bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel system for evaluation of drug mixtures for potential efficacy in treating multidrug resistant cancers
Authors: Tatosian Daniel A. , Shuler Michael L.
Published: December 2008

Multidrug resistant (MDR) cancer is difficult to treat. Chemicals that are effective MDR modulators have never exited clinical trials as FDA approved products due to side effects. It has been hypothesized that using a combination of chemotherapeutics with a mixture of MDR modulators (each with different side effects) may lead to useful treatment strategies. Because the experimental space for combination treatments can be large, this space may be impracticable to explore using animal studies. Here we describe an in vitro system based on microfabrication and cell culture that can potentially be used to explore large experimental spaces efficiently. The Microscale Cell Culture Analog (µCCA) concept mimics the body's response using interconnected compartments that represent various tissues or organs. A µCCA is based on the structure of an appropriate physiologically based pharmacokinetic (PBPK) model and emulates the body's dynamic response to exposure to various drugs and chemicals. For this problem we have chosen a µCCA with living cells representing the liver (HepG2/C3A), bone marrow (MEG‐01), uterine cancer (MES‐SA), and a MDR variant of uterine cancer (MES‐SA/DX‐5). In proof of concept experiments we found in 24 h “acute” exposures and 72 h treatments that the µCCA system predicts combining the chemotherapeutic, doxorubicin, with cyclosporine and nicardipine, as MDR modulators will have greater efficacy than using doxorubicin by itself or with either modulator alone. This combined strategy is selective in inhibiting MES‐SA/DX‐5 cell proliferation and may prove to be advantageous in vivo by specifically targeting MDR cancer with acceptable side‐effects. This cell specific synergy was not observed in traditional 96‐well plate assays. By combining the µCCA with a PBPK model, appropriate drug doses and area under the curve exposure for in vivo trials can be extrapolated directly from the results obtained with this device. This device and approach should be useful in screening potential drug/modulator combinations to determine candidate treatments for MDR cancer. Indeed this approach may be useful for in vitro evaluation of human response to a wide range of exposures to mixtures of chemicals or drugs. Biotechnol. Bioeng. 2009;103: 187–198. © 2008 Wiley Periodicals, Inc.
Monitoring of biomass composition from microbiological sources by means of FT‐IR spectroscopy
Authors: Pistorius Arthur M.A. , DeGrip Willem J. , Egorova‐Zachernyuk Tatjana A.
Published: December 2008

An FT‐IR spectroscopic method was developed for the simultaneous quantitative analysis of biomacromolecular components in biomass, originating from various microbiological sources. For the determination of protein, lipid and carbohydrate content, creatine phosphokinase, egg phosphatidyl choline and starch hydrolysate were chosen as external standards. This selection was based on spectral similarity and ease of availability. Protein content was based on the area under the amide II band profile around 1,545 cm−1. Because of the heterogeneous lipid composition in the different species, lipid content was determined using integration over the CH stretching vibrational population between 2,984 and 2,780 cm−1. Carbohydrate content was determined using integration over a CO and COC stretching band area between 1,180 and 1,133 cm−1. Linear regression analysis provided three calibration lines, according to which biomasses from ten species were analyzed. This approach showed good intra‐batch reproducibility. With this method we could demonstrate good reproducibility between batches of the same species with similar growth conditions while large differences in biomass composition were observed between the various species. Protein content as determined by FT‐IR spectroscopy compared well with the results obtained from elemental analysis. Biotechnol. Bioeng. 2009;103: 123–129. © 2008 Wiley Periodicals, Inc.
Automation of the acclimation phase in a sequencing batch reactor using dissolved oxygen regulation
Authors: Vargas Alejandro , Buitrón Germán
Published: December 2008

In this work, an automatic strategy for acclimation of biomass to toxic wastewater biodegradation in a sequencing batch reactor (SBR) is proposed and evaluated. It is based on monitoring the airflow needs for regulating the dissolved oxygen concentration at a desired set point. The regulation is achieved by using a PID controller with an input/output linearizing feedforward part, using the airflow rate as input variable. A simple algorithm to detect the end of the reaction phase using this manipulated signal is proposed. The strategy was tested on a laboratory bioreactor using 4‐chlorophenol as model toxic compound and two inocula from different sources. The results show that it is a viable alternative for automatic acclimation of biomass for toxic wastewater treatment in a SBR. Furthermore, the strategy is also suitable for the operation of the reactor after acclimation.
A methodology for the comparative evaluation of alternative bioseparation technologies
Authors: Tran Richard , Zhou Yuhong , Lacki Karol M. , Titchener‐Hooker Nigel J.
Published: December 2008

