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.

Large deviations for stochastic Volterra equations

This paper is devoted to prove a large-deviation principle for solutions to multidimensional stochastic Volterra equations.

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.

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.

Solar Powered Highway Delineator System, HR-367, 1993

Currently, many drivers experience some difficulty in viewing the road ahead of them during times of reduced visibility, such as rain, snow, fog, or the darkness of night- Recent studies done by the National Safety Council provide a detailed contrast between fatal accidents occurring during the day and night. Revealed was that the motor vehicle night death rate (4.41 deaths per 100 million miles driven) was sharply higher than the corresponding death rate during daylight hours (1.21). By providing a delineating system powered by the natural resource of solar power, a constant source of visibility may be maintained throughout the evening. Along with providing enough light to trace the outline of the road, other major goals defined in producing this delineator system are as follows: 1. A strong and durable design that would protect the internal components and survive extreme weather conditions. 2. A low maintenance system where components need few repairs or replacements. 3. A design which makes all components accessible in the event that maintenance is needed, but also prevents vandalism. 4. A design that provides greater visibility to drivers and will not harm a vehicle or its passengers in the event of a collision. This solar powered highway delineator consists of an adjustable solar array, a light fixture, and a standard delineator pole. The solar array houses and protects the solar panels, and can be easily adjusted to obtain a maximum amount of sunlight. The light fixture primarily houses the battery, the circuit and the light assembly. Both components allow for easy accessibility and reduce vandalism using internal connections for bolts and wires. The delineator mounting pole is designed to extensively deform in the event of a collision, therefore reducing any harm caused to the vehicle and/or the passengers. The cost of a single prototype to be produced is approximately $70.00 excluding labor costs. However, these material and labor costs will be greatly reduced if a large number of delineators are produced. It is recommended that the Iowa Department of Transportation take full advantage of the research and development put into this delineator design. The principles used in creating this delineator can be used to provide an outline for drivers to follow, or on a larger scale, provide actual roadway lighting in areas where it was never before possible or economically feasible. In either event, the number of fatal accidents will be decreased due to the improved driver visibility in the evening.

A conserved transcript pattern in response to a specialist and a generalist herbivore.

Transcript patterns elicited in response to attack reveal, at the molecular level, how plants respond to aggressors. These patterns are fashioned both by inflicted physical damage as well as by biological components displayed or released by the attacker. Different types of attacking organisms might therefore be expected to elicit different transcription programs in the host. Using a large-scale DNA microarray, we characterized gene expression in damaged as well as in distal Arabidopsis thaliana leaves in response to the specialist insect, Pieris rapae. More than 100 insect-responsive genes potentially involved in defense were identified, including genes involved in pathogenesis, indole glucosinolate metabolism, detoxification and cell survival, and signal transduction. Of these 114 genes, 111 were induced in Pieris feeding, and only three were repressed. Expression patterns in distal leaves were markedly similar to those of local leaves. Analysis of wild-type and jasmonate mutant plants, coupled with jasmonate treatment, showed that between 67 and 84% of Pieris-regulated gene expression was controlled, totally or in part, by the jasmonate pathway. This was correlated with increased larval performance on the coronatine insensitive1 glabrous1 (coi1-1 gl1) mutant. Independent mutations in COI1 and GL1 led to a faster larval weight gain, but the gl1 mutation had relatively little effect on the expression of the insect-responsive genes examined. Finally, we compared transcript patterns in Arabidopis in response to larvae of the specialist P. rapae and to a generalist insect, Spodoptera littoralis. Surprisingly, given the complex nature of insect salivary components and reported differences between species, almost identical transcript profiles were observed. This study also provides a robustly characterized gene set for the further investigation of plant-insect interaction.