Myocardial protection by ischemic preconditioning and delta-opioid receptor activation in the isolated working rat heart.

OBJECTIVE: delta-Opioid receptors are involved in the cardioprotective effect of ischemic preconditioning. This study was designed (1) to assess the protective capacities of ischemic preconditioning and the synthetic delta-opioid receptor agonist D-Ala(2)-D-Leu(5) enkephalin (DADLE) in a functionally oriented experimental model of ischemia and reperfusion and (2) to assess whether the effects of both protective measures are similarly blocked by naloxone, a nonspecific delta-opioid receptor antagonist. METHODS: Sixty-four isolated working rat hearts were subjected to 45 minutes of hypothermic ischemia at 30 degrees C followed by 25 minutes of normothermic reperfusion. Rats were pretreated with DADLE (1 mg/kg body weight intravenously), naloxone (3 mg/kg body weight intravenously), or a combination thereof within 60 minutes before onset of isolated heart perfusion. During the preischemic perfusion period, 8 hearts per group were preconditioned by one cycle of 5 minutes of normothermic global ischemia and subsequent reperfusion whereas another 8 served as nonpreconditioned controls. The postischemic functional recovery of hearts and their creatine kinase leakage were determined. RESULTS: Pretreatment with DADLE and ischemic preconditioning improved the postischemic recovery of aortic flow when compared with nonpreconditioning (57.7% +/- 4.0% and 60.8% +/- 4.3% vs 40.0% +/- 4.2% of preischemic baseline value, P <.001). Combined pretreatment with DADLE before ischemic preconditioning afforded additional aortic flow recovery compared with pretreatment with DADLE alone (68.6% +/- 3.3% vs 57.7% +/- 4.0% of preischemic baseline value; P =.038). With combined pretreatment, early postischemic creatine kinase release was lower than control in hearts without pretreatment (0.48 +/- 0.11 vs 0.80 +/- 0.12 IU/5 minutes per heart; P =.001). Naloxone abolished the beneficial functional effects of pretreatment with DADLE and ischemic preconditioning. CONCLUSIONS: Pharmacologic activation of delta-opioid receptors affords improvement of functional protection in isolated working rat hearts similar to that conferred by classic ischemic preconditioning. The combination of both pretreatments reduces ischemic cellular damage and further adds to postischemic functional recovery. These changes are reversed by naloxone, an observation providing evidence that ischemic preconditioning involves signaling through opioid receptors.

Prophylactic mitral reconstruction for mitral regurgitation.

BACKGROUND: Mitral regurgitation (MR) will produce myocardial dysfunction. The goal of this study was to review outcomes of mitral valve reconstruction in asymptomatic patients with severe MR. METHODS: From 1992 to 2000, 93 asymptomatic patients with degenerative disease and severe MR underwent mitral valve reconstruction. Mean preoperative left ventricular internal diameter diastole was 56 +/- 8 mm and ejection fraction was 60% +/- 6%. Mean age was 47 +/- 10 years and mean follow-up 23 +/- 27 months. All patients underwent complex reconstruction. RESULTS: There were no deaths and two late reoperations. One was for systolic anterior motion of the anterior leaflet of the mitral valve requiring valve replacement and one for hemolysis requiring re-repair. There was one perioperative transient ischemic attack and no late thromboembolic events. At follow-up all but 1 patient remains in NYHA class I and all had no MR except in 2 patients at 63 and 89 months. CONCLUSIONS: Mitral valve reconstruction for "asymptomatic" MR can be performed with no mortality and low morbidity before development of left ventricular dysfunction. Early prophylactic repair is advocated in the presence of severe MR if valve reparability is assured.

Opioids confer myocardial tolerance to ischemia: interaction of delta opioid agonists and antagonists.

