Position-orientation adaptive smoothing of diffusion weighted magnetic resonance data (POAS).

We introduce an algorithm for diffusion weighted magnetic resonance imaging data enhancement based on structural adaptive smoothing in both voxel space and diffusion-gradient space. The method, called POAS, does not refer to a specific model for the data, like the diffusion tensor or higher order models. It works by embedding the measurement space into a space with defined metric, in this case the Lie group of three-dimensional Euclidean motion SE(3). Subsequently, pairwise comparisons of the values of the diffusion weighted signal are used for adaptation. POAS preserves the edges of the observed fine and anisotropic structures. It is designed to reduce noise directly in the diffusion weighted images and consequently also to reduce bias and variability of quantities derived from the data for specific models. We evaluate the algorithm on simulated and experimental data and demonstrate that it can be used to reduce the number of applied diffusion gradients and hence acquisition time while achieving a similar quality of data, or to improve the quality of data acquired in a clinically feasible scan time setting.

Dissipative dark soliton in a complex plasma.

The observation of a dark soliton in a three-dimensional complex plasma containing monodisperse microparticles is presented. We perform our experiments using neon gas in the bulk plasma of an rf discharge. A gas temperature gradient of 500K/m is applied to balance gravity and to levitate the particles in the bulk plasma. The wave is excited by a short voltage pulse on the electrodes of the radio frequency discharge chamber. It is found that the wave propagates with constant speed. The propagation time of the dark soliton is approximately 20 times longer than the damping time.

[Feasibilities and limitations of high field parallel MRI]. / Möglichkeiten und Grenzen der parallelen MRT im Hochfeld.

In medical magnetic resonance imaging (MRI) it is standard to use MR scanners with a field strength of 1.5 Tesla. Recently, an ongoing development to higher field strength can be observed and a new clinical standard at 3.0 Tesla seems to be established. High field MRI with its intrinsic higher signal to noise ratio (SNR) can enable new applications of MRI in medical diagnosis, or can serve to improve existing methods. It is important to note, that the use of high field MRI is not without its limitations. Besides the SNR, other unwanted effects increase with a higher field strength. Without correction, these high field problems cause a serious loss in image quality. An elegant way to address these problems is the use of parallel imaging. In many clinical applications, parallel MRI (pMRI) is part of the standard protocol, because pMRI can enhance virtually every MRI application, without necessarily affecting the contrast behavior of the underlying imaging sequence. In high field MRI, besides the speed advantage of pMRI, the positive influence on high field specific problems and therefore on the image quality will be of major importance.

Resolution enhancement in lung 1H imaging using parallel imaging methods.

Resolution in (1)H lung imaging is limited mainly by the acquisition time. Today, half-Fourier acquisition single-shot turbo spin-echo (HASTE) sequences, with short echo time (TE) and short interecho spacing (T(inter)) have found increased use in lung imaging. In this study, a HASTE sequence was used in combination with a partially parallel acquisition (PPA) strategy to increase the spatial resolution in single-shot (1)H lung imaging. To investigate the benefits of using a combination of single-shot sequences and PPA, five healthy volunteers were examined. Compared to conventional imaging methods, substantially increased resolution is obtained using the PPA approach. Representative in vivo (1)H lung images acquired with a HASTE sequence in combination with the generalized autocalibrating partially parallel acquisition (GRAPPA) method, up to an acceleration factor of three, are presented.

VD-AUTO-SMASH imaging.

Recently a self-calibrating SMASH technique, AUTO-SMASH, was described. This technique is based on PPA with RF coil arrays using auto-calibration signals. In AUTO-SMASH, important coil sensitivity information required for successful SMASH reconstruction is obtained during the actual scan using the correlation between undersampled SMASH signal data and additionally sampled calibration signals with appropriate offsets in k-space. However, AUTO-SMASH is susceptible to noise in the acquired data and to imperfect spatial harmonic generation in the underlying coil array. In this work, a new modified type of internal sensitivity calibration, VD-AUTO-SMASH, is proposed. This method uses a VD k-space sampling approach and shows the ability to improve the image quality without significantly increasing the total scan time. This new k-space adapted calibration approach is based on a k-space-dependent density function. In this scheme, fully sampled low-spatial frequency data are acquired up to a given cutoff-spatial frequency. Above this frequency, only sparse SMASH-type sampling is performed. On top of the VD approach, advanced fitting routines, which allow an improved extraction of coil-weighting factors in the presence of noise, are proposed. It is shown in simulations and in vivo cardiac images that the VD approach significantly increases the potential and flexibility of rapid imaging with AUTO-SMASH.

