Transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG(4) encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 mug mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn(297) was not significantly affected by nocodazole during transient production by this method. (C) 2004 Wiley Periodicals, Inc.

GCMC-surface area of carbonaceous materials with N-2 and Ar adsorption as an alternative to the classical BET method

In this paper we apply a new method for the determination of surface area of carbonaceous materials, using the local surface excess isotherms obtained from the Grand Canonical Monte Carlo simulation and a concept of area distribution in terms of energy well-depth of solid–fluid interaction. The range of this well-depth considered in our GCMC simulation is from 10 to 100 K, which is wide enough to cover all carbon surfaces that we dealt with (for comparison, the well-depth for perfect graphite surface is about 58 K). Having the set of local surface excess isotherms and the differential area distribution, the overall adsorption isotherm can be obtained in an integral form. Thus, given the experimental data of nitrogen or argon adsorption on a carbon material, the differential area distribution can be obtained from the inversion process, using the regularization method. The total surface area is then obtained as the area of this distribution. We test this approach with a number of data in the literature, and compare our GCMC-surface area with that obtained from the classical BET method. In general, we find that the difference between these two surface areas is about 10%, indicating the need to reliably determine the surface area with a very consistent method. We, therefore, suggest the approach of this paper as an alternative to the BET method because of the long-recognized unrealistic assumptions used in the BET theory. Beside the surface area obtained by this method, it also provides information about the differential area distribution versus the well-depth. This information could be used as a microscopic finger-print of the carbon surface. It is expected that samples prepared from different precursors and different activation conditions will have distinct finger-prints. We illustrate this with Cabot BP120, 280 and 460 samples, and the differential area distributions obtained from the adsorption of argon at 77 K and nitrogen also at 77 K have exactly the same patterns, suggesting the characteristics of this carbon.

Preparation of immuno-stimulating complexes (ISCOMs) by ether injection

preparation of liposomes, as a new, continuous and potentially scaleable method for the preparation of ISCOMs. Phosphatidylcholine (PC) and cholesterol (Chol) were dissolved in ether, which was injected into an aqueous solution, maintained at 55 degrees C, containing Quil A. The influences of the following variables on ISCOM formation were investigated: ratio of PC:Quil A:Chol used, pumping rate, total lipid mass and concentration of buffer salts and Quil A in the aqueous phase. All samples were characterized by negative stain transmission electron microscopy, photon correlation spectroscopy and sucrose ultracentrifugation gradient. It was demonstrated that ISCOMs could be produced by this method but the homogeneity of the preparation was influenced by the conditions used. Homogeneous ISCOM preparations were consistently produced only when the weight ratio of PC:Quil A:Chol was 5:3:2 with a total lipid mass of 20 mg, the Quil A dissolved in a 0.01 M phosphate buffer at a concentration of 6 mg in 4 ml, and the ether solution injected into the warmed buffer solution at a rate of 0.2 ml/min. Changing any of these variables resulted in more heterogeneous preparations in which ISCOMs typically co-existed with other colloidal structures such as worm-like and helical micelles, liposomes, lamellae and lipidic particles. (C) 2005 Elsevier B.V. All rights reserved.

Inscription of in-fibre photonic devices by an infrared femtosecond laser

In this work, a point by point method for the inscription of fibre Bragg gratings using a tightly focused infrared femtosecond laser is implemented for the first time. Fibre Bragg gratings are wavelength-selective, retro-reflectors which have become a key component in optical communications as well as offering great potential as a sensing tool. Standard methods of fabrication are based on UV inscription in fibre with a photosensitive core. Despite the high quality of the gratings, a number of disadvantages are associated with UV inscription, in particular, the requirements of a photosensitive fibre, the low thermal stability and the need to remove the protective coating prior to inscription. By combining the great flexibility offered by the point by point method with the advantages inherent to inscription by an infrared femtosecond laser, the previous disadvantages are overcome. The method here introduced, allows a fast inscription process at a rate of ~1mm/s, gratings of lengths between 1cm and 2cm exhibiting reflections in excess of 99%. Physical dimensions of these gratings differ significantly from those inscribed by other methods, in this case the grating is confined to a fraction of the cross section of the core, leading to strong and controllable birefringence and polarisation dependent loss. Finally, an investigation of the potential for their exploitation towards novel applications is carried out, devices such as directional bend sensors inscribed in single-mode fibre, superimposed but non-overlapping gratings, and single-mode, single-polarisation fibre lasers, were designed, fabricated and characterised based on point by point femtosecond inscription.

