Mesenchymal Stromal Cell-Based Products: Challenges and Clinical Therapeutic Options.

Mesenchymal stromal cell (MSC)-based advanced therapy medicinal products (ATMPs) are being tried in a vast range of clinical applications. These cells can be isolated from different donor tissues by using several methods, or they can even be derived from induced pluripotent stem cells or embryonic stem cells. However, ATMP heterogeneity may impact product identity and potency, and, consequently, clinical trial outcomes. In this review, we discuss these topics and the need to establish minimal criteria regarding the manufacturing of MSCs so that these innovative therapeutics may be better positioned to contribute to the advancement of regenerative medicine.

Advanced cell and gene therapies in cardiology.

We review the evidence for the presence of stem/progenitor cells in the heart and the preclinical and clinical data using diverse cell types for the therapy of cardiac diseases. We highlight the failure of adult stem/progenitor cells to ameliorate heart function in most cardiac diseases, with the possible exception of refractory angina. The use of pluripotent stem cell-derived cardiomyocytes is analysed as a viable alternative therapeutic option but still needs further research at preclinical and clinical stages. We also discuss the use of direct reprogramming of cardiac fibroblasts into cardiomyocytes and the use of extracellular vesicles as therapeutic agents in ischemic and non-ischemic cardiac diseases. Finally, gene therapies and genome editing for the treatment of hereditary cardiac diseases, ablation of genes responsible for atherosclerotic disease, or modulation of gene expression in the heart are discussed.

Creating an HLA-homozygous iPS cell bank for the Brazilian population: Challenges and opportunities.

Identifying human leukocyte antigen (HLA) haplotype-homozygous donors for the generation of induced pluripotent stem (iPS) cell lines permits the construction of biobanks immunologically compatible with significant numbers of individuals for use in therapy. However, two questions must be addressed to create such a bank: how many cell lines are necessary to match most of the recipient population and how many people should be tested to find these donors? In Japan and the UK, 50 and 100 distinct HLA-A, -B, and -DRB1 triple-homozygous haplotypes would cover 90% of those populations, respectively. Using data from the Brazilian National Registry of Bone Marrow Donors (REDOME), encompassing 4,017,239 individuals, we identified 1,906 distinct triple-homozygous HLA haplotypes. In Brazil, 559 triple-homozygous cell lines cover 95% of the population, and 3.8 million people would have to be screened. Finally, we show the contribution of the 30 most frequent triple-homozygous HLA haplotypes in Brazil to populations of different countries.

Extracellular Vesicles and Cardiac Aging.

Global population aging is a major challenge to health and socioeconomic policies. The prevalence of diseases progressively increases with aging, with cardiovascular disease being the major cause of mortality among elderly people. The allostatic overload imposed by the accumulation of cardiac senescent cells has been suggested to play a pivotal role in the aging-related deterioration of cardiovascular function. Senescent cells exhibit intrinsic disorders and release a senescence-associated secretory phenotype (SASP). Most of these SASP compounds and damaged molecules are released from senescent cells by extracellular vesicles (EVs). Once secreted, these EVs can be readily incorporated by recipient neighboring cells and elicit cellular damage or otherwise can promote extracellular matrix remodeling. This has been associated with the development of cardiac dysfunction, fibrosis, and vascular calcification, among others. The molecular signature of these EVs is highly variable and might provide important information for the development of aging-related biomarkers. Conversely, EVs released by the stem and progenitor cells can exert a rejuvenating effect, raising the possibility of future anti-aging therapies.

Aging Triggers Mitochondrial Dysfunction in Mice.

