North American Biliary Stricture Management Strategies in Children After Liver Transplantation: A Multicenter Analysis From the Society of Pediatric Liver Transplantation (SPLIT) Registry.

Biliary strictures affect 4%-12% of pediatric liver transplantations. Biliary strictures can contribute to graft loss if left untreated; however, there remains no consensus on the best course of treatment. Study objectives included analyses of outcomes associated with biliary stricture management strategies via percutaneous transhepatic cholangiography (PTC), endoscopic retrograde cholangiopancreatography (ERCP), or surgery. We identified pediatric liver transplantation recipients (2011-2016) with biliary strictures from the Society of Pediatric Liver Transplantation (SPLIT) registry and retrieved imaging, procedural, and operative reports from individual centers. Subanalyses were performed to specifically evaluate PTC and ERCP for "optimal biliary outcome" (OBO), defined as graft survival with stricture resolution and without recurrence or surgery. A total of 113 children with a median follow-up of 3.9 years had strictures diagnosed 100 days (interquartile range, 30-290) after liver transplantation; 81% were isolated anastomotic strictures. Stricture resolution was achieved in 92% within 101 days, more frequently with isolated anastomotic strictures (96%). 20% of strictures recurred, more commonly in association with hepatic artery thrombosis (32%). Patient and graft survival at 1 and 3 years were 99% and 98% and 94% and 92%, respectively. In a subgroup analysis of 79 patients with extrahepatic strictures managed by PTC/ERCP, 59% achieved OBO following a median of 4 PTC, and 75% following a median of 3 ERCP (P < 0.001). Among patients with OBO, those with ERCP had longer time intervals between successive procedures (41, 47, 54, 62, 71 days) than for PTC (27, 31, 36, 41, 48 days; P < 0.001). Allograft salvage was successful across all interventions. Stricture resolution was achieved in 92%, with 20% risk of recurrence. Resolution without recurrence was highest in patients with isolated anastomotic strictures and without hepatic artery thrombosis.

The pediatric solid organ transplant experience with COVID-19: An initial multi-center, multi-organ case series.

The clinical course of COVID-19 in pediatric solid organ transplant recipients remains ambiguous. Though preliminary experiences with adult transplant recipients have been published, literature centered on the pediatric population is limited. We herein report a multi-center, multi-organ cohort analysis of COVID-19-positive transplant recipients ≤ 18 years at time of transplant. Data were collected via institutions' respective electronic medical record systems. Local review boards approved this cross-institutional study. Among 5 transplant centers, 26 patients (62% male) were reviewed with a median age of 8 years. Six were heart recipients, 8 kidney, 10 liver, and 2 lung. Presenting symptoms included cough (n = 12 (46%)), fever (n = 9 (35%)), dry/sore throat (n = 3 (12%)), rhinorrhea (n = 3 (12%)), anosmia (n = 2 (8%)), chest pain (n = 2 (8%)), diarrhea (n = 2 (8%)), dyspnea (n = 1 (4%)), and headache (n = 1 (4%)). Six patients (23%) were asymptomatic. No patient required supplemental oxygen, intubation, or ECMO. Eight patients (31%) were hospitalized at time of diagnosis, 3 of whom were already admitted for unrelated problems. Post-transplant immunosuppression was reduced for only 2 patients (8%). All symptomatic patients recovered within 7 days. Our multi-institutional experience suggests the prognoses of pediatric transplant recipients infected with COVID-19 may mirror those of immunocompetent children, with infrequent hospitalization and minimal treatment, if any, required.

Measles, mumps, rubella (vaccine) and varicella vaccines in pediatric liver transplant: An initial analysis of post-transplant immunity.

Varicella and measles infection represents a significant source of morbidity and mortality for pediatric LT recipients. We evaluated the prevalence and correlates of post-transplant immunity in pediatric LT recipients previously immunized against measles (n = 72) and varicella (n = 67). Sixteen of seventy-two (22%) patients were measles non-immune, and 42/67 (63%) were varicella non-immune after LT. Median time from LT to titers for measles and varicella was 4.0 and 3.3 years, respectively. In the measles cohort, non-immune patients received fewer pretransplant vaccine doses (P = 0.026) and were younger at both time of vaccination (P = 0.006) and LT (P = 0.004) compared with immune patients. Upon multivariable analysis, weight > 10 kg at LT (OR 5.91, 95% CI 1.27-27.41) and technical variant graft (OR 0.07, 95% CI 0.01-0.37) were independently, significantly associated with measles immunity. In the varicella cohort, non-immune patients received fewer pretransplant vaccine doses (P = 0.028), were younger at transplant (P = 0.022), and had less time lapse between their last vaccine and transplant (P = 0.012) compared with immune patients. Upon multivariate analysis, time > 1 year from last vaccine to LT was independently, significantly associated with varicella immunity (OR 3.78, CI 1.30-11.01). This study demonstrates that non-immunity to measles and varicella is a prevalent problem after liver transplantation in children and identifies 3 unique risk factors for non-immunity in this high-risk population.

