Quantification of peptides for the monitoring of protease-catalyzed reactions by matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrixes

Ionic liquid matrixes (ILM) have been shown to allow very homogeneous sample preparations, facilitating relative quantifications using internal standards in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In the present work, the ability to perform quantifications of peptides without using internal standards in these matrixes was investigated. Linear correlations between peptide amount and signal intensities could be observed when increased molar matrix-to-analyte ratios were applied. The dynamic range of linearity was approximately 1 order of magnitude. The method was applied successfully to monitor the time-dependent evolution of substrates and products in trypsin-catalyzed digests of single peptides and peptide mixtures. Thus, ionic liquid matrixes allow quantitative MALDI-MS without the need for internal standards, making the method a suitable tool for the fast screening of new enzymes or the search for substrates or inhibitors.     

Sensitive determination of volatile aliphatic aldehydes in autoxidized fats and oils by gas chromatographic/electron-capture detection of their O-pentafluorobenzyloxime derivatives

 This paper describes a novel method for the sensitive determination of the volatile aliphatic aldehydes formed by autoxidation in fats and oils. In a solution of fats, the aldehydes are derivatized to the O-pentafluorobenzyloximes (PFBOs) without any isolation procedure. From the mixture, the lipids are removed by silica gel column chromatography using pentane/dichloromethane (4+1) as the eluant. The eluate is evaporated to dryness and the PFBOs are extracted from the residue with a small volume of acetonitrile. They are separated by capillary column gas chromatography and are determined with high selectivity and sensitivity by using an electron-capture detector. The determination limit was as low as 0.01 mg/kg for hexanal. Analysis of a series of corn oil samples yielded high recoveries for 14 aldehydes added at the 1 mg/kg level, with low relative standard deviations of between 3 and 17%. Received: 9 May 1997     

Polychlorinated biphenyl residues in food and human milk: determination of co-planar and mono-ortho substituted congeners

An analytical method is described for determining the residues of coplanar as well as mono-ortho substituted PCB congeners both exhibiting toxicological relevance, in foods of animal origin and in human milk. The unsophisticated procedure, convenient for routine analyses, includes the extraction of lipids, clean-up steps using liquid/liquid partition and column chromatography, fractionation of the congeners using HPLC on a special carbon column with an optimal gradient elution, and capillary column gas chromatography with electron capture or mass spectrometric detection. As preliminary results indicate, the low-chlorinated PCB technical products contribute more to the actual contamination of environment and foods than has been estimated so far. Obviously the co-planar congeners are accumulated during the food chain in a portion different from that found for the other congeners.     

Exposure to secondhand smoke in Germany: air contamination due to smoking in German restaurants, bars, and other venues.

This study quantified exposure to secondhand smoke in German restaurants, bars, and entertainment venues by determining the concentration of respirable suspended particles measuring 2.5 microm or less (PM2.5) in indoor air. The measurements were taken using an inconspicuous device placed on the investigator's table in the venue. The concentration of particulate matter in the indoor air was measured for a minimum of 30 min. A total of 39 restaurants, 20 coffee bars, 12 bars, 9 discothèques, and 20 restaurant cars in trains were visited throughout Germany from September 30 to October 31, 2005. The readings disclosed a median PM2.5 of 260 microg/m3 and an arithmetic mean PM2.5 of 333 microg/m3. Median values were 378 microg/m3 in bars, 131 microg/m3 in cafes, and 173 microg/m3 in restaurants. The highest medians were measured in discothèques and restaurant cars, with values averaging 432 microg/m3 and 525 microg/m3 PM2.5, respectively. This study was the first to show the magnitude and extent of exposure to secondhand smoke on such an extensive scale in Germany. The contaminated air due to smoking is a human carcinogenic and major health hazard, which would be prevented most effectively and completely by implementing a ban on smoking. This study is important for the ongoing national debate in Germany as well as for debates in all countries without smoke-free air legislation, which includes most countries around the world.     

Effect of repeated episodes of generic clinical mastitis on milk yield in dairy cows

Our objective was to estimate the milk losses associated with multiple occurrences of generic bovine clinical mastitis (CM) within and across lactations. We studied 10,380 lactations from 5 large, high-producing dairy herds that used automatic recording of daily milk yields. Mixed models, with a random herd effect and an autoregressive covariance structure to account for repeated measurements, were used to quantify the effect of CM and other control variables (parity, week of lactation, other diseases) on milk yield. Many cows that developed CM were higher producers than their non-mastitic herdmates before CM occurred. Milk yield began to drop after diagnosis; the greatest loss occurred in the first weeks (up to 126 kg) and then gradually tapered to a constant value approximately 2 mo after CM. Mastitic cows often never recovered their potential yield. First-lactation cows lost 164 kg of milk for the first episode and 198 kg for the second in the 2 mo after CM diagnosis, compared with their potential yield. Among older cows, this estimate was 253 kg for the first, 238 kg for the second, and 216 kg for the third CM case. A cow that had 1 or more CM episodes in her previous lactation produced 1.2 kg/d less milk over the whole current lactation (95% confidence interval: 0.6, 1.7) than a cow without CM in her previous lactation. These findings provide dairy producers with information on the average milk loss associated with CM cases without considering the causative agent, and can be used for economic analysis.     