BACKGROUND: Mammalian hibernation biology is now known to be mediated by delta opioids. The altered myocellular physiology of hibernation closely parallels that of hypothermic ischemia used to protect the heart for cardiac surgery. METHODS AND RESULTS: The present study examined the interaction of delta opioid agonists and antagonists on myocardial tolerance to ischemia. By means of a nonhibernating isolated rabbit heart model, functional and metabolic myocardial parameters were assessed during nonischemic baseline and postischemic recovery periods. Control hearts with standard cardioplegic protection alone were compared with those with cardioplegia plus preperfusion with a delta opioid agonist, a delta opioid antagonist, or both. All hearts were then subjected to 2 hours of global ischemia. Compared with cardioplegia alone, postischemic left ventricular developed pressure, coronary flows, and myocardial oxygen consumption were all increased with administration of delta opioid agonists and decreased below baseline with delta opioid antagonists. Functional recovery of left ventricular developed pressure was improved with opioids (control hearts: 36 +/- 3 mm Hg vs hearts with cardioplegia plus delta opioid agonist: 65 +/- 5 mm Hg, P <.01) and inhibited with antagonists (control hearts: 36 +/- 3 mm Hg vs hearts with cardioplegia plus delta opioid antagonist: 17 +/- 5 mm Hg, P <.05), and true to form, the protective opioid effect was negated when combined with an antagonist (control hearts: 36 +/- 3 mm Hg vs hearts with cardioplegia plus delta opioid agonist and delta opioid antagonist: 42 +/- 4 mm Hg, P = not significant). CONCLUSIONS: This study demonstrates that cardiac tolerance to ischemia may be mediated by delta opioids.

Designing clinical trials on energy healing: ancient art encounters medical science.

Demand for energy healing is growing rapidly in the United States. Until recently, however, few clinical trials have been conducted to investigate its clinical efficacy, risks, and cost-effectiveness. This article discusses principles underlying the research design of clinical trials on energy healing, based on the experience of an interdisciplinary team conducting a large-sample clinical study on qigong funded by the National Institutes of Health. The first part overviews the background and contemporary practice of qigong therapy. The second addresses some difficulties and unique issues to be considered in designing a clinical trial on energy healing. These issues include research emphasis on outcome versus mechanism, randomization, control, expectations/placebo effects, staff and practitioner bias/conflict of interest, patients' belief, selection bias, intent-to-treat analysis, ethics, informed consent, sample size, and outcome report. The ultimate goal is to promote more scholarly and clinical discussion on the evaluation of energy healing.

Surgical alternatives for heart failure.

Heart failure is one of the leading causes of hospitalization in the United States. Congestive heart failure is a chronic, progressive disease and its central element is remodeling of the cardiac chamber associated with ventricular dilation. Secondary mitral regurgitation is a complication of end-stage cardiomyopathy and is associated with poor prognosis. Historically, these patients were not considered operative candidates because of their high morbidity and mortality. Heart transplantation is now considered standard treatment for select patients with end-stage heart disease; however, it is applicable only to a small number of patients. In an effort to address this problem, newer and alternative surgical approaches are evolving, including mitral valve annuloplasty, the Batista procedure, and other left ventricular shape changing technologies. Using these operative techniques to alter the shape of the left ventricle, in combination with optimal medical management for heart failure, improves survival, and patients may avoid or postpone transplantation.

Surgical treatment of atrial fibrillation using radiofrequency energy.

BACKGROUND: The Maze III procedure for atrial fibrillation (AF) is effective but has not been used widely due to its complexity, bleeding risk, and added operative time. Surgical radiofrequency ablation may simplify the procedure and make intraoperative correction of AF more accessible and widely performed. METHODS: Endocardial pulmonary venous isolation was performed on 48 patients with AF undergoing concurrent operation using temperature-controlled radiofrequency energy delivered through a hand-held flexible probe. Additional right-sided lesions were made at the surgeon's discretion. RESULTS: Forty-two patients were appropriate for analysis (6 died). These patients had an AF duration of 4.8 +/- 6.4 years. At a mean follow-up of 138 +/- 96 days, 34 patients were in sinus rhythm. We were unable to demonstrate a difference in outcome based on AF duration, left atrial size, or addition of right-sided lesions. CONCLUSIONS: Radiofrequency atrial ablation was effective in 81% of patients with AF at restoring sinus rhythm at an average follow-up of 4 months. This procedure is simple to perform and should broaden the number of patients that receive an AF treatment procedure during concurrent cardiac operation.

Mitral valve reconstruction in the patient with heart failure.

Secondary MR is a complication of end-stage cardiomyopathy and is associated with a poor prognosis and is due to progressive mitral annular dilation and alteration in LV geometry. A vicious cycle of continuing volume overload, ventricular dilation, progression of annular dilation, increased LV wall tension and worsening MR and CHF occur. The mainstay of medical therapy is diuretics and afterload reduction, and is associated with poor long-term survival in these patients with CHF and MR. However, surgical intervention in the form of undersized, 'overcorrecting' mitral valve repair has shown great promise and is an area of ongoing investigation.

