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Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum.

The enzyme activities of the valine biosynthetic pathway and their regulation have been studied in the valine-producing strain, Corynebacterium glutamicum 13032DeltailvApJC1ilvBNCD. In this micro-organism, this pathway might involve up to five enzyme activities: acetohydroxy acid synthase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxyacid dehydratase and transaminases B and C. For each enzyme, kinetic parameters (optimal temperature, optimal pH and affinity for substrates) were determined. The first enzyme of the pathway, AHAS, was shown to exhibit a weak affinity for pyruvate (K(m)=8.3 mM). It appeared that valine and leucine inhibited the three first steps of the pathway (AHAS, AHAIR and DHAD). Moreover, the AHAS activity was inhibited by isoleucine. Considering the kinetic data collected during this work, AHAS would be a key enzyme for further strain improvement intending to increase the valine production by C. glutamicum.

Acetolactate Synthase↗

[Helical CT of abdominal aorta].

The purpose was to illustrate that single or multirow spiral CT angiography has become the main modality for the examination of the abdominal aorta. Our multidisciplinary group investigates weekly 15 abdominal aortas, with a single slice spiral CT since 1995, and a 4 row detector spiral CT since 1999. CT scans protocols and parameters optimization for the 2 CT systems are described. Indications and results of CT to investigate the various abdominal aortic diseases are illustrated and discussed: aneurisms (pretherapeutic staging, selection of indications of endovascular stent-grafting); obstructive aorto-arteriopathies (pretherapeutic staging, follow up after endovascular revascularization); dissection (diagnosis, pretherapeutic staging, follow up); inflammatory diseases; normal patterns and principal complications after surgery. Situations where multislice CT is superior to single slice CT and compared advantages of MRI are discussed. CT is at the present time the main technique to image abdominal aorta. Multislice CT is especially interesting to explore the whole aorta, poly traumatisms, and to plan and follow up endovascular treatments.

Aortic Dissection↗

Electrothermal chondroplasty--monopolar.

Despite the lack of clinical studies, monopolar RF energy is currently used for the treatment of grade 2, 3, and 4 chondromalacia. Basic science studies have confirmed that monopolar RF can mechanically debride the articular surface by smoothing, potentially reducing further delamination. However, a zone of thermal injury in the areas of treatment has been observed with loss of chondrocyte viability. Therefore, further use of this procedure lies in the surgeon's understanding of the potential deleterious effects of temperature on chondrocyte viability and the limitations of alternate treatment options. Monopolar RF energy is a potential alternative to other cartilage removal or smoothing techniques, but more science is needed to define optimal parameters for thermal chondroplasty. Great care is currently proposed in the selection of operative parameters by clinicians when using monopolar RF chondroplasty until safety and efficacy are confirmed.

Cartilage↗

Ultrasound nucleolysis: an in vitro study.

Thermal intradiscal therapy for chronic low back pain, using a catheter inserted into the intervertebral disc, is becoming more popular in the treatment of low back pain. The aim of this study was to investigate the possibility of heating the nucleus pulposus of the intervertebral disc with high-intensity focused ultrasound (US) or HIFU. Two specific situations were considered, invasive transducers that would be in contact with the annulus fibrosus of the disc, and noninvasive transducers that could be used externally. Theoretical simulations were performed to find the optimal parameters of US transducers and then experimental studies were done using transducers made to these specifications. These experiments confirmed that it was possible to heat the discs with HIFU. Two orthogonal transducers resulted in a superior temperature distribution than using just one transducer. It is, therefore, feasible to consider thermal treatment of the nucleus pulposus of the disc using noninvasive US.

Animals↗

Potential of intensity-modulated radiotherapy to escalate doses to head-and-neck cancers: what is the maximal dose?

