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High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.
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Glioblastoma , Glioma , Enfermedad de Parkinson , Humanos , Glioblastoma/genética , Proteínas de la Membrana/genética , Células Endoteliales , Angiogénesis , Glioma/genética , Neuroglía , Neovascularización Patológica/genéticaRESUMEN
INTRODUCTION: Single-cell (SC) gene expression analysis is crucial to dissect the complex cellular heterogeneity of solid tumors, which is one of the main obstacles for the development of effective cancer treatments. Such tumors typically contain a mixture of cells with aberrant genomic and transcriptomic profiles affecting specific sub-populations that might have a pivotal role in cancer progression, whose identification eludes bulk RNA-sequencing approaches. We present scMuffin, an R package that enables the characterization of cell identity in solid tumors on the basis of a various and complementary analyses on SC gene expression data. RESULTS: scMuffin provides a series of functions to calculate qualitative and quantitative scores, such as: expression of marker sets for normal and tumor conditions, pathway activity, cell state trajectories, Copy Number Variations, transcriptional complexity and proliferation state. Thus, scMuffin facilitates the combination of various evidences that can be used to distinguish normal and tumoral cells, define cell identities, cluster cells in different ways, link genomic aberrations to phenotypes and identify subtle differences between cell subtypes or cell states. We analysed public SC expression datasets of human high-grade gliomas as a proof-of-concept to show the value of scMuffin and illustrate its user interface. Nevertheless, these analyses lead to interesting findings, which suggest that some chromosomal amplifications might underlie the invasive tumor phenotype and the presence of cells that possess tumor initiating cells characteristics. CONCLUSIONS: The analyses offered by scMuffin and the results achieved in the case study show that our tool helps addressing the main challenges in the bioinformatics analysis of SC expression data from solid tumors.
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Variaciones en el Número de Copia de ADN , Neoplasias , Humanos , Análisis de Expresión Génica de una Sola Célula , Neoplasias/genética , Transcriptoma , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodosRESUMEN
The synthesis of one-dimensional (1D) nanostructures in polymeric matrices has become the focus of much research, as the presence of these highly anisotropic domains determines the transport behaviour and mechanical properties of the resulting nanostructured polymers. In this work, 1D PEO nanocrystals were synthesized in situ from polystyrene-b-polyethylene oxide (PS-b-PEO) self-assembly in a polystyrene matrix. For this, three different block copolymers (BCP) were employed: L-BCP (PS = 32 000 Da and PEO = 11 000 Da), M-BCP, (PS = 59 000 Da and PEO = 31 000 Da), and H-BCP, (PS = 102 000 Da and PEO = 34 000 Da). The formation of 1D nanocrystals starts with the reaction-induced microphase separation of BCP during styrene photopolymerization at room temperature. Then, as matrix polymerizes, the primary crystalline micelles aggregate via epitaxial crystallization by end-to-end coupling. The morphology of the resulting nanocrystals was highly dependent on the BCP employed. While L-BCP self-assembles into 1D ribbon-like nanocrystals, M-BCP macro-phase separates and, H-BCP self-assembles into short disk-like nanocrystals. This dissimilar behavior was mainly associated to the length of the stabilizing corona block. In the case of H-BCP, it was found that 1D self-assembly occurred when the conditions for core thickening were given, that is, when a non-reactive period was introduced in the cure cycle. During such a period, core thickening clears the lateral surface of the nanocrystals, allowing end-to-end coupling. The driving force for crystallization was also modified. An increase in undercooling resulted in an elevated nucleation rate and accelerated crystal growth. This led to a narrower size distribution of shorter 1D nanocrystals. This knowledge will enable the synthesis of customized 1D nanocrystals in a thermoplastic matrix, through the precise selection of the BCP formulation and curing conditions.
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Conventional drug delivery systems often have several pharmacodynamic and pharmacokinetic limitations related to their low efficacy and bad safety. It is because these traditional systems cannot always be selectively addressed to their therapeutic target sites. Currently, target-specific and controlled drug delivery is one of the foremost challenges in the biomedical field. In this context, stimuli-responsive polymeric nanomaterials have been recognized as a topic of intense research. They have gained immense attention in therapeutics - particularly in the drug delivery area - due to the ease of tailorable behavior in response to the surroundings. Light irradiation is of particular interest among externally triggered stimuli because it may be specifically localized in a contact-free manner. Light-human body interactions may sometimes be harmful due to photothermal and photomechanical reactions that lead to cell death by photo-toxicity and/or photosensitization. However, these limitations may also be overcome by the use of photo-responsive polymeric nanostructures. This review summarizes recent developments in photo-responsive polymeric nanocarriers used in the field of drug delivery systems, including nanoparticles, nanogels, micelles, nanofibers, dendrimers, and polymersomes, as well as their classification and mechanisms of drug release.
