Establishment of a new pig model for auxiliary partial orthotopic liver transplantation

AIM: To establish a new pig model for auxiliary partial orthotopic liver transplantation (APOLT).METHODS: The liver of the donor was removed from its body. The left lobe of the liver was resected in vivo and the right lobe was used as a graft. After the left lateral lobe of the recipient was resected, end-to-side anastomoses of suprahepatic inferior vena cava and portal vein were performed between the donor and recipient livers,respectively. End-to-end anastomoses were made between hepatic artery of graft and splenic artery of the host.Outside drainage was placed in donor common bile duct.RESULTS: Models of APOLT were established in 5 pigs with a success rate of 80%. Color ultrasound examination showed an increase of blood flow of graft on 5th d compared to the first day after operation. When animals were killed on the 5th d after operation, thrombosis of hepatic vein (HV) and portal vein (PV) were not found. Histopathological examination of liver samples revealed evidence of damage with mild steatosis and sporadic necrotic hepatocytes and focal hepatic lobules structure disorganized in graft. Infiltration of inflammatory cells was mild in portal or central vein area. Hematologic laboratory values and blood chemical findings revealed that compared with group A (before transplantation), mean arterial pressure (MAP), central venous pressure (CVP), buffer base (BB), standard bicarbonate (SB) and K+ in group B (after portal vein was clamped) decreased (P＜0.01). After reperfusion of the graft, MAP, CVP and K+ restored gradually.CONCLUSION: Significant decrease of congestion in portal vein and shortened blocking time were obtained because of the application of in vitro veno-venous bypass during complete vascular clamping. This new procedure,with such advantages as simple vessel processing, quality anastomosis, less postoperative hemorrhage and higher success rate, effectively prevents ischemia reperfusion injury of the host liver and deserves to be spread.

Feasible management of median arcuate ligament syndrome in orthotopic liver transplantation recipients

BACKGROUND In orthotopic liver transplantation(OLT)recipients,median arcuate ligament syndrome(MALS)is considered a risk factor for hepatic arterial thrombosis(HAT),which is dreadful for OLT recipients.Different alternative surgical procedures have been proposed to overcome the impact of MALS on transplantation,but clinical evidence is still scarce.AIM To evaluate the feasible surgical management of MALS to reduce complications in OLT patients.METHODS Data for 288 consecutive patients who underwent OLT at The First Hospital of Jilin University between January 2017 and July 2020 were retrospectively reviewed.The surgical management of median arcuate ligament(MAL)and modifications to the arterial anastomosis were recorded.The perioperative and long-term prognosis of MALS recipients were noted.Detailed preoperative and postoperative data of patients were analyzed in a descriptive manner.RESULTS Eight patients with MALS were included in this study.The first patient with MALS received no intervention during the primary surgery and developed postoperative HAT.Salvage liver transplantation with MAL division was successfully performed.Gastroduodenal artery(GDA)preservation with splenic artery ligation was performed on three patients,only GDA preservation was performed on two patients,and no intervention was performed on two patients.No patient developed HAT after surgery and postoperative recovery was satisfactory.CONCLUSION The preservation of collateral circulation between the superior mesenteric artery and celiac trunk via the GDA with or without splenic artery ligation is a safe and feasible alternative to MAL division.

Transfusion and coagulation management in liver transplantation

There is wide variation in the management of coagulation and blood transfusion practice in liver transplantation. The use of blood products intraoperatively is declining and transfusion free transplantations take place ever more frequently. Allogenic blood products have been shown to increase morbidity and mortality. Primary haemostasis, coagulation and fibrinolysis are altered by liver disease. This, combined with intraoperative disturbances of coagulation, increases the risk of bleeding. Meanwhile, the rebalancing of coagulation homeostasis can put patients at risk of hypercoagulability and thrombosis. The application of the principles of patient blood management to transplantation can reduce the risk of transfusion. This includes: preoperative recognition and treatment of anaemia, reduction of perioperative blood loss and the use of restrictive haemoglobin based transfusion triggers. The use of point of care coagulation monitoring using whole blood viscoelastic testing provides a picture of the complete coagulation process by which to guide and direct coagulation management. Pharmacological methods to reduce blood loss include the use of anti-fibrinolytic drugs to reduce fibrinolysis, and rarely, the use of recombinant factor VIIa. Factor concentrates are increasingly used; fibrinogen concentrates to improve clot strength and stability, and prothrombin complex concentrates to improve thrombin generation. Non-pharmacological methods to reduce blood loss include surgical utilisation of the piggyback technique and maintenance of a low central venous pressure. The use of intraoperative cell salvage and normovolaemic haemodilution reduces allogenic blood transfusion. Further research into methods of decreasing blood loss and alternatives to blood transfusion remains necessary to continue to improve outcomes after transplantation.

