Addressing trypsin bias in large scale (phospho)proteome analysis by size exclusion chromatography and secondary digestion of large post-trypsin peptides.

In the vast majority of bottom-up proteomics studies, protein digestion is performed using only mammalian trypsin. Although it is clearly the best enzyme available, the sole use of trypsin rarely leads to complete sequence coverage, even for abundant proteins. It is commonly assumed that this is because many tryptic peptides are either too short or too long to be identified by RPLC-MS/MS. We show through in silico analysis that 20-30% of the total sequence of three proteomes (Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Homo sapiens) is expected to be covered by Large post-Trypsin Peptides (LpTPs) with M(r) above 3000 Da. We then established size exclusion chromatography to fractionate complex yeast tryptic digests into pools of peptides based on size. We found that secondary digestion of LpTPs followed by LC-MS/MS analysis leads to a significant increase in identified proteins and a 32-50% relative increase in average sequence coverage compared to trypsin digestion alone. Application of the developed strategy to analyze the phosphoproteomes of S. pombe and of a human cell line identified a significant fraction of novel phosphosites. Overall our data indicate that specific targeting of LpTPs can complement standard bottom-up workflows to reveal a largely neglected portion of the proteome.

Expression of MYC, IgM, As Well As Non-Germinal Centre B-Cell Like Immunophenotype and Positive Immunofish Index Predict a Worse Progression Free Survival and Overall Survival in a Series of 670 De Novo Diffuse Large B-Cell Lymphomas Included in Clinical Trials: A GELA Study of the 2003 Program

Introduction: Diffuse large B-cell lymphomas (DLBCL) represent a heterogeneous disease with variable clinical outcome. Identifying phenotypic biomarkers of tumor cells on paraffin sections that predict different clinical outcome remain an important goal that may also help to better understand the biology of this lymphoma. Differentiating non-germinal centre B-cell-like (non-GCB) from Germinal Centre B-cell-like (GCB) DLBCL according to Hans algorithm has been considered as an important immunohistochemical biomarker with prognostic value among patients treated with R-CHOP although not reproducibly found by all groups. Gene expression studies have also shown that IgM expression might be used as a surrogate for the GCB and ABC subtypes with a strong preferential expression of IgM in ABC DLBCL subtype. ImmunoFISH index based on the differential expression of MUM-1, FOXP1 by immunohistochemistry and on the BCL6 rearrangement by FISH has been previously reported (C Copie-Bergman, J Clin Oncol. 2009;27:5573-9) as prognostic in an homogeneous series of DLBCL treated with R-CHOP. In addition, oncogenic MYC protein overexpression by immunohistochemistry may represent an easy tool to identify the consequences of MYC deregulation in DLBCL. Our aim was to analyse by immunohistochemistry the prognostic relevance of MYC, IgM, GCB/nonGCB subtype and ImmunoFISH index in a large series of de novo DLBCL treated with Rituximab (R)-chemotherapy (anthracyclin based) included in the 2003 program of the Groupe d'Etude des Lymphomes de l'Adulte (GELA) trials. Methods: The 2003 program included patients with de novo CD20+ DLBCL enrolled in 6 different LNH-03 GELA trials (LNH-03-1B, -B, -3B, 39B, -6B, 7B) stratifying patients according to age and age-adjusted IPI. Tumor samples were analyzed by immunohistochemistry using CD10, BCL6, MUM1, FOXP1 (according to Barrans threshold), MYC, IgM antibodies on tissue microarrays and by FISH using BCL6 split signal DNA probes. Considering evaluable Hans score, 670 patients were included in the study with 237 (35.4%) receiving intensive R-ACVBP regimen and 433 (64.6%) R-CHOP/R-mini-CHOP. Results: 304 (45.4%) DLBCL were classified as GCB and 366 (54.6%) as non-GCB according to Hans algorithm. 337/567 cases (59.4%) were positive for the ImmunoFISH index (i.e. two out of the three markers positive: MUM1 protein positive, FOXP1 protein Variable or Strong, BCL6 rearrangement). Immunofish index was preferentially positive in the non-GCB subtype (81.3%) compared to the GCB subtype (31.2%), (p<0.001). IgM was recorded as positive in tumor cells in 351/637 (52.4%) DLBCL cases with a preferential expression in non-GCB 195 (53.3%) vs GCB subtype 100(32.9%), p<0.001). MYC was positive in 170/577 (29.5%) cases with a 40% cut-off and in 44/577 (14.2%) cases with a cut-off of 70%. There was no preferential expression of MYC among GCB or non-GCB subtype (p>0.4) for both cut-offs. Progression-free Survival (PFS) was significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p<0.0001), MYC positive tumor with a 40% threshold (p<0.001), ImmunoFISH positive index (p<0.002), non-GCB DLBCL subtype (p<0.0001). Overall Survival (OS) was also significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p=0.02), MYC positive tumor with a 40% threshold (p<0.01), ImmunoFISH positive index (p=0.02), non-GCB DLBCL subtype (p<0.0001). All significant parameters were included in a multivariate analysis using Cox Model and in addition to IPI, only the GCB/non-GCB subtype according to Hans algorithm predicted significantly a worse PFS among non-GCB subgroup (HR 1.9 [1.3-2.8] p=0.002) as well as a worse OS (HR 2.0 [1.3-3.2], p=0.003). This strong prognostic value of non-GCB subtyping was confirmed considering only patients treated with R- CHOP for PFS (HR 2.1 [1.4-3.3], p=0.001) and for OS (HR 2.3 [1.3-3.8], p=0.002). Conclusion: Our study on a large series of patients included in trials confirmed the relevance of immunohistochemistry as a useful tool to identify significant prognostic biomarkers for clinical use. We show here that IgM and MYC might be useful prognostic biomarkers. In addition, we confirmed in this series the prognostic value of the ImmunoFISH index. Above all, we fully validated the strong and independent prognostic value of the Hans algorithm, daily used by the pathologists to subtype DLBCL.

