabilities to efficiently identify the role of various feeder cells in guiding the fate of stem cells. Biotechnol. Bioeng. 2011;108: 2509–2516. © 2011 Wiley Periodicals, Inc.
Clonal analysis of the proliferation potential of human bone marrow mesenchymal stem cells as a function of potency
Authors: Russell Katie C. , Lacey Michelle R. , Gilliam Jennifer K. , Tucker H. Alan , Phinney Donald G. , O'Connor Kim C.
Published: May 2011

Human mesenchymal stem cells (MSCs) from bone marrow are a heterogeneous ensemble of progenitors and lineage‐committed cells, with a broad range of regenerative properties. Ex vivo expansion to produce sufficient quantities of MSCs is essential for most therapeutic applications. The present study resolves the relationship between proliferation potential of MSCs and their potency. Clonal analysis generated single‐cell derived colonies of MSCs that were classified according to their trilineage potential to exhibit adipo‐ (A), chondro‐ (C), and osteogenesis (O) as a measure of potency. Multipotent OAC clones were highly proliferative with colony‐forming efficiencies that ranged from 35% to 90%; whereas, O clones formed colonies with an efficiency of 5% or less (P < 0.01). Similar trends were evident during ex vivo expansion: for example, the median specific growth rate was 0.85 day−1 (20 h doubling time) for cultures inoculated with OAC clones and was 5‐fold less for inocula of O clones (P < 0.01). OA and OC clones had similar proliferation potentials. More than 75% of cells in subconfluent cultures inoculated with O clones stained positive for senescence‐associated β‐galactosidase activity vs. less than 10% for OAC clones (P < 0.001). Apoptotic cells were in the minority for all potency groups. Preliminary data generated during clonal analysis suggest that osteogenic potential of MSCs to produce mineralized matrix is a function of potency, as well. These results are discussed in the context of the preparation of efficacious MSC therapies by ex vivo expansion. Biotechnol. Bioeng. 2011;108: 2716–2726. © 2011 Wiley Periodicals, Inc.
Effect of O2:CO2 ratio on the primary metabolism of Chlamydomonas reinhardtii
Authors: Kliphuis Anna M.J. , Martens Dirk E. , Janssen Marcel , Wijffels René H.
Published: May 2011

High oxygen:carbon dioxide ratios may have a negative effect on growth and productivity of microalgae. To investigate the effect of O2 and CO2 concentrations and the ratio between these on the metabolism of Chlamydomonas reinhardtii we performed turbidostat experiments at different O2:CO2 ratios. These experiments showed that elevated O2 concentrations and the corresponding increase in the ratio of O2:CO2 common in photobioreactors led to a reduction of growth and biomass yield on light with 20–30%. This is most probably related to the oxygenase activity of Rubisco and the resulting process of photorespiration. Using metabolic flux modeling with measured rates for each experiment we were able to quantify the ratio of the oxygenase reaction to the carboxylase reaction of Rubisco and could demonstrate that photorespiration indeed can cause the reduction in biomass yield on light. The calculated ratio of the oxygenase reaction to the carboxylase reaction was 16.6% and 20.5% for air with 2% CO2 and 1% CO2, respectively. Thus photorespiration has a significant impact on the biomass yield on light already at conditions common in photobioreactors (air with 2% CO2). Biotechnol. Bioeng. 2011;108: 2390–2402. © 2011 Wiley Periodicals, Inc.
Predicting mab product yields from cultivation media components, using near‐infrared and 2D‐fluorescence spectroscopies
Authors: Jose Gledson E. , Folque Francisca , Menezes Jose C. , Werz Silke , Strauss Ulrike , Hakemeyer Christian
Published: May 2011