Trends in pediatric liver transplant donors and deceased donor circumstance of death in the United States, 2002-2015.

While much of the discussion regarding expanding the donor pool for pediatric liver transplantation has surrounded the use of technical variant grafts, little attention has been directed toward changes in the deceased donor population. The aim of this study was to investigate trends in the circumstance of the death of deceased donors used for pediatric liver transplantation. All pediatric liver transplant recipients transplanted between 2002 and 2015 were identified in the UNOS database and were categorized based on the donor circumstance of death. There was no significant correlation between year of transplantation and number of pediatric liver transplants performed, pediatric donors, split livers, or living donors. There was a significant downward trend in donors from motor vehicle fatalities and an upward trend in suicide, non-MVA, and death due to natural causes. There was also an upward trend in drowning, one of the most common mechanisms of death among non-MVA in 2015. While the number of donors who died in MVA has fallen, the number of deceased donors who died from suicide, natural causes, and non-MVA, especially drowning, has increased, maintaining the overall number of pediatric deceased donor livers transplanted.

Persistent Pulmonary Hypertension and Histologic Chorioamnionitis in Preterm Infants: Controlled Study.

Persistent pulmonary hypertension (PPHN) of the newborn is one of the most challenging acute disorders of postnatal transition with substantial morbidity and mortality. The aim of the study was to find if there is an association between persistent pulmonary hypertension and histologic chorioamnionitis in preterm infants. 27 preterm infants with echocardiographic evidence of PPHN within the first 3 days of life were eligible for the study. A matched control group of 27 patients was chosen according to gestational age, date of birth, and gender. Data collection included the need for respiratory support, use of nitric oxide oxygen supplementation, duration of rupture of membranes, blood culture, blood count, and C-reactive protein levels at birth and 12 h. Maternal clinical and laboratory data suggesting clinical chorioamnionitis Placentas of both groups were examined. Differences between groups were analyzed using two-tail t test, Kolmogorov-Smirnov test, Chi-square test. No statistically differences were found in all parameters compared between groups, except for a higher number of patients in the PPHN group who were treated by oxygen supplementation. An association was not found between the incidence of HCA and echocardiographic PPHN in preterm infants in the first 3 days of life.

Portosystemic shunt as a bridge to liver transplantation in infants: A comparison of two techniques.

Portosystemic shunts can serve as a bridge to liver transplantation in patients with end-stage liver disease by providing portal decompression to treat life-threatening variceal bleeding and prevent recurrent episodes until an organ becomes available. The conventional TIPS procedure, however, is technically challenging to perform in infants due to the small size of their intrahepatic vasculature. We report two cases of emergent creation of portosystemic shunts as a bridge to liver transplantation in infants with life-threatening variceal bleeding using a conventional TIPS technique in the first case and a percutaneous DIPS technique in the other. Both procedures were successful at reducing the portosystemic pressure gradient and preventing further variceal bleeds until a liver transplant could be performed. The novel percutaneous DIPS procedure is a valuable alternative to the conventional TIPS in infants, as it is better suited for small or challenging intrahepatic vascular anatomy.

Recommendations for Probiotic Use--2015 Update: Proceedings and Consensus Opinion.

This paper describes the consensus opinion of the participants in the 4th Triennial Yale/Harvard Workshop on Probiotic Recommendations. The recommendations update those of the first 3 meetings that were published in 2006, 2008, and 2011. Recommendations for the use of probiotics in necrotizing enterocolitis, childhood diarrhea, inflammatory bowel disease, irritable bowel syndrome and Clostridium difficile diarrhea are reviewed. In addition, we have added recommendations for liver disease for the first time. As in previous publications, the recommendations are given as A, B, or C ratings.

Time required to determine performance variables and production efficiency of lactating dairy cows.

