Background-We established an efficient preparation method to obtain endothelial-like cells (ECs) from human embryonic stem cells (hESCs) and tested whether these hESC-ECs would show therapeutic potential for treatment of hindlimb ischemia. Methods and Results-ECs differentiated from hESCs were obtained by mechanical isolation and cell sorting for von Willebrand factor. The isolated hESC-ECs maintained endothelial cell-specific characteristics such as endothelial marker expression and capillary formation. One day after surgical induction of hindlimb ischemia in athymic mice, hESC-ECs were injected intramuscularly into ischemic limbs. Four weeks after treatment, hESC-EC treatment significantly increased limb salvage (36%) compared with treatment with medium (0%). In addition, laser Doppler imaging showed that the ratio of blood perfusion (ischemic to normal limb) was increased significantly (PϽ0.01) by hESC-EC treatment (0.511Ϯ0.167) compared with medium injection (0.073Ϯ0.061). Capillary and arteriole densities were 658Ϯ190/mm 2 and 30Ϯ11/mm 2 in the hESC-EC group, respectively, whereas those in the medium group were 392Ϯ118/mm 2 and 16Ϯ8/mm

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, ␣-dystroglycan (␣-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown ␣-DG to be modified by both O-GalNAc-and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle ␣-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on ␣-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from ␣-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.Defects in protein glycosylation related to human disease were first reported in the 1980s, and since then, about 40 various types of congenital disorders of glycosylation have been reported (1). The term congenital disorders of glycosylation was first used to describe alterations of the N-glycosylation pathway and was later expanded to include the O-glycosylation pathways (1-3). The importance and complexity of O-linked glycosylation have only recently begun to be appreciated (1, 3, 4). In particular, mutations in genes encoding (putative) glycosyltransferases, which catalyze the addition and extension of O-linked mannose-initiated glycans, have garnered increased attention in the last decade given that they are causative for several forms of congenital muscular dystrophy (5, 6).The most common forms of O-glycosylation on secretory proteins are the mucin-like O-GalNAc structures that are initiated by polypeptide N-␣-acetylgalactosaminyltransferases in the endoplasmic reticulum-Golgi intermediate compartment and/or early cis-Golgi (7). Additionally, other O-linked structures are initiated with alternative monosaccharides, such as O-mannose, O-glucose, O-fucose, O-xylose, and O-GlcNAc onSer/Thr residues and the O-galactose modification of hydroxylysine residues in collagen domains (4). The diversity of O-mannosylated proteins in mammals, although quite abundant in some tissues (ϳ30% of O-glycans released from mouse brains (8)), has not been well characterized. The only clearly identified mamma...

Ischemia is a potentially fatal medical event that is associated with as many as 30 percent of all deaths. Stem cell therapy offers significant therapeutic promise, but poor survival following transplantation to ischemic tissue limits its efficacy. Here we demonstrate that nanosphere-mediated growth factor delivery can enhance the survival of transplanted human adipose-derived stromal cells (hADSCs) and secretion of human angiogenic growth factors per cell, and substantially improve therapeutic efficacy of hADSCs. In vitro, in hypoxic (1% oxygen) and serum-deprived conditions which simulate in vivo ischemia, fibroblast growth factor-2 (FGF2) significantly reduced hADSC apoptosis and enhanced angiogenic growth factor secretion. In vivo, hADSCs delivered intramuscularly into ischemic hindlimbs in combination with FGF2 resulted in significant improvements in limb survival and blood perfusion, as well as survival of the transplanted hADSCs and secretion of human angiogenic growth factors (i.e., vascular endothelial growth factor, hepatocyte growth factor, and FGF2). Interestingly, the majority of transplanted hADSCs were localized adjacent to the microvessels rather than being incorporated into them, suggesting that their major contribution to angiogenesis might be to increase paracrine secretion of angiogenic growth factors. This study demonstrates the potential of hADSCs in combination with growth factors for use in the treatment of ischemia.