Dynamic changes of cellular immune function and individualized adjustments of immunosup-pressant for the management of severe infection after liver transplantation

Objective To explore the dynamic changes of the cellular immune function in severe infection after liver transplantation,and to guide the individualized immunology adjustment. Methods 378 cases of liver transplantation were analyzed retrospectively. Seventy - four cases ( infection group) suffered serious infection,including 54 cases cured ( cure group) ,20 cases died (

Advances in endoscopic management of biliary complications after living donor liver transplantation: Comprehensive review of the literature

Apart from noticeable improvements in surgical techniques and immunosuppressive agents, biliary complications remain the major causes of morbidity and mortality after living donor liver transplantation(LDLT). Bile leakage and stricture are the predominant complications. The reported incidence of biliary complications is 15%-40%, and these are known to occur more frequently in living donors than in deceased donors. Despite the absence of a confirmed therapeutic algorithm, many approaches have been used for treatment, including surgical, endoscopic, and percutaneous transhepatic techniques. In recent years, nonsurgical approaches have largely replaced reoperation. Among these, the endoscopic approach is currently the preferred initial treatment for patients who undergo duct-to-duct biliary reconstruction. Previously, endoscopic management was achieved most optimally through balloon dilatation and single or multiple stents placement. Recently, there have been significant developments in endoscopic devices, such as novel biliary stents, as well as advances in endoscopic technologies, including deep enteroscopy, the rendezvous technique, magnetic compression anastomosis, and direct cholangioscopy. These developments have resulted in almost all patients being managed by the endoscopic approach. Multiple recent publications suggest superior long-term results, with overall success rates ranging from 58% to 75%. This article summarizes the advances in endoscopic management of patients with biliary complications after LDLT.

Effect of cardiac output-guided hemodynamic management on acute lung injury in pediatric living donor liver transplantation

BACKGROUND Acute lung injury(ALI)after liver transplantation(LT)may lead to acute respiratory distress syndrome,which is associated with adverse postoperative outcomes,such as prolonged hospital stay,high morbidity,and mortality.Therefore,it is vital to maintain hemodynamic stability and optimize fluid management.However,few studies have reported cardiac output-guided(CO-G)management in pediatric LT.AIM To investigate the effect of CO-G hemodynamic management on early postoperative ALI and hemodynamic stability during pediatric living donor LT.METHODS A total of 130 pediatric patients scheduled for elective living donor LT were enrolled as study participants and were assigned to the control group(65 cases)and CO-G group(65 cases).In the CO-G group,CO was considered the target for hemodynamic management.In the control group,hemodynamic management was based on usual perioperative care guided by central venous pressure,continuous invasive arterial pressure,urinary volume,etc.The primary outcome was early postoperative ALI.Secondary outcomes included other early postoperative pulmonary complications,readmission to the intense care unit(ICU)for pulmonary complications,ICU stay,hospital stay,and in-hospital mortality.RESULTS The incidence of early postoperative ALI was 27.7%in the CO-G group,which was significantly lower than that in the control group(44.6%)(P<0.05).During the surgery,the incidence of postreperfusion syndrome was lower in the CO-G group(P<0.05).The level of intraoperative positive fluid transfusions was lower and the rate of dobutamine use before portal vein opening was higher,while the usage and dosage of epinephrine during portal vein opening and vasoactive inotropic score after portal vein opening were lower in the CO-G group(P<0.05).Compared to the control group,serum inflammatory factors(interleukin-6 and tumor necrosis factor-α),cardiac troponin I,and N-terminal pro-brain natriuretic peptide were lower in the CO-G group after the operation(P<0.05).CONCLUSION CO-G hemodynamic management in pediatric living-donor LT decreases the incidence of early postoperative ALI due to hemodynamic stability through optimized fluid management and appropriate administration of vasopressors and inotropes.

