Analytical precision,biological variation,and mathematical normalization in high data density metabolomics

Metabolic serotypes sensitive to caloric intake may enable sera metabolomic profiles to validate epidemiological parameters and predict disease risk in humans. This long-range goal is complicated by the lack of known state markers and the requirement for simultaneous monitoring of multiple small changes. Therefore, analytical precision for appropriate high data density studies using HPLC separations coupled with coulometric array detectors was evaluated over a two month period in pooled rat sera samples (previously collected and stored at –80 °C), and in authentic biochemical standards. In sera, mean coefficients of variation (CV) of retention time and ratio accuracy within the established metabolic serotype varied within ±1% and ±3%, respectively. In sets of purified standards, the same parameters fluctuated, correspondently, in ranges of ±0.1% and ±1%. Median CV of the metabolite concentrations were ~13% in standards and ~11–19% in sera, and varied non-monotonically with the analytical system status and experimental design. These parameters were shown to be sufficiently controlled so as not to dominate intra-group biological variability in serum metabolomics studies. Continuation of experimental runs across an analytical breakpoint (column replacement) was associated with disproportionate changes in metabolite concentrations, independent of maintained analytical precision. These changes were sufficient to shift overall profile localization in megavariate projection analyses. We developed a mathematical approach to normalize this break and use partial least squares projection to latent structure discriminant analysis to confirm validity of this normalization approach. This generally applicable mathematical correction helps enable longer term high data density studies by removing a critical source of systemic variation.     

Sequence determinants of protein folding rates: positive correlation between contact energy and contact range indicates selection for fast folding

In comparison with intense investigation of the structural determinants of protein folding rates, the sequence features favoring fast folding have received little attention. Here, we investigate this subject using simple models of protein folding and a statistical analysis of the Protein Data Bank (PDB). The mean-field model by Plotkin and coworkers predicts that the folding rate is accelerated by stronger-than-average interactions at short distance along the sequence. We confirmed this prediction using the Finkelstein model of protein folding, which accounts for realistic features of polymer entropy. We then tested this prediction on the PDB. We found that native interactions are strongest at contact range l = 8. However, since short range contacts tend to be exposed and they are frequently formed in misfolded structures, selection for folding stability tends to make them less attractive, that is, stability and kinetics may have contrasting requirements. Using a recently proposed model, we predicted the relationship between contact range and contact energy based on buriedness and contact frequency. Deviations from this prediction induce a positive correlation between contact range and contact energy, that is, short range contacts are stronger than expected, for 2/3 of the proteins. This correlation increases with the absolute contact order (ACO), as expected if proteins that tend to fold slowly due to large ACO are subject to stronger selection for sequence features favoring fast folding. Our results suggest that the selective pressure for fast folding is detectable only for one third of the proteins in the PDB, in particular those with large contact order.     

A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells

Background

Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays.

Principal Findings

LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis.

Conclusion

LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.     

Human Haemato-Endothelial Precursors: Cord Blood CD34+ Cells Produce Haemogenic Endothelium

Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144−), triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45−) capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.     

Homogeneous asymmetric catalysis in fragrance chemistry

Opposite enantiomers of a chiral fragrance may exhibit different olfactory activities making a synthesis in high enantiomeric purity commercially and scientifically interesting. Accordingly, the asymmetric synthesis of four chiral odorants, Fixolide, Phenoxanol, Citralis, and Citralis Nitrile, has been investigated with the aim to develop practically feasible processes. In the devised synthetic schemes, the key step that leads to the formation of the stereogenic center is the homogeneous asymmetric hydrogenation of a prochiral olefin. By an appropriate choice of the catalyst and the reaction conditions, Phenoxanol, Citralis, and Citralis Nitrile were obtained in high enantiomeric purity, and odor profiles of the single enantiomers were determined.     

Anaerobic and aerobic relative contribution to total energy release during supramaximal effort in patients with left ventricular dysfunction.

Energetic metabolism during effort is impaired in patients with left ventricular dysfunction (Dysf), but data have been lacking up to now on the relative anaerobic vs. aerobic contribution to total energy release during supramaximal effort. Recently, the maximal accumulated oxygen deficit (MAOD) has been shown to be measurable in Dysf patients, making it possible to evaluate the anaerobic/aerobic interaction under conditions of maximal stress of both anaerobic and aerobic metabolic pathways in this population. Nineteen Dysf patients and 17 normal patients (N) underwent one ramp cardiopulmonary, three moderate-intensity constant-power, and three supramaximal constant-power (1- to 2-min, 2- to 3-min, and 3- to 4-min duration) exercise tests. MAOD was the difference between accumulated O(2) demand (accO(2)dem; estimated from the moderate-intensity O(2) uptake/watt relationship) and uptake during supramaximal tests. Percent anaerobic (%Anaer) and aerobic (%Aer) energetic release were [(MAOD/accO(2)dem).100] and 100 - %Anaer, respectively. MAOD did not vary between 1-2, 2-3, and 3-4 min supramaximal tests, whereas accO(2)dem increased significantly with and was linearly related to test duration in both Dysf and N. Consequently, %Anaer and %Aer decreased and increased, respectively, with increasing test duration but did not differ between Dysf and N in 1-2 min, 2-3 min, and 3-4 min tests. Our study demonstrates a similar relative anaerobic vs. aerobic contribution to total energy release during supramaximal effort in Dysf and N. This finding indicates that energetic metabolism during supramaximal exercise is exercise tolerance independent and that relative anaerobic vs. aerobic contribution in this effort domain remains the same within the physiology- or pathology-induced limits to individual peak exercise performance.     