Advances in upstream technologies and growing commercial demand have led to cell culture processes of ever larger volumes and expressing at higher product titers. This has increased the burden on downstream processing. Concerns regarding the capacity limitations of packed‐bed chromatography have led process engineers to begin investigating new bioseparation techniques that may be considered as “alternatives” to chromatography, and which could potentially offer higher processing capacities but at a lower cost. With the wide range of alternatives, which are currently available, each with their own strengths and inherent limitations, coupled with the time pressures associated with process development, the challenge for process engineers is to determine which technologies are most worth investigating. This article presents a methodology based on a multiattribute decision making (MADM) analysis approach, utilizing both quantitative and qualitative data, which can be used to determine the “industrial attractiveness” of bioseparation technologies, accounting for trade‐offs between their strengths and weaknesses. By including packed‐bed chromatography in the analysis as a reference point, it was possible to determine the alternatives, which show the most promise for use in large‐scale manufacturing processes. The results of this analysis show that although the majority of alternative techniques offer certain advantages over conventional packed‐bed chromatography, their attractiveness overall means that currently none of these technologies may be considered as viable alternatives to chromatography. The methodology introduced in this study may be used to gain significant quantitative insight as to the key areas in which improvements are required for each technique, and thus may be used as a tool to aid in further technological development.
External modulation of HT‐1080 human fibrosarcoma cells improves urokinase production
Authors: Khaparde Shilpa S. , Roychoudhury Pradip K. , Gomes James , Mukhopadhyay Asok
Published: December 2008

Urokinase was produced in a hollow fiber reactor using HT‐1080 human fibrosarcoma cells. External modulation comprised replenishing of the medium in the extracapillary space, reducing the serum concentration in the extracapillary space from 10% to 2% and increasing flow rate of the circulating medium in the intracapillary space from 20 to 80 mL/min, each according to a specific protocol. More than sixfold increase was observed in the cumulative urokinase production for two and three medium replenishing modulations of the extracapillary space. After 15 days of continuous operation, the highest cumulative urokinase obtained was 1.63 × 106 PU/mL. SDS‐PAGE and zymogram study established that the urokinase obtained was in the high molecular weight range of 54 kDa. The effect of external modulation on cumulative urokinase production was visualized as trajectories with respect to the ratio of lactic acid production rate (LPR) to the glucose uptake rate (GUR). The collective external modulation data showed two separate physiological regions in the cumulative urokinase vs. LPR/GUR plane. The HT‐1080 cells exhibited two distinct morphologies in these regions that may be related to acidosis and metastasis. These regions also correspond to low and high urokinase productivity.
Novel methods for storage stability and release of Bacillus spores
Authors: Sorokulova Iryna B. , Krumnow April A. , Pathirana Suram , Mandell Arnold J. , Vodyanoy Vitaly
Published: December 2008

Bacillus subtilis spores were immobilized in activated charcoal and tapioca and filled with acacia gum. These formulations were tested for spore stability during storage at temperatures ranging from 40°C to 90°C and for bacterial release. Thermodynamic analysis showed that immobilization of spores in acacia gum significantly increased their viability compared with unprotected spores. The viability was further increased when suspensions of spores in acacia gum were added to granules of charcoal and tapioca. The number of the spores released after storage was also increased when spores were treated with acacia gum prior to immobilization in tapioca and charcoal. Formulations of Bacillus spores with acacia gum and porous carriers (charcoal and tapioca) prolong the anticipated shelf‐life of spores even under ambient temperature and provide slow and steady bacterial release consistent with their high viability.
In situ extraction of polar product of whole cell microbial transformation with polyethylene glycol‐induced cloud point system
Authors: Wang Zhilong , Xu Jian‐He , Zhang Wenzhi , Zhuang Baohua , Qi Hanshi
Published: December 2008

A novel polyethylene glycol‐induced cloud point system (PEG‐CPS) was developed for in situ extraction of moderate polar product by setting a microbial transformation of benzaldehyde into L‐phenylacetylcarbinol (L‐PAC) with Saccharomyces cerevisiae (baker's yeast) as a model reaction. The biocompatibility of the microorganism in PEG‐CPS was comparatively studied with a series of water‐organic solvent two‐phase partitioning systems. The tolerance of microorganism to the toxic substrate benzaldehyde was increased and the moderate polar product L‐PAC was extracted into the surfactant‐rich phase in the PEG‐CPS. The novel PEG‐CPS fills the gap of in situ extraction of polar product in microbial transformation left by water‐organic solvent two‐phase partitioning system. At the same time, the application of PEG‐CPS in a microbial transformation also avoids expensive solvent when compared with that of aqueous two‐phase system or CPS.
Recovery of proteins from corn and soybean extracts by membrane adsorption
Authors: Menkhaus Todd J. , Roseland Jane
Published: December 2008

Efficient separation strategies for the recovery of high‐value proteins (native or recombinant) from plant agriculture are an important aspect of many different processes, from biopharmaceuticals to byproduct recovery during biofuel production. Here we report the use of membrane adsorption for the recovery of proteins from soybean and corn extracts, and compare the results with packed bed adsorption. Two alternative operating modes were investigated, a flowthrough strategy and a bind and elute method. Overall, membrane adsorption provided faster throughput, and had equal or slightly higher dynamic binding capacities compared with resin beads, without compromising yield and purity of the chosen target. Soybean was found to be an ideal plant host when capturing native protein on an anion exchange medium. This provided an opportunity to capture a large percentage (>80%) of native protein as the product, and/or allowed for elevated enrichment factors (>20) during purification of a recombinant target with pI > 7.0, using a flowthrough approach. On the other hand, for corn, a single ion‐exchange step was not able to capture more than 60% of the native protein. However, the bind and elute method with corn as the host for a recombinant product allowed for higher enrichment factors compared to soybean. In all cases, the concentration of a recombinant protein (as dictated by expression level) was found to play a significant role in the level of dynamic binding capacity, with higher concentration leading to elevated capacity. Likewise, a higher concentration of competing proteins was shown to decrease the overall capacity of a recombinant target.
pH‐insensitive glucose indicators
Authors: Garrett Jared R. , Wu Xinxin , Jin Sha , Ye Kaiming
Published: December 2008