Acute respiratory distress syndrome after liver transplantation: etiology, prevention and management

Objective: To study the etiology, prevention andmanagement of acute respiratory distress syndrome(ARDS) after liver transplantation.Methods: The clinical data of 104 patients with end-stage liver diseases who had had liver transplanta-tions were retrospectively reviewed.Results: Seventeen patients (16.3%, 17/104) alto-gether were diagnosed as having ARDS after livertransplantation. Ten of them developed ARDS within24 hours, of whom 1 died during the operation, and7 developed ARDS 3 or 4 days after they were extu-bated and when methylprednisolone was tapered.Fourteen of the 17 ARDS patients (14/17) werefound to have overloaded crystalloid infusion, mas-sive transfusion of blood or blood products such asplasma, platelets, in addition to a prolonged surgicaltime secondary to serious bleeding during the dis-eased liver removal without evidence of active infec-tion. One was found to have serious systemic infec-tion and operatively disseminated intravascular coag-ulation. Four of the recipients developed ARDS sud-denly when intravenous cyclosporine was given on the3rd day after operation. One patient of the 4 had allof the aforementioned conditions. Two patients suf-fered from gastric aspiration. Five (30%, 5/17) ofthem survived ARDS with the combined treatmentconsisting of positive end-expiratory pressure me-chanicai ventilation suctioning as much edema fluidor sputum as possible, administration of diuretics,bolus of corticosteroids, and culture-based antibiot-ics. Hemeodialysis was indicated for patients with ol-iguric renal failure.Conclusions: ARDS is a serious multifactoral compli-cation after liver transplantation with a high mortali-ty and fatality. The most likely cause is fluid over-load from crystalloid liquid infusion or massive trans-fusion. The other predisposing or contributing fac-tors include sepsis, Ⅳ use of cyclosporine, fast ta-pering of corticosteroids, and gastric aspiration.Other factors such as transfusion-related acute lunginjury (TRALI), and reperfusion syndrome of thenewly implanted liver may also contribute. Thoughthe treatment should primarily be supportive in na-ture, it is helpful to understand the predisposing andcontributing factors and to aid in prevention, man-agement and treatment.

Usefulness of Monitoring Stroke Volume Variations for Fluid Management During Pediatric Living-Donor Liver Transplantation

Purpose: Central venous pressure (CVP) is considered to be unsuitable as preload parameter. Stroke volume variation (SVV) has recently been reported to be effective as a preload and fluid responsiveness parameter, and its usefulness for fluid management during living-donor liver transplantation (LDLT). However, use of SVV has not been reported in children. Our aim is to evaluate the use of SVV as a target parameter of circulating blood volume during pediatric LDLT. Methods: This retrospective study was conducted in 40 consecutive patients aged between 5 and 109 months who underwent elective LDLT. Twenty patients underwent LDLT without FloTrac? (C group) and the rest patients underwent LDLT with the FloTrac? monitoring (F group). As a fluid management target, CVP was maintained at 10 mmHg in the C group and SVV at 10% in the F group. We compared MAP and CVP at the times of the greatest decrease within 5 minutes after reperfusion. Results: MAP after reperfusion was significantly decreased in both groups (P < 0.01), with the magnitude of decrease significantly greater in the C group compared with the F group (P = 0.02). MAP before and after reperfusion did not significantly differ between the groups. After reperfusion, CVP was nearly the same in both groups, with that in the C group slightly decreased and nearly no change in the F group. SVV after reperfusion was significantly increased (P < 0.001). Conclusion: When used as a target parameter for fluid management during pediatric LDLT, hemodynamic changes was less when SVV was used as the parameter of circulating blood volume.