Eccentric mitral regurgitation jets among patients having sustained inferior wall myocardial infarction.

A strong association has been recognized between partial or complete mitral leaflet flail and highly eccentric mitral regurgitation jets. In light of anecdotal observation of eccentric mitral regurgitation apparently due to geometric and functional changes accompanying inferior wall myocardial infarction, the present study was performed to systematically study the eccentricity of mitral regurgitation jets complicating nonacute inferior wall myocardial infarction. Forty-eight consecutive patients with evidence of prior isolated inferior wall myocardial infarction and at least moderate mitral regurgitation but without other valvular, annular, chordal, or ventricular pathology potentially contributory to mitral regurgitation were studied. Mitral regurgitation jets were characterized with respect to eccentricity and anterior versus posterior direction. Regurgitant jet and mitral leaflet position were quantified relative to the mitral annulus. Five of 48 patients (10.4%) had eccentric jets, of which four were directed posterior and one anterior. Although not reaching statistical significance, patients with eccentric jets tended to have somewhat smaller left atrial size (41.2 +/- 7.8 vs 47.2 +/- 9.3 mm, P = 0.17) and left ventricular size (51.5 +/- 3.4 vs 55.1 +/- 7.8 mm, P = 0.13), and higher left ventricular ejection fraction (0.52 +/- 0.11 vs 0.46 +/- 0.09, P = 0.25) compared with patients with noneccentric jets. Leaflet position relative to the mitral annulus was significantly different among patients with eccentric compared with noneccentric posterior jets (54 +/- 10 degrees vs 33 +/- 11 degrees, P = 0.02), implying greater leaflet restriction toward the left ventricular apex. In conclusion, approximately one in 10 patients with isolated inferior wall myocardial infarction and at least moderate mitral regurgitation was found to have marked eccentricity of the regurgitant jet. Leaflet position was more apically displaced among patients with eccentric jets, suggesting greater leaflet restriction in systole. The finding of a highly eccentric posterior mitral regurgitation jet can be due to inferior wall myocardial infarction with posterior leaflet restriction as well as partial or complete anterior mitral leaflet flail.

Effect of postoperative atrial fibrillation on length of stay after cardiac surgery (The Postoperative Atrial Fibrillation in Cardiac Surgery study [PACS(2)].

Atrial fibrillation (AF) after cardiac surgery is thought to increase length of stay (LOS). A clinical pathway focused on the management of postoperative AF, including prophylaxis with beta blockers, was implemented to assess the effect of AF on LOS after cardiac surgery. Data were obtained on consecutive cardiac surgery patients in preoperative normal sinus rhythm, no prior history of AF, and no chronic antiarrhythmic therapy from January to May 1995 (control) and November 1996 to June 1997 (pathway). Statistical analysis was performed to assess the effect of postoperative AF on the LOS, clinical outcomes, and cost after cardiac surgery. Despite the clinical pathway, the LOS (7 days for both periods; p = 0.12) and incidence of AF (28.9% vs 28.4%; p = 0.92) remained unchanged. Unadjusted direct costs were 15% higher in the pathway period (p <0.001). Increased rates of beta-blocker therapy had a marginal effect on the incidence of postoperative AF, except in the group who only underwent primary coronary artery bypass graft surgery (31.2% vs 25.3%; p = 0.31). Multivariate analysis revealed that AF contributed only 1 to 1.5 days to the LOS. Thus, this investigation represents the most recent analysis of the effects of postoperative AF on LOS, clinical outcomes, and cost after cardiac surgery. Unlike prior studies, the impact of postoperative AF is less prominent in the current era of cardiac surgical care regardless of the presence of a clinical pathway addressing AF.

Reoperation for Freestyle stentless aortic valves.

