Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Dasty3, a WEB framework for DAS

MOTIVATION: Dasty3 is a highly interactive and extensible Web-based framework. It provides a rich Application Programming Interface upon which it is possible to develop specialized clients capable of retrieving information from DAS sources as well as from data providers not using the DAS protocol. Dasty3 provides significant improvements on previous Web-based frameworks and is implemented using the 1.6 DAS specification. AVAILABILITY: Dasty3 is an open-source tool freely available at http://www.ebi.ac.uk/dasty/ under the terms of the GNU General public license. Source and documentation can be found at http://code.google.com/p/dasty/. CONTACT: hhe@ebi.ac.uk.     

A comparative study of MHC Class-II HLA-DRbeta1*0401-Col II and HLA-DRbeta1*0101-HA complexes: a theoretical point of view

A study was performed on the HLA-DRbeta1*0401-collagen II peptide complex using the computation of electronic multipolar variables proposed by us previously. Furthermore, these results were compared with those obtained for the HLA-DRbeta1*0101-haemaglutinin peptide complex studied by us with the same tools, confirming that Pocket 1 for this new complex is also the most important pocket for the interaction between the presenting molecule and the presented peptide. The pocket hierarchy established for HLA-DRbeta1*0401 allele was P1 > P9 approximately P7 > P6 > P4, whilst a P1 > P4 > P9 approximately P7>P6 pocket hierarchy was found for HLA-DRbeta1*0101, showing how the relative importance of the pockets distinguishes the two alleles. There are high correlation levels with experimental results (when possible), again confirming the validity of using calculated values for electronic multipolar variables as a useful tool for studying interactions between immune system molecules and peptides.     

Adolescent expectations of early death predict adult risk behaviors

Only a handful of public health studies have investigated expectations of early death among adolescents. Associations have been found between these expectations and risk behaviors in adolescence. However, these beliefs may not only predict worse adolescent outcomes, but worse trajectories in health with ties to negative outcomes that endure into young adulthood. The objectives of this study were to investigate perceived chances of living to age 35 (Perceived Survival Expectations, PSE) as a predictor of suicidal ideation, suicide attempt and substance use in young adulthood. We examined the predictive capacity of PSE on future suicidal ideation/attempt after accounting for sociodemographics, depressive symptoms, and history of suicide among family and friends to more fully assess its unique contribution to suicide risk. We investigated the influence of PSE on legal and illegal substance use and varying levels of substance use. We utilized the National Longitudinal Study of Adolescent Health (Add Health) initiated in 1994-95 among 20,745 adolescents in grades 7-12 with follow-up interviews in 1996 (Wave II), 2001-02 (Wave III) and 2008 (Wave IV; ages 24-32). Compared to those who were almost certain of living to age 35, perceiving a 50-50 or less chance of living to age 35 at Waves I or III predicted suicide attempt and ideation as well as regular substance use (i.e., exceeding daily limits for moderate drinking; smoking ≥ a pack/day; and using illicit substances other than marijuana at least weekly) at Wave IV. Associations between PSE and detrimental adult outcomes were particularly strong for those reporting persistently low PSE at both Waves I and III. Low PSE at Wave I or Wave III was also related to a doubling and tripling, respectively, of death rates in young adulthood. Long-term and wide-ranging ties between PSE and detrimental outcomes suggest these expectations may contribute to identifying at-risk youth.     

Quantitative patterns of Hsps in tubular adenoma compared with normal and tumor tissues reveal the value of Hsp10 and Hsp60 in early diagnosis of large bowel cancer

Large bowel carcinogenesis involves accumulation of genetic alterations leading to transformation of normal mucosa into dysplasia and, lastly, adenocarcinoma. It is pertinent to elucidate the molecular changes occurring in the pre-neoplastic lesions to facilitate early diagnosis and treatment. Heat shock proteins (Hsps), many of which are molecular chaperones, are implicated in carcinogenesis, and their variations with tumor progression encourage their study as biomarkers. There are many reports on Hsps and cancer but none to our knowledge on their systematic quantification in pre-neoplastic lesions of the large bowel. We performed immunohistochemical determinations of Hsp10, Hsp60, Hsp70, and Hsp90 in biopsies of large bowel tubular adenomas with moderate grade of dysplasia and compared to normal mucosa and adenocarcinoma with a moderate grade of differentiation (G2). A significant elevation of Hsp10 and Hsp60 only, i.e., in the absence of elevation of Hsp70 or Hsp90, in both epithelium and lamina propria was found in tubular adenoma by comparison with normal mucosa. In contrast, adenocarcinoma was characterized by the highest levels of Hsp10 and Hsp60 in epithelium and lamina propria, accompanied by the highest levels of Hsp70 only in epithelium and of Hsp90 only in lamina propria, by comparison with normal and tubular adenoma counterparts. Hsp10 and Hsp60 are promising biomarkers for early diagnosis of tubular adenoma and for its differentiation from more advanced malignant lesions. Hsp10 and Hsp60 may be implicated in carcinogenesis from its very early steps and, thus, are potentially convenient targets for therapy.