Key challenges of post-liver transplant weight management

Liver transplantation serves as a life-saving intervention for patients with endstage liver disease,yet long-term survival remains a challenge.Post-liver transplant obesity seems to have a significant contribution to this challenge and it emerges as a significant risk factor for graft steatosis,metabolic syndrome and denovo malignancy development.This review synthesizes current literature on prevalence,risk factors and management strategies for post-liver transplant obesity,emphasizing its impact on graft and patient survival.Literature review consultation was conducted in Medline/PubMed,SciELO and EMBASE,with the combination of the following keywords:Weight management,liver transplantation,immunosuppressive therapy,lifestyle interventions,bariatric surgery.Immunosuppressive therapy has a significant influence on long-term survival of liver transplant patients,yet it seems to have lesser effect on post-transplant obesity development than previously thought.However,it significantly contributes to the development of other components of metabolic syndrome.Key predisposing factors for post-transplant obesity development encompass elevated recipient and donor body mass index,a history of alcoholic liver disease,hepatocellular carcinoma,male gender,the absence of cellular rejection and the marital status of the recipient.Tailored immunosuppressive regimens,pharmacotherapy,lifestyle interventions and bariatric surgery represent key components in mitigating post-transplant obesity and improving long-term survival and quality of life in this group of patients.Timely identification and intervention thus hold paramount importance.Further research is warranted to refine optimal management strategies and enhance outcomes in this patient population.

Management of bacterial and fungal infections in end stage liver disease and liver transplantation: Current options and future directions

Patients with liver cirrhosis are susceptible to infections due to various mechanisms, including abnormalities of humoral and cell-mediated immunity and occurrence of bacterial translocation from the intestine. Bacterial infections are common and represent a reason for progression to liver failure and increased mortality. Fungal infections, mainly caused by Candida spp., are often associated to delayed diagnosis and high mortality rates. High level of suspicion along with prompt diagnosis and treatment of infections are warranted. Bacterial and fungal infections negatively affect the outcomes of liver transplant candidates and recipients, causing disease progression among patients on the waiting list and increasing mortality, especially in the early posttransplant period. Abdominal, biliary tract, and bloodstream infections caused by Gram-negative bacteria [e.g., Enterobacteriaceae and Pseudomonas aeruginosa(P. aeruginosa)] and Staphylococcus spp. are commonly encountered in liver transplant recipients. Due to frequent exposure to broad-spectrum antibiotics, invasive procedures, and prolonged hospitalizations, these patients are especially at risk of developing infections caused by multidrug resistant bacteria. The increase in antimicrobial resistance hampers the choice of an adequate empiric therapy and warrants the knowledge of the local microbial epidemiology and the implementation of infection control measures. The main characteristics and the management of bacterial and fungal infections in patients with liver cirrhosis and liver transplant recipients are presented.

Liver transplantation as a management of hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is one of the most common cancers worldwide and has a poor prognosis if untreated. It is ranked the third among the causes of cancer-related death. There are multiple etiologic factors that can lead to HCC. Screening for early HCC is challenging due to the lack of well specific biomarkers. However,early diagnosis through successful screening is very important to provide cure rate. Liver transplantation(LT) did not gain wide acceptance until the mid-1980 s,after the effective immunosuppression withcyclosporine became available. Orthotopic LT is the best therapeutic option for early,unresectable HCC. It is limited by both,graft shortage and the need for appropriate patient selection. It provides both,the removal of tumor and the remaining cirrhotic liver. In Milan,a prospective cohort study defined restrictive selection criteria known as Milan criteria(MC) that led to superior survival for transplant patients in comparison with any other previous experience with transplantation or other options for HCC. When transplantation occurs within the established MC,the outcomes are similar to those for nonmalignant liver disease after transplantation. The shortage of organs from deceased donors has led to the problems of long waiting times and dropouts. This has led to the adoption of extended criteria by many centers. Several measures have been taken to solve these problems including prioritization of patients with HCC,use of pretransplant adjuvant treatment,and living donor LT.

Endoscopic management of biliary strictures post-liver transplantation

Biliary complications play a significant role in morbidity of liver transplant recipients. Biliary strictures occur between 10%-25% of patients with a higher incidence in living donor recipients compared to deceased donors. Strictures can be classified as either anastomotic or non-anastomotic and may be related to ischemic events. Endoscopic management of biliary strictures in the posttransplant setting has become the preferred initial approach due to adequate rates of resolution of anastomotic and non-anastomotic strictures(NAS).However, several factors may increase complexity of the endoscopic approach including surgical anatomy, location, number, and severity of bile duct strictures.Many endoscopic tools are available, however, the approach to management of anastomotic and NAS has not been standardized. Multi-disciplinary techniques may be necessary to achieve optimal outcomes in select patients. We will review the risk factors associated with the development of bile duct strictures in the posttransplant setting along with the efficacy and complications of current endoscopic approaches available for the management of bile duct strictures.