Ten patients who initially underwent Freestyle stentless aortic valve implantation required reoperation. The goal of this study was to describe the reoperative techniques used and to review the outcomes of reoperation in patients with Freestyle stentless aortic valves. From September 1992 to April 2001, at the University of Michigan, a total of 552 Freestyle stentless aortic valves were implanted, and in 10 (1.8%) of these patients (7 men, 3 women) a reoperation was required. The mean age at the time of the initial implantation was 53.5 +/- 14.1 years. Implantation techniques included both modified inclusion root (7) and inclusion root (3). Reasons for reoperation included endocarditis (7), aortic aneurysm (1), valve dehiscence (1), and subvalvular outflow tract obstruction (1). Eight patients underwent homograft reimplantations and in 2 Freestyle reimplantations. In all cases, the previous aortotomy was re-entered, the pseudoendothelial layer over the distal suture line of the noncoronary sinus was incised and continued into the other 2 sinuses. Utilizing a ganglion knife, the Freestyle valve was freed from the native aortic tissue to the proximal suture line. The Dacron sewing ring was then separated using sharp dissection and the lower suture line excised. No calcification was noted in any case. The mean time interval between the first and second operative procedure was 13.4 +/- 21.5 months. There were no operative deaths and only one late death. Mean long-term follow-up was 43 +/- 29 months. Reoperation on a Freestyle stentless aortic valve, when necessary, can be accomplished without increased operative risk and with excellent survival.

An immortalized myocyte cell line, HL-1, expresses a functional delta -opioid receptor.

The present study characterizes opioid receptors in an immortalized myocyte cell line, HL-1. Displacement of [(3)H]bremazocine by selective ligands for the mu (mu), delta (delta), and kappa (kappa) receptors revealed that only the delta -selective ligands could fully displace specific [(3)H]bremazocine binding, indicating the presence of only the delta -receptor in these cells. Saturation binding studies with the delta -antagonist naltrindole afforded a B(max)of 32 fmols/mg protein and a K(D)value for [(3)H]naltrindole of 0.46 n M. The binding affinities of various delta ligands for the receptor in HL-1 cell membranes obtained from competition binding assays were similar to those obtained using membranes from a neuroblastomaxglioma cell line, NG108-15. Finally, various delta -agonists were found to stimulate the binding of [(35)S]GTP gamma S, confirming coupling of the cardiac delta -receptor to G-protein. DADLE (D-Ala-D-Leu-enkephalin) was found to be the most efficacious in this assay, stimulating the binding of [(35)S]GTP gamma S to 27% above basal level. The above results indicate that the HL-1 cell line contains a functionally coupled delta -opioid receptor and therefore provides an in vitro model by which to study the direct effects of opioids on cardiac opioid receptors.

Mitral valve repair in heart failure.

Mitral regurgitation (MR) is a frequent complication of end-stage heart failure. Historically, these patients were either managed medically or with mitral valve replacement, both associated with poor outcomes. Mitral valve repair via an 'undersized' annuloplasty repair is safe and effectively corrects MR in heart-failure patients. All of the observed changes contribute to reverse remodeling and restoration of the normal left-ventricular geometric relationship. Mitral valve repair offers a new strategy for patients with MR and end-stage heart failure.

Surgical approaches to dilated cardiomyopathy.

Heart failure is one of the leading causes of hospitalization in the United States today. Congestive heart failure is a chronic progressive disease with the common central element being the remodeling of the cardiac chamber associated with ventricular dilation. Secondary mitral regurgitation is a complication of end-stage cardiomyopathy and is associated with a poor prognosis. Historically, these patients were not considered operative candidates due to the high morbidity and mortality in this patient population. Heart transplantation is now considered the standard of treatment for select patients with end-stage heart disease, however, it is only applicable to a small number of patients. In an effort to address this problem, newer and alternative surgical approaches are evolving, including mitral valve annuloplasty, the Batista myoplasty, and cardiomyoplasty. When these operative techniques that alter the shape of the left ventricle are utilized, in combination with optimal medical management for heart failure, survival is improved and patients can avoid or postpone transplantation.

Current status of mitral valve reconstruction in patients with dilated cardiomyopathy.

Congestive heart failure (CHF) is one of the leading causes of hospitalization in the United States today and its incidence is increasing. Despite improvements with medical management approximately 50% of patients with CHF die within 3 years of presentation. Heart transplantation is now considered standard treatment for selected patients with severe CHF and end-stage heart disease; however, it is only applicable to a small percentage of patients. In an effort to solve this problem medical and surgical strategies are rapidly expanding and evolving. Mitral valve reconstruction represents an alternative surgical strategy in patients with dilated cardiomyopathy that will allow for preservation of the limited number of donor organs for those patients who have no other surgical or medical alternatives.