Influences of temperature and threshold effect of NaCl concentration on Alpias vulpinus OCT

Thermodynamic, circular dichroism (CD), and activity measurements have been used to characterize the different conformational states and the effects of NaCl concentrations (0.0-3.0 M) on thermal unfolding of ornithine carbamoyltransferase (OCT) from Alopias vulpinus. Furthermore conformational changes in whole enzyme structure have been monitored by titration of SH-groups. OCT unfolding process follows an irreversible two-state mechanism with a first-order kinetic of denaturation, without breaking-point. NaCl shows very little stabilization effects at low concentration and its action become very important over 1.5 M concentration. The presence of 3.0 M NaCl completely avoids OCT unfolding at 60, 64 and 66 degrees C. Kinetic and thermodynamic parameters are strongly influenced by the presence of high NaCl concentration. Our experiments showed that NaCl stabilization process involved changes in preferential binding, in electrostatic and van der Waals interactions and exposure of buried site and SH-groups. During thermal denaturation, UV-vis and CD spectroscopy show that high salts concentration preserves OCT activity, avoiding exposure of hydrophobic site and destruction of secondary and tertiary structure elements.     

Spatial scale and meiobenthic copepod recolonisation: testing the effect of disturbance size in a seagrass habitat

A central problem in relating disturbance to community structure lies in determining how community structure is affected by the size of disturbance events. In soft-bottom habitats, recovery rate and patterns of macrobenthic community are usually affected by the spatial scale of disturbance. Thus far, no studies have explicitly addressed these issues for meiobenthic copepods. To test the effects of the size of hypoxia/anoxia disturbance on the recovery of meiobenthic copepod communities in a vegetated (Ruppia cirrhosa) sediment, a field experiment was set up in Valle Smarlacca, a brackish lagoon on the northern Adriatic coast of Italy. Plots of three different sizes—small (40×40 cm), medium (80×80 cm) and large (160×160 cm)—were exposed to experimentally induced hypoxia/anoxia for 5 days. Control plots of 40×40 cm were added, for comparison with ambient abundance. Recolonisation and community recovery were then observed for 12 days, with samplings on days 0, 1, 3, 5, 7, 9 and 12. Sampling was designed to focus on the distance from source pools of colonists. The induced anoxia had a severe impact on the copepods, but the impact of the disturbance was independent of plot size. Copepod abundance increased linearly over time, and no differences in recolonisation rate among the differently sized plots were observed. Recolonisation comprised two phases: until day 3, abundances were low and very similar in all plot sizes; from day 5 onwards, abundances of the dominant species (which were the same in control and disturbed plots) increased, and a more diversified pattern among disturbance sizes was observed. Multivariate analyses showed a gradual response of community structure to disturbance size: copepod assemblages in small plots attained the same structure of the control plots at day 5, while for larger-sized plots this occurred later and was observed on the following sampling date. However, clear differences among the three disturbance sizes were never detected. Variability in community structure seemed to respond more to the overall impact of disturbance than to the size of the disturbed area. In the seagrass meadows of the Valle Smarlacca, several factors seem to influence the structural organisation of meiobenthic copepod communities during recolonisation processes. Among others, the recovery of the vegetated habitat to suitable conditions seems to play a much more relevant role than the size of the disturbed patches.     

Circulating hematopoietic progenitor cells in runners.

Because endurance exercise causes release of mediators and growth factors active on the bone marrow, we asked whether it might affect circulating hematopoietic progenitor cells (HPCs) in amateur runners [n = 16, age: 41.8 +/- 13.5 (SD) yr, training: 93.8 +/- 31.8 km/wk] compared with sedentary controls (n = 9, age: 39.4 +/- 10.2 yr). HPCs, plasma cortisol, interleukin (IL)-6, granulocyte colony-stimulating factor (G-CSF), and the growth factor fms-like tyrosine kinase-3 (flt3)-ligand were measured at rest and after a marathon (M; n = 8) or half-marathon (HM; n = 8). Circulating HPC counts (i.e., CD34(+) cells and their subpopulations) were three- to fourfold higher in runners than in controls at baseline. They were unaffected by HM or M acutely but decreased the morning postrace. Baseline cortisol, flt3-ligand, IL-6, and G-CSF levels were similar in runners and controls. IL-6 and G-CSF increased to higher levels after M compared with HM, whereas cortisol and flt3-ligand increased similarly postrace. Our data suggest that increased HPCs reflect an adaptation response to recurrent, exercise-associated release of neutrophils and stress and inflammatory mediators, indicating modulation of bone marrow activity by habitual running.