Direct analysis of isolated mitochondria from old mice enables a better understanding of heart senescence dysfunction. Despite a well-defined senescent phenotype in cardiomyocytes, the mitochondrial state in aged cardiomyocytes is still unclear. Here, we report data about mitochondrial function in old mice. Isolated cardiomyocytes' mitochondria were obtained by differential centrifugation from old and young mice hearts to perform functional analyses of mitochondrial O2 consumption, transmembrane potential, ROS formation, ATP production, and swelling. Our results show that mitochondria from old mouse hearts have reduced oxygen consumption during the phosphorylative states of complexes I and II. Additionally, these mitochondria produced more ROS and less ATP than those of young hearts. Mitochondria from old hearts also showed a depolarized membrane potential than mitochondria from young hearts and, as expected, a greater electron leak. Our results indicate that mitochondria from senescent cardiomyocytes are less efficient in O2 consumption, generating more ROS and producing less ATP. Furthermore, the phosphorylative state of complexes I and II presents a functional defect, contributing to greater leakage of protons and ROS production that can be harmful to the cell.

Empagliflozin reduces arrhythmogenic effects in rat neonatal and human iPSC-derived cardiomyocytes and improves cytosolic calcium handling at least partially independent of NHE1.

The antidiabetic agent class of sodium-glucose cotransporter 2 (SGLT2) inhibitors confer unprecedented cardiovascular benefits beyond glycemic control, including reducing the risk of fatal ventricular arrhythmias. However, the impact of SGLT2 inhibitors on the electrophysiological properties of cardiomyocytes exposed to stimuli other than hyperglycemia remains elusive. This investigation tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) affects cardiomyocyte electrical activity under hypoxic conditions. Rat neonatal and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes incubated or not with the hypoxia-mimetic agent CoCl2 were treated with EMPA (1 µM) or vehicle for 24 h. Action potential records obtained using intracellular microelectrodes demonstrated that EMPA reduced the action potential duration at 30%, 50%, and 90% repolarization and arrhythmogenic events in rat and human cardiomyocytes under normoxia and hypoxia. Analysis of Ca2+ transients using Fura-2-AM and contractility kinetics showed that EMPA increased Ca2+ transient amplitude and decreased the half-time to recover Ca2+ transients and relaxation time in rat neonatal cardiomyocytes. We also observed that the combination of EMPA with the Na+/H+ exchanger isoform 1 (NHE1) inhibitor cariporide (10 µM) exerted a more pronounced effect on Ca2+ transients and contractility than either EMPA or cariporide alone. Besides, EMPA, but not cariporide, increased phospholamban phosphorylation at serine 16. Collectively, our data reveal that EMPA reduces arrhythmogenic events, decreases the action potential duration in rat neonatal and human cardiomyocytes under normoxic or hypoxic conditions, and improves cytosolic calcium handling at least partially independent of NHE1. Moreover, we provided further evidence that SGLT2 inhibitor-mediated cardioprotection may be partly attributed to its cardiomyocyte electrophysiological effects.

Genetic Ablation and Pharmacological Blockade of Bradykinin B1 Receptor Unveiled a Detrimental Role for the Kinin System in Chagas Disease Cardiomyopathy.

Chagas disease, the parasitic infection caused by Trypanosoma cruzi, afflicts about 6 million people in Latin America. Here, we investigated the hypothesis that T. cruzi may fuel heart parasitism by activating B1R, a G protein-coupled (brady) kinin receptor whose expression is upregulated in inflamed tissues. Studies in WT and B1R-/- mice showed that T. cruzi DNA levels (15 days post infection-dpi) were sharply reduced in the transgenic heart. FACS analysis revealed that frequencies of proinflammatory neutrophils and monocytes were diminished in B1R-/- hearts whereas CK-MB activity (60 dpi) was exclusively detected in B1R+/+ sera. Since chronic myocarditis and heart fibrosis (90 dpi) were markedly attenuated in the transgenic mice, we sought to determine whether a pharmacological blockade of the des-Arg9-bradykinin (DABK)/B1R pathway might alleviate chagasic cardiomyopathy. Using C57BL/6 mice acutely infected by a myotropic T. cruzi strain (Colombian), we found that daily treatment (15-60 dpi) with R-954 (B1R antagonist) reduced heart parasitism and blunted cardiac injury. Extending R-954 treatment to the chronic phase (120-160 dpi), we verified that B1R targeting (i) decreased mortality indexes, (ii) mitigated chronic myocarditis, and (iii) ameliorated heart conduction disturbances. Collectively, our study suggests that a pharmacological blockade of the proinflammatory KKS/DABK/B1R pathway is cardioprotective in acute and chronic Chagas disease.

Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that causes accelerated aging and a high risk of cardiovascular complications. However, the underlying mechanisms of cardiac complications of this syndrome are not fully understood. This study modeled HGPS using cardiomyocytes (CM) derived from induced pluripotent stem cells (iPSC) derived from a patient with HGPS and characterized the biophysical, morphological, and molecular changes found in these CM compared to CM derived from a healthy donor. Electrophysiological recordings suggest that the HGPS-CM was functional and had normal electrophysiological properties. Electron tomography showed nuclear morphology alteration, and the 3D reconstruction of electron tomography images suggests structural abnormalities in HGPS-CM mitochondria, however, there was no difference in mitochondrial content as measured by Mitotracker. Immunofluorescence indicates nuclear morphological alteration and confirms the presence of Troponin T. Telomere length was measured using qRT-PCR, and no difference was found in the CM from HGPS when compared to the control. Proteomic analysis was carried out in a high-resolution system using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). The proteomics data show distinct group separations and protein expression differences between HGPS and control-CM, highlighting changes in ribosomal, TCA cycle, and amino acid biosynthesis, among other modifications. Our findings show that iPSC-derived cardiomyocytes from a Progeria Syndrome patient have significant changes in mitochondrial morphology and protein expression, implying novel mechanisms underlying premature cardiac aging.

Anthracycline-induced cardiotoxicity and cell senescence: new therapeutic option?

Anthracyclines are chemotherapeutic drugs widely used in the frontline of cancer treatment. The therapeutic mechanisms involve the stabilization of topoisomerase IIα, DNA, and the anthracycline molecule in a ternary complex that is recognized as DNA damage. Redox imbalance is another vital source of oxidative DNA damage. Together, these mechanisms lead to cytotoxic effects in neoplastic cells. However, anthracycline treatment can elicit cardiotoxicity and heart failure despite the therapeutic benefits. Topoisomerase IIß and oxidative damage in cardiac cells have been the most reported pathophysiological mechanisms. Alternatively, cardiac cells can undergo stress-induced senescence when exposed to anthracyclines, a state primarily characterized by cell cycle arrest, organelle dysfunction, and a shift to senescence-associated secretory phenotype (SASP). The SASP can propagate senescence to neighboring cells in an ongoing process that leads to the accumulation of senescent cells, promoting cellular dysfunction and extracellular matrix remodeling. Therefore, the accumulation of senescent cardiac cells is an emerging pathophysiological mechanism associated with anthracycline-induced cardiotoxicity. This paradigm also raises the potential for therapeutic approaches to clear senescent cells in treating anthracycline-induced cardiotoxicity (i,e, senolytic therapies).

Optimizing the Decellularized Porcine Liver Scaffold Protocol.

There are few existing methods for shortening the decellularization period for a human-sized whole-liver scaffold. Here, we describe a protocol that enables effective decellularization of the liver obtained from pigs weigh 120 ± 4.2 kg within 72 h. Porcine livers (approx. 1.5 kg) were decellularized for 3 days using a combination of chemical and enzymatic decellularization agents. After trypsin, sodium deoxycholate, and Triton X-100 perfusion, the porcine livers were completely translucent. Our protocol was efficient to promote cell removal, the preservation of extracellular matrix (ECM) components, and vascular tree integrity. In conclusion, our protocol is efficient to promote human-sized whole-liver scaffold decellularization and thus useful to generate bioengineered livers to overcome the shortage of organs.

Stem cell therapies in cardiac diseases: Current status and future possibilities.

