We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n = 5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ∼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.

Objectives: To indicate cardiogenic shock as a very rare but serious clinical consequence of untreated panhypopituitarism secondary to Sheehan's syndrome; to emphasize the importance of eliciting a detailed endocrine and obstetric history in women presenting with idiopathic heart failure; to indicate the diagnostic shortcomings of screening for thyroid dysfunction solely with TSH determinations; and to report the reversibility of severe heart failure induced by long-term pituitary insufficiency.Methods: Described is a case report of a 35 year old woman who presented with severe congestive heart failure, hypotension and confusion. Her two-dimensional echocardiogram revealed marked systolic and diastolic dysfunction. In screening for possible endocrine causes of heart failure, a normal TSH level of 0.72 (0.35-5.5 mcIU/ml) was unremarkable; but a profoundly low free T4 level of 0.12 (0.9-1.8 ng/dl) led clinicians to pursue a workup of central hypothyroidism.Results: Endocrine testing confirmed panhypopituitarism and adrenal insufficiency. Brain MRI noted empty sella syndrome. Further questioning of the patient revealed a history of extensive postpartum bleeding 15 years earlier, failure to lactate, and secondary amenorrhea; all consistent with undiagnosed Sheehan's syndrome. In the hospital, the patient was treated with intravenous corticosteroids and levothyroxine. Her mental status and symptomatic heart failure improved dramatically. Following nine months of oral levothyroxine and glucocorticoid therapy, the patient has remained asymptomatic and repeat echocardiography indicated completely normalized cardiac function.Conclusions: Severe heart failure and cardiogenic shock can be a very rare (but fortunately reversible) complication of longstanding panhypopituitarism resulting from undiagnosed Sheehan's syndrome.

BACKGROUND Hematopoietic stem cell (HSC) transplantation is an essential element of medical therapy, leading to cures of previously incurable hematological and nonhematological diseases. Many patients do not find matched donors in a timely manner, which has driven efforts to find alternative pools of transplantable HSCs. The use of umbilical cord blood (UCB) as a source of transplantable HSCs began more than two decades ago. However, the use of UCB as a reliable source of HSCs for transplantation still faces crucial challenges: the number of HSCs present in a unit of UCB is usually sufficient for younger children but not for adults, and the persistent delayed engraftment often seen can result in high rates of infection and mortality. STUDY DESIGN AND METHODS We propose a new approach to a solution of these problems: a potential increase of the limited number of UCB-HSCs available by harvesting HSCs contained in the placenta and the fetal chorionic membrane available at birth. RESULTS We investigated the presence of hematopoietic progenitors and HSCs in human placenta and chorion at different gestational ages. The characterization of these cells was performed by flow cytometry and immunolocalization, and their functional status was investigated by transplanting them into immunodeficient mice. CONCLUSION HSCs are present in extraembryonic tissues and could be banked in conjunction to the UCB-HSCs. This novel approach could have a large impact on the field of HSC banking and, more crucially, on the outcome of patients undergoing this treatment by greatly improving the use of life-saving hematopoietic transplants.

The implantation process begins with attachment of the trophectoderm (TE) of the blastocyst to the maternal endometrial epithelium. Herein we have investigated the transcriptome of mural TE cells from 13 human blastocysts and compared these with those of human embryonic stem cell (hESC)-derived-TE (hESC(troph)). The transcriptomes of hESC(troph) at Days 8, 10, and 12 had the greatest consistency with TE. Among genes coding for secreted proteins of the TE of human blastocysts and of hESC(troph) are several molecules known to be involved in the implantation process as well as novel ones, such as CXCL12, HBEGF, inhibin A, DKK3, WNT5A, follistatin. The similarities between the two lineages underscore some of the known mechanisms and offer discovery of new mechanisms and players in the process of the very early stages of human implantation. We propose that the hESC(troph) is a viable functional model of human trophoblasts to study trophoblast-endometrial interactions. Furthermore, the data derived herein offer the promise of novel diagnostics and therapeutics aimed at practical challenges in human infertility and pregnancy disorders associated with abnormal embryonic implantation.

We have developed an information-dependent, iterative MS/MS acquisition (IMMA) tool for improving MS/MS efficiency, increasing proteome coverage, and shortening analysis time for high-throughput proteomics applications based on the LC-MALDI MS/MS platform. The underlying principle of IMMA is to limit MS/MS analyses to a subset of molecular ions that are likely to identify a maximum number of proteins. IMMA reduces redundancy of MS/MS analyses by excluding from the precursor ion peak lists proteotypic peptides derived from the already identified proteins and uses a retention time prediction algorithm to limit the degree of false exclusions. It also increases the utilization rate of MS/MS spectra by removing "low value" unidentifiable targets like nonpeptides and peptides carrying large loads of modifications, which are flagged by their "nonpeptide" excess-to-nominal mass ratios. For some samples, IMMA increases the number of identified proteins by ∼20-40% when compared to the data dependent methods. IMMA terminates an MS/MS run at the operator-defined point when "costs"(e.g., time of analysis) start to overrun "benefits"(e.g., number of identified proteins), without prior knowledge of sample contents and complexity. To facilitate analysis of closely related samples, IMMA's inclusion list functionality is currently under development.

Placental trophoblasts are key determinants of in utero development. Mouse trophoblast (TB) stem cells, which were first derived over a decade ago, are a powerful cell culture model for studying their self-renewal or differentiation. Our attempts to isolate an equivalent population from the trophectoderm of human blastocysts generated colonies that quickly differentiated in vitro. This finding suggested that the human placenta has another progenitor niche. Here, we show that the chorion is one such site. Initially, we immunolocalized pluripotency factors and TB fate determinants in the early gestation placenta, amnion, and chorion. Immunoreactive cells were numerous in the chorion. We isolated these cells and plated them in medium containing fibroblast growth factor which is required for human embryonic stem cell self-renewal, and an inhibitor of activin/nodal signaling. Colonies of polarized cells with a limited lifespan emerged. Trypsin dissociation yielded continuously self-replicating monolayers. Colonies and monolayers formed the two major human TB lineages-multinucleate syncytiotrophoblasts and invasive cytotrophoblasts (CTBs). Transcriptional profiling experiments revealed the factors associated with the self-renewal or differentiation of human chorionic TB progenitor cells (TBPCs). They included imprinted genes, NR2F1/2, HMGA2, and adhesion molecules that were required for TBPC differentiation. Together, the results of these experiments suggested that the chorion is one source of epithelial CTB progenitors. These findings explain why CTBs of fully formed chorionic villi have a modest mitotic index and identify the chorionic mesoderm as a niche for TBPCs that support placental growth.

Preeclampsia (PE) impacts 8 million mother-infant pairs worldwide each year. This human pregnancy-specific disease characterized by hypertension and proteinuria accounts for significant maternal and neonatal morbidity and mortality. The current theory of the pathogenesis of PE as reviewed by Drs. Christopher Redman and Ian Sargent is thought to occur as a 2-stage process with poor placentation in the first half of pregnancy resulting in the maternal response in the second half of pregnancy. Our studies have focused on understanding the placental contribution to this serious disease by examining the gene expression profile of the deciduas basalis or basal plate, the region of the placenta involved in the "poor placentation". In this review we present summaries of our microarray datasets both of normal placentation and those gene expression changes resulting in the context of PE. Additionally, we have taken this opportunity to combine the data sets to provide a more comprehensive view of this region of the placenta. As defects in the basal plate are, in part, at the root of the disease process, we believe that understanding the pathobiology that occurs in this region will increase our ability to alter the development and/or course of PE.