There is an urgent need for developing a biosensor that can real‐time and noninvasively determine glucose concentration within living cells. In our previous study, we have engineered a glucose indicator protein (GIP) that can provide continuous glucose monitoring through a conformation change‐induced Förster resonance‐energy transfer measurement. Because of the pH‐sensitivity of the fluorescent proteins used in the GIP construction, the GIP made from these fluorescent proteins is less tolerant to a pH change, especially to the acidic environment. It has been well documented that intracellular pH does not always remain the same, and it fluctuates in metabolism and other cellular activities and also differs between cellular compartments. To address these issues, we developed a GIP that can tolerate to pH change. This GIP was constructed by flanking a glucose binding protein with a cyan fluorescent protein and a pH‐insensitive yellow fluorescent protein. Our experimental results indicated that the new GIP is more tolerant to pH change. The glucose response of this new GIP kept almost unchanged from pH 7.3 to 5.3, suggesting its capability of tolerating to acidic environment. This capability is desirable for intracellular glucose measurement.
Developing an industrial artemisinic acid fermentation process to support the cost‐effective production of antimalarial artemisinin‐based combination therapies
Authors: Lenihan Jacob R. , Tsuruta Hiroko , Diola Don , Renninger Neil S. , Regentin Rika
Published: December 2008

Artemisinin‐based combination therapies (ACTs) are currently unaffordable for many of the people who need them most. A major cost component of ACTs is the plant‐derived artemisinin. A fermentation process for a precursor to artemisinin might provide a viable second source to stabilize the artemisinin supply and therefore reduce price. The heterologous production of artemisinic acid, an artemisinin precursor, by Saccharomyces cerevisiae was improved 25‐fold from a 100 mg/L flask process to a 2.5 g/L process in bioreactors. A defined medium fed‐batch process with galactose as the carbon source and inducer was developed, with titers of 1.3 g/L. In this strain ERG9 was controlled with promoter Pmet3 so that methionine repressed the sterol biosynthesis pathway and increased precursor availability for artemisinic acid biosynthesis. Addition of methionine to the process increased artemisinic acid titers to 1.8 g/L. A dissolved oxygen‐stat algorithm was developed, which simultaneously controlled the agitation and feed pump. This improved process control and increased titers to 2.5 g/L.
Three‐dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles
Authors: Nilsang Suthasinee , Nehru Vishal , Plieva Fatima M. , Nandakumar Kutty Selva , Rakshit Sudip Kumar , Holmdahl Rikard , Mattiasson Bo , Kumar Ashok
Published: December 2008

Cell proliferation and long‐term production of monoclonal antibody IgG2b by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed‐bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7 × 9 mm2). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three‐dimensional (3D) cultivation using gelatin as a substrate layer. Optimizing productivity by hybridoma cell relies on understanding regulation of antibody production. In this study, the behavior of M2139 cells in two‐dimensional cultures on multiwell plate surfaces was also investigated. The effect of three different medium such as basal medium Dulbecco's modified Eagle's medium (D‐MEM) containing L‐glutamine or L‐glutamine + 2 mM α‐ketoglutarate or L‐alanyl‐glutamine (GlutaMAX™) was studied prior to its use in 3D cultivation. The kinetics of cell growth in basal medium containing L‐glutamine + α‐ketoglutarate was similar to cells grown on GlutaMAX containing medium, whereas D‐MEM containing L‐glutamine showed lower productivity. With the maximal viable cell density (6.85 × 106 cells mL−1) and highest specific mAb production rate (3.9 μg mL−1 10−4 viable cell day−1), D‐MEM‐GlutaMAX was further selected for 3D cultivation. Cells in MGPs were able to grow and secrete antibody for 30 days in packed‐bed batch reactor, before a fresh medium reservoir was replaced. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long‐term monoclonal antibody production.
Forecasting for fermentation operational decision making
Authors: Montague Gary A. , Martin Elaine B. , O'Malley Christopher J.
Published: December 2008

