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Exploiting the benefits of miniaturization for the enhancement of DNA microarrays

The present study demonstrates that the best way to enhance DNA microarray assays, both in terms of analysis speed and in final spot intensity, is to dissolve the available molar amount of sample in the smallest possible buffer volume and to subsequently convect this solution continuously across the surface of the array. The presently proposed shear-driven flow system is pre-eminently suited for this task, as it allows to induce strongly enhanced lateral transport rates, independently of the degree of miniaturization of the hybridization chamber. This transport enhancement method, however, only increases the hybridization rate and not the final spot intensity, as neither can any of the other transport enhancement methods already proposed in literature. A series of experiments with synthetic single-stranded (ssDNA) samples and an accompanying mass balance analysis are presented to demonstrate these points.     

Preparation and characterisation of synthetic mixtures of lithium isotopes

Re-certification of the absolute isotopic composition of the natural lithium isotopic reference material (IRM), IRMM-016, requires measurements calibrated by means of synthetic mixtures of highly enriched lithium isotopes. Ten such mixtures were prepared by weighing and mixing of two well characterised, isotopically enriched, Li₂CO₃ compounds. The starting materials, 99.9981% enriched ⁶Li, and 99.9937% enriched ⁷Li, were purified by ion exchange, and the purified materials converted from LiOH to Li₂CO₃ by reaction with CO₂. Ten new mixtures were prepared by mixing different weighed amounts of these dissolved Li₂CO₃ carrier compounds. The compounds had an estimated level of impurities of 100 ± 100 μg · g^–1 (expanded uncertainty with a coverage factor of 2). In the ten mixtures, the n(⁶Li)/n(⁷Li) ratio varies from 0.025 to 14 and the achieved expanded relative uncertainty on the amount ratio prepared is typically 2 · 10^–4. These mixtures were then used to determine the correction factor, K, for mass discrimination of the measurement procedure and instrument concerned.     

Preparation and characterisation of synthetic mixtures of lithium isotopes

Re-certification of the absolute isotopic composition of the natural lithium isotopic reference material (IRM), IRMM-016, requires measurements calibrated by means of synthetic mixtures of highly enriched lithium isotopes. Ten such mixtures were prepared by weighing and mixing of two well characterised, isotopically enriched, Li₂CO₃ compounds. The starting materials, 99.9981% enriched ⁶Li, and 99.9937% enriched ⁷Li, were purified by ion exchange, and the purified materials converted from LiOH to Li₂CO₃ by reaction with CO₂. Ten new mixtures were prepared by mixing different weighed amounts of these dissolved Li₂CO₃ carrier compounds. The compounds had an estimated level of impurities of 100 ± 100 μg · g^–1 (expanded uncertainty with a coverage factor of 2). In the ten mixtures, the n(⁶Li)/n(⁷Li) ratio varies from 0.025 to 14 and the achieved expanded relative uncertainty on the amount ratio prepared is typically 2 · 10^–4. These mixtures were then used to determine the correction factor, K, for mass discrimination of the measurement procedure and instrument concerned. Received: 6 November 1997 / Revised: 22 December 1997 / Accepted: 24 January 1998     

Epigenetic dysregulation of immune-related pathways in cancer: bioinformatics tools and visualization

Cancer immune evasion is one of the hallmarks of carcinogenesis. Cancer cells employ multiple mechanisms to avoid immune recognition and suppress antitumor immune responses. Recently, accumulating evidence has indicated that immune-related pathways are epigenetically dysregulated in cancer. Most importantly, the epigenetic footprint of immune-related pathways is associated with the patient outcome, underscoring the crucial need to understand this process. In this review, we summarize the current evidence for epigenetic regulation of immune-related pathways in cancer and describe bioinformatics tools, informative visualization techniques, and resources to help decipher the cancer epigenome.Subject terms: Genetics research, Biomarkers     

In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen

The aim of this work was to optimize the conditions for in vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose, using detergent extracts of membranes from hybrid aspen (Populus tremula × tremuloides) cells grown as suspension cultures. Callose was the only product synthesized when CHAPS extracts were used as a source of enzyme. The optimal reaction mixture for callose synthesis contained 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 8 mM Ca²⁺, and 20 mM cellobiose. The use of digitonin to extract the membrane-bound proteins was required for cellulose synthesis. Yields as high as 50% of the total in vitro products were obtained when cells were harvested in the stationary phase of the growth curve, callose being the other product. The optimal mixture for cellulose synthesis consisted of 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 1 mM Ca²⁺, 8 mM Mg²⁺, and 20 mM cellobiose. The in vitroβ-glucans were identified by hydrolysis of radioactive products, using specific enzymes. ¹³C-Nuclear magnetic resonance spectroscopy and transmission electron microscopy were also used for callose characterization. The (1→3)-β-D-glucan systematically had a microfibrillar morphology, but the size and organization of the microfibrils were affected by the nature of the detergent used for enzyme extraction. The discussion of the results is included in a short review of the field that also compares the data obtained with those available in the literature. The results presented show that the hybrid aspen is a promising model for in vitro studies on callose and cellulose synthesis.     

A kinetic study of the polymerization of pure meta-divinylbenzene and pure para-divinylbenzene

The kinetics of the polymerization of pure meta-divinylbenzene (DVB) and pure para-divinylbenzene at 70°C have been studied in the presence of toluene and 2-ethylhexanoic acid. The apparent rate constant ratios (k_p/k_t)^1/2 for these systems have been calculated. meta-Divinylbenzene polymerizes at a higher rate than the para-isomer in both toluene and 2-EHA, and the polymerization rates of meta-DVB and para-DVB before the gel point were both higher in the presence of 2-EHA than in toluene. The monomer conversion at the visual gel point is higher for para-DVB than for meta-DVB. The gel point has also been determined indirectly by size exclusion chromatography, and these results are consistent with the gel times observed visually. The conversion of pendant vinyl groups during the polymerization has been determined by bromination. It is found that the homopolymers of poly(para-DVB) have a substantially higher content of pendant vinyl groups than poly(meta-DVB) both during and at the end of the polymerization. The molecular weight distribution (MWD) prior to gelation has been determined by size exclusion chromatography (SEC). Weight average (M̄_w); and number average (M̄_n) molecular weight prior to gelation and of the sol fractions after gelation have also been measured by SEC. There are larger fractions of high molecular weight polymers prior to gelation, when the polymerization was run in the presence of toluene, than in 2-EHA, mainly due to the differences in solvating power of the two diluents. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 3345–3359, 1999