The yield of monoclonal antibody (Mab) production processes depends on media formulation, inocula quality, and process conditions. As in industrial processes tight cultivation conditions are used, and inocula quality and viable cell densities are controlled to reasonable levels, media formulation and raw materials lot‐to‐lot variability in quality will have, in those circumstances, the highest impact on process performance. In the particular Mab process studied, two different raw materials were used: a complex carbon and nitrogen source made of specific peptones and defined chemical media containing multiple components. Using different spectroscopy techniques for each of the raw material types, it was concluded that for the complex peptone‐based ingredient, near‐infrared (NIR) spectroscopy was more capable of capturing lot‐to‐lot variability. For the chemically defined media containing fluorophores, two‐dimensional (2D)‐fluorescence spectroscopy was more capable of capturing lot‐to‐lot variability. Because in Mab cultivation processes both types of raw materials are used, combining the NIR and 2D‐fluorescence spectra for each of the media components enabled predictive models for yield to be developed that out‐performed any other model involving either one raw material alone, or only one type of spectroscopic tool for both raw materials. For each particular raw material, the capability of each spectroscopy to detect lot‐to‐lot differences was demonstrated after spectra preprocessing and specific wavelength regions selection. The work described and the findings reported here open up several possibilities that could be used to feed‐forward control the process. These include, for example, enabling specific actions to be taken regarding media formulation with particular lots, and all types of predictive control actions aimed at increasing batch‐to‐batch yield and product quality consistency at harvest. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Process and economic evaluation for monoclonal antibody purification using a membrane‐only process
Authors: Varadaraju Hemanthram , Schneiderman Steven , Zhang Lifeng , Fong Hao , Menkhaus Todd J.
Published: May 2011

In recent years, the market for therapeutic monoclonal antibodies (mAb) has grown exponentially, and with this there has been a desire to reduce the costs associated with production and purification of these high‐value biological products. A typical mAb purification process involves three adsorption/chromatography steps [protein A, ion exchange (IEX), and hydrophobic interaction (HIC)], along with ultrafiltration, nanofiltration, and microfiltration. With the development of membrane adsorption/chromatography as a viable alternative to traditional pack bed systems, the opportunity exists to complete the entire downstream purification process using only membrane operations. In this study, the process simulation tool SuperPro Designer was used to evaluate the application of recently developed ultra‐high capacity electrospun nanofibrous adsorption membranes as a replacement for conventional chromatographic media in the downstream mAb production process. The simulation showed that nanofibrous adsorption membranes in place of the three packed bed chromatography steps reduced the required volume of protein A, IEX, and HIC adsorptive medium by 25, 80, and 80%, respectively. In addition, the membrane‐only process reduced the downstream processing time by 50%, decreased the number of labor hours associated with the purification steps by 40%, generated 40% less aqueous waste, and reduced the overall downstream process operating expenses per unit product by 23%. There were also significant savings in facility construction costs and the price of fixed equipment required for separations. With these savings not only is the membrane‐only process economically competitive with the traditional packed bed operations, but it offers the possibility of moving toward more disposable process.© 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Assembly of horseradish peroxidase within supported cationic bilayers
Authors: Leão‐Silva Ana Carolina , Naves Alliny F. , Pereira Edla M. A. , Petri Denise F. S. , Carmona‐Ribeiro Ana M.
Published: May 2011

The interaction between horseradish peroxidase (HRP) and dioctadecyldimethylammonium bromide (DODAB) bilayers supported on polystyrene microspheres (PSS) or on flat silicon wafers was evaluated from the following techniques: (1) dynamic light‐scattering for determining size distributions, zeta‐potentials and polydispersities for dispersions; (2) spectrophotometric determination of HRP concentration in supernatants of centrifuged mixtures; (3) in situ ellipsometry for mean thickness of deposited layers on wafers; (4) kinetics of product appearance for oxidation of 2,2′‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid by H2O2 in presence of free or immobilized enzyme. HRP incorporation (3.0 mg/m2) did not alter mean diameter and zeta‐potential of PSS/DODAB particles but reduced enzyme activity by 50%, though activity persisted after several rinsing steps. In situ ellipsometry could not detect any HRP layer on top of the DODAB bilayer. HRP insertion in the bilayer core explained all results for both systems. Useful biotechnological applications are anticipated for such assemblies. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
High‐pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: Refolding optimization, and feasibility assessment
Authors: Cothran Amber , John Richard J. St. , Schmelzer Charles H. , Pizarro Shelly A.
Published: May 2011