Etude de la variation interlaboratoire du test de dépistage prénatal des défauts de fermeture du tube neural [Interlaboratory variation in the prenatal screening test for neural tube closing defects].

Early detection of neural-tude defects is possible by determining Alpha-fetoprotein (AFP) in maternal serum. 16'685 pregnant women were observed. Three methods for the determination of the "normal" range are compared. The first one, already used in similar studies, makes use of a constant multiple of the median. The other two ones make use of robust estimates of location and scale. Their comparison shows the interest of the robust methods to reduce the interlaboratory variability.

Iron Supplementation Therapy in a Large Swiss Cohort of Inflammatory Bowel Disease Patients: Shift from Oral to Intravenous Therapy over Time

Background a nd A ims: T he 2 007 ECCO g uidelines o nanemia in inflammatory bowel disease (IBD) favour intravenous(iv) over oral (po) i ron supplementation due to bettereffectiveness and tolerance. Application of guidelines in clinicalpractice m ay r equire time. We a imed to determine thepercentage of IBD patients under iron supplementation therapyand its application mode over time in a large IBD cohort.Methods: Helsana, a leading Swiss health insurance companyprovides c overage f or approximately 18% of t he Swisspopulation, corresponding to about 1.2 million enrollees.Patients with Crohn's disease (CD) and ulcerative colitis (UC)were identified b y keyword search from t he a nonymisedHelsana database.Results: I n total, 6 29 CD ( 61% female) a nd 4 03 UC ( 56%female) patients w ere identified, mean retrospectiveobservation time w as 2 0.4 m onths f or CD and 13 m onths f orUC patients. Of t he entire study population, 29.3% wereprescribed iron. O ccurrence of iron prescription was 21.3% inmales a nd 31.2% in f emales ( odds r atio [OR] 1 .69, 95%-confidence interval [CI] 1.26-2.28). The prescription of iv i ronincreased from 2006/2007 ( 48.8% w ith iv i ron) to 2 008/2009(65.2% with iv iron) by a factor of 1.89.Conclusions: One third of the IBD population was treated withiron supplementation. A gradual s hift from oral t o iv iron wasobserved over time in a large Swiss IBD cohort. This switch inprescription habits g oes a long with the implementation of theECCO consensus guidelines on anemia in IBD.

