The impact of peptide abundance and dynamic range on stable-isotope-based quantitative proteomic analyses

Recently, mass spectrometry has been employed in many studies to provide unbiased, reproducible, and quantitative protein abundance information on a proteome-wide scale. However, how instruments' limited dynamic ranges impact the accuracy of such measurements has remained largely unexplored, especially in the context of complex mixtures. Here, we examined the distribution of peptide signal versus background noise (S/N) and its correlation with quantitative accuracy. With the use of metabolically labeled Jurkat cell lysate, over half of all confidently identified peptides had S/N ratios less than 10 when examined using both hybrid linear ion trap-Fourier transform ion cyclotron resonance and Orbitrap mass spectrometers. Quantification accuracy was also highly correlated with S/N. We developed a mass precision algorithm that significantly reduced measurement variance at low S/N beyond the use of highly accurate mass information alone and expanded it into a new software suite, Vista. We also evaluated the interplay between mass measurement accuracy and S/N; finding a balance between both parameters produced the greatest identification and quantification rates. Finally, we demonstrate that S/N can be a useful surrogate for relative abundance ratios when only a single species is detected.     

A drastic reduction in the life span of cystatin C L68Q carriers due to life-style changes during the last two centuries

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal dominant disease with high penetrance, manifest by brain hemorrhages in young normotensive adults. In Iceland, this condition is caused by the L68Q mutation in the cystatin C gene, with contemporary carriers reaching an average age of only 30 years. Here, we report, based both on linkage disequilibrium and genealogical evidence, that all known copies of this mutation derive from a common ancestor born roughly 18 generations ago. Intriguingly, the genealogies reveal that obligate L68Q carriers born 1825 to 1900 experienced a drastic reduction in life span, from 65 years to the present-day average. At the same time, a parent-of-origin effect emerged, whereby maternal inheritance of the mutation was associated with a 9 year reduction in life span relative to paternal inheritance. As these trends can be observed in several different extended families, many generations after the mutational event, it seems likely that some environmental factor is responsible, perhaps linked to radical changes in the life-style of Icelanders during this period. A mutation with such radically different phenotypic effects in reaction to normal variation in human life-style not only opens the possibility of preventive strategies for HCCAA, but it may also provide novel insights into the complex relationship between genotype and environment in human disease.     

Rapid and sensitive anthrone-sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: method development and validation.

The need for an accurate, fast and reliable analysis of carbohydrate test is crucial for numerous biological processes. In that sense, anthrone-sulfuric acid assay is one of the most efficient quantification techniques successfully applied to carbohydrate determination. In this paper, a sensitive and accurate anthrone-sulfuric acid microplate assay was developed and validated for the quantitative estimation of yeast carbohydrates in the production of hepatitis B virus surface antigen, and the main component of the recombinant vaccine HEBERBIOVAC HB. A response surface methodology was applied to design and optimize the assay in order to maximize the differences on the expected effect and to minimize the number of experiments. The proposed method was linear over the concentration range from 10 to 120 microg/mL for glucose, with values for the coefficient of determination >0.99. Intra- and inter-assay variation coefficient ranged between 0.45-4.79% and 2.48-8.94%, respectively. The Student t-test used in the interference study, revealed good parallelism among curves (T(obs)< or =T(0.05)), which indicates the lack of interference in the working range. Yields obtained in accuracy test for two concentration levels varied between 90 and 105%, confirming the assay's reliability. In conclusion, the validated method, which has successfully been used for the process control monitoring of several samples generated from the production of hepatitis B vaccine, allows the quality and purity of the final product.     

Involvement of the p38 MAPK-NF-kappaB signal transduction pathway and COX-2 in the pathobiology of meniscus degeneration in humans

Meniscal tears are attributed to either trauma or degeneration processes. Clinical data suggest that meniscal degeneration (MD) is associated with knee osteoarthritis; however, the molecular events underpinning the pathogenesis of MD in humans remain elusive. Here we immunohistochemically examined the expression of p38 MAPK, its phosphorylated/activated form (p-p38), its target NF-kappaB (p50-p65 dimer), and COX-2 in ruptured menisci and investigated their involvement in MD development. Our findings demonstrate increased expression of the p38-NF-kappaB axis elements and COX-2 in disintegrated fibrocartilage, suggesting a role of these molecules in the pathobiochemistry of MD and consequential rupture.     

The localized adherence pattern of an atypical enteropathogenic Escherichia coli is mediated by intimin omicron and unexpectedly promotes HeLa cell invasion

Enteropathogenic Escherichia coli (EPEC) forms attaching and effacing lesions in the intestinal mucosa characterized by intimate attachment to the epithelium by means of intimin (an outer membrane adhesin encoded by eae ). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC); only tEPEC carries the EAF (EPEC adherence factor) plasmid that encodes the bundle-forming pilus (BFP). Characteristically, after 3 h of incubation, tEPEC produces localized adherence (LA) (with compact microcolonies) in HeLa/HEp-2 cells by means of BFP, whereas most aEPEC form looser microcolonies. We have previously identified nine aEPEC strains displaying LA in extended (6 h) assays (LA6). In this study, we analysed the kinetics of LA6 pattern development and the role of intimin in the process. Transmission electron microscopy and confocal laser microscopy showed that the invasive process of strain 1551-2 displays a LA phenotype. An eae -defective mutant of strain 1551-2 prevented the invasion although preserving intense diffused adherence. Sequencing of eae revealed that strain 1551-2 expresses the omicron subtype of intimin. We propose that the LA phenotype of aEPEC strain 1551-2 is mediated by intimin omicron and hypothesize that this strain expresses an additional novel adhesive structure. The present study is the first to report the association of compact microcolony formation and an intense invasive ability in aEPEC.     

