Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus )

Abstract: Platelet-rich plasma (PRP) is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm) for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP) and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes. It was concluded that PRP was successfully obtained by double centrifugation protocol, which is able to increase the platelet concentration in the sample compared with whole blood, allowing its use in surgical procedures. Furthermore, the preparation time is appropriate to obtain PRP in just 40 minutes, and calcium gluconate is able to promote the activation of platelets.

Plasma rico em plaquetas empregado na cirurgia reconstrutiva em coelhos (Oryctolagus cuniculus): avaliação da exequibilidade da técnica, achados macroscópicos e histopatológicos

Pós-graduação em Cirurgia Veterinária - FCAV

The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts

Objectives:The aim of this study was to assess the biological rationale for the use of platelet-rich plasma (PRP) by evaluating the effect of different concentrations of PRP on osteoblasts (OB) and fibroblasts (FB) function in vitro. Materials and methods:PRP was obtained from volunteer donors using standard protocols. Primary human cultures of oral FBs and OBs were exposed to both activated and non-activated plasma as well as various concentrations of PRP (2.5 x, 3.5 x and max (4.2-5.5 x)). Cell proliferation was evaluated after 24 and 72 h using an MTT proliferation assay. Production of osteocalcin (OCN), osteoprotegerin (OPG) and transforming growth factor beta 1 (TGF-beta 1) was evaluated in OB after 24 and 72 h. Statistical analysis was performed using one-way ANOVA. Results:PRP-stimulated cell proliferation in both OBs and FBs. The effect of different PRP concentrations on cell proliferation was most notable at 72 h. The maximum effect was achieved with a concentration of 2.5 x, with higher concentrations resulting in a reduction of cell proliferation. Upregulation of OCN levels and downregulation of OPG levels were noted with increasing PRP concentrations at both 24 and 72 h. TGF-beta 1 levels were stimulated by increasing concentrations of PRP, with the increased levels being maintained at 72 h. Conclusions:PRP preparations exert a dose-specific effect on oral FBs and OBs. Optimal results were observed at a platelet concentration of 2.5 x, which was approximately half of the maximal concentrate that could be obtained. Increased concentrations resulted in a reduction in proliferation and a suboptimal effect on OB function. Hence, different PRP concentrations may have an impact on the results that can be obtained in vivo.

Association Between Increased Platelet P-Selectin Expression and Obesity in Patients With Type 2 Diabetes A BARI 2D (Bypass Angioplasty Revascularization Investigation 2 Diabetes) substudy

OBJECTIVE- To determine whether obesity increases platelet reactivity and thrombin activity in patients with type 2 diabetes plus stable coronary artery disease. RESEARCH DESIGN AND METHODS- We assessed platelet reactivity and markers of thrombin generation and activity in 193 patients from nine clinical sites of the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D). Blood taken at the time of enrollment was used for assay of the concentration of prothrombin fragment 1.2 (PT1.2, released when prothrombin is activated) and fibrinopeptide A (FPA, released when fibrinogen is cleaved). Platelet activation was identified with the use of flow cytometry in response to 0, 0.2, and 1 mu mol/l adenosine diphosphate (ADP). RESULTS- Concentrations of FPA, PT1.2, and platelet activation in the absence of agonist were low. Greater BMI was associated with higher platelet reactivity in response to 1 mu m ADP as assessed by surface expression of P-selectin (r = 0.29, P < 0.0001) but not reflected by the binding of fibrinogen to activated glycoprotein IIb-IIIa. BMI was not associated with concentrations of FPA or PT1.2. Platelet reactivity correlated negatively with A1C (P < 0.04), was not related to the concentration Of triglycerides in blood, and did not correlate with the concentration of C-reactive peptide. CONCLUSIONS- Among patients enrolled in this substudy of BARI 2D, a greater BMI was associated with higher platelet reactivity at the time of enrollment. Our results suggest that obesity and insulin resistance that accompanies obesity may influence platelet reactivity in patients with type 2 diabetes.

Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL ( PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents.

Role of platelet-activating factor in the pathogenesis of 5-fluorouracil-induced intestinal mucositis in mice

Gastrointestinal mucositis is a common side effect of cancer chemotherapy. Platelet-activating factor (PAF) is produced during gut inflammation. There is no evidence that PAF participates in antineoplastic-induced intestinal mucositis. This study evaluated the role of PAF in 5-fluorouracil (5-FU)-induced intestinal mucositis using a pharmacological approach and PAF receptor knockout mice (PAFR(-/-)). Wild-type mice or PAFR(-/-) mice were treated with 5-FU (450 mg/kg, i.p.). Other mice were treated with saline or BN52021 (20 mg/kg, s.c.), an antagonist of the PAF receptor, once daily followed by 5-FU administration. After the third day of treatment, animals were sacrificed and tissue samples from the duodenum were removed for morphologic evaluation. In addition, myeloperoxidase activity and the cytokine concentration were measured. 5-FU treatment decreased the duodenal villus height/crypt depth ratio, increased MPO activity, and increased the concentration of TNF-alpha, IL-1 beta and KC in comparison with saline-treated animals. In PAFR(-/-) mice and PAFR antagonist-treated mice, 5-FU-dependent intestinal damage was reduced and a decrease in duodenal villus height/crypt depth ratio was attenuated. However, the 5-FU-dependent increase in duodenum MPO activity was not affected. Without PAFR activation, 5-FU treatment did not increase the TNF-alpha, IL-1 beta and KC concentration. In conclusion, our study establishes the role of PAFR activation in 5-FU-induced intestinal mucositis. This study implicates treatment with PAFR antagonists as novel therapeutic strategy for this condition.

Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate

1 Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-l-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory effect with vasorelaxation, to determine the effects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-ajquinoxalin-1-one), and to investigate the possible role of activation of sarco-encloplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2 - 10 mug ml(-1)) and adenosine diphosphate (ADP; 2 mum) in platelet rich plasma. It was (i) more effective at inhibiting aggregation induced by collagen than by ADP, and (ii) less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3 ODQ (10 mum) caused only a small shift (approximately half a log unit) in the concentration-response curve to MAHMA NONOate irrespective of the aggregating agent. 4 The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzy] indazole; 1 - 100 mum), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1 - 1 mm), caused minimal inhibition. 5 On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 PM present) were abolished by thapsigargin (200 nm). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6 Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7 Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing : a prospective randomized clinical trial on skin graft donor sites

La cicatrisation cutanée requiert de nombreux mécanismes et un nombre toujours croissant de molécules participant à ces réactions sont identifiées. La compréhension de cette cinétique et des différents facteurs impliqués dans ce processus permet d'accélérer la guérison des plaies. Certains facteurs de croissance (PDGF, FGF, EGF) ont déjà été utilisés localement sur des plaies mais leurs effets relatés sont inconsistants et contradictoires, probablement en raison de l'absence de cinétique ou de concentration physiologique. Les plaquettes jouent un rôle fondamental dans la cicatrisation cutanée, principalement par la formation du clou plaquettaire et le relargage de divers facteurs de croissance et de cytokines. De même, les kératinocytes ont un rôle similaire dans l'épithélialisation des plaies. Ainsi, l'apport de plaquettes et de kératinocytes sur une plaie pourrait accélérer sa cicatrisation. L'étude rapportée ici tente de vérifier si une solution de kératinocytes autologues en suspension dans un concentré plaquettaire ou un concentré plaquettaire seul pourrait stimuler la cicatrisation cutanée. Pour ce faire, nous avons comparé trois groupes de 15 patients bénéficiant, sur une prise de greffe de profondeur similaire, d'un traitement standard fait de pansement simple, d'un concentré plaquettaire seul ou de kératinocytes autologues suspendus dans un concentré plaquettaire. Sur ces plaies, la durée de cicatrisation ainsi que la douleur au cours de la guérison ont été étudiés. Nos résultats montrent une réduction significative de la durée de cicatrisation dans le groupe traité avec un concentré plaquettaire, passant de 13.9 +/- 0.5 à 7.2 +/- 0.2 jours. Cet effet est encore plus marqué dans le groupe traité avec des kératinocytes en suspension dans un concentré plaquettaire passant de 13.9 +/- 0.5 à 5.7 +/- 0.2 jours. De même, la douleur évaluée au cinquième jour montre une nette diminution dans les deux groupes traités avec des cellules. En conclusion, notre travail montre que l'application de plaquettes autologues ou de kératinocytes en suspension dans un concentré plaquettaire permet d'accélérer la cicatrisation cutanée et de diminuer les douleurs, sans aucun effet indésirable constaté. Notre étude montre également qu'un concentré plaquettaire peut être utilisé comme vecteur pour une suspension de kératinocytes. L'identification de la cinétique d'apparition et de la concentration de chacun des facteurs de croissance lors de la cicatrisation cutanée permettrait dans le futur d'analyser plus finement et de traiter physiologiquement des plaies chroniques.

The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

Development of a cost-effective method for platelet-rich plasma (PRP) preparation for topical wound healing.

Platelet-rich plasma (PRP) is a volume of plasma fraction of autologous blood having platelet concentrations above baseline whole-blood values due to processing and concentration. PRP is used in various surgical fields to enhance soft-tissue and bone healing by delivering supra-physiological concentrations of autologous platelets at the site of tissue damage. These preparations may provide a good cellular source of various growth factors and cytokines, and modulate tissue response to injury. Common clinically available materials for blood preparations combined with a two-step centrifugation protocol at 280g each, to ensure cellular component integrity, provided platelet preparations which were concentrated 2-3 fold over total blood values. Costs were shown to be lower than those of other methods which require specific equipment and high-cost disposables, while safety and traceability can be increased. PRP can be used for the treatment of wounds of all types including burns and also of split-thickness skin graft donor sites, which are frequently used in burn management. The procedure can be standardized and is easy to adapt in clinical settings with minimal infrastructure, thus enabling large numbers of patients to benefit from a form of cellular therapy.

Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells

What is already known about this subject center dot Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins. center dot In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets. center dot Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid-TP interaction inhibits signalling downstream TP has not been shown. What this study adds center dot This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells. center dot Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TP alpha- and TP beta-transfected HEK 293T cells was explored using binding assays and the TP antagonist H-3-SQ29548. Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC50 10-30 mu M). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by > 50% H-3-SQ29548 binding to different cell types. These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.

Long-term monounsaturated fatty acid diets reduce platelet aggregation in healthy young subjects

The aim of the present study was to compare the response of a range of atherogenic and thrombogenic risk markers to two dietary levels of saturated fatty acid (SFA) substitution with monounsaturated fatty acids (MUFA) in students living in a university hall of residence. Although the benefits of such diets have been reported for plasma lipoproteins in high-risk groups, more needs to be known about effects of more modest SFA-MUFA substitutions over the long term and in young healthy adults. In a parallel design over 16 weeks, fifty-one healthy young subjects were randomised to one of two diets: (1) a moderate-MUFA diet in which 16 g dietary SFA/100 g total fatty acids were substituted with MUFA (n 25); (2) a high-MUFA diet in which 33 g dietary SFA/100 g total fatty acids were substituted with MUFA (n 26). All subjects followed an 8-week run-in diet (reference diet), with a fatty acid composition close to the UK average values. There were no differences in plasma lipid responses between the two diets over 16 weeks of the study with similar reductions in total cholesterol (P<0.001) and LDL-cholesterol (P<0.01) in both groups; a small but significant reduction in HDL-cholesterol was also observed in both groups (P<0.01). Platelet responses to ADP (P<0.01) and arachidonic acid (P<0.05) differed with time on the two diets; at 16 weeks, platelet aggregatory response to ADP was significantly lower on the high-MUFA than the moderate-MUFA (P<0.01) diet; ADP responses were also significantly lower within this group at 8 (P< 0.05) and 16 (P< 0.01) weeks compared with baseline. There were no differences in fasting factor VII activity (factors VIII and VIIag), fibrinogen concentration or tissue-type plasminogen activator activity between the diets. There were no differences in postprandial factor VIII responses to a standard meal (area under the curve) between the diets after 16 weeks, but postprandial factor VIII response was lower than on the high-MUFA diet compared with baseline (P<0.01). In conclusion, a high-MUFA diet sustains potentially beneficial effects on platelet aggregation and postprandial activation of factor VII. Moderate or high substitution of MUFA for SFA achieves similar reductions in fasting blood lipids in young healthy subjects.

Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway

Background: The regulation of platelet function by pharmacological agents that modulate platelet signaling haspharmacolo proven a successful approach to the prevention of thrombosis. A variety of molecules present in the diet have been shown to inhibit platelet activation, including the antioxidant quercetin. Objectives: In this report we investigate the molecular mechanisms through which quercetin inhibits collagen-stimulated platelet aggregation. Methods: The effect of quercetin on platelet aggregation, intracellular calcium release, whole cell tyrosine phosphorylation and intracellular signaling events including tyrosine phosphorylation and kinase activity of proteins involved in the collagen-stimulated glycoprotein (GP) signaling pathway were investigated. Results: We report that quercetin inhibits collagen-stimulated whole cell protein tyrosine phosphorylation and intracellular mobilization of calcium, in a concentration-dependent manner. Quercetin was also found to inhibit various events in signaling generated by the collagen receptor GPVI. This includes collagen-stimulated tyrosine phosphorylation of the Fc receptor gamma-chain, Syk, LAT and phospholipase Cgamma2. Inhibition of phosphorylation of the Fc receptor gamma-chain suggests that quercetin inhibits early signaling events following stimulation of platelets with collagen. The activity of the kinases that phosphorylate the Fc receptor gamma-chain, Fyn and Lyn, as well as the tyrosine kinase Syk and phosphoinositide 3-kinase was also inhibited by quercetin in a concentration-dependent manner, both in whole cells and in isolation. Conclusions: The present results provide a molecular basis for the inhibition by quercetin of collagen-stimulated platelet activation, through inhibition of multiple components of the GPVI signaling pathway, and may begin to explain the proposed health benefits of high quercetin intake.

Tangeretin regulates platelet function through inhibition of phosphoinositide 3-Kinase and cyclic nucleotide signaling

OBJECTIVE: Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. APPROACH AND RESULTS: Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner. It inhibited agonist-induced integrin αIIbβ3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagen-coated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. CONCLUSIONS: This study provides support for the ability and mechanisms of action of dietary flavonoids to modulate platelet signaling and function, which may affect the risk of thrombotic disease.

Fucoidan is a novel platelet agonist for the C-type lectin-like receptor 2 (CLEC-2)

Fucoidan, a sulfated polysaccharide from Fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylations of Syk and Phospholipase C-γ2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ-chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets.