Use of 18O Labels to Monitor Deamidation during Protein and Peptide Sample Processing

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c· + 58 and z − 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)(n−1)+· − 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of 18O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (̃4 h) trypsin digestion process generates no additional detectable peptide deamidations

Ecological assessment of groundwater ecosystems disturbed by recharge systems using organic matter quality, biofilm characteristics and bacterial diversity

International audienc

On the evaluation of photogrammetric methods for dense 3D surface reconstruction in a metrological context.

This paper discusses a methodology to evaluate the accuracy of recently developed image-based 3D modelling techniques. So far, the emergence of these novel methods has not been supported by the definition of an internationally recognized standard which is fundamental for user confidence and market growth. In order to provide an element of reflection and solution to the different communities involved in 3D imaging, a promising approach is presented in this paper for the assessment of both metric quality and limitations of an open-source suite of tools (Apero/MicMac), developed for the extraction of dense 3D point clouds from a set of un- ordered 2D images. The proposed procedural workflow is performed within a metrological context, through inter-comparisons with \u2018reference\u2019 data acquired with two hemispherical laser scanners, one total station, and one laser tracker. The methodology is applied to two case studies, designed in order to analyse the software performances in dealing with both outdoor and environmentally controlled conditions, i.e. the main entrance of Cath\ue9drale de la Major (Marseille, France) and a custom-made scene located at National Research Council of Canada 3D imaging Metrology Laboratory (Ottawa). Comparative data and accuracy evidence produced for both tests allow the study of some key factors affecting 3D model accuracy

A rat model of early stage osteonecrosis induced by glucocorticoids

<p>Abstract</p> <p>Background</p> <p>Glucocorticoid (GC)-induced osteonecrosis (ON) is an important complication of medical therapy. The exact pathomechanisms of ON has not been clearly elucidated. There is a need for a reproducible animal model that better approximates the clinical scenario.</p> <p>Methods</p> <p>To determine the genetic susceptibility of rats to develop GC-induced femoral head ON, we evaluated 5 different inbred strains of rats (Spontaneous Hypertensive Rat, Wistar Kyoto, Wistar Furth, SASCO Fisher and Lewis). Prednisone pellets (dosage of 1.82-2.56 mg/kg/day) were implanted subcutaneously for 90. After 90 days, the femurs were resected and examined histologically and radiographically. Pathological and histological examination was performed. Hematoxylin and eosin (H & E) staining was used to delineate the femoral head osteonecrosis lesions as well as abnormalities of articular cartilage and growth plate.</p> <p>Results</p> <p>The greatest differences in H & E staining were seen in the Wistar Kyoto and Wistar Furth groups. In these groups 4 out of 5 and 3 out of 5, respectively, steroid-induced rats revealed growth plate disruption with acellular areas. The TUNEL apoptosis staining assay for apoptosis revealed that 4 out of 5 of Wistar Kyoto rats, 5 out of 5 of Wistar Furth, 2 out of 4 of surviving Lewis and 2 out of 2 of the surviving spontaneous hypertensive rats had apoptotic osteocytes in trabeculae, whereas none of the Fisher rats showed apoptotic osteocytes.</p> <p>Conclusions</p> <p>We postulate that Wistar Kyoto, Wistar Furth and spontaneous hypertensive rats may be strains of rats more susceptible to develop ON of the femoral head while Fisher rats were the most resistant.</p

Long-Lived Electron Capture Dissociation Product Ions Experience Radical Migration via Hydrogen Abstraction

To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the α-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the α-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c·, and they also show c·-1 peaks in the deuterated peptides only. The presence of the c·-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H· and −CO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments

Radiation shielding of protoplanetary discs in young star-forming regions

Protoplanetary discs spend their lives in the dense environment of a star forming region. While there, they can be affected by nearby stars through external photoevaporation and dynamic truncations. We present simulations that use the AMUSE framework to couple the Torch model for star cluster formation from a molecular cloud with a model for the evolution of protoplanetary discs under these two environmental processes. We compare simulations with and without extinction of photoevaporation-driving radiation. We find that the majority of discs in our simulations are considerably shielded from photoevaporation-driving radiation for at least 0.5 Myr after the formation of the first massive stars. Radiation shielding increases disc lifetimes by an order of magnitude and can let a disc retain more solid material for planet formation. The reduction in external photoevaporation leaves discs larger and more easily dynamically truncated, although external photoevaporation remains the dominant mass loss process. Finally, we find that the correlation between disc mass and projected distance to the most massive nearby star (often interpreted as a sign of external photoevaporation) can be erased by the presence of less massive stars that dominate their local radiation field. Overall, we find that the presence and dynamics of gas in embedded clusters with massive stars is important for the evolution of protoplanetary discs.Comment: 23 pages, 22 figures, 1 table, accepted for publication in MNRA

Selection of nitrogen-fixing deficient Burkholderia vietnamiensis strains by cystic fibrosis patients: involvement of nif gene deletions and auxotrophic mutations

Burkholderia vietnamiensis is the third most prevalent species of the Burkholderia cepacia complex (Bcc) found in cystic fibrosis (CF) patients. Its ability at fixing nitrogen makes it one of the main Bcc species showing strong filiations with environmental reservoirs. In this study, 83% (29 over 35) of the B. vietnamiensis CF isolates and 100% of the environmental ones (over 29) were found expressing the dinitrogenase complex (encoded by the nif cluster) which is essential in N 2 fixation. Among the deficient strains, two were found growing with ammonium chloride suggesting that they were defective in N 2 fixation, and four with amino acids supplements suggesting that they were harbouring auxotrophic mutations. To get insights about the genetic events that led to the emergence of the N 2 -fixing defective strains, a genetic analysis of B. vietnamiensis nitrogen-fixing property was undertaken. A 40-kb-long nif cluster and nif regulatory genes were identified within the B. vietnamiensis strain G4 genome sequence, and analysed. Transposon mutagenesis and nifH genetic marker exchanges showed the nif cluster and several other genes like gltB (encoding a subunit of the glutamate synthase) to play a key role in B. vietnamiensis ability at growing in nitrogen-free media. nif cluster DNA probings of restricted genomic DNA blots showed a full deletion of the nif cluster for one of the N 2 -fixing defective strain while the other one showed a genetic organization similar to the one of the G4 strain. For 17% of B. vietnamiensis clinical strains, CF lungs appeared to have favoured the selection of mutations or deletions leading to N 2 -fixing deficiencies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75167/1/j.1462-2920.2007.01240.x.pd

Early-Stage Metastasis Requires Mdm2 and Not p53 Gain of Function

Metastasis of cancer cells to distant organ systems is a complex process that is initiated with the programming of cells in the primary tumor. The formation of distant metastatic foci is correlated with poor prognosis and limited effective treatment options. We and others have correlated Mouse double minute 2 (Mdm2) with metastasis; however, the mechanisms involved have not been elucidated. Here, it is reported that shRNA-mediated silencing of Mdm2 inhibits epithelial–mesenchymal transition (EMT) and cell migration. In vivo analysis demonstrates that silencing Mdm2 in both post-EMT and basal/triple-negative breast cancers resulted in decreased primary tumor vasculature, circulating tumor cells, and metastatic lung foci. Combined, these results demonstrate the importance of Mdm2 in orchestrating the initial stages of migration and metastasis