Line-by-line fiber Bragg grating made by femtosecond laser

In this letter, we report on the inscription of a fourth-order fiber Bragg grating made line-by-line in the optical fiber using a femtosecond laser. Strong Bragg resonance (~17 dB) and low insertion loss (~0.5 dB) were obtained with only 2000 periods. Measured refractive index change of these inscribed lines reaches up to 7 × 10-3. The grating was fully characterized and the low insertion loss together with low polarization-dependent loss were realized compared to gratings made by the point-by-point method. The high temperature annealing experiment shows the grating can survive up to at least 800°C.

Microstructured waveguides in z-cut LiNbO3 by high-repetition rate direct femtosecond laser inscription

We report on the operational parameters that are required to fabricate buried, microstructured waveguides in a z-cut lithium niobate crystal by the method of direct femtosecond laser inscription using a highrepetition-rate, chirped-pulse oscillator system. Refractive index contrasts as high as −0.0127 have been achieved for individual modification tracks. The results pave the way for developing microstructured WGs with low-loss operation across a wide spectral range, extending into the mid-infrared region up to the end of the transparency range of the host material.

Control of the properties of micro-structured waveguides in LiNbO3 fabricated by direct femtosecond laser inscription

This paper reports on buried waveguides fabricated in lithium niobate (LN) by the method of direct femtosecond (fs) laser inscription. 5% MgO doped LiNbO3 was chosen as the host material because of its high quality and damage threshold, as well as relatively low cost. Direct fs inscription by astigmatically shaped beam in crystals usually produces multiple 'smooth' tracks (with reduced refractive index), which encircle the light guiding 'core', thus creating a depressed cladding WG. A high-repetition rate fs laser system was used for inscription at a depth of approximately 500 μm. Using numerical modelling, it was demonstrated that the properties of fs-written WGs can be controlled by the WG geometry. Buried, depressed-cladding WGs in LN host with circular cross-section were also demonstrated. Combining control over the WG dispersion with quasi-phase matching will allow various ultralow-pump-power, highly-efficient, nonlinear light-guiding devices - all in an integrated optics format.

Evolution of lace knitting stitch patterns by genetic programming

In this paper we study the generation of lace knitting stitch patterns by using genetic programming. We devise a genetic representation of knitting charts that accurately reflects their usage for hand knitting the pattern. We apply a basic evolutionary algorithm for generating the patterns, where the key of success is evaluation. We propose automatic evaluation of the patterns, without interaction with the user. We present some patterns generated by the method and then discuss further possibilities for bringing automatic evaluation closer to human evaluation. Copyright 2007 ACM.

Pulse width modulation (PWM) method for power components estimation in power meters

The scope of this paper is to present the Pulse Width Modulation (PWM) based method for Active Power (AP) and Reactive Power (RP) measurements as can be applied in Power Meters. Necessarily, the main aim of the material presented is a twofold, first to present a realization methodology of the proposed algorithm, and second to verify the algorithm’s robustness and validity. The method takes advantage of the fact that frequencies present in a power line are of a specific fundamental frequency range (a range centred on the 50 Hz or 60 Hz) and that in case of the presence of harmonics the frequencies of those dominating in the power line spectrum can be specified on the basis of the fundamental. In contrast to a number of existing methods a time delay or shifting of the input signal is not required by the method presented and the time delay by n/2 of the Current signal with respect to the Voltage signal required by many of the existing measurement techniques, does not apply in the case of the PWM method as well.

Method for Analytical Representation of the Maximum Inaccuracies of Indirectly Measurable Variable

Let us have an indirectly measurable variable which is a function of directly measurable variables. In this survey we present the introduced by us method for analytical representation of its maximum absolute and relative inaccuracy as functions, respectively, of the maximum absolute and of the relative inaccuracies of the directly measurable variables. Our new approach consists of assuming for fixed variables the statistical mean values of the absolute values of the coefficients of influence, respectively, of the absolute and relative inaccuracies of the directly measurable variables in order to determine the analytical form of the maximum absolute and relative inaccuracies of an indirectly measurable variable. Moreover, we give a method for determining the numerical values of the maximum absolute and relative inaccuracies. We define a sample plane of the ideal perfectly accurate experiment and using it we give a universal numerical characteristic – a dimensionless scale for determining the quality (accuracy) of the experiment.