An awareness of the likely future behavior of a batch or a fed‐batch fermentation process is valuable information that can be exploited to improve product consistency and maximize profitability. For example, by making operational policy changes in a feedforward control sense, improved consistency can be facilitated, while prior knowledge of batch productivity, or the end time, can help determine the downstream processing configuration and upstream process scheduling. In this article, forecasting methods based on multivariate batch statistical data analysis procedures are contrasted with case‐based reasoning (CBR). Additionally, the importance of appropriate statistical data prescreening and the choice of a suitable metric for case selection are investigated. Two industrial case studies are considered, a fed‐batch pharmaceutical fermentation and a batch beer fermentation process. It is demonstrated that, following appropriate statistical prescreening of the data, in terms of forecasting performance, CBR is comparable to linear projection to latent structures (PLS), for the more straightforward problem, i.e., the batch beer fermentation, while for the more complex case—the pharmaceutical process—CBR exhibits enhanced performance over PLS.
Biomimetic macroporous hydrogel scaffolds in a high‐throughput screening format for cell‐based assays
Authors: Dainiak Maria B. , Savina Irina N. , Musolino Isabella , Kumar Ashok , Mattiasson Bo , Galaev Igor Yu.
Published: December 2008

Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell‐based assays. In this study, the possibility of combination of three‐dimensional (3D) cell culture with a miniaturized screening format was demonstrated on human colon cancer HCT116, human acute myeloid leukemia KG‐1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm × 7.1 mm I.D.) in a 96‐minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine‐based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm‐containing and collagen‐MHs but not on plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG‐1 cells grown as aggregates were more resistant to the treatment with cis‐diaminedichloroplatinum (II) (cisplatin) and cytosine 1‐β‐D‐arabinofuranoside (Ara‐C), respectively, during the first 18–24 h of incubation, than single cells grown on unmodified MH. HCT116 cells grown as 2D cultures in conventional 96‐well tissue culture plates were 1.5‐ to 3.5‐fold more sensitive to the treatment with 70 μM cisplatin than cells in 3D cultures in functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures on ECM‐modified MHs may provide a realistic in vitro experimental model for high‐throughput toxicity tests.
Phenylalanine containing hydrophobic nanospheres for antibody purification
Authors: Türkmen Deniz , Öztürk Nevra , Akgöl Sinan , Elkak Assem , Denizli Adil
Published: December 2008

In this study, novel hydrophobic nanospheres with an average size of 158 nm utilizing N‐methacryloyl‐(L)‐phenylalanine methyl ester (MAPA) as a hydrophobic monomer were produced by surfactant free emulsion polymerization of 2‐hydroxyethyl methacrylate (HEMA) and MAPA conducted in an aqueous dispersion medium. MAPA was synthesized using methacryloyl chloride and L‐phenylalanine methyl ester. Specific surface area of the nonporous nanospheres was found to be 1874 m2/g. Poly(HEMA–MAPA) nanospheres were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Average particle size, size distribution, and surface charge measurements were also performed. Elemental analysis of MAPA for nitrogen was estimated as 0.42 mmol/g polymer. Then, poly(HEMA–MAPA) nanospheres were used in the adsorption of immunoglobulin G (IgG) in batch system. Higher adsorption values (780 mg/g) were obtained when the poly (HEMA–MAPA) nanospheres were used from both aqueous solutions and human plasma. The adsorption phenomena appeared to follow a typical Langmuir isotherm. It was observed that IgG could be repeatedly adsorbed and desorbed without significant loss in adsorption amount. These findings show considerable promise for this material as a hydrophobic support in industrial processes.
Evaluation of the antimicrobial activity of cationic polyethylenimines on dry surfaces
Authors: Koplin Stephen A. , Lin Shirley , Domanski Tammy
Published: December 2008

The antimicrobial activity of cationic N‐dodecyl‐N‐methylpolyethylenimine (PEI+) against S. aureus, A. baumannii, and E. coli was evaluated when the polymer was applied as a coating to various surfaces using a paint‐like procedure. Antimicrobial activity of PEI+ as a function of time was determined using an assay for long‐term survival involving placement of single drops of various bacterial concentrations on dry surfaces. These data were compared with an assay method where bacteria were applied by spraying and surfaces were incubated overnight under agar. PEI+‐coated surfaces were found to be highly bactericidal after 30 min when bacteria were sprayed onto surfaces. However, when bacteria were applied as single drops, PEI+‐coated surfaces were less biocidal at short contact times particularly for A. baumannii and E. coli. The observations are explained in the context of the difference in drying time between drops deposited on uncoated surfaces and PEI+‐coated surfaces and the sensitivity of bacterial survival to dehydration. These results demonstrate that PEI+‐coated surfaces are not effectively biocidal for some types of bacteria under certain conditions and that the method of assaying bactericidal efficiency can greatly affect the results obtained.
The characterization of pretreated lignocellulosic substrates prior to enzymatic hydrolysis, part 1: A modified Simons' staining technique
Authors: Chandra Richard , Ewanick Shannon , Hsieh Carmen , Saddler Jack N.
Published: December 2008

To date, there is limited knowledge available regarding the key features of pretreated lignocellulosic substrates that promote the effective enzymatic hydrolysis of the cellulose component to glucose during bioconversion processes to produce ethanol. Fundamentally, cellulase enzymes require access to the cellulose to carry out effective hydrolysis. Porosity and the overall surface area of substrates have major structural features influencing the hydrolysis of pretreated substrates by cellulases. Simons' Stain (SS) is a potentially useful semiquantitative method for estimating the available surface area of lignocellulosic substrates. In this study, a modified, rapid SS method was developed, where the processing time was decreased from >50 to 6 h and the maximum dye adsorbed on the substrate was calculated using the adsorption isotherm for the orange and blue components of the dye mixture. The modified SS test readily measures the decrease in accessibility and hydrolyzability of a steam pretreated substrate that had been dried under three different drying regimes. For each of the lignocellulosic substrates, the total dye adsorption correlated well with the hydrolysis yields resulting in a correlation coefficient of r2 = 0.95. The modified SS procedure is an effective tool for assessing how lignocellulosic substrates might be potentially hydrolyzed by cellulases.
Complete sequence of biosynthetic gene cluster responsible for producing triostin A and evaluation of quinomycin‐type antibiotics from Streptomyces triostinicus
Authors: Praseuth Alex P. , Wang Clay C. C. , Watanabe Kenji , Hotta Kinya , Oguri Hiroki , Oikawa Hideaki
Published: December 2008

Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary metabolite. Surprisingly, this strain also produced a trace amount of echinomycin. We sequenced the entire triostin A biosynthetic gene cluster from S. triostinicus, and found that this 36 kilobase‐long gene cluster contained an ORF homologous to ecm18 that encodes a thioacetal‐forming enzyme responsible for the triostin A‐to‐echinomycin bioconversion. These findings indicate that, unlike previously thought, S. triostinicus is capable of producing not only triostin A but also echinomycin. Our observation suggests potential value in careful re‐analysis for metabolites from previously characterized natural product producers with the current technologies.
Microcontact printing of P‐selectin increases the rate of neutrophil recruitment under shear flow
Authors: Lee Dooyoung , King Michael R.
Published: December 2008

The local variation of P‐selectin expression on inflamed endothelial layers affects leukocyte recruitment in vivo. As an initial study of the spatially heterogeneous presentation of P‐selectin in vitro, the influence of microcontact printing (μCP) of P‐selectin on a planar surface in neutrophil recruitment was investigated using a parallel‐plate flow chamber. Microline patterned and nonpatterned P‐selectin were prepared using PDMS, Poly(dimethylsiloxane) (PMDS) stamps and isolated neutrophils perfused over the surface to quantify the level of neutrophil recruitment. We first found a significant increase in cell rolling flux and a decrease in cell rolling velocity on the microcontact printed P‐selectin‐surfaces compared with a randomly adsorbed P‐selectin‐surface. However, the increase in rolling adhesion under shear on the surfaces prepared by μCP was not proportional to the number of functional sites of P‐selectin transferred using immunofluorescent labeling. Interestingly, the relative immunofluorescent intensities of the nonfunctional regions of microcontact printed P‐selectin‐surfaces were substantially lower than that that of randomly adsorbed P‐selectin. Taken together, these data indicate that the μCP of selectin increases the transfer rate of the adhesion molecule on a surface in the functionally correct orientation and, consequently, improves the recruitment of leukocytes to the selectin surface under flow. It is concluded that μCP may be a more general technique to control protein orientation on a substrate.
A novel three‐stage light irradiation strategy in the submerged fermentation of medicinal Mushroom Ganoderma lucidum for the efficient production of ganoderic acid and Ganoderma polysaccharides
Authors: Zhang Wei , Tang Ya‐Jie
Published: December 2008

A novel three‐stage light irradiation strategy in the submerged fermentation of medicinal mushroom Ganoderma lucidum for the efficient production of bioactive metabolites ganoderic acid (GA) and Ganoderma polysaccharides was developed. Significance of light quality, i.e., blue light (390–500 nm, λmax = 470 nm), red light (560–700 nm, λmax = 625 nm), and white light (400–740 nm, λmax = 550 nm), was studied at first. Interestingly, there was a gradual decrease trend of GA content after the culture of day 2 when the maximal GA content was obtained, while GA content decreased slowly under white light irradiation after day 6. The dark environment was favorable to the specific GA biosynthesis (i.e., GA content) before day 6, and after that the optimum was white light irradiation. A relatively lower irradiation density of white light (i.e., 0.94 and 2.82 W/m2) was beneficial for the specific GA biosynthesis before day 6, while GA content was higher under higher irradiation density of white light (i.e., 4.70 and 9.40 W/m2) at the later‐stage of cultivation. 4.70 W/m2 white light irradiation culture was the best from the viewpoint of GA accumulation. Therefore, a two‐stage light irradiation strategy by combing the first 2 days dark culture with the following 4.70 W/m2 white light irradiation culture was developed. The highest GA production in the two‐stage culture was 276.0 ± 12.5 mg/L, which was increased by 19% compared to 4.70 W/m2 white light irradiation culture (i.e., 232.4 ± 15.8 mg/L) and by 178% compared to the dark culture (i.e., 99.4 ± 1.0 mg/L). Although there still existed a gradual decrease trend of GA content after day 2 when the maximal GA content was obtained in the two‐stage culture. Following three‐stage light irradiation strategy was further demonstrated in order to turn around the sharp decrease of GA content after day 2. The first‐stage was the 2‐day dark culture; the second‐stage was the following six‐day 0.94 W/m2 white light irradiation culture, and the third‐stage was 4.70 W/m2 white light irradiation culture until the end of fermentation. During the three‐stage culture of G. lucidum, the gradual decrease trend of GA content after day 2 was turned around, which suggested that 0.94 W/m2 white light irradiation was beneficial for the metabolic flux towards the GA biosynthesis. The maximal GA content of 3.1 ± 0.1 mg/100 mg DW was obtained, which was higher by 41% compared to the two‐stage culture. The maximal GA production (i.e., 466.3 ± 24.1 mg/L) and productivity (i.e., 38.9 mg/L per day) in the three‐stage culture were 69 and 101% higher than those obtained in the two‐stage culture. This is the first report investigating the significance of light irradiation on the medicinal mushroom submerged fermentation. Such work is very helpful to other mushroom fermentations for useful metabolite production.
Homogeneous esterification by lipase from Burkholderia cepacia in the fluorinated solvent
Authors: Shipovskov S.
Published: December 2008

