Functionality of macrophages encapsulated in porcine decellularized adipose matrix hydrogels and interaction with Candida albicans.

Extracellular matrix hydrogels are considered one of the most suitable biomaterials for tissue regeneration due to their similarity with the extracellular microenvironment of the native tissue. Their properties are dependent on their composition, material concentration, fiber density and the fabrication approaches, among other factors. The encapsulation of immune cells in this kind of hydrogels, both in absence or presence of a pathogen, represents a promising strategy for the development of platforms that mimic healthy and infected tissues, respectively. In this work, we have encapsulated macrophages in 3D hydrogels of porcine decellularized adipose matrices (pDAMs) without and with the Candida albicans fungus, as 3D experimental models to study the macrophage immunocompetence in a closer situation to the physiological conditions and to mimic an infection scenario. Our results indicate that encapsulated macrophages preserve their functionality within these pDAM hydrogels and phagocytose live pathogens. In addition, their behavior is influenced by the hydrogel pore size, inversely related to the hydrogel concentration. Thus, larger pore size promotes the polarization of macrophages towards M2 phenotype along the time and enhances their phagocytosis capability. It is important to point out that encapsulated macrophages in absence of pathogen showed an M2 phenotype, but macrophages coencapsulated with C. albicans can switch towards an M1 inflammatory phenotype to resolve the infection, depending on the fungus quantity. The present study reveals that pDAM hydrogels preserve the macrophage plasticity, demonstrating their relevance as new models for macrophage-pathogen interaction studies that mimic an infection scenario with application in regenerative medicine research.

Effects of graphene oxide and reduced graphene oxide nanomaterials on porcine endothelial progenitor cells.

Graphene oxide (GO) and reduced graphene oxide (rGO) have been widely used in the field of tissue regeneration and various biomedical applications. In order to use these nanomaterials in organisms, it is imperative to possess an understanding of their impact on different cell types. Due to the potential of these nanomaterials to enter the bloodstream, interact with the endothelium and accumulate within diverse tissues, it is highly relevant to probe them when in contact with the cellular components of the vascular system. Endothelial progenitor cells (EPCs), involved in blood vessel formation, have great potential for tissue engineering and offer great advantages to study the possible angiogenic effects of biomaterials. Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates vascular permeability, mainly activating VEGFR2 on endothelial cells. The effects of GO and two types of reduced GO, obtained after vacuum-assisted thermal treatment for 15 min (rGO15) and 30 min (rGO30), on porcine endothelial progenitor cells (EPCs) functionality were assessed by analyzing the nanomaterial intracellular uptake, reactive oxygen species (ROS) production and VEGFR2 expression by EPCs. The results evidence that short annealing (15 and 30 minutes) at 200 °C of GO resulted in the mitigation of both the increased ROS production and decline in VEGFR2 expression of EPCs upon GO exposure. Interestingly, after 72 hours of exposure to rGO30, VEGFR2 was higher than in the control culture, suggesting an early angiogenic potential of rGO30. The present work reveals that discrete variations in the reduction of GO may significantly affect the response of porcine endothelial progenitor cells.

Effects of Graphene Oxide and Reduced Graphene Oxide Nanostructures on CD4+ Th2 Lymphocytes.

The activation of T helper (Th) lymphocytes is necessary for the adaptive immune response as they contribute to the stimulation of B cells (for the secretion of antibodies) and macrophages (for phagocytosis and destruction of pathogens) and are necessary for cytotoxic T-cell activation to kill infected target cells. For these issues, Th lymphocytes must be converted into Th effector cells after their stimulation through their surface receptors TCR/CD3 (by binding to peptide-major histocompatibility complex localized on antigen-presenting cells) and the CD4 co-receptor. After stimulation, Th cells proliferate and differentiate into subpopulations, like Th1, Th2 or Th17, with different functions during the adaptative immune response. Due to the central role of the activation of Th lymphocytes for an accurate adaptative immune response and considering recent preclinical advances in the use of nanomaterials to enhance T-cell therapy, we evaluated in vitro the effects of graphene oxide (GO) and two types of reduced GO (rGO15 and rGO30) nanostructures on the Th2 lymphocyte cell line SR.D10. This cell line offers the possibility of studying their activation threshold by employing soluble antibodies against TCR/CD3 and against CD4, as well as the simultaneous activation of these two receptors. In the present study, the effects of GO, rGO15 and rGO30 on the activation/proliferation rate of these Th2 lymphocytes have been analyzed by studying cell viability, cell cycle phases, intracellular content of reactive oxygen species (ROS) and cytokine secretion. High lymphocyte viability values were obtained after treatment with these nanostructures, as well as increased proliferation in the presence of rGOs. Moreover, rGO15 treatment decreased the intracellular ROS content of Th2 cells in all stimulated conditions. The analysis of these parameters showed that the presence of these GO and rGO nanostructures did not alter the response of Th2 lymphocytes.

