Amniotic fluid is a dynamic and complex mixture that reflects the physiological status of the developing fetus. In this study, the human amniotic fluid (AF) proteome of a 16 -18-week normal pregnancy was profiled and analyzed to investigate the composition and functions of this fluid. Due to the complexity of AF, we utilized three different fractionation strategies to provide greater coverage. Two types of two-dimensional LC/MS/MS as well as an LC-SDS-PAGE-LC-MS/MS platform were used. A total of 16 AF samples between gestational ages of 16 and 18 weeks from women carrying chromosomally normal fetuses were analyzed by one of the three fractionation methods followed by a common reverse phase LC-MS/MS step. Mascot and The Global Proteome Machine engines were used to search the International Protein Index human database for peptide sequence identification. The list of proteins was generated by combining the results of both engines through the PeptideProphet of Scaffold software. All identified proteins were combined to generate the AF proteome comprising 1,026 unique gene matches or 842 non-redundant proteins. This list includes most of the currently used biomarkers for pregnancy-associated pathologic conditions such as preterm delivery, intra-amniotic infection, and chromosomal anomalies of the fetus. The subcellular localization, tissue expression, functions, and networks of the AF proteome were analyzed by various bioinformatic tools. These data will contribute to the better understanding of amniotic fluid function and to the discovery of novel biomarkers for prenatal diagnosis of fetal abnormalities.

It has been suggested that cancer-associated fibroblasts (CAFs) positioned at the desmoplastic areas of various types of cancer are capable of executing a migratory program, characterized by accelerated motility and collective configuration. Since CAFs are reprogrammed derivatives of normal progenitors, including quiescent fibroblasts, we hypothesized that such migratory program could be context-dependent, thus being regulated by specific paracrine signals from the adjacent cancer population. Using the traditional scratch assay setup, we showed that only specific colon cancer cell lines (i.e. HT29) were able to induce collective CAF migration. By performing quantitative proteomics (SILAC), we identified a 2.7-fold increase of claudin-11, a member of the tight junction apparatus, in CAFs that exerted such collectivity in their migratory pattern. Further proteomic investigations of cancer cell line secretomes revealed a specific signature, involving TGF-β, as potential mediator of this effect. Normal colonic fibroblasts stimulated with TGF-β exerted myofibroblastic differentiation, occludin (OCLN) and claudin-11 (CLDN11) overexpression and cohort formation. Subsequently, inhibition of TGF-β attenuated all the previous effects. Immunohistochemistry of the universal tight junction marker occludin in a cohort of 30 colorectal adenocarcinoma patients defined a CAF subpopulation expressing tight junctions. Overall, these data suggest that cancer cells may induce CLDN11 overexpression and subsequent collective migration of peritumoral CAFs via TGF-β secretion.

BackgroundDown syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways.ResultsChromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression.ConclusionsThe most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21.