PURPOSE: To investigate the potential of intensity-modulated radiotherapy (IMRT) to escalate doses to head-and-neck cancer and find the maximal dose that could be prescribed to the target volume with IMRT while doses to critical organs were maintained at their currently acceptable levels. The secondary goal was to search for limits in current IMRT technology. METHODS AND MATERIALS: For a group of 12 head-and-neck cancer patients with different tumor locations and shapes, we performed IMRT planning using a simultaneous integrated boost strategy, that is, the gross tumor volume (GTV), clinical target volume (CTV), and electively treated nodes were treated simultaneously at different dose levels. The critical structures involved in the treatment field that needed to be spared included the brainstem, spinal cord, and parotid glands, depending on the disease site. Nine coplanar 6-MV photon beams were used for planning with the IMRT system developed at our institution, and dose-volume criteria were used for optimization. By varying the optimization parameters, we gradually increased the dose to the GTV while keeping the dose to the critical structures at less than the acceptable tolerance level. The criteria for accepting the plan included the following: (1) the prescription dose to the GTV had to cover 99% of the volume, and the dose homogeneity of the GTV needed to be <10%; (2) the prescription to the CTV (which was set either at 60 Gy or 10 Gy less than that of the GTV) had to cover 95% of the volume, and the same amount of normal tissue outside the CTV received the CTV prescription dose as in the current acceptable plan; (3) the prescription to the electively treated lymph nodes needed to cover 90% of the volume; and (4) the maximal dose to the brainstem and spinal cord had to be <55 Gy and 45 Gy, respectively. For parotid glands, the dose needed to be as low as possible without compromising the target doses. The deliverable plans as determined by the actual multileaf collimator leaf sequences were used for the final evaluation. To verify that the acceptable plans were deliverable, the experimental measurements of planar dose distribution were performed in phantom with film. RESULTS: The maximal dose to the GTV varied from 86 to 176 Gy if the CTV dose increased with the GTV dose. It was reduced to 76-82 Gy if the CTV dose was kept at 60 Gy. The competing criteria usually are the requirements of the tolerance doses to the critical organs and target dose homogeneity, not the target prescription dose. Using more beams only increased the dose marginally. The results could change significantly if a different set of criteria for the plan evaluation were used. Dosimetric measurements confirmed that such a high dose and dose gradient could be delivered accurately with dynamic multileaf collimators. Statistical analyses showed no significant correlations between the maximal doses and the number of GTVs and volume of GTVs and CTVs. CONCLUSION: Doses to head-and-neck cancers with simultaneous integrated boost IMRT can be escalated to a greater level than currently prescribed clinically. The limit of IMRT in head-and-neck cancer has not been reached at the current prescription level of 70 Gy. Such high total and fractionated doses should be carefully evaluated before being prescribed clinically.

Adult↗

Optimisation of AP-PCR fingerprinting discriminatory power for clinical isolates of Pseudomonas aeruginosa.

Recently methods based on analysis of arbitrarily amplified target sites of microorganism genomes have been extensively applied in microbiological studies. The range of their applications is limited by problems with discrimination and reproducibility resulting from lack of standardised and reliable methods of optimisation. By orthogonal-array optimisation most advantageous and optimal parameters for highly discriminatory primers (CagA2+CMVin2) were selected and efficient AP-PCR (arbitrarily primed-polymerase chain reaction) fingerprinting conditions for Pseudomonas aeruginosa isolates were set up. Stable and multiplex amplicon profiles obtained in this study revealed high level of intraspecies DNA polymorphism among 20 analysed clinical strains of P. aeruginosa proving optimised AP-PCR fingerprinting to be useful in epidemiological typing of the species.

DNA Fingerprinting↗

Application of N-PCR for diagnosis of distemper in dogs and fur animals.