Benefits in the Macrophage Response Due to Graphene Oxide Reduction by Thermal Treatment.

Graphene and its derivatives are very promising nanomaterials for biomedical applications and are proving to be very useful for the preparation of scaffolds for tissue repair. The response of immune cells to these graphene-based materials (GBM) appears to be critical in promoting regeneration, thus, the study of this response is essential before they are used to prepare any type of scaffold. Another relevant factor is the variability of the GBM surface chemistry, namely the type and quantity of oxygen functional groups, which may have an important effect on cell behavior. The response of RAW-264.7 macrophages to graphene oxide (GO) and two types of reduced GO, rGO15 and rGO30, obtained after vacuum-assisted thermal treatment of 15 and 30 min, respectively, was evaluated by analyzing the uptake of these nanostructures, the intracellular content of reactive oxygen species, and specific markers of the proinflammatory M1 phenotype, such as CD80 expression and secretion of inflammatory cytokines TNF-α and IL-6. Our results demonstrate that GO reduction resulted in a decrease of both oxidative stress and proinflammatory cytokine secretion, significantly improving its biocompatibility and potential for the preparation of 3D scaffolds able of triggering the appropriate immune response for tissue regeneration.

Effects of Human and Porcine Adipose Extracellular Matrices Decellularized by Enzymatic or Chemical Methods on Macrophage Polarization and Immunocompetence.

The decellularized extracellular matrix (ECM) obtained from human and porcine adipose tissue (AT) is currently used to prepare regenerative medicine bio-scaffolds. However, the influence of these natural biomaterials on host immune response is not yet deeply understood. Since macrophages play a key role in the inflammation/healing processes due to their high functional plasticity between M1 and M2 phenotypes, the evaluation of their response to decellularized ECM is mandatory. It is also necessary to analyze the immunocompetence of macrophages after contact with decellularized ECM materials to assess their functional role in a possible infection scenario. In this work, we studied the effect of four decellularized adipose matrices (DAMs) obtained from human and porcine AT by enzymatic or chemical methods on macrophage phenotypes and fungal phagocytosis. First, a thorough biochemical characterization of these biomaterials by quantification of remnant DNA, lipids, and proteins was performed, thus indicating the efficiency and reliability of both methods. The proteomic analysis evidenced that some proteins are differentially preserved depending on both the AT origin and the decellularization method employed. After exposure to the four DAMs, specific markers of M1 proinflammatory and M2 anti-inflammatory macrophages were analyzed. Porcine DAMs favor the M2 phenotype, independently of the decellularization method employed. Finally, a sensitive fungal phagocytosis assay allowed us to relate the macrophage phagocytosis capability with specific proteins differentially preserved in certain DAMs. The results obtained in this study highlight the close relationship between the ECM biochemical composition and the macrophage's functional role.

An Immunological Approach to the Biocompatibility of Mesoporous SiO2-CaO Nanospheres.

Mesoporous bioactive glass nanospheres (NanoMBGs) have high potential for clinical applications. However, the impact of these nanoparticles on the immune system needs to be addressed. In this study, the biocompatibility of SiO2-CaO NanoMBGs was evaluated on different mouse immune cells, including spleen cells subsets, bone marrow-derived dendritic cells (BMDCs), or cell lines like SR.D10 Th2 CD4+ lymphocytes and DC2.4 dendritic cells. Flow cytometry and confocal microscopy show that the nanoparticles were rapidly and efficiently taken up in vitro by T and B lymphocytes or by specialized antigen-presenting cells (APCs) like dendritic cells (DCs). Nanoparticles were not cytotoxic and had no effect on cell viability or proliferation under T-cell (anti-CD3) or B cell (LPS) stimuli. Besides, NanoMBGs did not affect the balance of spleen cell subsets, or the production of intracellular or secreted pro- and anti-inflammatory cytokines (TNF-α, IFN-γ, IL-2, IL-6, IL-10) by activated T, B, and dendritic cells (DC), as determined by flow cytometry and ELISA. T cell activation surface markers (CD25, CD69 and Induced Costimulator, ICOS) were not altered by NanoMBGs. Maturation of BMDCs or DC2.4 cells in vitro was not altered by NanoMBGs, as shown by expression of Major Histocompatibility Complex (MHC) and costimulatory molecules (CD40, CD80, CD86), or IL-6 secretion. The effect of wortmannin and chlorpromazine indicate a role for phosphoinositide 3-kinase (PI3K), actin and clathrin-dependent pathways in NanoMBG internalization. We thus demonstrate that these NanoMBGs are both non-toxic and non-inflammagenic for murine lymphoid cells and myeloid DCs despite their efficient intake by the cells.