Cardiovascular diseases represent the world's leading cause of death. In this heterogeneous group of diseases, ischemic cardiomyopathies are the most devastating and prevalent, estimated to cause 17.9 million deaths per year. Despite all biomedical efforts, there are no effective treatments that can replace the myocytes lost during an ischemic event or progression of the disease to heart failure. In this context, cell therapy is an emerging therapeutic alternative to treat cardiovascular diseases by cell administration, aimed at cardiac regeneration and repair. In this review, we will cover more than 30 years of cell therapy in cardiology, presenting the main milestones and drawbacks in the field and signaling future challenges and perspectives. The outcomes of cardiac cell therapies are discussed in three distinct aspects: The search for remuscularization by replacement of lost cells by exogenous adult cells, the endogenous stem cell era, which pursued the isolation of a progenitor with the ability to induce heart repair, and the utilization of pluripotent stem cells as a rich and reliable source of cardiomyocytes. Acellular therapies using cell derivatives, such as microvesicles and exosomes, are presented as a promising cell-free therapeutic alternative.

Inclusivity and diversity: Integrating international perspectives on stem cell challenges and potential.

Regenerative medicine has great potential. The pace of scientific advance is exciting and the medical opportunities for regeneration and repair may be transformative. However, concerns continue to grow, relating to problems caused both by unscrupulous private clinics offering unregulated therapies based on little or no evidence and by premature regulatory approval on the basis of insufficient scientific rationale and clinical evidence. An initiative by the InterAcademy Partnership convened experts worldwide to identify opportunities and challenges, with a focus on stem cells. This was designed to be inclusive and consensus outputs reflected the diversity of the global research population. Among issues addressed for supporting research and innovation while protecting patients were ethical assessment; pre-clinical and clinical research; regulatory authorization and medicines access; and engagement with patients, policy makers, and the public. The InterAcademy Partnership (IAP) identified options for action for sharing good practice and building collaboration within the scientific community and with other stakeholders worldwide.

Cell-Based Therapies for Heart Failure.

Heart failure has reached epidemic proportions with the advances in cardiovascular therapies for ischemic heart diseases and the progressive aging of the world population. Efficient pharmacological therapies are available for treating heart failure, but unfortunately, even with optimized therapy, prognosis is often poor. Their last therapeutic option is, therefore, a heart transplantation with limited organ supply and complications related to immunosuppression. In this setting, cell therapies have emerged as an alternative. Many clinical trials have now been performed using different cell types and injection routes. In this perspective, we will analyze the results of such trials and discuss future perspectives for cell therapies as an efficacious treatment of heart failure.

New Cardiomyokine Reduces Myocardial Ischemia/Reperfusion Injury by PI3K-AKT Pathway Via a Putative KDEL-Receptor Binding.

Background CDNF (cerebral dopamine neurotrophic factor) belongs to a new family of neurotrophic factors that exert systemic beneficial effects beyond the brain. Little is known about the role of CDNF in the cardiac context. Herein we investigated the effects of CDNF under endoplasmic reticulum-stress conditions using cardiomyocytes (humans and mice) and isolated rat hearts, as well as in rats subjected to ischemia/reperfusion (I/R). Methods and Results We showed that CDNF is secreted by cardiomyocytes stressed by thapsigargin and by isolated hearts subjected to I/R. Recombinant CDNF (exoCDNF) protected human and mouse cardiomyocytes against endoplasmic reticulum stress and restored the calcium transient. In isolated hearts subjected to I/R, exoCDNF avoided mitochondrial impairment and reduced the infarct area to 19% when administered before ischemia and to 25% when administered at the beginning of reperfusion, compared with an infarct area of 42% in the untreated I/R group. This protection was completely abrogated by AKT (protein kinase B) inhibitor. Heptapeptides containing the KDEL sequence, which binds to the KDEL-R (KDEL receptor), abolished exoCDNF beneficial effects, suggesting the participation of KDEL-R in this cardioprotection. CDNF administered intraperitoneally to rats decreased the infarct area in an in vivo model of I/R (from an infarct area of ≈44% in the I/R group to an infarct area of ≈27%). Moreover, a shorter version of CDNF, which lacks the last 4 residues (CDNF-ΔKTEL) and thus allows CDNF binding to KDEL-R, presented no cardioprotective activity in isolated hearts. Conclusions This is the first study to propose CDNF as a new cardiomyokine that induces cardioprotection via KDEL receptor binding and PI3K/AKT activation.

