Anti-Human CD9 Antibody Fab Fragment Impairs the Internalization of Extracellular Vesicles and the Nuclear Transfer of their Cargo Proteins.

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell-derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti-CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9-green fluorescent protein fusion protein and various melanoma cell lines and bone marrow-derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab-mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-melanoma therapeutic strategies

HIV-1-induced nuclear invaginations mediated by VAP-A, ORP3, and Rab7 complex explain infection of activated T cells

The mechanism of human immunodeficiency virus 1 (HIV-1) nuclear entry, required for productive infection, is not fully understood. Here, we report that in HeLa cells and activated CD4+ T cells infected with HIV-1 pseudotyped with VSV-G and native Env protein, respectively, Rab7+ late endosomes containing endocytosed HIV-1 promote the formation of nuclear envelope invaginations (NEIs) by a molecular mechanism involving the VOR complex, composed of the outer nuclear membrane protein VAP-A, hyperphosphorylated ORP3 and Rab7. Silencing VAP-A or ORP3 and drug-mediated impairment of Rab7 binding to ORP3-VAP-A inhibited the nuclear transfer of the HIV-1 components and productive infection. In HIV-1-resistant quiescent CD4+ T cells, ORP3 was not hyperphosphorylated and neither VOR complex nor NEIs were formed. This new cellular pathway and its molecular players are potential therapeutic targets, perhaps shared by other viruses that require nuclear entry to complete their life cycle

Haematopoietic stem cell differentiation promotes the release of prominin-1/CD133-containing membrane vesicles—a role of the endocytic–exocytic pathway

The differentiation of stem cells is a fundamental process in cell biology and understanding its mechanism might open a new avenue for therapeutic strategies. Using an ex vivo co-culture system consisting of human primary haematopoietic stem and progenitor cells growing on multipotent mesenchymal stromal cells as a feeder cell layer, we describe here the exosome-mediated release of small membrane vesicles containing the stem and cancer stem cell marker prominin-1 (CD133) during haematopoietic cell differentiation. Surprisingly, this contrasts with the budding mechanism underlying the release of this cholesterol-binding protein from plasma membrane protrusions of neural progenitors. Nevertheless, in both progenitor cell types, protein–lipid assemblies might be the essential structural determinant in the release process of prominin-1. Collectively, these data support the concept that prominin-1-containing lipid rafts may host key determinants necessary to maintain stem cell properties and their quantitative reduction or loss may result in cellular differentiation

Itraconazole inhibits nuclear delivery of extracellular vesicle cargo by disrupting the entry of late endosomes into the nucleoplasmic reticulum

Extracellular vesicles (EVs) are mediators of intercellular communication under bothhealthy and pathological conditions, including the induction of pro-metastatic traits,but it is not yet known how and where functional cargoes of EVs are delivered to theirtargets in host cell compartments. We have described that after endocytosis, EVsreach Rab+late endosomes and a fraction of these enter the nucleoplasmic reticu-lum and transport EV biomaterials to the host cell nucleoplasm. Their entry thereinand docking to outer nuclear membrane occur through a tripartite complex formedby the proteins VAP-A, ORP and Rab (VOR complex). Here, we report that theantifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZor ketoconazole, disrupts the binding of Rab to ORP–VAP-A complexes, leadingto inhibition of EV-mediated pro-metastatic morphological changes including cellmigration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhi-bition of the VOR complex was maintained, although the ICZ moieties responsiblefor antifungal activity and interference with intracellular cholesterol distributionwere removed. Knowing that cancer cells hijack their microenvironment and thatEVs derived from them determine the pre-metastatic niche, small-sized inhibitors ofnuclear transfer of EV cargo into host cells could nd cancer therapeutic applications,particularly in combination with direct targeting of cancer cell

Attitude to pregnant pain during childbirth

Bakalářská práce se zabývá metodami tlumení porodní bolesti a postojem těhotných žen k bolesti při porodu. V teoretické části jsou uvedeny dosavadní poznatky z odborné literatury uvádějící současné farmakologické a nefarmakologické metody tlumení porodní bolesti, definici porodu a charakteristiku porodních bolestí. Výzkumná část zjišťuje pomocí nestandardizovaného anonymního dotazníkového šetření informovanost těhotných žen o metodách tlumení porodních bolestí. Dále zjišťuje míru obav těhotných žen z bolesti při porodu, a jak velkou bolest očekávají těhotné ženy během porodu.This thesis deals with labor pain control methods and attitudes of pregnant women to the pain of childbirth. The theoretical part consists of existing knowledge from the literature showing the current pharmacological and non-pharmacological pain control methods birth, childbirth definition and characteristics of labor pains. The research part determined using non-standardized anonymous questionnaire informing pregnant women about methods of birth control pain. It also investigates the level of concern of pregnant women pain in childbirth, and how much they expect pain during childbirth.Katedra porodní asistence a zdravotně sociální práceHodnocení vedoucího: výborně Hodnocení oponenta: výborně Doplňující otázky k obhajobě: 1. Z Vašeho šetření vyplynulo, že těhotné ženy získávají nejvíce informací o tlumení bolestí z jiných zdrojů než od porodních asistentek. Měla byste nějaký návrh, aby informace byly získávané ve větší míře od porodních asistentek? Obhajoba bakalářské práce s prezentací výborná