Macrophage inflammatory and metabolic responses to graphene-based nanomaterials differing in size and functionalization.

The preparation of graphene-based nanomaterials (GBNs) with appropriate stability and biocompatibility is crucial for their use in biomedical applications. In this work, three GBNs differing in size and/or functionalization have been synthetized and characterized, and their in vitro biological effects were compared. Pegylated graphene oxide (GO-PEG, 200-500 nm) and flavin mononucleotide-stabilized pristine graphene with two different sizes (PG-FMN, 200-400 nm and 100-200 nm) were administered to macrophages, chosen as cellular model due to their key role in the processing of foreign materials and the regulation of inflammatory responses. The results showed that cellular uptake of GBNs was mainly influenced by their lateral size, while the inflammatory potential depended also on the type of functionalization. PG-FMN nanomaterials (both sizes) triggered significantly higher nitric oxide (NO) release, together with some intracellular metabolic changes, similar to those induced by the prototypical inflammatory stimulus LPS. NMR metabolomics revealed that macrophages incubated with smaller PG-FMN displayed increased levels of succinate, itaconate, phosphocholine and phosphocreatine, together with decreased creatine content. The latter two variations were also detected in cells incubated with larger PG-FMN nanosheets. On the other hand, GO-PEG induced a decrease in the inflammatory metabolite succinate and a few other changes distinct from those seen in LPS-stimulated macrophages. Assessment of TNF-α secretion and macrophage surface markers (CD80 and CD206) further corroborated the low inflammatory potential of GO-PEG. Overall, these findings revealed distinct phenotypic and metabolic responses of macrophages to different GBNs, which inform on their immunomodulatory activity and may contribute to guide their therapeutic applications.

Incorporation and effects of mesoporous SiO2-CaO nanospheres loaded with ipriflavone on osteoblast/osteoclast cocultures.

Mesoporous nanospheres in the system SiO2-CaO (NanoMBGs) with a hollow core surrounded by a radial arrangement of mesopores were characterized, labeled with FITC (FITC-NanoMBGs) and loaded with ipriflavone (NanoMBG-IPs) in order to evaluate their incorporation and their effects on both osteoblasts and osteoclasts simultaneously and maintaining the communication with each other in coculture. The influence of these nanospheres on macrophage polarization towards pro-inflammatory M1 or reparative M2 phenotypes was also evaluated in basal and stimulated conditions through the expression of CD80 (as M1 marker) and CD206 (as M2 marker) by flow cytometry and confocal microscopy. NanoMBGs did not induce the macrophage polarization towards the M1 pro-inflammatory phenotype, favoring the M2 reparative phenotype and increasing the macrophage response capability against stimuli as LPS and IL-4. NanoMBG-IPs induced a significant decrease of osteoclast proliferation and resorption activity after 7 days in coculture with osteoblasts, without affecting osteoblast proliferation and viability. Drug release test demonstrated that only a fraction of the payload is released by diffusion, whereas the rest of the drug remains within the hollow core after 7 days, thus ensuring the local long-term pharmacological treatment beyond the initial fast IP release. All these data ensure an appropriate immune response to these nanospheres and the potential application of NanoMBG-IPs as local drug delivery system in osteoporotic patients.

High glucose alters the secretome of mechanically stimulated osteocyte-like cells affecting osteoclast precursor recruitment and differentiation.

Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte-driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro-survival effect conferred to osteocytic MLO-Y4 cells and osteoblastic MC3T3-E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre-exposure prevented both cell survival and ERK and ß-catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO-Y4 and MC3T3-E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO-Y4 cell-conditioned medium (CM), but not by MC3T3-E1 cell-CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP-1α, MIP-1ß MCP-1, and GM-CSF in MLO-Y4 cell-CM. RAW264.7 proliferation was inhibited by MLO-Y4 CM under static or HG conditions, but it increased by FF-CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG-CM in static condition allowed formation of osteoclast-like cells, which were unable to resorb hydroxyapatite. In contrast, FF-CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL-6 secretion or their inhibition induced by FF in MLO-Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte-osteoclast communication.

