Identification and quantification of the glucose degradation product glucosone in peritoneal dialysis fluids by HPLC/DAD/MSMS

Glucose degradation products (GDPs) formed during heat sterilization of peritoneal dialysis (PD) fluids exert cytotoxic effects and promote the formation of advanced glycation end-products in the peritoneal cavity. As a result, long-term application of continuous ambulatory peritoneal dialysis is limited. The composition and concentration of GDPs in PD fluids must be known to evaluate their biological effects. The present study describes a targeted screening for novel GDPs in PD fluids. For this purpose, dicarbonyl compounds were converted with o-phenylenediamine to give the respective quinoxaline derivatives, which were selectively monitored by HPLC/diode array detector. Glucosone was thereby identified as a novel major GDP in PD fluids. Product identity was confirmed by LC/MSMS analysis using independently synthesized glucosone as a reference compound. Furthermore, a method was developed to quantify glucosone in PD fluids by HPLC/UV after derivatization with o-phenylenediamine. The method's limit of detection was 0.6 μM and the limit of quantitation 1.1 μM. A linear calibration curve was obtained between 1.1 and 113.9 μM (R² = 0.9999). Analyzed at three different concentration levels, recovery varied between 95.6% and 102.0%. The coefficient of variation ranged between 0.4% and 4.7%. The method was then applied to the measurement of glucosone in typical PD fluids. Glucosone levels in double chamber bag PD fluids varied between not detectable and 6.7 μM. In single chamber bag fluids, glucosone levels ranged between 28.7 and 40.7 μM.     

An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography–tandem mass spectrometry

A fully automated screening using liquid chromatography–mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100 μg/l (67%). The developed LC–MS–MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.     

The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts

Cardiac diseases such as myocardial infarction and heart failure are among the leading causes of death in western societies. Therapeutic angiogenesis has been suggested as a concept to combat these diseases. The biology of angiogenic factors expressed in the heart such as vascular endothelial growth factor (VEGF) is well studied, whereas data on anti-angiogenic mediators in the heart are scarce. Here we study the expression of the anti-angiogenic factor pigment epithelium-derived factor (PEDF) in the human heart and in human cardiac cells. PEDF expression could be detected in human cardiac tissue on the protein and mRNA levels. PEDF mRNA levels were significantly lower in explanted human ischemic hearts as compared to healthy hearts. Our in vitro experiments showed that human adult cardiac myocytes and fibroblasts constitutively secrete PEDF. In addition to anoxic conditions, cobalt chloride, 2,2'dipyridyl and dimethoxally glycine, which stabilize hypoxia inducible factor-α decreased PEDF expression. Furthermore we show that PEDF inhibits VEGF-induced sprouting. We have identified PEDF in healthy and ischemic human hearts and we show that PEDF expression is down-regulated by low oxygen levels. Therefore, we suggest a role for PEDF in the regulation of angiogenesis in the heart and propose PEDF as a possible therapeutic target in heart disease.     