BKCa Channel Activation Attenuates the Pathophysiological Progression of Monocrotaline-Induced Pulmonary Arterial Hypertension in Wistar Rats.

PURPOSE: In the present study, the therapeutic efficacy of a selective BKCa channel opener (compound X) in the treatment of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) was investigated. METHODS: PAH was induced in male Wistar rats by a single injection of MCT. After two weeks, the MCT-treated group was divided into two groups that were either treated with compound X or vehicle. Compound X was administered daily at 28 mg/kg. Electrocardiographic, echocardiographic, and haemodynamic analyses were performed; ex vivo evaluations of pulmonary artery reactivity, right ventricle (RV) and lung histology as well as expression levels of α and ß myosin heavy chain, brain natriuretic peptide, and cytokines (TNFα and IL10) in heart tissue were performed. RESULTS: Pulmonary artery rings of the PAH group showed a lower vasodilatation response to acetylcholine, suggesting endothelial dysfunction. Compound X promoted strong vasodilation in pulmonary artery rings of both control and MCT-induced PAH rats. The untreated hypertensive rats presented remodelling of pulmonary arterioles associated with increased resistance to pulmonary flow; increased systolic pressure, hypertrophy and fibrosis of the RV; prolongation of the QT and Tpeak-Tend intervals (evaluated during electrocardiogram); increased lung and liver weights; and autonomic imbalance with predominance of sympathetic activity. On the other hand, treatment with compound X reduced pulmonary vascular remodelling, pulmonary flow resistance and RV hypertrophy and afterload. CONCLUSION: The use of a selective and potent opener to activate the BKCa channels promoted improvement of haemodynamic parameters and consequent prevention of RV maladaptive remodelling in rats with MCT-induced PAH.

Therapy with Cardiomyocytes Derived from Pluripotent Cells in Chronic Chagasic Cardiomyopathy.

Chagas disease discovered more than a century ago remains an incurable disease. The objective of this work was to investigate the therapeutic potential of cardiomyocytes derived from mouse embryonic stem cells (CM-mESC) in a model of chronic Chagasic cardiomyopathy (CCC). Mouse embryonic stem cells (mESC) were characterized, transduced with luciferase, and submitted to cardiac differentiation. CM-mESC were labeled with superparamagnetic iron oxide particles. To induce CCC, mice were infected with Brazil strain trypomastigotes. At 150 days post-infection (dpi), infected animals were treated with CM-mESC or PBS. Cells were detected by magnetic resonance imaging (MRI) and bioluminescence. Cardiac function was evaluated by MRI and electrocardiogram at 150 and 196 dpi. CCC mice showed significant differences in MRI and ECG parameters compared to non-infected mice. However, no differences were observed in contractile and electrical parameters between cell and PBS injected groups, 45 days after cell transplantation. Cells were detected 24 h after transplantation by MRI. CM-mESC bioluminescence tracking demonstrated over 90% decrease in signal 8 days after treatment. Nevertheless, the Infected + CM-mESC group showed a significant reduction in the percentage of collagen fibers when compared to the Infected + PBS group. In conclusion, CM-mESC therapy was not effective in reversing cardiac functional changes induced by Chagas disease despite some improvement in myocardial fibrosis.

Exogenous 10 kDa-Heat Shock Protein Preserves Mitochondrial Function After Hypoxia/Reoxygenation.