The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells.

Protein hydrolysates as substitutes for serum havebeen employed by many in cell culture mediumformulation, especially with the shift to low proteinor protein-free media. More recently, vegetablehydrolysates have also been added as nutritionalsupplements to fortify the amino acid content in smallpeptide form for batch and fed-batch fermentations. Several of these new hydrolysates (peptones of soy,rice, wheat gluten etc.) were tested as protein-freemedium supplements for the production of a recombinanttherapeutic protein. Multiple peptone-supplemented,continuous perfusion bioreactor experiments wereconducted, varying dilution rates and basal mediumcomposition over the various runs. Cell specificrates and product quality studies were obtained forthe various peptones and compared with peptone-freemedium. The potential for peptones to decreaseintrinsic and proteolytic degradation of the productwas also investigated.It was found that peptones confer a nutritionalbenefit, especially at low dilution rates, for therecombinant BHK cell line used in this investigation.The specific productivity increased 20-30% comparedto the peptone-free controls. However, this benefitwas also fully delivered by using fortified medium inplace of the peptone-enriched media. Therefore, whilepeptones may be considered as useful medium additiveswhen development time is limited, their addition maybe avoided by systematic medium development ifpermitted by the time line of the project.

Effects of dissolved oxygen levels and the role of extra- and intracellular amino acid concentrations upon the metabolism of mammalian cell lines during batch and continuous cultures.

The effects of dissolved oxygen and the concentration of essential amino acids upon the metabolism of two mammalian cell lines (rCHO producing human active (t-PA) and a mouse-mouse hybridoma) were investigated in batch, chemostat, and perfusion cultures. Intracellular amino acid concentrations were measured for both cell lines during repeated batch cultures and the K(S)-values for the essential amino acids were calculated using Monod equations via computer simulation. The K(S)-values were in the range of 10 mmol L(-1) and the pool of most intracellular amino acids remained constant at about 10-100 fold higher in concentration than in the medium. No significant differences were observed between the hybridoma and CHO cell. The specific nutrient uptake rates corresponded with the cell specific growth rate and the effects of reduced dissolved oxygen concentrations only became evident when the DO dropped below 5% of air saturation (critical concentration below 1%). Nevertheless, a correlation between nutrient concentration and specific oxygen uptake was detected.

Production of recombinant human antithrombin III on 20-L bioreactor scale: correlation of supernatant neuraminidase activity, desialylation, and decrease of biological activity of recombinant glycoprotein.

Chinese hamster ovary (CHO) cells producing the recombinant glycoprotein human antithrombin III (rhAT III) were batch cultivated in a 20-L bioreactor for 13 days. Neuraminidase activity in cell-free supernatant was monitored during cultivation and free sialic acid was determined by HPLC. Neu5Acalpha(2-->3)Gal-specific Maackia amurensis and Galbeta(1-->4)GlcNAc-specific Datura stramonium agglutinin were used for determination of sialylated and desialylated rhAT III, respectively. A commercial test kit was used for evaluation of functional rhAT III activity. Supernatant neuraminidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increased neuraminidase activity, which seemed to be principally due to cell lysis, resulting in release of cytosolic neuraminidase. Loss of terminally alpha(2-->3) linked sialic acids of the oligosaccharide portions of rhAT III, analyzed in lectin-based Western blot and lectin-adsorbent assays, correlated with a decrease of activity of rhAT III produced throughout long-term batch cultivation. Thus, structural oligosaccharide integrity as well as the functional activity of recombinant glycoprotein depend on the viability and mortality of the bioreactor culture, and batches with a high number of viable cells are required to guarantee production of glycoproteins with maximum biological activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 441-448, 1997.