Criminal law aspects of data electronization in healthcare

Criminal law aspects of data electronization in healthcare Abstract Continuous spread of information and communication technologies into all aspects of everyday life goes hand in hand with importance of data and information. This trend can be also observed in the healthcare sector which has recently experienced massive electronization. However, along with electronization, respective criminal law aspects are developing as well. Relevancy of this issue can be clearly demonstrated on the recent cyber-attacks on healthcare providers, which seriously jeopardize valuable data in healthcare. The aim of this work is to set forth comprehensive summary of criminal aspects of data electronization in healthcare with emphasis on the importance of electronization phenomenon, the issue of cybercrime and selected crimes that may be committed in connection with data electronization. This work also aims at highlighting negative impact of electronization in the global information society. In terms of content, the work consists of five structured chapters, in which the author defined relevant theory behind the issue and subsequently performed her own analysis thereof. The first chapter defines the term "data" as well as its categories and legislative framework with emphasis on area of criminal law and related protection of..

Osteogenic Differentiation of Human Dental Pulp-derived Stem Cells under Various Ex-vivo Culture Conditions

Dental pulp stem cells (DPSCs) can be easily isolated and cultured in low-serum containing medium supplemented with growth factors PDGF-BB and EGF while exhibiting multipotency and immature phenotypic characteristics. In the present study, we investigated their potential to differentiate towards osteogenic lineages using various culture conditions in order to optimize their therapeutic use. DPSCs were cultured either as a cell monolayer or as three-dimensional (3D) micro-mass structures. Monolayers preincubated with bFGF and valproic acid for one week prior their differentiation were cultured in serum containing standard osteodifferentiation medium for four weeks, which resulted in multilayered nodule formation. Micro-mass structures were cultured for same period either in serum containing medium or under serum-free conditions supplemented with TGF-β3 with or without BMP-2. Histochemically, we detected massive collagen I and weak calcium phosphate depositions in multilayered nodules. When culture 3D-aggregates in either standard osteodifferentiation medium or serum-free medium containing TGF-β3, only small amount of collagen I fibres was observed and almost no deposits of calcium phosphate were detected. In contrast, in presence of both TGF-β3 and BMP-2 in the serum- free medium a significant amount of collagen I fibers/bundles and calcification were detected, which is in line with osteogenic effect of BMP-2. Thus, our data indicate that certain environmental cues can enhance differentiation process of DPSCs into osteogenic lineage, which suggest their possible utilization in tissue engineering

CD133 and membrane microdomains: Old facets for future hypotheses

Understanding all facets of membrane microdomains in normal and cancerous cells within the digestive tract is highly important, not only from a clinical point of view, but also in terms of our basic knowledge of cellular transformation. By studying the normal and cancer stem cell-associated molecule CD133 (prominin-1), novel aspects of the organization and dynamics of polarized epithelial cells have been revealed during the last decade. Its association with particular membrane microdomains is highly relevant in these contexts and might also offer new avenues in diagnosis and/or targeting of cancer stem cells

Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention

Differentiation of Neural Stem Cells Into Cells of Oligodendroglial Lineage

We described three different conditions that induce differentiation of dissociated neural stem cells derived from mouse embryonic CNS. In the first set of experiments, where the cell differentiation was triggered by cell adhesion, removal of growth factors and serum-supplemented medium, only sporadic neuronal and astroglial cells survived longer than two weeks and the latter formed a monolayer. When differentiation was induced in serum-free medium supplemented with retinoic acid, rapid and massive cell death occurred. A prolonged survival was observed in cultivation medium supplemented with serum and growth factors EGF plus FGF-2. One third of the cells did not express cell differentiation markers and were responsible for an increase in cell numbers. The remaining cells differentiated and formed the astrocytic monolayer on which occasional neuronal cells grew. One third of the differentiated phenotypes were represented by cells of oligodendroglial lineage. Differentiation of oligodendroglial cells occurred in a stepwise mechanism because the culture contained all successive developmental stages, including oligodendrocyte progenitors, preoligodendrocytes and immature and mature oligodendrocytes. Maturing oligodendrocytes displayed immunocytochemical and morphological features characteristic of cells that undergo physiological development. The cultivation conditions that supported growth and differentiation of neural stem cells were optimal for in vitro developmental studies and the production of oligodendroglial cells