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Sistemas de Liberación de Medicamentos , Preparaciones Farmacéuticas , Humanos , Procesos FotoquímicosRESUMEN
A dispersion of elongated nanostructures with a high aspect ratio in polymer matrices has been reported to provide a material with valuable properties such as mechanical strength, barrier effect and shape memory, among others. In this study, we show the procedure to achieve a distribution of elongated crystalline nanodomains in a PS matrix employing the self-assembly of amphiphilic block copolymers (BCP). The selected BCP was polystyrene-block-polyethylene oxide (PS-b-PEO). It was dissolved at 10 wt% in a styrene (St) monomer and the blend was slowly photopolymerized over four days at room temperature, until the reaction was arrested by vitrification. This blend was initially homogeneous and nanostructuration took place in an early stage of the polymerization as a result of the microphase separation (MS) of PEO blocks. Due to its high tendency to crystallize, demixed PEO blocks crystallized almost concomitantly with MS triggering the growing of the nanostructures. Thus, the time window between the onset of crystallization and the vitrification of the matrix was almost four days, allowing all micelles to have the opportunity to couple to a growing nanostructure. As a result, a population of nanoribbons with average lengths surpassing 10 µm dispersed in a PS matrix was obtained. It was demonstrated that these ribbon-like nanostructures are preserved as long as the heating temperature is located below the Tg of the matrix. If the material is heated above this temperature, softening of the matrix allows the breakup of the molten PEO nanoribbons due to Plateau-Rayleigh instability.
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Metal/polymer nanocomposites have attracted much attention in recent years due to their exceptional properties and wide range of potential applications. A key challenge to obtain these materials is to stabilize the metal nanoparticles in the matrix, avoiding uncontrolled aggregation processes driven by the high surface free energy of nanosized particles. Here, we investigate the aggregation mechanism of primary particles in gold-epoxy nanocomposites prepared via light-assisted in situ synthesis, under different irradiation conditions. The growth and aggregation of gold nanoparticles were monitored in situ by time-resolved small-angle X-ray scattering experiments, whereas spectroscopic measurements were performed to interpret how matrix polymerization influences the aggregation process. It was found that light intensity has a greater influence on the reduction rate than on the polymerization rate. Under irradiation, gold nanostructures evolve through five time-defined stages: nuclei-mass fractals-surface fractals-spherical nanoparticles-aggregates. If the maximum in the polymerization rate is reached before the aggregation step, individual primary nanoparticles will be preserved in the polymer matrix due to diffusional constraints imposed by the reaction medium. Because the light intensity has a different influence on the reduction rate than on the polymerization rate, this parameter can be used as a versatile tool to avoid aggregation of gold nanoparticles into the polymer matrix.
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It has been previously reported that poly(ethylene) (PE)-based block copolymers self-assemble in certain thermosetting matrices to form a dispersion of one-dimensional (1D) nanoribbons. Such materials exhibit exceptional properties that originate from the high aspect ratio of the elongated nano-objects. However, the ability to prepare 1D assemblies with well-controlled dimensions is limited and represents a key challenge. Here, we demonstrate that the length of ribbon-like nanostructures can be precisely controlled by regulating the mobility of the matrix during crystallization of the core-forming PE block. The selected system to prove this concept was a poly(ethylene-block-ethylene oxide) (PE-b-PEO) block copolymer in an epoxy monomer based on diglycidyl ether of bisphenol A (DGEBA). The system was activated with a dual thermal- and photo-curing system, which allowed us to initiate the epoxy polymerization at 120 °C until a certain degree of conversion, stop the reaction by cooling to induce crystallization and micellar elongation, and then continue the polymerization at room temperature by visible-light irradiation. In this way, crystallization of PE blocks took place in a matrix whose mobility was regulated by the degree of conversion reached at 120 °C. The mechanism of micellar elongation was conceptualized as a diffusion-limited colloid aggregation process which was induced by crystallization of PE cores. This assertion was supported by the evidence obtained from in situ small-angle X-ray scattering (SAXS), in combination with differential scanning calorimetry (DSC) and transmission electron microscopy (TEM).