The formation of noncovalent complexes between lipase from Burkholderia cepacia and the fluorinated ionic surfactant Kryto Development Product 4606 KDP was shown to promote the solubilization of the enzyme in the fluorinated solvent perfluoro(methylcyclohexane) (PFMC) and its operation as a catalyst in the fluorous PFMC/hexane biphasic system (FBS). In the reaction of esterification of 1‐phenylethanol and vinyl acetate, the solubilized lipase showed high stereospecificity (ca. 99%) with a catalytic efficiency up to 56 nmol/(U h) with a high operational and storage stability. Temperature modulation of the FBS miscibility allowed the separation and recovery of the solubilized lipase for at least three times. The results are of practical importance for the development of recoverable biocatalysts soluble and active in the FBS.
Thiopropyl‐agarose as a solid phase reducing agent for chemical modification of IgG and F(ab′)2
Authors: Ferraz Natalia , Leverrier Juliana , Batista‐Viera Francisco , Manta Carmen
Published: December 2008

Selective reduction of native disulfide bonds in immunoglobulins is one of the best methods for introducing reactive groups on to the protein surface. Additionally, the thiol groups so generated may allow oriented conjugation at a specific site of the immunoglobulin. Solid‐phase reducing agents have many advantages over soluble ones (including ease of separation of excess reagent from reduced protein by filtration, and the potential for regeneration and multiple reuse). In this work we report a comparative study of the reduction of rabbit IgG and its F(ab′)2 fragments, with mercaptohydroxypropylether‐agarose (thiopropyl‐agarose), a solid phase reducing agent, and dithiothreitol. The effect of different parameters on the process, such as the amount of reducing agent, incubation period, and temperature, was assessed by titration of thiol groups and SDS‐PAGE analysis. Optimized reduction with thiopropyl‐agarose introduced six thiol groups in the F(ab′)2 fragment (mol/mol). Native IgG was less reactive, probably due to steric effects, as only an average of three thiol groups were introduced. However, by increasing reaction temperature from 22 to 37°C, six thiol groups could be introduced in native IgG (mol/mol). Reduction with dithiothreitol also introduced six thiol groups in F(ab′)2 fragments (mol/mol) but led to higher thiol content for the whole IgG. These results demonstrated that thiopropyl‐agarose can be a very useful tool for exercising more precise control over the reduction treatment, and for selecting which disulfide bridges are to be broken. After 6 h incubation with reducing agent containing 8 and 16 μmoles SH per mg of protein, the resulting reduced IgG retained the same biological activity as the native immunoglobulin. The controlled modification of native disulfides achieved with thiopropyl‐agarose will be useful for the development of soluble and insoluble immunoglobulin conjugates.
Facile evaluation of cell disruption efficiency using pH‐controlled fluorescence resonance energy transfer
Authors: Kim Young Soo , Cha Hyung Joon
Published: December 2008

The evaluation of cell disruption efficiency is important in monitoring durability and diverse cellular physiologies of host cells and improving recovery yield of intracellular products. On the basis of our previously suggested pH‐controlled fluorescence resonance energy transfer (FRET) system, we herein demonstrated that easy and effective estimation of the degree of cell disruption under certain stress environments is possible using a simple measurement of changes in fluorescence spectra of two fused fluorescent proteins, because its unique linear correlation was established with cell disruption efficiency from the conventional colony counting method.
Augmentation of EB‐directed hepatocyte‐specific function via collagen sandwich and SNAP
Authors: Novik Eric I. , Barminko Jeffery , Maguire Tim J. , Sharma Nripen , Wallenstein Eric J. , Schloss Rene S. , Yarmush Martin L.
Published: December 2008

