Double standards in quantitative proteomics: direct comparative assessment of difference in gel electrophoresis and metabolic stable isotope labeling

Quantitative protein expression profiling is a crucial part of proteomics and requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. In this report we evaluate in a direct comparative assessment two state-of-the-art quantitative proteomic approaches, namely difference in gel electrophoresis (DiGE) and metabolic stable isotope labeling. Therefore, Saccharomyces cerevisiae was grown under well defined experimental conditions in chemostats under two single nutrient-limited growth conditions using (14)N- or (15)N-labeled ammonium sulfate as the single nitrogen source. Following lysis and protein extraction from the two yeast samples, the proteins were fluorescently labeled using different fluorescent CyDyes. Subsequently, the yeast samples were mixed, and the proteins were separated by two-dimensional gel electrophoresis. Following in-gel digestion, the resulting peptides were analyzed by mass spectrometry using a MALDI-TOF mass spectrometer. Relative ratios in protein expression between these two yeast samples were determined using both DiGE and metabolic stable isotope labeling. Focusing on a small, albeit representative, set of proteins covering the whole gel range, including some protein isoforms and ranging from low to high abundance, we observe that the correlation between these two methods of quantification is good with the differential ratios determined following the equation R(Met.Lab.) = 0.98R(DiGE) with r(2) = 0.89. Although the correlation between DiGE and metabolic stable isotope labeling is exceptionally good, we do observe and discuss (dis)advantages of both methods as well as in relation to other (quantitative) approaches.     

3D reconstruction techniques of human coronary bifurcations for shear stress computations

Background

Heterogeneity in plaque composition in human coronary artery bifurcations is associated with blood flow induced shear stress. Shear stress is generally determined by combing 3D lumen data and computational fluid dynamics (CFD). We investigated two new procedures to generate 3D lumen reconstructions of coronary artery bifurcations for shear stress computations.

Methods

We imaged 10 patients with multislice computer tomography (MSCT) and intravascular ultrasound (IVUS). The 3D reconstruction of the main branch was based on the fusion of MSCT and IVUS. The proximal part of side branch was reconstructed using IVUS data or MSCT data, resulting in two different reconstructions of the bifurcation region. The distal part of the side branch was based on MSCT data alone. The reconstructed lumen was combined with CFD to determine the shear stress. Low and high shear stress regions were defined and shear stress patterns in the bifurcation regions were investigated.

Results

The 3D coronary bifurcations were successfully generated with both reconstruction procedures. The geometrical features of the bifurcation region for the two reconstruction procedures did not reveal appreciable differences. The shear stress maps showed a qualitative agreement, and the low and high shear stress regions were similar in size and average shear stress values were identical. The low and high shear stress regions showed an overlap of approximately 75%.

Conclusion

Reconstruction of the side branch with MSCT data alone is an adequate technique to study shear stress and wall thickness in the bifurcation region. The reconstruction procedure can be applied to further investigate the effect of shear stress on atherosclerosis in coronary bifurcations.     

Comparison of predator–prey interactions with and without intraguild predation by manipulation of the nitrogen source

Theory predicts that intraguild predation leads to different community dynamics than the trophic cascades of a linear food chain. However, experimental comparisons of these two food‐web modules are rare. Mixotrophic plankton species combine photoautotrophic and heterotrophic nutrition by grazing upon other phytoplankton species. We found that the mixotrophic chrysophyte Ochromonas can grow autotrophically on ammonium, but not on nitrate. This offered a unique opportunity to compare predator–prey interactions in the presence and absence of intraguild predation, without changing the species composition of the community. With ammonium as nitrogen source, Ochromonas can compete with its autotrophic prey for nitrogen and therefore acts as intraguild predator. With nitrate, Ochromonas acts solely as predator, and is not in competition with its prey for nitrogen. We parameterized a simple intraguild predation model based on chemostat experiments with monocultures of Ochromonas and the toxic cyanobacterium Microcystis. Subsequently, we tested the model predictions by inoculating Ochromonas into the Microcystis monocultures, and vice versa. The results showed that Microcystis was a better competitor for ammonium than Ochromonas. In agreement with theoretical predictions, Microcystis was much more strongly suppressed by intraguild predation on ammonium than by top–down predation on nitrate. Yet, Microcystis persisted at very low population densities, because the type III functional response of Ochromonas implied that the grazing pressure upon Microcystis became low when Microcystis was rare. Our results provide experimental support for intraguild predation theory, and indicate that intraguild predation may enable biological control of microbial pest species.     

