High-throughput data analysis for detecting and identifying differences between samples in GC/MS-based metabolomic analyses

In metabolomics, the objective is to identify differences in metabolite profiles between samples. A widely used tool in metabolomics investigations is gas chromatography-mass spectrometry (GC/MS). More than 400 compounds can be detected in a single analysis, if overlapping GC/MS peaks are deconvoluted. However, the deconvolution process is time-consuming and difficult to automate, and additional processing is needed in order to compare samples. Therefore, there is a need to improve and automate the data processing strategy for data generated in GC/MS-based metabolomics; if not, the processing step will be a major bottleneck for high-throughput analyses. Here we describe a new semiautomated strategy using a hierarchical multivariate curve resolution approach that processes all samples simultaneously. The presented strategy generates (after appropriate treatment, e.g., multivariate analysis) tables of all the detected metabolites that differ in relative concentrations between samples. The processing of 70 samples took similar time to that of the GC/TOFMS analyses of the samples. The strategy has been validated using two different sets of samples: a complex mixture of standard compounds and Arabidopsis samples.     

Zur Bestimmung der Wasseraufnahmefähigkeit pulverförmiger Proteinrohstoffe 1. Mitt. Die Kapillarsaugmethode

Die Kapillarsaugmethode ermöglicht eine einfache und schnelle Bestimmung der Wasseraufnahmefähigkeit pulverförmiger Proteinrohstoffe. In Untersuchungen an 10 verschiedenen Proteinrohstoffen wird ge-zeigt. daß sich mit zunehmender Einwaage (Schichtdicke) die in g Wasser/g Pulver ausgedrückte Wasseraufnahme leicht erhöht. Bei Einwaagen zwischen 50 und 200 mg ist die Abhängigkeit jedoch so gering, daß sie vernachlässigt werden kann. Mit der Korngröße nimmt die Wasseraufnahme exponentiell zu. Nur bei Korngrößen zwischen 50 und 110 μm Durchmesser kann dies außer acht gelassen werden. Die Art des Proteinrohstoffs, Temperatur, pH-Wert und Ionenmilieu wirken sich unterschiedlich aus. Die Standardabweichung der Methode ist bei Proteinrohstoffen unabhängig von der Größe des Meßwertes und betragt 0,075 g je g Die Bestimmungsgrenze des Verfahrens liegt für eine Einzelbestimmung bei 0,28 und für eine Dreifach-bestimmung bei 0,16 g/g. Die mit der Kapillarsaugmethode verfolgte Wasseraufnahme von Proteinrohstoffen kann nach einer Kinetik nullter (Vorgang mit konstanter Wasseraufnahmerate, z. B. Quellung) oder erster Ordnung (Adsorption) verlaufen, bzw. sie geht in eine gemischte Kinetik über. L. HEINEVFTTER, J. KROLL, B. GASSMANN and K. HOPPE: Determination of the capability of water uptaking by powdery protein raw materials. Part 1. The capillary suction method. The capillary suction method permits simple and rapid determination of the capability of water uptaking by powdery protein raw materials. Investigations with 10 different protein raw materials have demonstrated, that the water uptake expressed in gram water per gram powder is weakly increased, if the amount of sample taken and the layer thickness respectively has been increased. At amounts in the range of 50 to 200 mg however this dependence is insignificantly, so it may be neglected. The water uptake increases exponentially with increasing particle size. Only in case of particle sizes with diameters between 50 and 110 μm this may be ignored. Kind of protein material, temperature, pH value and ion relationships have diverse effects. The Standard deviation of the method related to protein raw materials is independent of the value order measured and amounts 0,075 g/g. The detection limit of the method in case of single determinations is about 0.28 g/g and in case of triple determinations it is 0,16 g/g. The water uptake by protein raw materials followed up with the capillary suction method can be represented by a zero order kinetics (process with constant water uptake rate, i.e. swelling) or by a first order kinetics (water adsorption), otherwise the water uptake rate-may change over to a mixed order kinetics.     

The cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (FKBP59)

Using a rapid single-step affinity chromatography procedure we have isolated the unactivated estrogen receptor from bovine uterus. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analyses for protein extracts recovered from affinity chromatography of receptor cytosols, either preincubated or untreated with estradiol, suggest a component structure for the intact oligomeric receptor which includes hsp90, hsp70, p59, a 40-kDa cyclophilin-related protein, and an uncharacterized 22-kDa protein species. We have chemically determined the amino acid sequences of eight peptides derived from the 40-kDa component and now report the cloning and primary sequence of a cDNA encoding this protein, which is designated estrogen receptor-binding cyclophilin (ERBC). Homology analyses confirm that ERBC is a new member of the cyclophilin family and contains a C-terminal domain with significant sequence homology to an internal region of p59, a binding protein for the immunosuppressant FK506 (FKBP59). This conserved region includes a 3-unit tetratricopeptide repeat domain bounded at the C terminus by a putative calmodulin binding site. We propose that the tetratricopeptide repeat domain mediates the protein interaction properties of ERBC and p59. Both immunophilins may have important roles in receptor assembly and may represent a new category of ligand- and calcium-dependent modulators of protein function.     

Engineered superoxide dismutase monomers for superoxide biosensor applications

Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasi-reversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by approximately 1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold.     

Upregulation of Anti-Oxidative Stress Response Improves Metabolic Changes in L-Selectin-Deficient Mice but Does Not Prevent NAFLD Progression or Fecal Microbiota Shifts

(1) Background: Non-alcoholic fatty liver disease (NAFLD) is a growing global health problem. NAFLD progression involves a complex interplay of imbalanced inflammatory cell populations and inflammatory signals such as reactive oxygen species and cytokines. These signals can derive from the liver itself but also from adipose tissue or be mediated via changes in the gut microbiome. We analyzed the effects of a simultaneous migration blockade caused by L-selectin-deficiency and an enhancement of the anti-oxidative stress response triggered by hepatocytic Kelch-like ECH-associated protein 1 (Keap1) deletion on NAFLD progression. (2) Methods: L-selectin-deficient mice (Lsel^−/−Keap1^flx/flx) and littermates with selective hepatic Keap1 deletion (Lsel^−/−Keap1^Δhepa) were compared in a 24-week Western-style diet (WD) model. (3) Results: Lsel^−/−Keap1^Δhepa mice exhibited increased expression of erythroid 2-related factor 2 (Nrf2) target genes in the liver, decreased body weight, reduced epidydimal white adipose tissue with decreased immune cell frequencies, and improved glucose response when compared to their Lsel^−/−Keap1^flx/flx littermates. Although WD feeding caused drastic changes in fecal microbiota profiles with decreased microbial diversity, no genotype-dependent shifts were observed. (4) Conclusions: Upregulation of the anti-oxidative stress response improves metabolic changes in L-selectin-deficient mice but does not prevent NAFLD progression and shifts in the gut microbiota.     

Thermoset IM-LSI-based C/C-SiC: Influence of flow direction and weld lines on microstructure and mechanical properties

The production of ceramic matrix composites (CMC) based on C/C-SiC is still very cost-intensive and therefore only economical for a few applications. The fabrication of the preforms involves many costs that need to be reduced. In this work, the shaping of the CFRP-preforms is realized by thermoset injection molding, which enables large-scale production. The polymeric matrix used is a multi-component matrix consisting of novolak resin, curing agent and lubricant. Six millimeter chopped carbon fiber with a proportion of 50 wt.% were used as a reinforcement. These ingredients are processed by an industrial equipment for compounding and injection molding in order to manufacture a CFRP demonstrator representing a brake disc. Test specimens are cut out of the demonstrator in different directions in order to investigate influences of flow direction and weld lines on microstructural and mechanical properties. Afterward, the CFRP samples were converted to C/C-SiC composites by the liquid silicon infiltration process. The article addresses the flow behavior of the compound during the injection molding and the building of the weld lines in the demonstrator. In addition, results of the directional dependence of the microstructural and mechanical properties within the fabricated disc in the different production steps are presented.