Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessaryprior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.     

Feather steroid hormone concentrations in relation to age,sex, and molting time in a long‐distance migratory passerine

In birds, concentrations of testosterone (T) and corticosterone (Cort) are closely connected with many morphological, behavioral, and other physiological traits, including reproduction, metabolism, immunity, and fitness. The direction of the effect of these hormones on above‐mentioned traits, and the potential feedback between hormones are in general unclear; in addition, knowledge on how age and sex can affect T and Cort concentrations is still inconsistent. Our study used a novel method to analyze testosterone and corticosterone in feathers (T_f, Cort_f) based on the precolumn chemical derivatization of hormones before liquid chromatography–tandem mass spectrometry (LC‐MS/MS) analysis. Unlike previously used methods (RIA, EIA), our analytical procedure allows simultaneous analysis of both hormones from small amounts of feathers (4–25 mg) and, thus, overcomes the problem of insufficient detection limits. We applied this method to reveal associations between T_f and Cort_f hormone concentrations and feather growth, age, and sex in feathers grown during the postbreeding (flanks) and prebreeding (tails) periods in barn swallows (Hirundo rustica). There was neither a correlation between prebreeding and postbreeding T_f, nor between prebreeding and postbreeding Cort_f. Tail Cort_f concentrations were negatively associated with tail feather growth rates. Feather hormone concentrations were correlated in the prebreeding period, negatively in males but positively in females. Both Cort_f and T_f were higher in young birds compared to older ones, indicating either an age‐related decrease in hormone concentrations within individuals, or the selective disappearance of individuals with high steroid concentrations. Males and females did not differ in Cort_f, but T_f concentrations were higher in males than females, particularly during the prebreeding period. In this study, we provide an effective method for analyzing hormones in feathers in an ecological context, especially in situations when the total amount of feathers available for the analysis is limited.     

Evaluation of risk profiles by subcutaneous adipose tissue topography in obese juveniles

Objective: To compare subcutaneous adipose tissue topography (SAT‐top) in obese juveniles with age‐matched normal‐weight controls. Research Methods and Procedures: The optical device LIPOMETER (European Patent EP 0516251) enables the non‐invasive, rapid, safe, and precise measurement of the thickness of subcutaneous adipose tissue. Fifteen defined body sites (1 = neck to 15 = calf) characterize the individual SAT‐top like an individual fingerprint. SAT‐top of 1351 juveniles (obese: 42 boys, 59 girls, normal weight: 680 boys, 570 girls) from 7 to 19 years of age were measured. For visual comparison, the 15‐dimensional SAT‐top information was condensed by factor analysis into a two‐dimensional factor plot. Results: Both female and male obese juveniles had markedly increased adipose tissue layers at 7 = upper abdomen, 8 = lower abdomen, 5 = front chest, and 6 = lateral chest. The pubertal changes of body shape and fat distribution of the normal‐weight boys and girls (boys show thinner adipose tissue layers on their legs, whereas girls had thicker adipose tissue layers at the extremities) were not seen in the obese group. Independently of age and sex, all of the obese juveniles showed a similar, more android body fat distribution with increased trunk fat. Discussion: SAT‐top of the obese juveniles is similar to that of patients with type 2 diabetes, polycystic ovary syndrome, and coronary heart disease. Patients with these metabolic disorders and obese juveniles are located in the factor plot in the same area. This body shape may indicate a risk profile for developing polycystic ovary syndrome (women), type 2 diabetes, and early atherosclerosis (both sexes).     

Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.     

Proteome analysis of chemically induced mouse liver tumors with different genotype

Mouse liver tumors frequently harbor mutations in Ha-ras, B-raf, or Ctnnb1 (encoding beta-catenin). We conducted a proteome analysis with protein extracts from normal mouse liver and from liver tumors which were induced by a single injection of N-nitrosodiethylamine (DEN) as initiator followed by multiple injections of two different polychlorinated biphenyls (PCBs) as tumor promoters, or corn oil as a control. Liver tumors were stratified into two classes: they were either mutated in Ctnnb1 and positive for the marker glutamine synthetase (GS(+)), or they lacked Ctnnb1 mutations and were therefore GS-negative (GS(-)). Proteome analysis by 2-DE and MS revealed 98 significantly deregulated proteins, 44 in GS(+) and 54 in GS(-) tumors. Twelve of these proteins showed expression changes in both tumor types, but only seven of them were deregulated in the same direction. Several of the identified enzymes could be assigned to fundamental metabolic or other cellular pathways with characteristically different alterations in GS(+) and GS(-) tumors such as ammonia and amino acid turnover, cellular energy supply, and calcium homeostasis. Our data suggest that GS(+) and GS(-) tumor cells show a completely different biology and use divergent evolutionary strategies to gain a selective advantage over normal hepatocytes.