Effectiveness of indoleacetic acid,indolebutyric acid and naphthaleneacetic acid during adventitious root formation in vitro in Malus ‘Jork 9’

The production of an antifungal spirostanol saponin designated SC-1 has been detected in cell suspension cultures of the Mexican species Solanum chrysotrichum. Batch cultures of a cell suspension obtained from hypocotyl derived calluses of this species were grown for 25 days in shake flasks containing Murashige & Skoog (MS) medium. Throughout the growth cycle, fresh and dry weight, SC-1 yield, and uptake of sucrose, glucose and fructose were determined. The effects of inoculum size and sucrose concentration on the biomass accumulation and synthesis of the active metabolite, were studied. The maximum SC-1 production, above 14 mg.g⁻¹ (which was fifty times that of field grown plants), was reached after 20 days using a 2% inoculum and complete MS medium supplemented with 2 mgl⁻¹ 2,4-D, 2 mg l⁻¹kinetin, and sucrose between 30 and 45 gl⁻¹. . This revised version was published online in June 2006 with corrections to the Cover Date.     

Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells.

Intercellular communication of epithelial cells was examined by measuring changes in intracellular calcium concentration ([Ca2+]i). Mechanical stimulation of respiratory tract ciliated cells in culture induced a wave of increasing Ca2+ that spread, cell by cell, from the stimulated cell to neighboring cells. The communication of these Ca2+ waves between cells was restricted or blocked by halothane, an anesthetic known to uncouple cells. In the absence of extracellular Ca2+, the mechanically stimulated cell showed no change or a decrease in [Ca2+]i, whereas [Ca2+]i increased in neighboring cells. Iontophoretic injection of inositol 1,4,5-trisphosphate (IP3) evoked a communicated Ca2+ response that was similar to that produced by mechanical stimulation. These results support the hypothesis that IP3 acts as a cellular messenger that mediates communication through gap junctions between ciliated epithelial cells.     

Intelligence May Moderate the Cognitive Profile of Patients with ASD

BackgroundThe intelligence of individuals with Autism Spectrum Disorder (ASD) varies considerably. The pattern of cognitive deficits associated with ASD may differ depending on intelligence. We aimed to study the absolute and relative severity of cognitive deficits in participants with ASD in relation to IQ.MethodsA total of 274 children (M age = 12.1, 68.6% boys) participated: 30 ASD and 22 controls in the below average Intelligence Quotient (IQ) group (IQ<85), 57 ASD and 54 controls in the average IQ group (85<IQ<115) and 41 ASD and 70 controls in the above average IQ group (IQ>115). Matching for age, sex, Full Scale IQ (FSIQ), Verbal IQ (VIQ), Performance IQ (PIQ) and VIQ-PIQ difference was performed. Speed and accuracy of social cognition, executive functioning, visual pattern recognition and basic processing speed were examined per domain and as a composite score.ResultsThe composite score revealed a trend significant IQ by ASD interaction (significant when excluding the average IQ group). In absolute terms, participants with below average IQs performed poorest (regardless of diagnosis). However, in relative terms, above average intelligent participants with ASD showed the most substantial cognitive problems (particularly for social cognition, visual pattern recognition and verbal working memory) since this group differed significantly from the IQ-matched control group (p < .001), whereas this was not the case for below-average intelligence participants with ASD (p = .57).ConclusionsIn relative terms, cognitive deficits appear somewhat more severe in individuals with ASD and above average IQs compared to the below average IQ patients with ASD. Even though high IQ ASD individuals enjoy a certain protection from their higher IQ, they clearly demonstrate cognitive impairments that may be targeted in clinical assessment and treatment. Conversely, even though in absolute terms ASD patients with below average IQs were clearly more impaired than ASD patients with average to above average IQs, the differences in cognitive functioning between participants with and without ASD on the lower end of the IQ spectrum were less pronounced. Clinically this may imply that cognitive assessment and training of cognitive skills in below average intelligent children with ASD may be a less fruitful endeavour. These findings tentatively suggest that intelligence may act as a moderator in the cognitive presentation of ASD, with qualitatively different cognitive processes affected in patients at the high and low end of the IQ spectrum.     

Mass spectrometric determination of glycosylation sites and oligosaccharide composition of insect-expressed mouse interleukin-3

The primary structure of Baculovirus-expressed mouse interleukin-3 produced in infected Bombyx mori larvae was characterized by liquid secondary ion mass spectrometry and ²⁵²Cf-plasma desorption mass spectrometry in combination with selected protein microchemical reactions. Interleukin-3 was found to consist of at least two glycoprotein species of ca. 17 000 dalton. Characterization of tryptic and S. aureus V8 protease peptides by Edman degradation combined with plasma desorption mass spectrometry showed that two N-glycosylation sites, Asn-16 and Asn-86, were present. N-Glycan residues were shown by liquid secondary ion mass spectrometry and high-performance liquid chromatography to consist of mannose, fucose, and glucosamine. The presence of galactosamine indicated that O-glycosylated residues were present, in addition to the N-glycosylated residues. Glucose was also present, which indicated incomplete processing of the insect-expressed N-linked oligosaccharides.