The development of implantable engineered liver tissue constructs and ex vivo hepatocyte‐based therapeutic devices are limited by an inadequate hepatocyte cell source. In our previous studies, embryoid body (EB)‐mediated stem cell differentiation spontaneously yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB) and cytokeratin‐18 (CK18). However, these cultures neither yielded a homogenous hepatocyte lineage population nor exhibited detoxification function typical of a more mature hepatocyte lineage cell. In this study, secondary culture configurations were used to study the effects of collagen sandwich culture and oncostatin‐M (OSM) or S‐nitroso‐N‐acetylpenicillamine (SNAP) supplementation of EB‐derived hepatocyte‐lineage cell function. Quantitative immunofluorescence and secreted protein analyses were used to provide insights into the long‐term maintenance and augmentation of existing functions. The results of these studies suggest that SNAP, independent of the collagen supplementation, maintained the highest levels of ALB expression, however, mature liver‐specific CK18 was only expressed in the presence of gel sandwich culture supplemented with SNAP. In addition, albumin secretion and cytochrome P450 detoxification studies indicated that this condition was the best for the augmentation of hepatocyte‐like function. Maintenance and augmentation of hepatocyte‐like cells isolated from heterogeneous EB cell populations will be a critical step in generating large numbers of functional differentiated cells for therapeutic use.
Preparation and characterization of polysulfone affinity membranes bearing a synthetic peptide ligand for the separation of murine immunoglobulins
Authors: Boi Cristiana , Algeri Cristian , Sarti Giulio C.
Published: December 2008

Affinity membranes have been prepared by immobilizing D‐PAM, a synthetic ligand that exhibits affinity for the Fc portion of antibodies, onto poliethersulfone microporous membranes. The ligand density has been measured and the ligand utilization was evaluated and compared with literature data available for chromatographic beads. The resulting new affinity membranes have been experimentally characterized and tested by using pure murine IgG solutions and mouse serum. Equilibrium and kinetic parameters have been obtained in batch experiments using pure protein solutions. The highest binding capacity measured for murine IgG was 45 μg/cm2 obtained at 1.2 mg/mL protein concentration at equilibrium, while the maximum static binding capacity calculated with the Langmuir model was 81 μg/cm2. The adsorption of murine IgG on the affinity membranes was described using different isotherms: Freundlich and Temkin models have been considered and critically compared with the Langmuir adsorption model. A dynamic binding capacity of 21 μg/cm2 was obtained by feeding a solution of 0.3 mg/mL of murine IgG, confirming the results obtained in batch experiments at the same concentration. The affinity membranes considered are endowed with good binding capacity for murine IgG and good selectivity for immunoglobulins and can be considered for the capturing step of an antibody production process.
Microbeads display of proteins using emulsion PCR and cell‐free protein synthesis
Authors: Gan Rui , Yamanaka Yumiko , Kojima Takaaki , Nakano Hideo
Published: December 2008

We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell‐free protein synthesis in emulsion. A PCR mixture containing streptavidin‐coated microbeads was compartmentalized by water‐in‐oil (w/o) emulsion with estimated 0.5 template molecules per droplet. The template molecules were amplified and immobilized on beads via bead‐linked reverse primers and biotinylated forward primers. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti‐glutathione S‐transferase (GST) antibody. The pool of beads was then subjected to cell‐free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We mixed two types of DNA templates of Histidine6 tag (His6)‐fused and FLAG tag‐fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. After incubation with fluorescein isothiocyanate (FITC)‐labeled anti‐His6 (C‐term) antibody, the beads with the His6 gene were enriched 917‐fold in a single‐round screening by using flow cytometry. A library with a theoretical diversity of 106 was constructed by randomizing the middle four residues of the His6 tag. After a two‐round screening, the randomized sequences were substantially converged to peptide‐encoding sequences recognized by the anti‐His6 antibody.
Towards area‐based in vitro metabolic engineering: Assembly of Pfs enzyme onto patterned microfabricated chips
Authors: Lewandowski Angela T. , Bentley William E. , Yi Hyunmin , Rubloff Gary W. , Payne Gregory F. , Ghodssi Reza
Published: December 2008

We report an approach for spatially selective assembly of an enzyme onto selected patterns of microfabricated chips. Our approach is based on electrodeposition of the aminopolysaccharide chitosan onto selected electrode patterns and covalent conjugation of a target enzyme to chitosan upon biochemical activation of a genetically fused “pro‐tag.” We report assembly of S‐adenosylhomocysteine nucleosidase (Pfs) fused with a C‐terminal pentatyrosine pro‐tag. Pfs is a member of the bacterial autoinducer‐2 biosynthesis pathway, catalyzing the irreversible cleavage of S‐adenosylhomocysteine. The assembled Pfs retains its catalytic activity and structure, as demonstrated by retained antibody recognition. Assembly is controlled by the electrode area, resulting in reproducible rates of catalytic conversion for a given area, and thus allowing for area‐based manipulation of catalysis and small molecule biosynthesis. Our approach enables optimization of small molecule biosynthesis in 1‐step as well as multistep enzymatic reactions, including entire metabolic pathways, and we envision a wide variety of potential applications.
Production of a His‐tagged canecystatin in transgenic sugarcane and subsequent purification
Authors: Ribeiro Carolina W. , Soares‐Costa Andrea , Falco Maria Cristina , Chabregas Sabrina M. , Ulian Eugênio C. , Cotrin Simone S. , Carmona Adriana K. , Santana Lucimeire A. , Oliva Maria Luiza V. , Henrique‐Silva Flavio
Published: December 2008