Liver Transplantation in the Elderly: An Evidence Based Review on Patient Outcome, Selection and Management Strategies

There has been a steady increase in the age of potential recipients for liver transplantation over the past two decades. While patients older than age 60, on average, did not do as well as younger patients after transplantation, optimizing patient selection could minimize this discrepancy. It is appropriate to evaluate functional elderly patients without significant medical comorbidities for liver transplantation for the same indications as younger patients. For elderly patients with hepa-tocellular carcinoma and otherwise compensated liver disease, however, it is important to consider alternative treatment modalities that could provide similar survival. Elderly candidates who became too ill should be compassionately counseled away from transplantation based on existing prognostic models that can accurately predict post-transplant mortality. Finally, elderly transplant candidates and recipients may benefit from a more tailored management strategy in terms of donor risk matching and less immunosuppression.

Analysis of critical issues in case management for liver retransplantation

We report a case of a patient who underwent successful combined liver-kidney transplant after two prior liver transplantations. The topic of liver retransplantation is very controversial. Given the critical organ shortage, the question arises as to whether hepatic retransplantation should be offered liberally despite its greater cost, and inevitable denial of access to primary transplantation for the other patients on the waiting list. We suggest that careful selection of the retransplant candidates will improve outcomes and allow rational use of the limited organ supply. Analysis of the available literature allows us to identify the main predictors of morbidity and mortality for this patient population. It also enables development of a detailed plan for perioperative management. The role of transesophageal echocardiography (TEE) as a monitor in the complex liver transplant cases is also discussed. Our report is a significant contribution to the very limited data available on the subject of multiple liver retransplants.

Details determining the success in establishing a mouse orthotopic liver transplantation model

Liver transplantation(LT)is currently the only effective treatment option for endstage liver disease.The importance of animal models in transplantation is widely recognized among researchers.Because of the well-characterized mouse genome and the greater diversity and availability of both genetically modified animals and research reagents,mouse orthotopic LT(MOLT)has become an ideal model for the investigation of liver biology,tissue injury,regulation of alloimmunity and tolerance induction,and the pathogenesis of specific liver diseases.However,due to its complicated and technically demanding procedure,the model has merely been used by only a few research groups in the world for years.For a new learner,training lasting at least a couple of months or even years is required.Most of the investigators have emphasized the importance of elaborate techniques and dedicated instruments in establishing a MOLT model,but some details are often neglected.The nontechnical details are also significant,especially for researchers who have little experience in mouse microsurgery.Here,we review and summarize the crucial technical and nontechnical details in establishing the model of MOLT based on scientific articles and our experience in six aspects:animal selection,anesthesia,perioperative management,organ procurement,back-table preparation,and implantation surgery.We aim to enable research groups to shorten the learning curve and implement the mouse LT procedure with high technical success.

Experiences relating to management of biliary tract complications following liver transplantation in 96 cases

To investigate best diagnosing methods and therapy for patients with biliary tract complications after liver transplantation and analyze related factors Methods A review was made of data collected from 96 patients, and confirmed by retrospective case notes examination Results A total of 94 patients (97 grafts) survived more than 2 days after transplantation; of whom, 92 had an end to end biliary anastomosis with a T tube The average follow up was 5 8 months (range: 0 3-10 2 months) Among the 94 patients, eight (8 5%, 8/94) had complications: leakage during T tube removal (2 patients), leakage at an earlier stage (2), simultaneous stricture and leak (2) and just stricture (2) Six patients with biliary tract complications had predisposing factors including hepatic artery stenosis (2 patients, including one hepatic artery stenosis combined with severe rejection, hepatic artery thrombosis (3), and donor recipient bile duct mismatch (1) There was no difference in cold ischemic time With hepatic artery thrombosis and/or stenosis 】50%, five patients were re transplanted; without hepatic artery thrombosis and/or stenosis 【50%, three patients required endoscopic stenting and radiological percutaneous drainage of bile collection with or without balloon dilation All patients survived Conclusions Biliary strictures occur later than leaks after surgery Without hepatic artery thrombosis and/or stricture, there is no need for surgery; with hepatic artery thrombosis and/or stricture 】50%, re transplantation is needed as early as possible

Using old liver grafts for liver transplantation: Where are the limits?