High‐pressure has been established as an effective technique for refolding proteins at high concentrations. In this study, high hydrostatic pressure (1–3 kbar) was utilized to refold a homodimeric protein from inclusion bodies and the process was evaluated for large‐scale manufacturing feasibility. This research focused on increasing protein concentration while maximizing yield and product quality. Refolding yields of 29–42% were achieved in the absence of urea at 2 kbar and at a protein concentration of 6 g/L. Optimization of the refolding buffer composition via multivariate design of experiments and other process parameters such as refolding pressure, gas sparging, and time under pressure are discussed. Although high‐pressure refolding can be considered a viable technology for manufacturing if the gains are clearly identified, in this particular case, the benefits that the high‐pressure technology offers do not compensate for the drawbacks of implementing new equipment in an existing facility, and unknown impact of scale‐up for this molecule. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Protein adsorption and transport in cation exchangers with a rigid backbone matrix with and without polymeric surface extenders
Authors: Almodóvar Ernie X. Pérez , Tao Yinying , Carta Giorgio
Published: May 2011

We compare the properties and protein adsorption characteristics of two polymeric cation exchangers: UNOsphere S, which has an open macroporous architecture, and Nuvia S, which is based on a very similar backbone matrix but contains sulfonated polymeric surface extenders. A monoclonal IgG and lysozyme were used as model adsorbates. The characteristic pore sizes, determined by inverse size exclusion chromatography, were about 140 nm for UNOsphere S, and only about 10 nm for Nuvia S, indicating that the polymeric extenders occupy a substantial portion of the base matrix pores. Greater exclusion limits were found for Nuvia S in 1 M NaCl and for a similar matrix containing uncharged surface extenders, suggesting that the polymeric extenders collapse partially at high ionic strength or when they are uncharged. Large equilibrium binding capacities were obtained for Nuvia S, approaching 320 ± 10 mg/mL of particle volume for both proteins in comparison with the UNOsphere S values of 170 ± 10 and 120 ± 10 mg/mL for lysozyme and IgG, respectively. Much higher adsorption rates were also found for Nuvia S, and the rate was nearly independent of protein concentration in solution. Confocal laser scanning microscopy showed very sharp intraparticle protein concentration profiles for UNOsphere S, consistent with a pore diffusion mechanism but diffuse concentration profiles for Nuvia S, consistent with a solid diffusion mechanism. The improved capacity and transport afforded by the polymeric extenders provide substantial potential benefits for bioprocess applications without sacrificing the desirable flow properties of the backbone matrix. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Assessment of the manufacturability of Escherichia coli high cell density fermentations
Authors: Perez‐Pardo M. A. , Ali S. , Balasundaram B. , Mannall G. J. , Baganz F. , Bracewell D. G.
Published: May 2011

The physical and biological conditions of the host cell obtained at the end of fermentation influences subsequent downstream processing unit operations. The ability to monitor these characteristics is central to the improvement of biopharmaceutical manufacture. In this study, we have used a combination of techniques such as adaptive focus acoustics (AFA) and ultra scale‐down (USD) centrifugation that utilize milliliter quantities of sample to obtain an insight into the interaction between cells from the upstream process and initial downstream unit operations. This is achieved primarily through an assessment of cell strength and its impact on large‐scale disc stack centrifugation performance, measuring critical attributes such as viscosity and particle size distribution. An Escherichia coli fed‐batch fermentation expressing antibody fragments in the periplasm was used as a model system representative of current manufacturing challenges. The weakening of cell strength during cultivation time, detected through increased micronization and viscosity, resulted in a 2.6‐fold increase in product release rates from the cell (as measured by AFA) and approximately fourfold decrease in clarification performance (as measured by USD centrifugation). The information obtained allows for informed harvest point decisions accounting for both product leakages during fermentation and potential losses during primary recovery. The clarification performance results were verified at pilot scale. The use of these technologies forms a route to the process understanding needed to tailor the host cell and upstream process to the product and downstream process, critical to the implementation of quality‐by‐design principles. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture
Authors: Goudar Chetan T. , Piret James M. , Konstantinov Konstantinov B.
Published: May 2011