Reactivity of antiglioma monoclonal antibodies for a large panel of cultured gliomas and other neuroectoderm derived tumors.

We produced three monoclonal antibodies, BF7, GE2 and CG12, against cultured human glioma cells. Their specificity was tested by an indirect antibody-binding radioimmunoassay on a panel of glial and non-glial tumor cell lines. BF7 and GE2 react preferentially with glioma cells and, except for one colon carcinoma line, they do not bind to the control non-neuroectodermal cells; they appear to be directed against common malignant glioma associated antigens. CG12, the third monoclonal antibody, binds to the great majority of tumor cell lines of neuroectodermal origin and does not bind to any other cell lines tested.

Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways.

Through genome-wide association meta-analyses of up to 133,010 individuals of European ancestry without diabetes, including individuals newly genotyped using the Metabochip, we have increased the number of confirmed loci influencing glycemic traits to 53, of which 33 also increase type 2 diabetes risk (q < 0.05). Loci influencing fasting insulin concentration showed association with lipid levels and fat distribution, suggesting impact on insulin resistance. Gene-based analyses identified further biologically plausible loci, suggesting that additional loci beyond those reaching genome-wide significance are likely to represent real associations. This conclusion is supported by an excess of directionally consistent and nominally significant signals between discovery and follow-up studies. Functional analysis of these newly discovered loci will further improve our understanding of glycemic control.

Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium.

Like numerous other eukaryotic organelles, the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. Organelle fission and fusion processes must be balanced to ensure organelle integrity. Coordination of vacuole fission and fusion depends on the interactions of vacuolar SNARE proteins and the dynamin-like GTPase Vps1p. Here, we identify a novel factor that impinges on the fusion-fission equilibrium: the vacuolar H(+)-ATPase (V-ATPase) performs two distinct roles in vacuole fission and fusion. Fusion requires the physical presence of the membrane sector of the vacuolar H(+)-ATPase sector, but not its pump activity. Vacuole fission, in contrast, depends on proton translocation by the V-ATPase. Eliminating proton pumping by the V-ATPase either pharmacologically or by conditional or constitutive V-ATPase mutations blocked salt-induced vacuole fragmentation in vivo. In living cells, fission defects are epistatic to fusion defects. Therefore, mutants lacking the V-ATPase display large single vacuoles instead of multiple smaller vacuoles, the phenotype that is generally seen in mutants having defects only in vacuolar fusion. Its dual involvement in vacuole fission and fusion suggests the V-ATPase as a potential regulator of vacuolar morphology and membrane dynamics.

Detection of HIV drug resistance during antiretroviral treatment and clinical progression in a large European cohort study.

OBJECTIVE(S): To investigate the relationship between detection of HIV drug resistance by 2 years from starting antiretroviral therapy and the subsequent risk of progression to AIDS and death. DESIGN: Virological failure was defined as experiencing two consecutive viral loads of more than 400 copies/ml in the time window between 0.5 and 2 years from starting antiretroviral therapy (baseline). Patients were grouped according to evidence of virological failure and whether there was detection of the International AIDS Society resistance mutations to one, two or three drug classes in the time window. METHODS: Standard survival analysis using Kaplan-Meier curves and Cox proportional hazards regression model with time-fixed covariates defined at baseline was employed. RESULTS: We studied 8229 patients in EuroSIDA who started antiretroviral therapy and who had at least 2 years of clinical follow-up. We observed 829 AIDS events and 571 deaths during 38,814 person-years of follow-up resulting in an overall incidence of new AIDS and death of 3.6 per 100 person-years of follow-up [95% confidence interval (CI):3.4-3.8]. By 96 months from baseline, the proportion of patients with a new AIDS diagnosis or death was 20.3% (95% CI:17.7-22.9) in patients with no evidence of virological failure and 53% (39.3-66.7) in those with virological failure and mutations to three drug classes (P = 0.0001). An almost two-fold difference in risk was confirmed in the multivariable analysis (adjusted relative hazard = 1.8, 95% CI:1.2-2.7, P = 0.005). CONCLUSION: Although this study shows an association between the detection of resistance at failure and risk of clinical progression, further research is needed to clarify whether resistance reflects poor adherence or directly increases the risk of clinical events via exhaustion of drug options.