Transgenic plants have been used widely as expression systems of recombinant proteins in recent years. This process can be an efficient alternative for the large‐scale production of proteins. In this work, we present the establishment of transgenic sugarcane expressing a His‐tagged canecystatin under the control of the maize ubiquitin promoter. A number of studies have demonstrated that cystatins, which are natural inhibitors of cysteine proteinases, can be used for protection against insect attacks. A transformed sugarcane plant that presented high levels of HISCaneCPI‐1 expression, was selected for the purification of this protein through affinity chromatography in a nickel column. This purified HISCaneCPI‐1 was immunodetected using a polyclonal antibody, which was also able to detect the HISCaneCPI‐1 in a crude extract from transgenic plant leaves. Assays of inhibitory activity performed with the purified HISCaneCPI‐1 revealed its ability to inhibit the catalytic activity of midgut cysteine proteinase partially purified from the sugarcane weevil Sphenophorus levis and human cathepsin L in nanomolar order. These studies demonstrate that sugarcane is a viable expression system for recombinant protein production.
Production of recombinant porcine colipase secreted by Pichia pastoris and its application to improve dietary fat digestion and growth of postweaning piglets
Authors: Liu Fang‐Chueh , Chen Hsiao‐Ling , Chong Kowit‐Yu , Hsu A‐Li , Chen Chuan‐Mu
Published: December 2008

Recombinant porcine pancreatic colipase (pCoL) was produced in the methylotrophic yeast Pichia pastoris. A synthetic yeast secretion cassette was constructed with the constitutive promoter of the glyceraldehyde‐3‐phosphate‐dehydrogenase (GAPDH) gene and the yeast α‐mating factor signal peptide. The pCoL cDNA corresponding in the coding sequence, excluding a 16‐amino acid segment of the native signal sequence, was cloned into the pGAPZαB vector and integrated into the genome of P. pastoris. Yeast transformants were cultured and bioactive pCoL protein was detected in the supernatant at a high‐level of 126.8 mg/L after 3 days of culture. The transformed yeast containing the highest recombinant colipase level (pCoL yeast) and native yeast GS 115 not containing pCoL (non‐pCoL yeast, as a control group) were separately cultured and the supernatants were adsorbed by dried skim milk. In an animal trial, two concentrations of colipase activity (0 vs. 5,000 U/kg in the diet) were blended with the pig corn‐soybean basal diet and fed to weaned piglets for 4 weeks. The pCoL‐administrated test group gained significantly more weight than piglets in the control group when measured at Day 15 (11.84 ± 0.70 vs. 10.59 ± 0.39 kg, P < 0.05), Day 22 (15.84 ± 0.95 vs. 14.32 ± 0.59 kg, P < 0.01), and Day 28 (20.19 ± 1.47 vs. 18.54 ± 0.92 kg, P < 0.01) after weaning. The blood triglyceride (TG) concentrations were significantly increased in the experimental test group that received recombinant colipase on the 28th day of postweaning when compared with that of the control group (32.50 vs. 16.37 mg/dL; P < 0.0001). These experimental data suggest that the use of recombinant porcine colipase as a dietary supplement provides an alternative approach for improving fat digestion and enhancing growth in postweaning piglets.
The establishment of a controllable expression system in baculovirus: Stimulated overexpression of polyhedrin promoter by LEF‐2
Authors: Wu Yueh‐Lung , Chao Yu‐Chan
Published: December 2008

Previously, controllable gene expression in baculovirus was not possible using an insect system. We found that this was due to a high background activation of minimal promoter by the viral polyhedrin upstream (pu) sequence. Here, by truncation of the pu sequence, regulatory gene expression was established through the tetracycline regulatory expression system. This novel system was used to test the stimulatory function of the polyhedrin promoter by the controlled expression of the late expression factor‐2 (lef‐2). To efficiently trace lef‐2 expression and analyze suppression of this gene, the coding sequences of lef‐2 and enhanced green fluorescent protein (egfp) were ligated together to generate a fusion protein, and an ∼100‐fold suppression of egfp‐lef‐2 expression was achieved by doxycycline treatment. A very low level expression of lef‐2 was found to be sufficient for proper expression of polyhedrin promoter; however, progressively higher levels of lef‐2 expression could stimulate much higher‐than‐original polyhedrin promoter expression in the viral genome. This system was found to exhibit significantly better suppression than the double‐stranded RNA (dsRNA) strategy, and would be useful for expression of foreign or viral genes whose functions require the interaction of multiple and/or unknown baculovirus gene products.
Ultrathin silver nanowires produced by amyloid biotemplating
Authors: Malisauskas Mantas , Meskys Rolandas , Morozova‐Roche Ludmilla A.
Published: December 2008

By using a self‐assembled amyloid from lysozyme as biotemplate we produced an ultrathin silver wire of 1 nm diameter and up to 2 μm in length, which is at the limit attainable in nanobiotechnological manufacturing. We showed that 2,2,2‐trifluoroethanol produces a dual effect: it reduces ionic silver to colloidal nanoparticles with a regular size, depending on the length of incubation, and induces fibrillar assembly into the amyloid scaffold, forming the hollow channel filled with silver.