Vegetation mapping and nature conservation: a case study in Terceira Island (Azores)

In order to emphasize the importance of vegetation mapping for nature conservation purposes a case study in Terceira island (Azores) is presented, in which the importance of the natural vegetation of the eastern slope of Santa Bárbara volcano (which is part of the Site of Community Importance of Santa Bárbara–Pico Alto) is evaluated through the elaboration of its vegetation map. Fourteen (14) different natural vegetation types were identified: grasslands (1 type), peat bogs (2 types), scrubs (2), forests (5), successional vegetation (3) and vegetation of rocky slopes (1). All communities are protected under the Habitat and Species Directive (EC/92/43) and most of them are endemic to the Azores Islands. This fact, together with the significant number of Azorean endemic taxa (18), Macaronesian endemic taxa (5) and species protected under the Habitat and Species Directive (7), gives this area an important conservation value that justifies future protection actions. Vegetation mapping is an important tool for the characterization, evaluation and implementation of managing plans of natural areas of the Azores islands. The use of a floristic-based classification, supported by multivariate analysis and structural data, is an efficient methodology for the construction of these maps. The data collected comprise an important set of information about the distribution and abundance of natural vegetation types and endemic and rare species. This information was not available until now and is indispensable for the elaboration of management plans of Special Zones for Conservation that will be part of the NATURA 2000 network.     

The guanine nucleotide exchange factors Sec2 and PRONE: candidate synapomorphies for the Opisthokonta and the Archaeplastida

Although recent multigene phylogenetic analyses support close relationship of Metazoa and Fungi (the eukaryotic supergroup Opisthokonta) and monophyly of eukaryotes with the primary plastid, that is, Chloroplastida, Rhodophyta, and Glaucophyta (the supergroup Archaeplastida or Plantae), some authors still challenge this scheme. I found that 2 particular types of guanine nucleotide exchange factors (GEFs, i.e., cofactors of GTPases) might provide a new piece of evidence to resolve this controversy. An exhaustive analysis of available sequence data revealed that Sec2-related proteins, known to serve as GEF for exocytic GTPases of the Rab8/Sec4 subfamily, are restricted to opisthokonts, whereas proteins with the PRONE domain, recently described as novel plant-specific GEFs for RHO family GTPases, occur only in Chloroplastida and Rhodophyta. The results thus point to possible evolutionary innovations in the exocytic apparatus of the ancestral opisthokonts and reveal the probably first plastid-independent trait (i.e., a unique mode of RHO GTPase regulation) exclusive for Chloroplastida + Rhodophyta, further supporting monophyly of these 2 groups.     

Water deprivation increases Fos expression in hypothalamic corticotropin-releasing factor neurons induced by right atrial distension in awake rats

Atrial mechanoreceptors, sensitive to stretch, contribute in regulating heart rate and intravascular volume. The information from those receptors reaches the nucleus tractus solitarius and then the paraventricular nucleus (PVN), known to have a crucial role in the regulation of cardiovascular function. Neurons in the PVN synthesize CRF, AVP, and oxytocin (OT). Stimulation of atrial mechanoreceptors was performed in awake rats implanted with a balloon at the junction of the superior vena cava and right atrium. Plasma ACTH, AVP, and OT concentrations and Fos, CRF, AVP, and OT immunolabeling in the PVN were determined after balloon inflation in hydrated and water-deprived rats. The distension of the balloon increased the plasma ACTH concentrations, which were higher in water-deprived than in hydrated rats (P < 0.05). In addition, the distension in the water-deprived group decreased plasma AVP concentrations (P < 0.05), compared with the respective control group. The distension increased the number of Fos- and double-labeled Fos/CRF neurons in the parvocellular PVN, which was higher in the water-deprived than in the hydrated group (P < 0.01). There was no difference in the Fos expression in magnocellular PVN neurons after distension in hydrated and water-deprived groups, compared with respective controls. In conclusion, parvocellular CRF neurons showed an increase of Fos expression induced by stimulation of right atrial mechanoreceptors, suggesting that CRF participates in the cardiovascular reflex adjustments elicited by volume loading. Activation of CRF neurons in the PVN by cardiovascular reflex is affected by osmotic stimulation.     

Tandem use of X-ray crystallography and mass spectrometry to obtain ab initio the complete and exact amino acids sequence of HPBP, a human 38-kDa apolipoprotein

The Human Phosphate Binding Protein (HPBP) is a serendipitously discovered apolipoprotein from human plasma that binds phosphate. Amino acid sequence relates HPBP to an intriguing protein family that seems ubiquitous in eukaryotes. These proteins, named DING according to the sequence of their four conserved N-terminal residues, are systematically absent from eukaryotic genome databases. As a consequence, HPBP amino acids sequence had to be first assigned from the electronic density map. Then, an original approach combining X-ray crystallography and mass spectrometry provides the complete and a priori exact sequence of the 38-kDa HPBP. This first complete sequence of a eukaryotic DING protein will be helpful to study HPBP and the entire DING protein family.