Influence of the covalent immobilization of graphene oxide in poly(vinyl alcohol) on human osteoblast response.

The differences in the response of human Saos-2 osteoblasts to nanocomposites of poly(vinyl alcohol) (PVA) and 1.5wt.% graphene oxide (GO) prepared by covalent linking (PVA/GO-c) and simple blending (PVA/GO-m) have been evaluated through different biocompatibility parameters. The effects produced on osteoblasts by these two nanocomposites were analysed in parallel and compared with the direct action of GO and with the effect of PVA films without GO. The intracellular content of reactive oxygen species (ROS) and the levels of interleukin-6 (IL-6) were measured to evaluate oxidative stress induction and protective response, respectively. The results demonstrate that the combination of GO with PVA reduces both the proliferation delay and the internal cell complexity alterations induced by GO on human osteoblasts. Moreover, the covalent attachment of GO to the PVA chains increases both cell viability and IL-6 levels, reducing both apoptosis and intracellular ROS content when compared to simple blending of both materials. The use of this strategy to modulate the biointerface reduces the toxic effects of graphene while preserving the reinforcement characteristics for application in tissue engineering scaffolds, and has enormous interest for polymer/graphene biomaterials development.

Early in vitro response of macrophages and T lymphocytes to nanocrystalline hydroxyapatites.

HYPOTHESIS: Synthetic hydroxyapatite (HA) and Si substituted hydroxyapatite (SiHA) are calcium phosphate ceramics currently used in the field of dentistry and orthopaedic surgery. The preparation of both biomaterials as polycrystalline solid pieces or grains formed by nanocrystallites has awakened a great interest to enhance the bioactive behavior due to the microstructural defects and the higher surface area. The study of the macrophage and lymphocyte behavior in contact with nanocrystalline HA and SiHA will allow to elucidate the immune response which conditions the success or rejection of these biomaterials. EXPERIMENTS: HA and SiHA granules (with sizes of tens of microns) have been prepared by controlled aqueous precipitation avoiding subsequent high temperature sintering. HA and SiHA granules were constituted by crystallites smaller than 50 nm. The effects of both nanocrystalline materials on immune system have been evaluated with macrophages (main components of innate immune system) and T lymphocytes (specific cells of adaptive response) after short-term culture as in vitro models of the early immune response. FINDINGS: Significant decreases of macrophage proliferation and phagocytic activity, increased production of inflammatory cytokines (IL-6, TNF-α) and T lymphocyte apoptosis, were induced by these nanocrystalline ceramics suggesting that, after in vivo implantation, they induce significant effects on immune responses, including an early activation of the innate immune system.

The effects of graphene oxide nanosheets localized on F-actin filaments on cell-cycle alterations.

Graphene oxide (GO) is considered to be a promising nanomaterial for biomedical applications due to its small two-dimensional shape besides its electrical and mechanical properties. However, only a few data concerning the cell responses to this material have been described and the GO biocompatibility has not been yet fully assessed. In the present study, graphene oxide nanosheets (GOs) decorated with 1-arm (1-GOs) and 6-arm (6-GOs) poly(ethylene glycol-amine) (PEG) have been incubated with cultured Saos-2 osteoblasts, MC3T3-E1 preosteoblasts and RAW-264.7 macrophages to analyze several key cell markers for in vitro biocompatibility evaluation. The results demonstrate that, after internalization, GO nanosheets are localized on F-actin filaments inducing cell-cycle alterations, apoptosis and oxidative stress in these cell types. The observed GOs effects must be considered in further studies focused on photothermal cancer therapy as a synergistic factor.

Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells.

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering.

In vitro evaluation of glass-glass ceramic thermoseed-induced hyperthermia on human osteosarcoma cell line.

The use of biomaterials as implantable thermoseeds under the action of an external magnetic field is a very interesting methodology to focus the heat into the target tumors as osteosarcoma. In this study, biocompatible and bioactive G15GC85 thermoseeds, tailored through the combination of sol-gel glasses (G) with a magnetic glass ceramic (GC), were used to induce hyperthermia on cultured human osteosarcoma cells after exposition to alternating magnetic field (MF, 100 kHz/200 Oe). G15GC85 magnetic glass-glass ceramic thermoseeds induced in vitro effective hyperthermia with drastic reduction in proliferation of human osteosarcoma Saos-2 cells and high increase of apoptotic cells after two 40 min consecutive sessions of MF. Deep cell morphology alterations were observed after this hyperthermic treatment, and the proteomic analysis revealed modification of gamma actin molecular properties related to cytoskeleton alterations. These results indicate that G15GC85 thermoseeds allow to induce in vitro effective hyperthermia on human osteosarcoma cells.