Humoral factors released during ischemic preconditioning (IPC) protect the myocardium against ischemia/reperfusion (I/R) injury. We have recently identified 10 kDa-heat shock protein (HSP10) and a fraction of small 5-10 kDa peptides (5-10-sP) in the coronary effluent of IPC-treated hearts and demonstrated their cardioprotective potential. We here used our isolated mitochondria model to characterize the impact of exogenous HSP10 and 5-10-sP on mitochondria function from myocardium subjected to I/R injury. Isolated perfused rat hearts were submitted to 30-min global ischemia and 10-min reperfusion. Before ischemia, isolated hearts were infused with saline or 5-10-sP, with or without a mitochondrial ATP-sensitive-K+-channel blocker (5HD 10 µmol·L-1) or PKC inhibitor (chelerythrine 10 µmol·L-1), before I/R. HSP10 (1 µmol·L-1) was infused into isolated hearts before I/R without blockers. At 10-min reperfusion, the mitochondria were isolated and mitochondrial function was assessed. In a subset of experiments, freshly isolated mitochondria were directly incubated with HSP10 or 5-10-sP with or without 5HD or chelerythrine before in vitro hypoxia/reoxygenation. Infusion of 5-10-sP (n = 5) and HSP10 (n = 5) into isolated hearts before I/R improved mitochondrial ADP-stimulated respiration, ATP production and prevented mitochondrial ROS formation compared to the I/R group (n = 5); this effect was abrogated by 5HD and chelerythrine. In freshly isolated mitochondria with in vitro hypoxia/reoxygenation, HSP10 (n = 16) and 5-10-sP (n = 16) incubation prevented reductions of mitochondrial ADP-stimulated respiration (91.5 ± 5.1 nmol O2/min/mg PTN), ATP production (250.1 ± 9.3 µmol ATP/200µg PTN), and prevented mitochondrial ROS production (219.7 ± 9.0 nmol H2O2/200µg PTN) induced by hypoxia/reoxygenation (n = 12, 51.5 ± 5.0 nmol O2/min/mg PTN; 187 ± 21.7 µmol ATP/200 µg PTN; 339.0 ± 14.3 nmol H2O2/200 µg PTN, p < 0.001, respectively). 5HD reduced the ADP-stimulated respiration in the HSP10 group (65.84 ± 3.3 nmol O2/min/mg PTN), ATP production (193.7 ± 12.1 µmol ATP/200µg PTN) and increased ROS in the 5-10-sP group (274.4 ± 21.7 nmol H2O2/200 µg PTN). Mitochondria are a target of the cardioprotection induced by 5-10-sP and HSP10. This protection is dependent of PKC and mKATP activation. HSP10 can act directly on mitochondria and protects against hypoxia/reoxygenation injury by mKATP activation.

Different Signatures of High Cardiorespiratory Capacity Revealed With Metabolomic Profiling in Elite Athletes.

PURPOSE: High cardiorespiratory capacity is a key determinant of human performance and life expectancy; however, the underlying mechanisms are not fully understood. The objective of this pilot study was to investigate biochemical signatures of endurance-performance athletes using high-resolution nontargeted metabolomics. METHODS: Elite long-distance runners with similar training and anthropometrical records were studied. After athletes' maximal oxygen consumption (V˙O2max) was measured, they were divided into 2 groups: low V˙O2max (<65 mL·kg-1·min-1, n = 7) and high V˙O2max (>75 mL·kg-1·min-1, n = 7). Plasma was collected under basal conditions after 12 hours of fasting and after a maximal exercise test (nonfasted) and analyzed by high-resolution LC-MS. Multivariate and univariate statistics were applied. RESULTS: A total of 167 compounds were putatively identified with an LC-MS-based metabolomics pipeline. Partial least-squares discriminant analysis showed a clear separation between groups. Significant variations in metabolites highlighted group differences in diverse metabolic pathways, including lipids, vitamins, amino acids, purine, histidine, xenobiotics, and others, either under basal condition or after the maximal exercise test. CONCLUSIONS: Taken together, the metabolic alterations revealed in the study affect cellular energy use and availability, oxidative stress management, muscle damage, central nervous system signaling metabolites, nutrients, and compound bioavailability, providing new insights into metabolic alterations associated with exercise and cardiorespiratory fitness levels in trained athletes.

R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome.

Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.