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During embryonic development, new arteries, and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis.
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Proliferación Celular , Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica , Receptores Notch/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Secuencia Conservada , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Receptores Notch/genética , Transducción de Señal , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 232: 1262-1269, 2017. © 2016 Wiley Periodicals, Inc.
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Enfermedad , Edición Génica , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , ARN/metabolismo , Diferenciación Celular , Reprogramación Celular , Descubrimiento de Drogas , Vectores Genéticos/metabolismo , Humanos , Medicina de PrecisiónRESUMEN
Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self-renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi-lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self-renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570-584, 2017. © 2016 Wiley Periodicals, Inc.
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Proliferación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Neoplasias Mamarias Animales/metabolismo , Células Madre Neoplásicas/metabolismo , Organoides/metabolismo , Animales , Perros , Femenino , Neoplasias Mamarias Animales/patología , Células Madre Neoplásicas/patología , Organoides/patología , Células Tumorales CultivadasRESUMEN
Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by Nω-propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2.NEW & NOTEWORTHY Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.
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Intestino Delgado/irrigación sanguínea , Plexo Mientérico/metabolismo , Óxido Nítrico/metabolismo , Factores de Transcripción Otx/metabolismo , Daño por Reperfusión/metabolismo , Animales , Arginina/análogos & derivados , Arginina/farmacología , Tránsito Gastrointestinal/efectos de los fármacos , Tránsito Gastrointestinal/fisiología , Masculino , Plexo Mientérico/patología , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Ratas , Ratas Wistar , Daño por Reperfusión/patologíaRESUMEN
A detailed understanding of the processes taking place during the in situ synthesis of metal/polymer nanocomposites is crucial to manipulate the shape and size of nanoparticles (NPs) with a high level of control. In this paper, we report an in-depth time-resolved analysis of the particle formation process in silver/epoxy nanocomposites obtained through a visible-light-assisted in situ synthesis. The selected epoxy monomer was based on diglycidyl ether of bisphenol A, which undergoes relatively slow cationic ring-opening polymerization. This feature allowed us to access a full description of the formation process of silver NPs before this was arrested by the curing of the epoxy matrix. In situ time-resolved small-angle X-ray scattering investigation was carried out to follow the evolution of the number and size of the silver NPs as a function of irradiation time, whereas rheological experiments combined with near-infrared and ultraviolet-visible spectroscopies were performed to interpret how changes in the rheological properties of the matrix affect the nucleation and growth of particles. The analysis of the obtained results allowed us to propose consistent mechanisms for the formation of metal/polymer nanocomposites obtained by light-assisted one-pot synthesis. Finally, the effect of a thermal postcuring treatment of the epoxy matrix on the particle size in the nanocomposite was investigated.
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We have incorporated push-pull azobenzene units into diblock-copolymer micelles by supramolecular assembly. Specifically, we encapsulated a phenol-functionalized chromophore, DO13, within PS-b-P4VP micelles in toluene by means of H-bond interactions developed between DO13 molecules and pyridine groups of P4VP block. The solutions were spin-coated onto glass substrates resulting in multi- or mono-layered thin films of micelles with P4VP(DO13) core and PS corona. We show that the use of DO13 as a building block of micellar aggregates allowed us to manipulate the developed nanostructures. Spherical to cylindrical micellar transition was found when we increased the degree of chromophore complexation. Also, it was found that the polymer concentration in the solution plays an important role in determining the micellar nanostructures. The chain extension and change in composition of the P4VP core in the presence of the chromophore may be responsible for the structural changes observed in the micelles. The optical properties of the thin films have been investigated focusing on the effect of the micellar morphology over the photoinduced birefringence. The optical anisotropy (Δn) increased with respect to the analogous homogeneous system P4VP(DO13), indicating that the protective micelle environment can enhance the optical properties of the embedded chromophores significantly. Furthermore, we show very interesting new results in which we have related changes in optical properties to the film morphology (spheres to cylinders). This can be exploited for producing optical devices having improved optoelectronic properties and stability.
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TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue.