The scarcity of ideal liver grafts for orthotopic liver transplantation (OLT) has led transplant teams to investigate other sources of grafts in order to augment the donor liver pool. One way to get more liver grafts is to use marginal donors, a not well-defined group which includes mainly donors &#x0003e; 60 years, donors with hypernatremia or macrosteatosis &#x0003e; 30%, donors with hepatitis C virus or hepatitis B virus positive serologies, cold ischemia time &#x0003e; 12 h, non-heart-beating donors, and grafts from split-livers or living-related donations. Perhaps the most practical and frequent measure to increase the liver pool, and thus to reduce waiting list mortality, is to use older livers. In the past years the results of OLT with old livers have improved, mainly due to better selection and maintenance of donors, improvements in surgical techniques in donors and recipients, and intra- and post-OLT management. At the present time, sexagenarian livers are generally accepted, but there still exists some controversy regarding the use of septuagenarian and octogenarian liver grafts. The aim of this paper is to briefly review the aging process of the liver and reported experiences using old livers for OLT. Fundamentally, the series of septuagenarian and octogenarian livers will be addressed to see if there is a limit to using these aged grafts.

Role of cardiovascular intervention as a bridge to liver transplantation

End stage liver disease(ESLD) is associated with many specific derangements in cardiovascular physiology, which influence perioperative outcomes and may pro-foundly influence diagnostic and management strate-gies in the preoperative period. This review focuses on evidence-based diagnosis and management of coro-nary, hemodynamic and pulmonary vascular disease in this population with an emphasis on specific strategies that may provide a bridge to transplantation. Specifi-cally, we address the underlying prevalence of cardio-vascular disease states in the ESLD population, and relevant diagnostic criteria thereof. We highlight tradi-tional and non-traditional predictors of cardiovascular outcomes following liver transplant, as well as data to guide risk-factor based diagnostic strategies. We go on to discuss the alterations in cardiovascular physiology which influence positive- and negative-predictive values of standard noninvasive testing modalities in the ESLD population, and review the data regarding the safetyand efficacy of invasive testing in the face of ESLD and its co-morbidities. Finally, based upon the totality of available data, we outline an evidence-based ap-proach for the management of ischemia, heart failure and pulmonary vascular disease in this population. It is our hope that such evidence-driven strategies can be employed to more safely bridge appropriate candidates to liver transplant, and to improve their cardiovascular health and outcomes in the peri-operative period.

Perioperative treatment of liver transplantation

A case of liver transplantation in the department of general surgery of the third affiliated hospital of Inner Mongolia medical school was collected and analyzed on the basis of diagnosis,physical examination and treatment.Liver transplantation was very complicated,accompanied by various postoperative complications.So this paper aims to share experience of perioperative management of liver transplantation among physicians.

Non-alcoholic fatty liver disease and liver transplantation:Outcomes and advances

Non-alcoholic fatty liver disease(NAFLD)is one of the most prevalent causes of chronic liver disease worldwide.In the last decade it has become the third most common indication for liver transplantation in the United States.Increasing prevalence of NAFLD in the general population also poses a risk to organ donation,as allograft steatosis can be associated with non-function of the graft.Post-transplant survival is comparable between NAFLD and non-NAFLD causes of liver disease,although long term outcomes beyond 10 year are lacking.NAFLD can recur in the allograft frequently although thus far post transplant survival has not been impacted.De novo NAFLD can also occur in the allograft of patients transplanted for non-NAFLD liver disease.Predictors for NAFLD post-transplant recurrence include obesity,hyperlipidemia and diabetes as well as steroid dose after liver transplantation.A polymorphism in PNPLA3 that mediates triglyceride hydrolysis and is linked to pre-transplant risk of obesity and NAFLD has also been linked to post transplant NAFLD risk.Although immunosuppression side effects potentiate obesity and the metabolic syndrome,studies of immunosuppression modulation and trials of specific immunosuppression regimens post-transplant are lacking in this patient population.Based on pre-transplant data,sustained weight loss through diet and exercise is the most effective therapy for NAFLD.Other agents occasionally utilized in NAFLD prior to transplantation include vitamin E and insulin-sensitizing agents.Studies of these therapies are lacking in the post-transplant population.A multimodality and multidisciplinary approach to treatment should be utilized in management of post-transplant NAFLD.