The Biosynthesis of Branched Dialkylpyrazines in Myxobacteria

The biosynthesis of the volatiles 2,5‐ and 2,6‐diisopropylpyrazine ( 2 and 3 , resp.) released by the myxobacteria Nannocystis exedens subsp. cinnabarina (Na c29) and Chondromyces crocatus (strains Cm c2 and Cm c5) was studied. Isotopically labeled precursors and proposed pathway intermediates were fed to agar plate cultures of the myxobacteria. Subsequently, the volatiles were collected by use of a closed loop stripping apparatus (CLSA), and incorporation into the pyrazines was followed by GC/MS analysis. [²H₈]Valine was smoothly incorporated into both pyrazines clearly establishing their origin from the amino acid pool. The cyclic dipeptide valine anhydride ( 16 ) – a potential intermediate on the biosynthetic pathway to branched dialkylpyrazines – was synthesized containing ²H₁ labels in specific positions. Feeding of [²H₁₆]‐ 16 and [²H₁₂]‐ 16 in both valine subunits mainly resulted in the formation of pyrazines derived from only one labeled amino acid, whereas only traces of the expected pyrazines with two labeled subunits were found. To investigate the origin of nitrogen in the pyrazines, a feeding experiment with [¹⁵N]valine was performed, resulting in the incorporation of the ¹⁵N label. The results contradict a biosynthetic pathway via cyclic dipeptides, but rather point to a pathway on which valine is reduced to valine aldehyde. Its dimerization to 2,5‐diisopropyldihydropyrazine 36 and subsequent oxidation results in 2 . The proposed biosynthetic pathway neatly fits the results of earlier labeling studies and also explains the formation of the regioisomer 2,6‐diisopropylpyrazine 3 by isomerization during the first condensation step of two molecules valine aldehyde. A general biosynthetic pathway to different classes of pyrazines is presented.     

Madin–Darby canine kidney cells are increased in aerobic glycolysis when cultured on flat and stiff collagen‐coated surfaces rather than in physiological 3‐D cultures

We investigate the influence of the dimensionality and the biochemistry of the culture system on the cellular functionality by analyzing the protein expression levels in Madin–Darby canine kidney (MDCK) cells grown in 3‐D and 2‐D substrates. We cultured MDCK cells on a hard and flat 2‐D uncoated plastic surface, on a 2‐D collagen‐coated plastic surface and in 3‐D collagen gel and employed 2‐D gel electrophoresis, MALDI‐TOF‐MS, and LC‐MS/MS analysis to identify the differentially regulated proteins. We found significant differences in the expression of antioxidant proteins, actin‐binding proteins, glycolytic enzymes, and heat‐shock proteins/chaperons among the three types of cultures. While MDCK cells cultured in 3‐D collagen up‐regulate antioxidant proteins and proteins involved in the dynamic remodeling of the actin cytoskeleton, 2‐D collagen‐coated plastic surfaces induce the up‐regulation of glycolytic enzymes. Our data shows that the culture conditions have profound effects on the physiology of the cell. Culture in 3‐D collagen induces a differentiated polarized phenotype. In contrast, collagen‐coated 2‐D substrates favor a tumor‐like phenotype with increased glycolysis. Thus, the suitability of 2‐D cultures to study the physiological behavior of cells, especially in drug discovery, bioprocessing, and toxicology, should be carefully reconsidered.     

Elevation-induced variations of pollen assemblages in the North-western Alps: An analysis of their value as temperature indicators

Seventy-seven modern pollen samples from various elevations (350-2680 m a.s.l.) in two different areas of the north-western Alps (the Aosta Valley, Italy and the Taillefer Massif, France) were statistically analyzed to derive correlations between pollen assemblages, elevation and temperature at the sampled points. Numerical classifications were performed on pollen data to judge similarities between the two areas. The results show that a strong relationship exists between altitude and variations in pollen taxa percentages despite some floristic differences between the two areas. As a test, transfer functions from pollen percentages to elevation and temperature were calculated from pollen data. The reconstruction appears to be reliable, with a higher reliability at sites located over 1000m. This analysis aims to serve as a basis for further quantitative reconstruction of temperature changes during the Holocene based on fossil pollen data from sensitive regions that encompass a significant altitudinal gradient.     

Wie sich Oasen‐Bananen gegen ihre Feinde wehren. Uralter Toleranzmechanismus im Oman entdeckt

Until recently little was known about the crop diversity in the Sultanate of Oman, situated at the NE tip of the Arabian Peninsula. Interdisciplinary research in the often millenia‐old oases provide evidence for their role as reservoirs for plant genetic resources of ancient varieties of wheat and banana. Two newly discovered banana clones show a highly efficient biochemical defense mechanisms against some of the most devastating pests and diseases of banana. If these mechanisms can be properly understood and exploited in breeding programs, may have major impact on the commercial production of edible banana.     