Registration-by-regression of coronary CTA and X-ray angiography

We evaluate the integration of 3D preoperative computed tomography angiography of the coronary arteries with intraoperative 2D X-ray angiographies by a recently proposed novel registration-by-regression method. The method relates image features of 2D projection images to the transformation parameters of the 3D image. We compared different sets of features and studied the influence of preprocessing the training set. For the registration evaluation, a gold standard was developed from eight X-ray angiography sequences from six different patients. The alignment quality was measured using the 3D mean target registration error (mTRE). The registration-by-regression method achieved moderate accuracy (median mTRE of 15 mm) on real images. It does therefore not provide yet a complete solution to the 3D–2D registration problem but it could be used as an initialisation method to eliminate the need for manual initialisation.

Investigation on possibility of survival and growth enhancing by selected seaweeds from Persian Gulf in post larvae shrimp Litopenaeus vannamei

The use of antibiotics in aquaculture has been limited. Scientifics seeking for natural substitutes to prevent of aquatic animals diseases. Considering seaweeds are rich of nutritions and bioactive compounds, the purpose of this study is: investigation the potential and use possibility of native seaweeds from Persian Gulf in shrimp aquculture industry to improve growth, survival of postlarvae and to resistance against pathogens such as vibriosis. For this propose 7 macroalgae species from Bushehr province coast, inclouding: green algae (C. iyengarii), brown algae (S. angutifolium and S. ilicifolium) and red algae (L. snyderiae, K. alvarezii and G. corticata) were collected and identified. Then seaweed extracts abtained by Water, Ethanol, Methanol and Chloroform solvents by soaking method. In vitro antibacterial activity of extracts against Gr+ bacteria (S. aureus and B. subtilis) and Gr- bacteria (V. harveyi, V. alginolyticus and E. coli) was conducted by Agar diffusion, MIC and MBC methods. Antioxidant activity also by DPPH and EC50 methods was investigated. According to results of these two tests four seaweeds species (S. angutifolium, L. snyderiae, K. alvarezii and G. corticata) were selected for use in shrimp postlarvae (PL22) diets by Bio-Encapsulation (Artemia enrichment). Before of enrichment, toxicity effect of extracts to Artemia nauplii were evaluated by determination of LC50 24 h method. From results of this section Ethanol extracts were selected to bioencapsulation. After encapsulation shrimp postlarvae divided to 12 groups in triplicate, namely: C-, C+, S (200), S (400), S (600), L(200), L(400), L(600), G(300), G(600), K(300) and K(600). During 30 days of reared period C- and C+ use of basal diet and unenriched Artemia, but the other groups use of basal diet and enriched Artemia. Except C-, the shrimps in first day of culture put in 107 cfu/ml v. harveyi suspension for 30 minutes, and after water exchange 10 ml of this dose was added to reared aquaria. After 30 days survival percentage, obtained weight and SGR% were investigated. To evaluate vibrio loading, every 10 days 5 postlarvae were sampled randomly for vibrio count. Results showed that vibrio count in C- was less than the others and in C+ was more than the others. In treatments vibrio count in L(200) was the most and L(600) was the less. Survival rate in C- was the most and after that G(600) with 79.4±6.6% and then S(300) and K(600) were 73.3±7.3% and 70.6±6.6% respectively that were significantly compare the other (P < 0.01). Also the C+ was the less with 33.3±6.6% that difference was significant (P< 0.01). In this study growth parameters of all groups that fed by enriched Artemia were better than C+ (P<0.05). After cultre period 10 shrimp of every aquarium disinfected and reared for 10 days like before treatment. After 10 days the shrimps were challenged by 3×108 cfu/ml V. harveyi and mortality was recorded for 7 days. The all of animals in C- were survive but more than 90% of C+ were dead. And survival in all of treatments were better the C+ (P<0.05). The study showed the ethanol extracts of selected seaweed from Persian Gulf is a good source for growth, Survival and disease control in shrimp larviculture.

