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Integrin subunits αV and β3 promote the osteogenic differentiation of umbilical cord blood mesenchymal stem cells.

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are multipotent cells that have self-renewal properties and can differentiate into osteocytes, adipocytes, cartilage and extoderm. Bone regeneration and repair are important for the repair of bone injury, skeletal development or continuous remodeling throughout adult life. Thus, investigating the factors influencing osteocyte regeneration from hUCMSCs could be conducive to advancements in skeletal repair and the repair of bone injury. Previous reports have demonstrated that single integrin subunits (αV, β3, α5) and collagen I contribute to the osteogenic differentiation of human mesenchymal stem cells (hMSCs). However, the functions of the vitronectin receptor αV and β3 in the osteogenic differentiation of hUCMSCs and bone regeneration remain unclear. Run-related transcription factor 2 (RUNX2) is considered to be an early osteoblastic gene that is upregulated during the osteogenic differentiation of hUCMSCs. Meanwhile, bone sialoprotein (BSP) and collagen I are the most common early markers of osteoblast differentiation. Herein, we found that the mRNA and protein expression of αV, β3, RUNX2 and collagen I were upregulated during the osteogenic differentiation of hUCMSCs. Overexpression of αV and β3 in hMSCs increased the levels of RUNX2, BSP, and collagen I, decreased the number of adipocytes and promoted the osteogenic differentiation of hUCMSCs. Meanwhile, downregulation of αV and β3 decreased the levels of RUNX2, BSP, and collagen I, increased the number of adipocytes and blocked the osteogenic differentiation of hUCMSCs. In conclusion, the integrin subunits αV and β3 can promote the osteogenic differentiation of hUCMSCs and encourage bone formation.

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Laminin is the ECM niche for trophoblast stem cells.

The niche is a specialized microenvironment for tissue stem cells in vivo. It has long been emphasized that niche ECM molecules act on tissue stem cells to regulate their behavior, but the molecular entities of these interactions remain to be fully elucidated. Here, we report that laminin forms the in vivo ECM niche for trophoblast stem cells (TSCs), the tissue stem cells of the placenta. TSCs expressed fibronectin-binding, vitronectin-binding, and laminin-binding integrins, whereas the integrin ligands present in the TSC niche were collagen and laminin. Therefore, the only niche integrin ligand available for TSCs in vivo was laminin. Laminin promoted TSC adhesion and proliferation in vitro in an integrin binding-dependent manner. Importantly, when the integrin-binding ability of laminin was genetically ablated in mice, the size of the TSC population was significantly reduced compared with that in control mice. The present findings underscore an ECM niche function of laminin to support tissue stem cell maintenance in vivo.

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Endodermal differentiation of human induced pluripotent stem cells using simple dialysis culture system in suspension culture.

A differentiation of human induced pluripotent stem cells (hiPSCs) into definitive endoderm linage is required for a preparation of metabolic organ derived cells. The differentiation consumed high-priced cytokines and small molecules, which have hampered the manufacturability of differentiated cells. Although the cytokines and small molecules are remained or cells produce the autocrine factors, daily culture medium change should be proceeded to remove toxic metabolites generated from cells. In this study, we developed a simple dialysis culture system to refine the medium during definitive endodermal differentiation. We demonstrated that dialysis culture prevented cell damage to remove lactate. The hiPSCs cultured with dialysis also differentiated similarly as usual differentiation without dialysis even if they were not supplied Activin A for latter culture days in the differentiation. With this dialysis culture system, hiPSCs were differentiated into endodermal lineage with medium refinement and recycling and autocrine factors as well as cytokines, which may lead to reduce differentiation cost.

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Molecular mechanism of two nanobodies that inhibit PAI-1 activity reveals a modulation at distinct stages of the PAI-1/plasminogen activator interaction.

Plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasminogen activators (PAs) tissue-type PA (tPA) and urokinase-type PA (uPA) plays a crucial role in many (patho)physiological processes, e.g. cardiovascular disease, tissue fibrosis, as well as in many age-related pathologies. Therefore, much effort has been put into the development of small molecule or antibody-based PAI-1 inhibitors.

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Novel Associations between Related Proteins and Cellular Effects of High-Density Lipoprotein.

Recent studies have examined the structure-function relationship of high-density lipoprotein (HDL). This study aimed to identify and rank HDL-associated proteins involved in several biological function of HDL.

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The cell surface adhesins of Mycobacterium tuberculosis.

Bacterial cell surface adhesins play a major role in facilitating host colonization and subsequent establishment of infection. The surface of Mycobacterium tuberculosis, owing to the complex architecture of its cell envelope, expresses numerous adhesins with varied chemical nature, including proteins, lipids, lipoproteins, glycoproteins and glycopolymers. Studies on mycobacterial adhesins show that they bind with multifarious host receptors and extracellular matrix (ECM) components. In this review we have highlighted the adhesins that are abundantly present on the mycobacterial surface and their interactions with host receptors. M. tuberculosis interacts with various host cell surface receptors such as toll like receptors, C-type lectin receptors, scavenger receptors, and Fc and complement receptors. Apart from these, ECM components like fibronectin, collagen, elastin, laminin, fibrillin and vitronectin also provide binding sites for surface adhesins of the tubercle bacilli. M. tuberculosis adhesins include proteins with and without signal peptide sequence and transmembrane proteins. Other surface adhesin macromolecules of M. tuberculosis comprises of lipids, glycolipids and glycopolymers. The interaction between the mycobacterial adhesins and their host receptors result in adhesion of the microbe to the host cells, induction of immune response and aid in the pathogenesis of the disease. A thorough understanding of the different M. tuberculosis surface adhesins and host receptors will provide a better picture of interaction between them at molecular level. The information gained on adhesins and host receptors will prove beneficial in developing novel therapeutic strategies such as the use of anti-adhesin molecules to hinder the adhesion of bacteria to the host cells, thereby preventing establishment of infection. The surface molecules discussed in this review will also benefit in identification of new drug targets, diagnostic markers or vaccine candidates against the deadly pathogen.