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IL1b is a central regulator of systemic inflammatory response in breast cancer, but the precise regulatory mechanisms that dictate the overproduction of IL1b are largely unsolved. Here, we show that IL1b secretion is increased by the coculture of human monocyte-like cells and triple-negative breast cancer (TNBC) cells. In addition, macrophages robustly produced IL1b when exposed to the conditioned media of TNBC cells. Consistent with these observations, macrophage depletion decreased serum IL1b and reduced breast cancer progression in an orthotopic breast cancer mouse model. Profiling the secretome of human breast cancer cells revealed that the CD44 antigen was the most differentially released protein in basal conditions of TNBC cells. Antibody-mediated neutralization of CD44 abrogated IL1b production in macrophages and inhibited the growth of primary tumors. These results suggest IL1b-mediated oncogenic signaling is triggered by breast cancer cell membrane-derived soluble CD44 (sCD44) antigen, and targeting sCD44 antigen may provide an alternative therapeutic strategy for breast cancer treatment by modulating inflammatory tumor microenvironment.Significance: A novel positive feedback loop between IL1b and CD44 promotes TNBC malignant progression.

Purpose Lung cancer is among the most common cancers. Bronchoalveolar lavage fluid (BALF) can be easily obtained from patients with lung cancers. The aim is to develop a novel proteomic method of the molecule‐based sensitive detection of biomarkers from BALF. Experimental Design BALF samples are collected from segmental bronchus of 14 patients with lung cancers from Kyung Hee University Hospital. First, BALF proteome is depleted using a depletion column, and then peptides are prepared from the enriched low abundant proteins and fractionated by high pH reverse phase liquid chromatography prior to LC‐MS/MS. Data are available via ProteomeXchange with identifier PXD012645. Results A novel method is developed for in‐depth proteomic analysis of BALF by combining antibody‐based depletion of high abundant proteins from BALF with high pH peptide fractionation. Peptides are analyzed on a high resolution Orbitrap Fusion mass spectrometer. MaxQuant search result in the identification of 4615 protein groups mapped to 4534 genes. Conclusions and Clinical Relevance It is found that the method outperformed conventional BALF proteomic methods and it is believed that this method will facilitate the biomarker research for lung cancer. In addition, it is shown that BALF will be a great source of biomarkers of lung diseases.

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BackgroundAlcohol is traditionally known to have a relaxing effect. However, persons who consume alcohol in excessive amounts suffer from poor sleep quality and patients with alcohol use disorders commonly report insomnia. In this study, we aimed to evaluate the effects of alcohol use on sleep quality.MethodsA questionnaire-based cross-sectional survey was conducted with 234 men and 159 women who had visited a general hospital. We used structured questionnaires, including Alcohol Use Disorder Identification Test-Korean revised version (AUDIT-KR) and the Pittsburgh Sleep Quality Index-Korean version (PSQI-K). We analyzed the association between scores for all subcategories of the PSQI-K and the AUDIT-KR and then analyzed the correlation between AUDIT-KR and global PSQI-K scores.ResultsThe global PSQI-K score for men was positively correlated with the AUDIT-KR score (P=0.008) after adjusting for age, chronic disease, tobacco use, exercise, depression, and anxiety. The AUDIT-KR score was significantly associated with subjective sleep quality (P=0.005), sleep duration (P=0.047), and sleep disturbance (P=0.048); it was not associated with sleep latency, sleep efficiency, or daytime dysfunction. Sleep disturbances due to snoring were significantly associated with total AUDIT-KR score (P=0.008). There was no correlation between the global PSQI-K and AUDIT-KR scores for women (P=0.333). However, daytime dysfunction showed a significant association with total AUDIT-KR score (P=0.048).ConclusionMen with higher AUDIT-KR scores tended to suffer from poor sleep quality. AUDIT-KR scores showed significant correlations with subjective sleep quality, sleep duration, and sleep disturbances in men.