Iron supplementation in a large Swiss Cohort of Inflammatory Bowel Disease patients demonstrates a shift form oral to intravenous therapy over time

Background and Aims: The 2007 European Crohn's and Colitis Organization guidelines on anemia in inflammatory bowel disease (IBD) favour intravenous (iv) over oral (po) iron supplementation due to better effectiveness and tolerance. We aimed to determine the percentage of IBD patients under iron supplementation therapy and the dynamics of prescription habits (iv versus po) over time. Methods: Helsana, a leading Swiss health insurance company provides coverage for approximately 18% of the Swiss population, corresponding to about 1.2 million enrollees. Patients with Crohn's disease (CD) and ulcerative colitis (UC) were analyzed from the anonymised Helsana database. Results: In total, 629 CD (61% female) and 398 UC (57% female) patients were identified, mean observation time was 31.8 months for CD and 31.0 months for UC patients. Of the entire study population, 27.1% were prescribed iron (21.1% in males and 31.1% in females). Patients treated with IBD-specific drugs (steroids, immunomodulators, anti-TNF agents) were more frequently treated with iron compared to patients without any medication (35.0% vs. 20.9%, OR 1.91, 95%-CI 1.41-2.61). The prescription of iv iron increased from 2006/2007 (48.8% of all patients receiving any iron priscription) to 65.2% in 2008/2009 by a factor of 1.89. Conclusions: One third of the IBD population was treated with iron supplementation. A gradual shift from oral to iv iron was observed over time. This switch in prescription habits goes along with the implementation of the ECCO consensus guidelines on anemia in IBD.

Dicer1 depletion in male germ cells leads to infertility due to cumulative meiotic and spermiogenic defects.

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis.

Recent human-induced trophic change in the large and deep perialpine Lake Lucerne (Switzerland) compared to historical geochemical variations

This study investigates the sedimentological and geochemical changes that occurred during the last 2200 years in the meromictic Lake Lucerne (Switzerland), one of the largest freshwater lakes of Central Europe. The stable isotope composition (delta C-13 and delta O-18 values) of bulk carbonates is compared to changes in grain-size distribution (clay and silt fraction), natural trace element input (titanium and thorium concentrations), and organic material abundance (C-org, nitrogen and phosphorus) and composition (C/N ratios and hydrogen and oxygen indexes). A drop in carbonate accumulation and in the delta O-18 values of sediments between ca. AD 500 and 700 followed a large and consistent rise in chemical weathering, marked by increases in the silicate-clay fraction and in crustal element concentrations. During the following millennium, there was a long-term decreasing trend in the lithogenic trace element input and in the phosphorus loading, suggesting decreasing terrigeneous input from runoff water. The major sedimentological change over the studied period occurred after ca. AD 1800 with a significant increase in the erosion-driven silt-fraction and in the sedimentation rate. During the last century, human-induced increase in nutrient input to the lake highly enhanced the accumulation of organic matter in sediment. Changes in nutrients and oxygen conditions in the hypolimnion of Lake Lucerne during the eutrophication period (i.e., the last 40 years) highly modified the geochemical fluxes compared to the relatively stable oligotrophic conditions that prevailed during the previous 2000 years. Before the 19th century, climate driven meromixis had a limited impact on the organic matter flux to the sediments, but the accumulation of carbonate considerably decreased during periods of lower mechanical erosion rates and high chemical weathering rates. (C) 2012 Elsevier B.V. All rights reserved.

Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).

Modelling hydrodynamics in the Rio Parana, Argentina : an evaluation and inter-comparison of reduced-complexity and physics based models applied to a large sand-bed river

Depth-averaged velocities and unit discharges within a 30 km reach of one of the world's largest rivers, the Rio Parana, Argentina, were simulated using three hydrodynamic models with different process representations: a reduced complexity (RC) model that neglects most of the physics governing fluid flow, a two-dimensional model based on the shallow water equations, and a three-dimensional model based on the Reynolds-averaged Navier-Stokes equations. Row characteristics simulated using all three models were compared with data obtained by acoustic Doppler current profiler surveys at four cross sections within the study reach. This analysis demonstrates that, surprisingly, the performance of the RC model is generally equal to, and in some instances better than, that of the physics based models in terms of the statistical agreement between simulated and measured flow properties. In addition, in contrast to previous applications of RC models, the present study demonstrates that the RC model can successfully predict measured flow velocities. The strong performance of the RC model reflects, in part, the simplicity of the depth-averaged mean flow patterns within the study reach and the dominant role of channel-scale topographic features in controlling the flow dynamics. Moreover, the very low water surface slopes that typify large sand-bed rivers enable flow depths to be estimated reliably in the RC model using a simple fixed-lid planar water surface approximation. This approach overcomes a major problem encountered in the application of RC models in environments characterised by shallow flows and steep bed gradients. The RC model is four orders of magnitude faster than the physics based models when performing steady-state hydrodynamic calculations. However, the iterative nature of the RC model calculations implies a reduction in computational efficiency relative to some other RC models. A further implication of this is that, if used to simulate channel morphodynamics, the present RC model may offer only a marginal advantage in terms of computational efficiency over approaches based on the shallow water equations. These observations illustrate the trade off between model realism and efficiency that is a key consideration in RC modelling. Moreover, this outcome highlights a need to rethink the use of RC morphodynamic models in fluvial geomorphology and to move away from existing grid-based approaches, such as the popular cellular automata (CA) models, that remain essentially reductionist in nature. In the case of the world's largest sand-bed rivers, this might be achieved by implementing the RC model outlined here as one element within a hierarchical modelling framework that would enable computationally efficient simulation of the morphodynamics of large rivers over millennial time scales. (C) 2012 Elsevier B.V. All rights reserved.

IRM cardiaque en évolution: de grandes études multicentriques en 2012 [Cardiac MR in development: the large multicenter CMR studies in 2012].

The results of several large multicenter CMR studies were reported in 2012, thus, constantly corroborating the evidence on CMR performance. In this review, we present results of the MR-IMPACT programme and the CE-MARC study, which demonstrated the superiority of perfusion-CMR over gated SPECT for the workup of suspected CAD, the currently available data from the European CMR registry, comprising almost 30,000 patients from 57 participating centers in 15 European countries, and finally, the results of the Advisa-MRI study, which documented the safety of a MRI-compatible pacemaker system. These large trials and others set the basis for the recommendations in the new European guidelines on heart failure to use CMR as a first line method if echocardiographic quality is inadequate or the etiology of heart failure is unclear.

The co-evolution of social institutions, demography, and large-scale human cooperation.

Human cooperation is typically coordinated by institutions, which determine the outcome structure of the social interactions individuals engage in. Explaining the Neolithic transition from small- to large-scale societies involves understanding how these institutions co-evolve with demography. We study this using a demographically explicit model of institution formation in a patch-structured population. Each patch supports both social and asocial niches. Social individuals create an institution, at a cost to themselves, by negotiating how much of the costly public good provided by cooperators is invested into sanctioning defectors. The remainder of their public good is invested in technology that increases carrying capacity, such as irrigation systems. We show that social individuals can invade a population of asocials, and form institutions that support high levels of cooperation. We then demonstrate conditions where the co-evolution of cooperation, institutions, and demographic carrying capacity creates a transition from small- to large-scale social groups.