To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early differentiation with the PAF1 (polymerase-associated factor 1) transcriptional elongation complex, which binds to promoters of the pluripotency and known DNMT target genes encoding OCT4 and NANOG, thereby providing a possible molecular link for the silencing of these genes during differentiation.

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations. Molecular & Cellular Proteomics 8:959 -970, 2009. Human embryonic stem cells (hESCs)1 are stem cells derived from the blastocyst inner cell mass. They are pluripotent; thus they are able to differentiate into any human cell type. The self-renewal capacity and pluripotency make hESCs an ideal system to study the processes of cell development and differentiation. Moreover hESC research is highly relevant for regenerative medicine, which aims at replacing or restoring tissue damaged by disease or injury through transplantation of functional hESCs (1, 2). However, factors responsible for maintaining the undifferentiated and pluripotent nature of hESCs are still largely unknown. Before hESCs can be used for transplantation into the human body, reliable and reproducible protocols for differentiating them into specific cell types are needed. To create such protocols we need to develop a thorough understanding of the mechanisms maintaining the undifferentiated pluripotent nature of hESCs and those guiding their differentiation into specific lineages.A number of factors involved in the maintenance of pluripotency have been described over the last few years (3). It has also been demonstrated that overexpression of some of these factors in somatic cells is sufficient to turn them into pluripotent stem cells very similar to hESCs (4 -8). However, it is apparent that the processes occurring during such transformation are extremely complex. A large number of factors and pathways are involved in maintaining the pluripotent state and regulat...

Our present liposomes selectively target monocytes in fresh blood, enabling delivery of TLR7 agonists to the intracellular TLR7 receptor, with subsequent monocyte activation and boost in secretion of proinflammatory cytokines. We envision this technology as a promising tool in future cancer immunotherapy.

Economical field development strategy often implies tie-in of subsea satellite fields to nearby host installations. This leads to a whole new set of benefits and challenges considering design and material selection, production volumes and limitations, company strategies, holistic management and multi-disciplinary approaches. Operation of complex systems with multiple fluid streams demands a broader understanding of the chemical processes taking place when different fluids are mixed. Typical challenges include mineral scale and "soft scale" deposits. To ensure optimum production and provide flow assurance through chemical management, proper monitoring is essential. Guidelines and best practices are even more required if the tie-in to the host includes several operators and service companies. Over the years, the Statoil operated Oseberg asset has through close cooperation with its chemical supplier M-I SWACO systematically improved the sampling and analysis procedures to strengthen the quality of data used in system monitoring. The supplier needs to have a strong focus on flow assurance related to chemical management and provide a range of onshore and offshore monitoring techniques and tools. Challenges from the North Sea Oseberg Field Centre installation with subsea tie-ins have been discussed. Laboratory and field data from bottle tests, chemical analysis, preservation techniques and scaling potential simulations are presented. The results have been used to plan for side stream tests, develop guidelines for early identification of flow assurance challenges, sampling and monitoring of complex fluid systems and chemical management to avoid process upsets and production losses.