Thirty-five lactating dairy cows throughout weeks of lactation (WOL) 16 to 30 were used to determine optimal time needed for reliable measurement of performance variables, and to classify the cows into high-, medium-, and low-efficiency groups. Individual performance variables [body weight (BW), dry matter intake (DMI), and milk production] were measured daily with a computerized monitoring system. Body condition was visually scored weekly and used to calculate retained or depleted body energy as a result of fat content change (REF). Milk composition was analyzed weekly. Body weight, DMI, and total recovered energy (RE), which represents energy in milk production plus REF, were summarized weekly. Efficiency was calculated as RE/DMI and as residual feed intake (RFI; i.e., the difference between actual and expected DMI), which was calculated from multiple linear regression of DMI dependence on BW(0.75) and RE. Unexpectedly, it was found that BW did not affect DMI and RE/DMI. Changes and relative changes in phenotypic coefficient of variation and correlations among data from shortened tests ranging from 1 wk (WOL 16) to a sequence of 15-wk tests were used to determine optimal test period durations for 5 traits: BW, DMI, RE, RE/DMI, and RFI. Traits were fitted into a mixed model with repeated measures. For each week, the traits were summarized as a sequence of cumulative data, starting from WOL 16 and cumulated over periods that increased in 1-wk steps up to WOL 16 to 29. Weekly cumulations were compared with those for entire test period (WOL 16 to 30). Consistency of each cow's efficiency classification as high, medium, or low was tested by the total-agreement procedure; the kappa index P-value was used. Throughout WOL 16 to 30, the effects of increasing test period duration on between-animal coefficient of variation differed with respect to the various performance variables and RE/DMI: it tended to change with respect to BW, did not change with respect to DMI, and decreased with respect to RE and RE/DMI. In conclusion, compared with a 15-wk study, a 2-wk study can classify RFI and RE/DMI to 3 efficiency levels, with an individual correlation coefficient of 0.6. When the study was carried out over 3 wk or more, the lowest significant index of the classification was P<0.004, the lowest individual correlation coefficient was 0.65, and its lowest significance was P<0.01. The current study indicated that the insignificant effect of the BW of dairy lactating cows on their DMI should be validated in more studies.

International Liver Transplantation Society Global Census: First Look at Pediatric Liver Transplantation Activity Around the World.

BACKGROUND: Over 16 000 children under the age of 15 died worldwide in 2017 because of liver disease. Pediatric liver transplantation (PLT) is currently the standard of care for these patients. The aim of this study is to describe global PLT activity and identify variations between regions. METHODS: A survey was conducted from May 2018 to August 2019 to determine the current state of PLT. Transplant centers were categorized into quintile categories according to the year they performed their first PLT. Countries were classified according to gross national income per capita. RESULTS: One hundred eight programs from 38 countries were included (68% response rate). 10 619 PLTs were performed within the last 5 y. High-income countries performed 4992 (46.4%) PLT, followed by upper-middle- (4704 [44·3%]) and lower-middle (993 [9·4%])-income countries. The most frequently used type of grafts worldwide are living donor grafts. A higher proportion of lower-middle-income countries (68·7%) performed ≥25 living donor liver transplants over the last 5 y compared to high-income countries (36%; P = 0.019). A greater proportion of programs from high-income countries have performed ≥25 whole liver transplants (52.4% versus 6.2%; P = 0.001) and ≥25 split/reduced liver transplants (53.2% versus 6.2%; P < 0.001) compared to lower-middle-income countries. CONCLUSIONS: This study represents, to our knowledge, the most geographically comprehensive report on PLT activity and a first step toward global collaboration and data sharing for the greater good of children with liver disease; it is imperative that these centers share the lead in PLT.

Prenatal diagnosis of a pulmonary arteriovenous malformation.

Pulmonary arteriovenous malformations (PAVMs) are caused by abnormal communications between pulmonary arteries and pulmonary veins. They are most often congenital, and may occur as isolated lesions or part of complex congenital syndromes. We describe a case of PAVM in a 16-week-old fetus. To the best of our knowledge this is the first case to be described in the literature of diagnosis, at such an early gestational age, of PAVM with the shunt found between the main pulmonary artery and pulmonary vein.

Hypotension during gradual blood loss: waveform variables response and absence of tachycardia.

BACKGROUND: Variation in arterial pressure and plethysmographic waveforms has been shown to be predictors of cardiac output response to fluid challenge. The objective of this study was to evaluate the ability of arterial and plethysmographic waveform variables to predict hypotension during blood loss. METHODS: Patients undergoing autologous haemodilution were studied. After anaesthesia induction, blood was withdrawn in steps of 2% of estimated circulating blood volume (ECBV). Arterial and plethysmographic waveforms were recorded and analysed offline at each step of blood withdrawal. RESULTS: Thirty-four (29%) out of 118 studied patients tolerated 20% ECBV withdrawal without hypotension. Patients who tolerated 20% ECBV withdrawal were younger than those who did not [mean (sd): 53.8 (11.1) vs 62.7 (10.7); P<0.0001]. Patients with hypertension developed hypotension earlier than healthier patients did. There were no differences at the baseline in arterial and plethysmographic waveform variables between those who did and those who did not tolerate 20% of ECBV withdrawal. All values of variables increased significantly from the baseline after the withdrawal of 4% of ECBV (P<0.005). There were no changes in heart rate (HR), 73 (12) at the baseline and 76 (13) after 20% of ECBV withdrawal (P=0.4). CONCLUSIONS: Arterial and plethysmographic waveform variables were augmented with increasing blood loss in all patients. Older patients, patients who received anti-hypertensive drugs, or both developed hypotension earlier than others. Baseline values were weak predictors of hypotension during stepwise blood withdrawal. No clinically significant increase in HR was observed, regardless of tolerance of arterial pressure to blood withdrawal.