The immunofluorescence test, routinely used for laboratory diagnosis of canine distemper virus (CDV) in Poland, is not sufficiently sensitive and specific. Therefore, the application of reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR (N-PCR) and Southern blot hybridization for detection of phosphoprotein (P) gene of CDV in peripheral blood mononuclear cells (PBMCs) or internal organs of dogs and fur animals was the aim of these studies. The optimal parameters for two-step PCR were elaborated for reference strains of distemper virus and used for testing biological samples collected from dogs, foxes, ferret and mink with spontaneous distemper. PCR product of 1069bp was obtained in one out of 10 dog blood samples, three out of 14 homogenates of internal organs of dogs and one out of five homogenates of internal organs of fox. Reamplification with the use of CDVa and CDVb primers demonstrated the 429bp fragment in six samples, negative by PCR: two samples collected from dogs, two from foxes, one from mink and one from ferret. The specificity of N-PCR was confirmed by Southern blot hybridization. We conclude that two-step PCR is sensitive and specific method for diagnosis of CDV infection.

Animals↗

Validated method for the determination of hydroquinone in human urine by high-performance liquid chromatography-coulometric-array detection.

The paper describes the computer aided method development and validation for the determination of hydroquinone in human urine from a clinical study on renal excretion of hydroquinone metabolites and the release of free hydroquinone in the urinary tract in order to evaluate the proposed urine disinfecting concept. The presented method uses high-performance liquid chromatography on reversed-phase material with a polar endcapping (Aqua-C18, 250x4.6 mm). Selective and sensitive determination (LOQ= 12.5 ng on-column) of the target compound was achieved by electrochemical array detection (CoulArray). Gradient and parameter optimization were supported by DryLab software in order to minimize efforts of the expensive and time-consuming method development. Specificity and selectivity were carried out by separation experiments involving the prodrug arbutin and the metabolites hydroquinone, hydroquinone glucuronide, and hydroquinone sulfate, respectively. Hydroquinone glucuronide reference standard was obtained from in vitro glucuronidation in a rat liver microsomes assay. The method was validated according to the criteria for validation of pharmaceutical bioanalytical methods as drafted by the US Department of Health and Human Services, 1998.

Animals↗

Rapid method for the isolation of hydroxylysylpyridinoline and lysylpyridinoline from concentrated bone hydrolysates by liquid chromatographic techniques.

A rapid method for the isolation of hydroxylysylpyridinoline and lysylyridinoline from bone by liquid chromatographic methods is described. Decalcified bone is hydrolysed in 7 M hydrochloric acid. After evaporation of the acid, the high molecular mass and dark coloured degradation products are removed by adsorption on non-polar adsorbents. The pyridinolines are separated from the majority of the amino acids by adsorption on cellulose. Separation of HP and LP is performed either by cation-exchange chromatography or by reversed-phase ion-pair chromatography. The pyridinoline containing fractions are desalted by size-exclusion chromatography. The progress of the hydrolytic cleavage of collagen and the optimal parameters for purification and separation were examined. As a result the existing method allows the isolation of high amounts of pyridinolines with low amounts of adsorbents and chemicals within a short time.

Amino Acids↗

Optimization of intercalation dye concentration for short tandem repeat allele genotyping using capillary electrophoresis with laser-induced fluorescence detection.

DNA analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection requires that polymerase chain reaction products either be prepared using primers with fluorescent molecules covalently bonded to them, or stained with a fluorescent intercalation dye following amplification. The intercalation technique has the advantage of allowing fluorescence detection of any double-stranded DNA (dsDNA) product regardless of the amplification primers used. The increased sensitivity of LIF detection is sometimes compromised by the intercalation dye changing the mass to charge ratio of the DNA. The purpose of this study was to evaluate the changes of migration rate, resolution and fluorescent intensity of dye-DNA complexes during electrophoretic separations, and to establish the optimal parameters for short tandem repeats alleles profiling. The alleles of three STR loci THO1, F13A01 and vWFA31 were intercalated with the monomeric dyes TOPRO-1 and YOPRO-1, and their corresponding dimers, TOTO-1 and YOYO-1 (Molecular Probes, Eugene, OR, USA). Alleles intercalated before injection onto the CE column resulted in loss of resolution and sensitivity when compared to the on-column labeling technique. The results of this experimentation were then applied to a STR typing assay using a commercially available polymer and buffer matrix. This assay included development of a unique internal standard used for migration time normalization assignment of alleles. Consequently, the 9 allele and the 9.3 microvariant of the THOI locus were separated and typed correctly with a resolution of 0.49 in less than 20 min, and the only sample preparation necessary after amplification was a dilution step.