Differential In Vitro Effects of SGLT2 Inhibitors on Mitochondrial Oxidative Phosphorylation,Glucose Uptake and Cell Metabolism

(1) The cardio-reno-metabolic benefits of the SGLT2 inhibitors canagliflozin (cana), dapagliflozin (dapa), ertugliflozin (ertu), and empagliflozin (empa) have been demonstrated, but it remains unclear whether they exert different off-target effects influencing clinical profiles. (2) We aimed to investigate the effects of SGLT2 inhibitors on mitochondrial function, cellular glucose-uptake (GU), and metabolic pathways in human-umbilical-vein endothelial cells (HUVECs). (3) At 100 µM (supra-pharmacological concentration), cana decreased ECAR by 45% and inhibited GU (IC5o: 14 µM). At 100 µM and 10 µM (pharmacological concentration), cana increased the ADP/ATP ratio, whereas dapa and ertu (3, 10 µM, about 10× the pharmacological concentration) showed no effect. Cana (100 µM) decreased the oxygen consumption rate (OCR) by 60%, while dapa decreased it by 7%, and ertu and empa (all 100 µM) had no significant effect. Cana (100 µM) inhibited GLUT1, but did not significantly affect GLUTs’ expression levels. Cana (100 µM) treatment reduced glycolysis, elevated the amino acids supplying the tricarboxylic-acid cycle, and significantly increased purine/pyrimidine-pathway metabolites, in contrast to dapa (3 µM) and ertu (10 µM). (4) The results confirmed cana´s inhibition of mitochondrial activity and GU at supra-pharmacological and pharmacological concentrations, whereas the dapa, ertu, and empa did not show effects even at supra-pharmacological concentrations. At supra-pharmacological concentrations, cana (but not dapa or ertu) affected multiple cellular pathways and inhibited GLUT1.     

Computational Light Painting Using a Virtual Exposure

Light painting is an artform where a light source is moved during a long‐exposure shot, creating trails resembling a stroke on a canvas. It is very difficult to perform because the light source needs to be moved at the intended speed and along a precise trajectory. Additionally, images can be corrupted by the person moving the light. We propose computational light painting, which avoids such artifacts and is easy to use. Taking a video of the moving light as input, a virtual exposure allows us to draw the intended light positions in a post‐process. We support animation, as well as 3D light sculpting, with high‐quality results.     

Quad‐Based Fourier Transform for Efficient Diffraction Synthesis

Far‐field diffraction can be evaluated using the Discrete Fourier Transform (DFT) in image space but it is costly due to its dense sampling. We propose a technique based on a closed‐form solution of the continuous Fourier transform for simple vector primitives (quads) and propose a hierarchical and progressive evaluation to achieve real‐time performance. Our method is able to simulate diffraction effects in optical systems and can handle varying visibility due to dynamic light sources. Furthermore, it seamlessly extends to near‐field diffraction. We show the benefit of our solution in various applications, including realistic real‐time glare and bloom rendering.     

Hydrodynamic modeling of ephemeral flow in the Iishana channel systems of the Cuvelai Basin—Northern Namibia

The transboundary region of the Iishana system in the western Cuvelai Basin, between southern Angola and northern Namibia, is frequently affected by floods at irregular intervals. As a result, the predominantly rural, subsistence farming population has experienced crop failures, human, and economic losses. To date, very little is known about the generation of floods, flood concentration, and stormwater drainage dynamics in this region. In this study, 2D-hydrodynamic modeling was applied to reconstruct one of the latest major flood events during the rainy season from November 2008 to March 2009 in order to study the runoff behavior and interconnectivity of the Iishana system. The model focused on the eastern part of the Iishana system, which was most affected by floods and flood damage due to the high population density in and around Oshakati, the regional capital. Two main streams were identified noteworthy because they merge and subsequently affect Oshakati. Regarding the simulated flood event water depths vary from 0.1 m to 14 m, with an average of 0.2 m, while water depths above 5 m were attributed to borrow pits. The inundation area ranged up to 1860 km² and the amount of water left after the rainy season on March 25th, 2009, was determined between 0.116 and 0.547 km³, depending on the amount of evapotranspiration considered in the model. Thus, in the Angolan part of the Iishana system, significantly larger quantities of water are available for longer periods of time during the subsequent dry season, whereas the system in Namibia stores less water, resulting in a shorter water retention period.     

Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine**

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay‘s potential for epigenetic evaluation of clinical samples, benefiting research and patient management.