We present robust methods for online estimation of cell specific oxygen uptake and carbon dioxide production rates (qO2 and qCO2, respectively) during perfusion cultivation of mammalian cells. Perfusion system gas and liquid phase mass balance expressions for oxygen and carbon dioxide were used to estimate qO2, qCO2 and the respiratory quotient (RQ) for Chinese hamster ovary (CHO) cells in perfusion culture over 12 steady states with varying dissolved oxygen (DO), pH, and temperature set points. Under standard conditions (DO = 50%, pH = 6.8, T = 36.5°C), qO2 and qCO2 ranges were 5.14–5.77 and 5.31–6.36 pmol/cell day, respectively, resulting in RQ values of 0.98–1.14. Changes to DO had a slight reducing effect on respiration rates with qO2 and qCO2 values of 4.64 and 5.47, respectively, at DO = 20% and 4.57 and 5.12 at DO = 100%. Respiration rates were lower at low pH with qO2 and qCO2 values of 4.07 and 4.15 pmol/cell day at pH = 6.6 and 4.98 and 5.36 pmol/cell day at pH = 7. Temperature also impacted respiration rates with respective qO2 and qCO2 values of 3.97 and 4.02 pmol/cell day at 30.5°C and 5.53 and 6.25 pmol/cell day at 37.5°C. Despite these changes in qO2 and qCO2 values, the RQ values in this study ranged from 0.98 to 1.23 suggesting that RQ was close to unity. Real‐time qO2 and qCO2 estimates obtained using the approach presented in this study provide additional quantitative information on cell physiology both during bioprocess development and commercial biotherapeutic manufacturing. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Development of a mathematical model for evaluating the dynamics of normal and apoptotic Chinese hamster ovary cells
Authors: Naderi Saeideh , Meshram Mukesh , Wei Catherine , McConkey Brendan , Ingalls Brian , Budman Hector , Scharer Jeno
Published: May 2011

A metabolic flux based methodology was developed for modeling the metabolism of a Chinese hamster ovary cell line. The elimination of insignificant fluxes resulted in a simplified metabolic network which was the basis for modeling the significant metabolites. Employing kinetic rate expressions for growing and non‐growing subpopulations, a logistic model was developed for cell growth and dynamic models were formulated to describe culture composition and monoclonal antibody (MAb) secretion. The model was validated for a range of nutrient concentrations. Good agreement was obtained between model predictions and experimental data. The ultimate goal of this study is to establish a comprehensive dynamic model which may be used for model‐based optimization of the cell culture for MAb production in both batch and fed‐batch systems. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Synthesis and performance of 3D‐Megaporous structures for enzyme immobilization and protein capture
Authors: Bibi Noor Shad , Gavara Poondi Rajesh , Espinosa Silvia L. Soto , Grasselli Mariano , Fernández‐Lahore Marcelo
Published: May 2011

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross‐linker agent. Ion‐exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation‐induced grafting. The total ionic capacity of such systems was 1.5 mmol H+/g, 1.4 mmol H+/g, and 17 mmol H+/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ≅7.2 mg bovine serum albumin/g, ≅7.4 hen egg‐white lysozyme (HEWL) mg/g, and ≅108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow‐through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D‐megaporous materials described in this work. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Effects of temperature and pH on growth and antioxidant content of the microalga Scenedesmus obliquus
Authors: Guedes A. Catarina , Amaro Helena M. , Pereira Ricardo D. , Malcata F. Xavier
Published: May 2011

Reactive forms of oxygen can damage DNA (among other molecules), thus triggering, e.g., atherogenesis and carcinogenesis. However, such dietary antioxidants as lutein and β‐carotene can effectively inactivate them; these compounds were found to high levels in a novel strain (M2‐1) of the microalga Scenedesmus obliquus. The independent and combined effects of pH and temperature on its rates of growth and production of antioxidants were experimentally assessed, via a full factorial experimental design; the effects of each parameter independently, and of their interactions were accordingly quantified by ANOVA. Our results indicated that temperature plays a more important role on the maximum specific growth rate than pH; in terms of antioxidant content, pH and, to a lesser extent, temperature also have relevant effects. Consequently, the highest rate of biomass specific growth (0.294 ± 0.013 day−1) and biomass productivity (0.837 ± 0.054 mg L−1 day−1) were associated with relatively low pH (6) and relatively high temperature (30°C). Conversely, the antioxidant production rate increased with pH; hence, the highest productivity (0.638 mg L−1 day−1) was attained at pH 8 and 30°C. At the best operating conditions for antioxidant content, the levels of lutein and β‐carotene were 203.57 ± 1.41 and 18.20 ± 0.33 mg mL−1, respectively; the maximum production of either one occurred at the early exponential phase. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011