Alleles↗

Development and optimisation of alginate-PMCG-alginate microcapsules for cell immobilisation.

Mechanical stability, uniformity of size, complete encapsulation of cells and optimal microenvironment are major challenges in the design and development of microcapsules for cell immobilisation purposes. In this work, a novel microcapsule chemistry based on polyelectrolyte complexation between alginate and poly(methylene-co-guanidine) (PMCG) is presented. We have characterised the effect of PMCG concentration and time of exposure on microcapsule diameter and membrane thickness, selecting a PMCG concentration of 0.5% (v/v) and an exposure time of 1 min as optimal parameters for a correct coating. Afterwards, the mechanically most resistant alginate-PMCG-alginate (A-PMCG-A) microcapsule type was chosen according to two different stability studies. Beads with a solid core and an inhomogeneous internal configuration resulted in stronger microcapsules. Further, the selected A-PMCG-A beads presented both an increased stability compared to classical Ca(2+)/alginate and alginate-poly-L-lysine-alginate (APA) microcapsules, and had an adequate microenvironment for cell viability. This new chemistry allows the controlled adjustment of microcapsule size and wall thickness, offering new alternatives for cell transplantation.

Alginates↗

The influence of temperature on the high performance liquid chromatographic separation of steroids using mobile phases modified with beta-cyclodextrin.

The effect of temperature on the retention and multiple separation of hydrocortisone, testosterone, 17 alpha-methyltestosterone, prednisone, cortisone and 17 alpha-hydroxyprogesterone in reversed-phase liquid chromatography has been studied. Capacity factors (k') of the steroids were measured using a mobile phase modified with different concentrations of beta-cyclodextrin (from 0-16 mM), a fixed solvent composition (acetonitrile-water) and a wide range of column temperatures (from 5-80 degrees C). The plots of capacity factors vs. reciprocal of absolute temperature are nonlinear in every case when mobile phase modified with beta-cyclodextrin was used. Particularly strong nonlinearity was observed at lower temperature and at higher beta-cyclodextrin concentration. The complex chromatograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k'max), the smallest resolution between adjacent peaks (Rs; min) and relative resolution product (r). The results presented describe precisely the role of temperature in high performance liquid chromatography systems in which mobile phases modified with cyclodextrin were used.

17-alpha-Hydroxyprogesterone↗

Comparison of liquid and supercritical fluid chromatography for the separation of enantiomers on chiral stationary phases.

Comparisons of liquid (LC) and supercritical fluid chromatography (SFC) were conducted using commercially available chiral stationary phases (CSPs) bearing three different types of chiral selectors. Chiral compounds of pharmaceutical and agricultural interest were used to probe advantages of limitations of SFC relative to LC for enantiomeric separations. Column equilibrium and parameter optimization were generally accomplished more rapidly in SFC than in LC. Although improved resolution was often observed in SFC, analysis times were not always lower in SFC than in LC. In some instances, SFC provided separation capabilities not readily accessible in LC.

Chromatography, Liquid↗

The influence of temperature on the multiple separation of estrogenic steroids using mobile phases modified with beta-cyclodextrin in high-performance liquid chromatography.

The effect of temperature on the retention and multiple separation of six estrogenic steroids in reversed-phase liquid chromatography has been studied. Capacity factors (k') of estriol, 17 beta-estradiol, 17 alpha-estradiol, d-equilenin, equilin and estrone were measured using mobile phase modified with different concentrations of beta-cyclodextrin (from 0-16 mM), a fixed solvent composition (acetonitrile-water) and a wide range of column temperatures (from 5 to 80 degrees C). The plots of capacity factors vs. reciprocal of absolute temperature are nonlinear in each case when mobile phase modified with beta-cyclodextrin was used. Particularly strong nonlinearity was observed at lower temperature and at higher beta-cyclodextrin concentration. The complex chromatograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k'max), the smallest resolution between adjacent peaks (Rs,min) and relative resolution product (r). The results presented describe precisely the role of temperature in high-performance liquid chromatography systems in which mobile phases modified with cyclodextrin were used. Moreover, the elution order of estrogenic steroids on modified and unmodified mobile phases has been discussed.