Hormonal concentrations in bitches with primary uterine inertia

Normal labor is accompanied by sequential changes in blood concentrations of prostaglandin F2α (measured as 15-ketodihydro-PGF2α = PGFM), progesterone, estradiol, oxytocin, vasopressin, and of elevated cortisol levels. The aim of this study was to investigate hormone concentrations in dogs diagnosed with primary uterine inertia before and during treatment by cesarian section. The hypothesis was the dogs would have abnormally low plasma concentrations in one or several of the hormones involved in parturition. The study comprised seven bitches with total primary uterine inertia (dystocia group) treated with cesarian section and six healthy bitches (control group) subjected to planned cesarean section. Blood samples were taken before anesthesia, before surgery started, on delivery of the first puppy and on delivery of the last puppy. The progesterone:PGFM ratio in plasma was higher in the dystocia group than in the control group, but the serum estradiol concentration did not differ between groups. The plasma concentrations of oxytocin and vasopressin increased in both groups when the first puppies were delivered, but both hormones were more elevated in the control group than in the dystocia group on delivery of the last puppies. The plasma cortisol concentration increased to the same level in both groups. In conclusion, the ratio between progesterone and PGFM was higher and the oxytocin and vasopressin concentrations lower in the dystocia dogs than in the control dogs. The findings indicate that these hormones are involved in the pathophysiology of total primary uterine inertia in bitches.     

A new soft-tissue simulation strategy for cranio-maxillofacial surgery using facial muscle template model

We propose a computationally efficient, bio-mechanically relevant soft-tissue simulation method for cranio-maxillofacial (CMF) surgery. Special emphasis is given to comply with the current clinical workflow. A template-based facial muscle prediction was introduced to avoid laborious segmentation from medical images. In addition, transversely isotropic mass-tensor model (MTM) was applied to realize the directional behavior of facial muscles in short computation time. Finally, sliding contact was incorporated to mimic realistic boundary condition in error-sensitive regions. Mechanical simulation result was compared with commercial finite element software. And retrospective validation study with post-operative scan of four CMF cases was performed.     

Ecophysiology of Fe-cycling bacteria in acidic sediments

Using a combination of cultivation-dependent and -independent methods, this study aimed to elucidate the diversity of microorganisms involved in iron cycling and to resolve their in situ functional links in sediments of an acidic lignite mine lake. Using six different media with pH values ranging from 2.5 to 4.3, 117 isolates were obtained that grouped into 38 different strains, including 27 putative new species with respect to the closest characterized strains. Among the isolated strains, 22 strains were able to oxidize Fe(II), 34 were able to reduce Fe(III) in schwertmannite, the dominant iron oxide in this lake, and 21 could do both. All isolates falling into the Gammaproteobacteria (an unknown Dyella-like genus and Acidithiobacillus-related strains) were obtained from the top acidic sediment zones (pH 2.8). Firmicutes strains (related to Bacillus and Alicyclobacillus) were only isolated from deep, moderately acidic sediment zones (pH 4 to 5). Of the Alphaproteobacteria, Acidocella-related strains were only isolated from acidic zones, whereas Acidiphilium-related strains were isolated from all sediment depths. Bacterial clone libraries generally supported and complemented these patterns. Geobacter-related clone sequences were only obtained from deep sediment zones, and Geobacter-specific quantitative PCR yielded 8 × 10(5) gene copy numbers. Isolates related to the Acidobacterium, Acidocella, and Alicyclobacillus genera and to the unknown Dyella-like genus showed a broad pH tolerance, ranging from 2.5 to 5.0, and preferred schwertmannite to goethite for Fe(III) reduction. This study highlighted the variety of acidophilic microorganisms that are responsible for iron cycling in acidic environments, extending the results of recent laboratory-based studies that showed this trait to be widespread among acidophiles.