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Effect of cell culture biomaterials for completely xeno-free generation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) were generated on several biomaterials from human amniotic fluid in completely xeno-free and feeder-free conditions via the transfection of pluripotent genes using a nonintegrating RNA Sendai virus vector. The effect of xeno-free culture medium on the efficiency of the establishment of human amniotic fluid stem cells from amniotic fluid was evaluated. Subsequently, the effect of cell culture biomaterials on the reprogramming efficiency was investigated during the reprogramming of human amniotic fluid stem cells into hiPSCs. Cells cultured in laminin-511, laminin-521, and Synthemax II-coated dishes and hydrogels having optimal elasticity that were engrafted with specific oligopeptides derived from vitronectin could be reprogrammed into hiPSCs with high efficiency. The reprogrammed cells expressed pluripotency proteins and had the capability to differentiate into cells derived from all three germ layers in vitro and in vivo. Human iPSCs could be generated successfully and at high efficiency (0.15-0.25%) in completely xeno-free conditions from the selection of optimal cell culture biomaterials.

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Integrins expressed on the surface of human endometrial stromal cells derived from a female patient experiencing spontaneous abortion.

Here, as a basic study in revealing the correlation between extracellular matrix components and spontaneous abortion, we defined the types of integrins expressed on the surface of endometrial stromal (ES) cells retrieved from the uterus of a patient experiencing spontaneous abortion. For these, the types of integrin subunits in the ES cells retrieved from a woman with spontaneous abortion were identified at the transcriptional and translational levels, and functional assay was conducted for confirming the combinations of integrin α and β subunits. Among the genes encoding 25 integrin subunits, significantly high transcription was seen in integrins α, α, α, α, α, α, β, β, and β. Translation of integrins α, α, α, α, and β on the cell surface was detected in almost all ES cells, whereas integrins α, α, β, and β were expressed translationally only in some ES cells. Subsequently, ES cells showed significantly increased adhesion to collagen I, laminin, fibronectin, and vitronectin, and functional blocking of integrin α, α, α, and α significantly inhibited adhesion to these molecules. These results demonstrated that active heterodimers composed of integrins αβ, αβ, αβ, and αβ were co-localized on the surface of ES cells derived from a patient experiencing spontaneous abortion.

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Screening and analysis of agouti signaling protein interaction partners in Pelodiscus sinensis suggests a role in lipid metabolism.

Agouti signaling protein (ASP) is a secreted paracrine protein that has been widely reported to function in melanogenesis and obesity and could potentially be a core protein that regulates the color and fatty phenotype of P. sinensis. In this study, we screened out interacting proteins of ASP by combined co-immunoprecipitation mass spectrometry (CoIP-MS), yeast two hybrid (Y2H) analysis, and computational predictions. We performed docking of ASP with its well-known receptor melanocortin receptor 4 (MC4R) to predict the binding capacity and to screen out actual ASP interacting proteins, CoIP-MS was performed where identified 32 proteins that could bind with ASP and Y2H confirmed seven proteins binding with ASP directly. CoIP-MS and Y2H screening results including PPI prediction revealed that vitronectin (VTN), apolipoprotein A1 (APOA1), apolipoprotein B (APOB), and filamin B (FLNB) were the key interacting proteins of ASP. VTN, APOA1, and APOB are functional proteins in lipid metabolism and various skin disorders, suggesting ASP may function in lipid metabolism through these partners. This study provided protein-protein interaction information of ASP, and the results will promote further research into the diverse roles of ASP, as well as its binding partners, and their function in different strains of P. sinensis.

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Characterization of an Extensive Interface on Vitronectin for Binding to Plasminogen Activator Inhibitor-1: Adoption of Structure in an Intrinsically Disordered Region.

Small-angle neutron scattering (SANS) measurements were pursued to study human vitronectin, a protein found in tissues and the circulation that regulates cell adhesion/migration and proteolytic cascades that govern hemostasis and pericellular proteolysis. Many of these functions occur via interactions with its binding partner, plasminogen activator inhibitor-1 (PAI-1), the chief inhibitor of proteases that lyse and activate plasminogen. We focused on a region of vitronectin that remains uncharacterized from previous X-ray scattering, nuclear magnetic resonance, and computational modeling approaches and which we propose is involved in binding to PAI-1. This region, which bridges the N-terminal somatomedin B (SMB) domain with a large central β-propeller domain of vitronectin, appears unstructured and has characteristics of an intrinsically disordered domain (IDD). The effect of osmolytes was evaluated using circular dichroism and SANS to explore the potential of the IDD to undergo a disorder-to-order transition. The results suggest that the IDD favors a more ordered structure under osmotic pressure; SANS shows a smaller radius of gyration () and a more compact fold of the IDD upon addition of osmolytes. To test whether PAI-1 binding is also coupled to folding within the IDD structure, a set of SANS experiments with contrast variation were performed on the complex of PAI-1 with a vitronectin fragment corresponding to the N-terminal 130 amino acids (denoted the SMB-IDD because it contains the SMB domain and IDD in linear sequence). Analysis of the SANS data using the Ensemble Optimization Method confirms that the SMB-IDD adopts a more compact configuration when bound to PAI-1. Calculated structures for the PAI-1:SMB-IDD complex suggest that the IDD provides an interaction surface outside of the primary PAI-1-binding site located within the SMB domain; this binding is proposed to lead to the assembly of higher-order structures of vitronectin and PAI-1 commonly found in tissues.

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