Immobilization and bioactivity evaluation of FGF-1 and FGF-2 on powdered silicon-doped hydroxyapatite and their scaffolds for bone tissue engineering.

Fibroblast growth factors (FGFs) are polypeptides that control the proliferation and differentiation of various cell types including osteoblasts. FGFs are also strong inducers of angiogenesis, necessary to obtain oxygen and nutrients during tissue repair. With the aim to incorporate these desirable FGF biological properties into bioceramics for bone repair, silicon substituted hydroxyapatites (Si-HA) were used as materials to immobilize bioactive FGF-1 and FGF-2. Thus, the binding of these growth factors to powdered Si-HA and Si-HA scaffolds was carried out efficiently in the present study and both FGFs maintained its biological activity on osteoblasts after its immobilization. The improvement of cell adhesion and proliferation onto Si-HA scaffolds suggests the potential utility of these FGF/scaffolds for bone tissue engineering.

N-terminal negatively charged residues in CD3varepsilon chains as a phylogenetically conserved trait potentially yielding isoforms with different isoelectric points: analysis of human CD3varepsilon chains.

CD3varepsilon chains are essential to the structure, expression and signaling of T cell receptors. Here, we extend to human CD3varepsilon our previous data in mouse CD3varepsilon showing that, in T cells, proteolytic processing of the acidic N-terminal sequence of CD3varepsilon chains generate distinct polypeptide species that can be identified by two-dimension (IEF-SDS PAGE) electrophoresis and immunoblot. This was shown first by showing the processing of a fusion protein of GFP and the extracellular domain of mouse CD3varepsilon (mCD3GFP) expressed in Jurkat cells. Secondly, pI heterogeneity was also found in human CD3varepsilon chains immunoprecipitated from the surface of Jurkat cells or PHA blasts of human blood T lymphocytes. Comparison of CD3varepsilon chains from 27 different species shows that their N-terminal sequences share a strong acidic nature, despite the large differences in terms of length and composition, even among closely related species. Our results suggest that generation of CD3varepsilon chain isoforms with different N-terminal sequence and pI is a general phenomenon. Thus, as previously observed in the mouse, the relative abundance of CD3varepsilon chain species might regulate TCR/CD3 structure and function, including the strength of the interactions between CD3 dimers and the TCR clonotypic receptors, as well as TCR/CD3 activation thresholds. Interestingly, CD3varepsilon chains from 7 out of 27 species studied have putative N-glycosylation (NxS or NxT) motifs in their Ig extracellular domain. Their location, plus the conservation of residues involved in domain organization, the interactions with other CD3 chains, or the TCR, and signal triggering add new data useful to establish a permissive topology for the interaction between CD3 dimers and the TCR chains.

CD46 (membrane cofactor protein) acts as a human epithelial cell receptor for internalization of opsonized uropathogenic Escherichia coli.

Escherichia coli is a common urinary pathogen whose uptake into epithelial cells is mediated by attachment through type 1 fimbriae. In this study, we show by using using human urinary tract epithelial cells that maximal internalization of E. coli is achieved only when bacteria are opsonized with complement. The concentrations of complement proteins in the urine rise sufficiently during infection to allow bacterial opsonization. The complement regulatory protein, CD46 (membrane cofactor protein), acts in cohort with fimbrial adhesion to promote the uptake of pathogenic E. coli. This uptake is inhibited by RNA interference to lower the expression of CD46 and by soluble CD46 that will competitively inhibit opsonized bacteria binding to cell surface CD46. We propose that efficient internalization of uropathogenic E. coli by the human urinary tract depends on cooperation between fimbrial-mediated adhesion and C3 receptor (CD46)-ligand interaction. Complement receptor-ligand interaction could pose a new target for interrupting the cycle of reinfection due to intracellular bacteria.

The TCR/CD3 complex: molecular interactions in a changing structure.

The T cell receptor-CD3 (TCR/CD3) complex is a multichain structure in charge of antigen recognition in T cells. Despite many genetic, structural, and functional data obtained in recent years, essential questions concerning the TCR/CD3 complex still remain open, including: 1) the precise number of polypeptides in each TCR/CD3 complex, their interactions and spatial arrangement, 2) the role(s) of each polypeptide in antigen recognition and/or in receptor signal transmission, and 3) the relationship between the TCR/CD3 complex and other membrane or cytoplasmic molecules involved in downstream signaling. In this work we shall review data concerning some of these issues, proposing a model of the overall structure of the TCR/CD3 complex to explain its known features.