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Metaloproteínas , Humanos , Metaloproteínas/metabolismo , Metaloproteínas/genética , Análisis de la Célula Individual , Autoinmunidad , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Animales , Perfilación de la Expresión GénicaRESUMEN
We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotype in tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactone scaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids and patient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.
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Envejecimiento , Organoides , Humanos , Organoides/metabolismo , Envejecimiento/metabolismo , Proteínas de la Membrana/metabolismo , Senescencia Celular , Femenino , Andamios del Tejido/química , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/citologíaRESUMEN
Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.
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Glioma , Gliosis , Proteínas de la Membrana , Humanos , Proteínas de la Membrana/metabolismo , Glioma/metabolismo , Glioma/patología , Gliosis/metabolismo , Gliosis/patología , Animales , Receptores de PéptidosRESUMEN
Thermal annealing was used for the bottom-up fabrication of morphologically controlled gold-block-copolymer (Au-BC) nanocomposites. Three different blends formed by polystyrene (PS) homopolymer and PS-coated gold nanoparticles (PSSH@Au) were used as modifiers of asymmetric polystyrene-b-polymethylmethacrylate (PS-b-PMMA): PS26/PS26SH@Au, PS75/PS75SH@Au and PS167/PS167SH@Au (where the subscripts refer to the number of styrene monomeric units).The results indicated that all three blends used as modifiers (PSn/PSnSH@Au) were successfully located in the PS phase during thermally induced BC self-assembly for a composition range from 5 to 43 wt% without macro-phase separation. The PSnSH@Au moiety experienced molecular desorption, nanocrystal core coalescence and partial molecular re-encapsulation processes during thermal annealing, leading to sphere-like gold NPs with a larger average size (without exceeding an interdomain distance). Ligand chain length regulated the degree of coalescence and re-encapsulation, defining ultimate core size. Furthermore, proper combination of chain length and composition enabled tuning of NP partitioning and arrangement on different length scales through thermally activated cooperative assembly processes. These results have not only significant impact for establishing thermal processing as a useful tool for the precise control of NP size and distribution, but also much broader implications for many nanoparticle-based technologies.
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Oro/química , Metacrilatos/química , Nanocompuestos/química , Nanopartículas/química , Nanotecnología/métodos , Poliestirenos/química , Nanocompuestos/ultraestructura , Nanopartículas/ultraestructura , TemperaturaRESUMEN
Congenital Central Hypoventilation Syndrome (CCHS) is a rare disorder of the autonomic nervous system (ANS), characterized by inadequate control of autonomic ventilation and global autonomic dysfunction. Heterozygous polyalanine repeat expansion mutations in exon 3 of the transcription factor Paired-like homeobox 2B (PHOX2B) gene occur in 90% of CCHS cases. In this study, we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines from female CCHS patients carrying a heterozygous + 5 alanine expansion mutation. The generated iPSC lines show a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.
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Células Madre Pluripotentes Inducidas , Femenino , Proteínas de Homeodominio/genética , Humanos , Hipoventilación/congénito , Mutación/genética , Péptidos , Apnea Central del Sueño , Factores de Transcripción/genéticaRESUMEN
Neuroligins constitute a family of transmembrane proteins localized at the postsynaptic side of both excitatory and inhibitory synapses of the central nervous system. They are involved in synaptic function and maturation and recent studies have linked mutations in specific human Neuroligins to mental retardation and autism. We isolated the human Neuroligin homologs in Danio rerio. Next, we studied their gene structures and we reconstructed the evolution of the Neuroligin genes across vertebrate phyla. Using reverse-transcriptase polymerase chain reaction, we analyzed the expression and alternative splicing pattern of each gene during zebrafish embryonic development and in different adult organs. By in situ hybridization, we analyzed the temporal and spatial expression pattern during embryonic development and larval stages and we found that zebrafish Neuroligins are expressed throughout the nervous system. Globally, our results indicate that, during evolution, specific subfunctionalization events occurred within paralogous members of this gene family in zebrafish.
Asunto(s)
Empalme Alternativo , Evolución Molecular , Familia de Multigenes , Proteínas del Tejido Nervioso/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Clonación Molecular , Desarrollo Embrionario , Exones , Perfilación de la Expresión Génica , Humanos , Hibridación in Situ , Intrones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Filogenia , Estructura Secundaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of ß1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.