Chromatography, High Pressure Liquid↗

Continuous-wave diode laser thermokeratoplasty: first clinical experience in blind human eyes.

PURPOSE: To evaluate the safety and stability of laser thermokeratoplasty (LTK) with a continuous-wave diode laser in blind human eyes and to optimize parameters for a study in sighted eyes. SETTING: Department of Ophthalmology, Medical University Lübeck, Germany. METHODS: A continuous-wave diode laser was set to emit radiation with a wavelength of 1.854 microns (Group 1, n = 4) or 1.870 microns (Group 2, n = 4) and 100 to 150 mW power for 10 seconds. A focusing handpiece was coupled with an application mask and fixed by partial vacuum to the conjunctiva or cornea. The radiation was focused into the corneal stroma between 400 and 600 microns in Group 1 and set to 1000 microns in Group 2. Eight (Group 1, single ring) or 16 (Group 2, double ring) coagulations were applied. RESULTS: The refractive change increased with higher laser power and smaller ring diameters. Two rings of coagulations provided higher and more stable refractive changes of up to 5.66 diopters (D) than a single ring. The refractive effect stabilized between 3 and 6 months postoperatively. At 1 year, mean refractive change was +0.99 D +/- 0.39 (SD) in Group 1 and +2.32 +/- 2.24 D in Group 2. Extensive endothelial damage occurred in Group 1 but was minimal in Group 2. CONCLUSIONS: Diode LTK was used to treat hyperopia safely and effectively. Regression occurred mainly in the first 3 postoperative months. With a wavelength of 1.870 microns, corneal endothelial damage was limited.

Adult↗

A small-size neural network for computing with strange attractors.

A small-size model for a chaotic neural network is proposed using strange attractors for computation. This network has a chaotic ground state and is capable of responding to external stimuli by constraining the network dynamics to specific parts of the ground state attractor. For parameter optimization, bifurcation diagrams are evaluated.

Journal Article↗

Radiofrequency tumor ablation: principles and techniques.

Radiofrequency (RF) tumor ablation has been demonstrated as a reliable method for creating thermally induced coagulation necrosis using either a percutaneous approach with image-guidance or direct surgical placement of thin electrodes into tissues to be treated. Early clinical trials with this technology have studied the treatment of hepatic, cerebral, and bony malignancies. However, more recently this technology has been used to treat a host of malignant processes throughout the body. This article will discuss the principles and technical considerations of RF ablation with the goal of defining optimal parameters for the therapy of focal lesions. This includes technologic innovations that permit large volume tumor ablation (i.e., hooked and internally cooled electrodes), as well as methods and adjuvant therapies that can modulate tumor biophysiology to permit improved tumor destruction (i.e, altered tissue conductivity and blood flow). Potential biophysical limitations to RF induced coagulation, such as perfusion mediated tissue cooling (vascular flow) will likewise be discussed. Lastly, the principles governing safe usage of the system, such as proper grounding pad placement, will be adressed.

Animals↗

Technical considerations in the application of intensity-modulated radiotherapy as a concomitant integrated boost for locally-advanced cervix cancer.

The technical aspects of IMRT applied to cervix cancer are discussed in this paper, as well as issues related to tumor delineation, target volume definitions, inverse planning, and IMRT delivery. A theoretical example illustrating how IMRT can accurately mimic dose distributions obtained using conventional planning plus HDR brachytherapy is also shown. The notion of clinical optimization parameters is introduced to account for the radiation delivery variables, which affect the overall treatment time. This is especially relevant to the possible introduction of intrafractional movement and resulting inaccuracy, as well as facility efficiency.

Brachytherapy↗