Regulatory role of Th-2 cytokines, IL-10 and IL-4, in cardiac allograft rejection.

The host response to alloantigen results in T- and B-cell activation, upregulation of Class II MHC antigens, and cytokine production by Th-1 cells, resulting in generation of IL-2 and IFN gamma. Th-2 cell responses produce IL-4 and IL-10 which may shift the immune response from the Th-1 pathway to Th-2 responses, favoring Ig production. This could imply that Th-2-related cytokines protect allografts. In the following studies, employing cardiac heterotopic allografts in rats (Brown Norway into Lewis), we investigated regulatory roles of Th-2-related cytokines IL-4 and IL-10. Two strategies were used in animals receiving allografts: antibody-induced blocking of endogenous IL-4 or IL-10 and exogenous administration of either interleukin. Antibody to IL-4 failed to alter the rejection time, whereas anti-IL-10 greatly accelerated the rejection process. Northern blot analysis of RNA from allografted hearts revealed mRNA for both IL-4 and IL-10, while immunostaining showed strong staining for IL-10 and very weak staining for IL-4. Exogenous administration of either IL-4 or 10 caused prolongation of allograft rejection times. These findings suggest that in rat cardiac allografts intrinsic IL-10 functions to attenuate the rejection process.

Calcification and degeneration following mitral valve reconstruction in patients requiring chronic dialysis.

BACKGROUND AND AIM OF THE STUDY: Abnormal calcium homeostasis in patients with end-stage renal failure results in dystrophic calcification; this limits the use of heterograft tissue valve prostheses in patients on chronic dialysis. Mitral valve reconstruction offers advantages over mitral replacement in many patients without renal failure, and offers theoretical advantages in patients requiring dialysis. This study was performed to determine the outcome of mitral valve reconstruction in patients with renal failure requiring chronic dialysis. METHODS: Ten patients with end-stage renal failure and on chronic dialysis who underwent mitral valve repair were identified retrospectively and followed for clinical and echocardiographic outcome. All patients had good results immediately following surgical valve mitral repair, with no more than mild mitral regurgitation and low transmitral gradients on intraoperative transesophageal echocardiography. RESULTS: Clinical and echocardiographic follow up was available for eight patients at an average of 2.3 +/- 1.4 years after surgery. Despite there being no significant valve calcification at the time of surgery, visible mitral leaflet calcification was evident in seven of these patients, and the transmitral gradient for the group was significantly increased (from 4.8 +/- 1.7 mmHg to 8.3 +/- 3.9 mmHg, p = 0.04). Two patients required reoperation for failed mitral repair; one at six months due to chordal rupture, and one at 15 months due to mitral calcification with stenosis. CONCLUSION: Despite good early surgical results, there was accelerated calcification of the repaired mitral valve, a rapid increase in postoperative mitral gradients, and a high incidence of failure of the reconstruction. Additional prospective studies are required to evaluate the optimal intervention for patients with end-stage renal failure who require mitral valve surgery.

Bedside chest radiography as part of a postcardiac surgery critical care pathway: a means of decreasing utilization without adverse clinical impact.

OBJECTIVE: To evaluate the use of bedside chest radiography and patient outcome before and after implementation of a cardiac surgery critical care pathway that included guidelines for bedside radiography. DESIGN: A cohort observational study. SETTING: A university hospital in the midwest. PATIENTS: Three groups, of 100 patients each, undergoing cardiac surgery in 1990, 1991, and 1995. INTERVENTION: Introduction of a critical care pathway. MEASUREMENTS: Medical records were retrospectively reviewed in three groups of 100 patients each: before the introduction of the critical care pathway; 2 months after introduction of the pathway in 1991; and 4 yrs after introduction in 1995. Data were analyzed to determine operative risk for each group. Subsequent analyses determined bedside radiography use, total length of hospital stay, and patient outcome (mortality rate, complications requiring intervention, and reoperation) during hospitalization and at outpatient follow-up 15-30 days postdischarge. RESULTS: Total length of hospital stay was shorter for the 1995 group (7.6+/-6.6 days) compared with other groups (prepathway, 11.1+/-10.3 days; 1991 postpathway, 10.2+/-9.6 days; p<.05). The mean numbers of radiographs per patient were as follows: prepathway, 5.1; 1991 postpathway, 5.2; and 1995 postpathway, 3.3. The mean number of radiographs in the 1995 group was significantly lower (p = .02). More patients had the proposed number of two bedside radiographs described in the pathway in the 1995 group compared with the other groups (prepathway, p<.0001; the two-month postpathway group, p = .01). Twenty-three malpositioned catheters/tubes were found in the prepathway and 1991 groups compared with 11 in the 1995 group (p = .02). No statistically significant difference was found in inpatient complications (mediastinal bleeding, pneumothoraces, and pleural effusions), postdischarge complications, reoperations, or mortality rate. CONCLUSION: Introduction of a critical care pathway can decrease the use of bedside radiography without adversely affecting near-term patient outcomes.