Lysis-free separation of hybridoma cells by continuous disc stack centrifugation.

The non-destructive removal of hybridoma cells from fermentation broth with an improved disc stack centrifuge (CSA1, Westfalia Separator AG, Oelde, Germany) was investigated. The centrifuge was equipped with a hydrohermetic feed system, which allowed a gentle, shearless acceleration of the cells inside the bowl. No significant cell damage was observed during the separation of hybridoma cells from repeated batch fermentation in 100 liter scale. In the clarified liquid phase there was no increase in Lactate-Dehydrogenase (LDH) activity. Consequently, there was no increased exposure of the product to intracellular components.Due to continuous operation with a periodic and automatic discharge of sediment, a high throughput was achieved without any considerable loss of product. The clarification for mammalian cells was in the range of 99% to 99.9%, depending on the operating conditions. The content of cell debris and other small particles decreased about 30 to 50%, depending on the particle load in the feed stream. The centrifuge was fully contained; cleaning and sterilizing in place possible. Therefore, the decice could be integrated easily into the fermentation process.

Investigations into the efficacy of doramectin on reactivated somatic larvae of Ancylostoma caninum Ercolani 1859 (Ancylostomatidae) in pregnant bitches.

The aim of this investigation was to examine whether reactivated somatic Ancylostoma caninum larvae can be eliminated by the administration of doramectin to pregnant bitches. Four pregnant bitches experimentally infected with 20,000 third-stage larvae of A. caninum were treated subcutaneously with 1 mg doramectin per kg body weight on day 55 of the pregnancy (5-8 days before parturition). Another four experimentally infected pregnant bitches served as controls. The single doramectin treatment completely prevented galactogenic infections in the puppies. Neither intestinal stages nor somatic larvae could be found. The administration of doramectin caused no local or generalized side-effects in the bitches. All 16 puppies of the treated bitches were born healthy and remained so during the whole trial period. Beginning during the third week after birth, all 20 puppies of the untreated bitches developed a severe microcytic, hypochromic anaemia. They had to be killed between 17 and 35 days after birth. Post mortem, they revealed a total of 8649 intestinal stages of A. caninum.

Re-use of spent cell culture medium in pilot scale and rapid preparative purification with membrane chromatography.

Based on experiments in bench scale, a recycling of spent cell culture medium was performed in a 100-1 pilot scale bioreactor. The cell cultivation has been done as a repeated batch procedure after the initial batch in the following four repeated batches spent medium from the previous batch was partially re-used. After microfiltration and ultrafiltration a part of the filtrate was mixed with a concentrate of amino acids and glucose, sterile filtered and subsequently filled back into the bioreactor. Up to 65% of the harvested cell- and product-free spent medium was re-used in each repeated batch. This procedure results in a saving of pure and waste water volume and saving of supplemented proteins as transferrin, insulin and lipoproteins and, therefore, also in a reduction of the production costs. A strongly acidic membrane ion exchanger was evaluated for the ability to purify the monoclonal antibodies from the pilot scale cultivation. Within minutes, gram quantities of product could be purified in a high flux system, especially developed for this purpose, achieving purities of 80%. The capacity of the acidic membrane ion exchanger was found in former investigations to be 1 mg cm-2 with recoveries up to 96%. Final purification was carried out by gel column filtration.

The Super-Spinner: a low cost animal cell culture bioreactor for the CO2 incubator.

The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner. Into a 1 1 standard Duran flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration. The aeration was facilitated by using the CO2 conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump. The maximal oxygen transfer rate (OTRmax) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTRmax was measured by the dynamic method. The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density. A balanced nutrient supply resulted in an optimal formation and yield of products.

Concentrations of isosorbide dinitrate, isosorbide-2-mononitrate and isosorbide-5-mononitrate in human vascular and muscle tissue under steady-state conditions.

The concentrations of isosorbide dinitrate (ISDN), isosorbide-5-mononitrate (IS-5-MN) and isosorbide-2-mononitrate (IS-2-MN) were determined in plasma (PL), saphenous vein wall (SV) and pectoral muscle (PM) from 8 patients undergoing coronary bypass surgery. The patients were pretreated for 2 days with ISDN 240 mg per day (standard release formulation) in 4 doses of 40 mg and one dose of 80 mg. The plasma and tissue samples were obtained during the operation, 10-12 h after the last dose. Isosorbide-2-mononitrate and isosorbide-5-mononitrate were present in plasma and tissues in the same concentration ranges with molar concentration ratios of 0.88 (IS-2-MN: PM/PL), 0.85 (IS-5-MN: PM/PL), 0.99 (IS-2-MN: SV/PL) and 1.06 (IS-5-MN: SV/PL). Mean ISDN concentrations in tissue were considerably higher than in plasma; the molar concentration ratios were 4.9 (SM/PL) and 7.21 (SV/PL). The accumulation of ISDN in vessel walls may contribute to its greater vascular action compared to the mononitrates, but it may also facilitate the development of tolerance during long-term treatment.

[Bioavailability of delayed-action and non-delayed-action preparations of isosorbide-5-nitrate]. / Bioverfügbarkeit von retardierten und nicht-retardierten Isosorbid-5-nitrat-Präparaten.

In a randomized crossover study on twelve healthy test subjects, the plasma concentration of isosorbide-5-nitrate (IS-5-N) after oral administration of 40 mg IS-5-N daily from two standard preparations (two tablets Ismo 20 or Corangin 20) and two sustained release preparations with 40 mg or 60 mg IS-5-N (one tablet Corangin 40 or Corangin 60) once a day was determined. The concentrations were measured in the first and in the third dose interval (24 hours). The respective pharmacokinetic parameters (AUC, Cmax and tmax) of the first and the third day only differed slightly. The two standard formulations proved to be bioequivalent. With the two sustained release preparations, maximum plasma concentrations attained after five to six hours were measured which were lower by roughly the factor 2. The sustained release preparations showed a low loss of bioavailability of 13-19% as compared to the nonretarded standard formulations.

[Serum concentration of glyceryl trinitrate (GTN) in transdermal application of GTN dressings of various origins]. / Serumkonzentration von Glyceroltrinitrat (GTN) bei transdermaler Applikation von GTN-Pflastern unterschiedlicher Provenienz.

Serum levels of glyceryl trinitrate have been determined in eight healthy subjects following transdermal application of four different glyceryl trinitrate plasters using a randomised crossover design. The plasters investigated were Deponit 10, Nitradisc 10, Nitroderm TTS 10 and Nitro-Pflaster-ratiopharm 10. The medians of serum concentrations arranged in decreasing order were 0.17-0.23 ng/ml for Nitroderm TTS 10, 0.13-0.21 ng/ml for Nitradisc 10, 0.08-0.13 ng/ml for Deponit 10, and 0.04-0.09 ng/ml for Nitro-Pflaster-ratiopharm 10. Corresponding areas under the concentration-time curves (AUC 0-24) were 23.15 nmol . h/l (Nitroderm TTS), 20.32 nmol . h/l (Nitradisc), 10.39 nmol . h/l (Deponit), and 5.7 nmol . h/l (Nitro-Pflaster-ratiopharm).

A van der Waals-type equation of state for fluids with associating molecules.

The basic assumptions of van der Waals theory are contained in two well-known concepts: excluded volume (repulsive forces) and a homogeneous, isotropic field potential (attractive forces). We have superimposed on these, one more well-known concept: the existence of dimers, trimers, etc., at chemical equilibrium. With reasonable simplifying assumptions, we obtain a closed-form equation of state, applicable to all fluid densities, and potentially useful for fluids containing strongly polar or hydrogen-bonded molecules. At all temperatures and at high densities, the equation of state suggests a phase transition where, because of extensive association, a new (solid-like) phase is in equilibrium with a "normal" fluid phase. The boundary between these phases has no critical point.