Effect of the CeZrNd mixed oxide synthesis method in the catalytic combustion of soot

Ce0.64Zr0.27Nd0.09Oδ mixed oxides have been prepared by three different methods (nitrates calcination, coprecipitation and microemulsion), characterized by N2 adsorption, XRD, H2-TPR, Raman spectroscopy and XPS, and tested for soot combustion in NOx/O2. The catalyst prepared by microemulsion method is the most active one, which is related to its high surface area (147 m2/g) and low crystallite size (6 nm), and the lowest activity was obtained with the catalyst prepared by coprecipitation (74 m2/g; 9 nm). The catalyst prepared by nitrates precursors calcination is slightly less active to that prepared by microemulsion, but the synthesis procedure is very straightforward and surfactants or other chemicals are not required, being very convenient for scaling up and practical utilization. The high activity of the catalyst prepared by nitrates calcination can be attributed to the better introduction of Nd cations into the parent ceria framework than on catalysts prepared by coprecipitation and microemulsion, which promotes the creation of more oxygen vacancies.

Physicochemical and antioxidant properties of the pequi (Caryocar brasiliense Camb.) almond oil obtained by handmade and cold-pressed processes.

Abstract: The aim of this study is to characterize physical and chemically and determine the antioxidant capacity of pequi almond oils (PAO) extracted by handmade and by cold-pressing. Both oils showed good quality by acid, peroxide and thiobarbituric acid values. The fatty acid (FA) profile showed a significant presence of monounsaturated FA, mainly oleic acid (53.48 to 55.41%); saturated FA, such as palmitic acid (33.30 to 35.89 %); and polyunsaturated FA (PUFA), such as linoleic acid (5.85 to 7.23%). The total phenolic (TP) and carotenoid content ranged in concentration from 87.56 to 392.00 mg GAE/100 g and 36.03 to 262.40 mg/100 g, respectively. The tocopherol and phytosterol results indicated the predominant presence of α-tocopherol (52 to 67%) and stigmasterol (63 to 68 %). The antioxidant capacity of PAO as measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH?) method oscillated from 58.48 mg/mL to 76.46 mg/mL (IC50), from 10.61 to 40.46 µmol TE/g by the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS?+) method, and from 113.93 to 280.85 µmol TE/100 g and 164.49 to 277.86 µmol TE/100 g, by the lipophilic and hydrophilic oxygen radical absorbance capacity (ORAC) methods, respectively. The oils presented a good oxidative and thermal stability by Rancimat method (IP of 7.33 a 15.91 h) and curves thermogravimetric and differential scanning calorimetry (To 337-363 °C and 159-184 °C, respectively). The results confirmed the presence of compounds that conferred antioxidant capacity and oxidative and thermal resistance for PAO made by handmade or cold-pressing, indicating that these oils can potentially be used for food and non-food applications.

Up-regulation Of Dnam-1 And Nkp30, Associated With Improvement Of Nk Cells Activation After Long-term Culture Of Mononuclear Cells From Patients With Ovarian Neoplasia.

This study aimed at evaluating the functional activation and activating receptors expression on resting, short- and long-term NK and NK-like T cells from blood of ovarian neoplasia patients. Blood from patients with adnexal benign alterations (n = 10) and ovarian cancer (grade I-IV n = 14) were collected after signed consent. Effector cells activation was evaluated by the expression of the CD107a molecule. Short-term culture was conducted overnight with IL-2 and long-term culture for 21 days, by a method designed to expand CD56(+) lymphocytes. Short-term culture significantly increased NK cells activation compared to resting NK cells (p<0.05), however, the long-term procedure supported an even higher increase (p<0.001). Resting NK-like T cells showed poor activation, which was not altered by the culture procedures. The long-term culture effectively increased the expression of the activating receptors on NK and NK-like T cells, either by increasing the number of cells expressing a given receptor and/or by up-regulating their expression intensity. As a conclusion, the long-term culture system employed, resulted in a high number of functional NK cells. The culture system was particularly efficient on the up-regulation of NKp30 and DNAM-1 receptors on NK cells.