Management strategies for high-risk cardiac surgery: improving outcomes in patients with heart failure.

BACKGROUND: Surgical heart failure management is the fastest growing aspect of cardio-vascular surgery. Advances in cardiac surgical techniques have changed the number and types of operations permitted physicians and thus broadened the complexity of patients recommended for operation. METHODS: Surgeons, anesthesiologists and cardiologists face hemodynamic and patho-physiological challenges that can be optimally overcome only by modifying treatment strategies. Because many treatment standards are still evolving in this rapidly advancing field, a team of cardiovascular surgeons and anesthesiologists convened to share clinical experience and impressions and discuss practical issues related to high-risk patients undergoing heart surgery. RESULTS: Heart failure pathophysiology, surgical heart failure management, including mitral reconstruction and left ventricular remodeling, cardiopulmonary bypass weaning, inotropic support, transesophageal echocardiography and acute cardiovascular collapse after cardiac surgery are discussed. CONCLUSION: This article is intended to guide clinicians to improve patient care and outcomes in this special population by providing specific guidance on the appropriate use of inotropic and mechanical support in patients undergoing high-risk procedures using innovative techniques.

RSR13, a synthetic allosteric modifier of hemoglobin, improves myocardial recovery following hypothermic cardiopulmonary bypass.

BACKGROUND: During hypothermic blood cardioplegia, oxygen delivery to myocytes is minimal with ineffective anaerobic metabolism predominating. RSR13, 2-[4-[[(3,5-dimethylanilino) carbonyl]methyl]phenoxy]-2-methylpropionic acid, a synthetic allosteric modifier of hemoglobin (Hb), increases release of oxygen from Hb, increasing oxygen availability to hypoxic tissues, and reverses the hypothermia-dependent increase in Hb oxygen affinity. We studied recovery of myocardial mechanical and metabolic function and examined myocardial morphology after cardioplegia, comparing RSR13 (1.75 mmol/L)-supplemented blood (RSR13-BC) to standard blood cardioplegia (BC). METHODS AND RESULTS: Twelve dogs underwent 15 minutes of 37 degrees C global ischemia on cardiopulmonary bypass, followed by 75 minutes of hypothermic cardioplegia (13 degrees C) with either BC (n=6) or RSR13-BC (n=6). There were no differences in baseline function between groups. Cardiac function was assessed after 30 minutes of 37 degrees C reperfusion (BC versus RSR13-BC, respectively) by measuring: % return to normal sinus rhythm (0/100%), % of baseline+dP/dt (33.7+/-1.7/76.3+/-1.9), % of baseline-dP/dt (26.6+/-2.0/81.1+/-1.6), stroke volume (3.5+/-0.5/7.1+/-0.9 mL), cardiac output (340+/-20/880+/-40.3 mL/min), and LVEDP (11.3+/-2.2/0. 3+/-2.9 mm Hg). Postischemic oxidative and metabolic parameters including myocardial lactate, pyruvate, ATP content, and percent water content also were determined. Histological analysis demonstrated preservation of endothelial and myocyte morphology in hearts receiving RSR13-BC compared with BC. CONCLUSIONS: These results indicate that in the setting of hypothermic cardiopulmonary bypass, RSR13 improves recovery of myocardial mechanical and metabolic function compared with standard hypothermic BC. Findings from this study suggest that RSR13-BC, by decreasing hemoglobin oxygen affinity, improves oxidative metabolism and preserves cellular morphology, resulting in significantly improved contractile recovery on reperfusion.