Hepatopulmonary syndrome in children - is conventional liver transplantation always needed?

BACKGROUND: Hepatopulmonary syndrome (HPS) is the association of liver disease, hypoxemia, and intrapulmonary vascular dilatations. There are little data on the management of HPS in children other than conventional orthotopic liver transplantation (OLT). AIMS: To describe the patient characteristics, mode of diagnosis, treatment, and outcomes of children with HPS at our center. METHODS: Retrospective review of patients diagnosed with HPS between 1997 and 2007 after IRB approval. RESULTS: There were 10 patients, six females; median age at diagnosis of HPS was 12 yr. Six with cirrhosis underwent OLT and had subsequent resolution of HPS and are stable at last follow-up. Of the remaining four, two had cirrhosis. HPS resolved without conventional OLT in the following four patients: hepatitis C after antiviral treatment, biliary atresia with portal hypertension after transjugular intrahepatic portosystemic shunting, Abernethy syndrome after auxiliary partial OLT, and in a child with splenic vein thrombosis after splenectomy. CONCLUSIONS: Our series shows resolution of HPS in all patients and 100% survival after conventional OLT. Four children had resolution of HPS after surgical or medical treatments other than conventional OLT. Careful review of clinical status and underlying pathophysiology and anatomy at diagnosis of HPS should inform treatment decisions.

Developmental and disease-related influences on self-management acquisition among pediatric liver transplant recipients.

Pediatric LT recipients are vulnerable to disruptions in their healthcare management and transitioning to self-managed care. This study aimed to examine whether age at transplant and indication for transplant (acute vs. chronic liver disease) influence later self-management skills. Sixty-three LT recipients, aged 14 and older (M = 17.68, s.d. = 3.01), were recruited and asked to complete a healthcare management survey, the Developmentally Based Skills Checklist, adapted for transplant patients, listing 22 behaviors that medically ill adolescents should progressively master. While there were no significant differences between those who received an LT owing to an acute disease vs. those who received an LT owing to a chronic disease, the age at which patients received their transplant did yield significant results, although, overall, these findings were attenuated by current age. However, our findings indicated that males transplanted at a younger age struggled with mastery over their healthcare responsibilities relative to males transplanted later and females in both age groups. There are many possible reasons why the experience of transplant at a younger age could negatively affect or derail healthcare transitions. Future research is necessary to further untangle this relationship; yet, it seems as though longer time living with LT may make transition harder for families.

Roux-en-Y loop varices in children with portal hypertension after liver transplantation: an unusual cause of "obscure" gastrointestinal bleeding.

PHALT may result from graft dysfunction, portal vein thrombosis, arterio-venous fistulas and can lead to GIB, commonly from bleeding esophageal varices. We present three children with GIB requiring multiple blood transfusions that were diagnosed with RY Loop bleeding. Routine EGD, colonoscopy, and CE failed to reveal the bleeding source. However, enteroscopy revealed large varices at the site of hepaticojejunostomy anastomosis in all. Our experience demonstrates that RY loop varices in children with PHALT are a rare and treatable cause of obscure GI bleeding.

Benign liver masses and lesions in children: 53 cases over 12 years.

BACKGROUND: Primary liver masses in children may require intervention because of symptoms or concern about malignant transformation. OBJECTIVES: To review the management and outcome of benign liver masses in children. METHODS: We conducted a retrospective chart review of children with liver masses referred to our institution during the period 1997-2009. RESULTS: Benign liver masses were identified in 53 children. Sixteen of these children (30%) had hemangioma/infantile hepatic hemangioendothelioma (IHH) and 15 (28%) had focal nodular hyperplasia. The remainder had 6 cysts, 4 hamartomas, 3 nodular regenerative hyperplasia, 2 adenomas, 2 vascular malformations, and one each of polyarteritis nodosa, granuloma, hepatic hematoma, lymphangioma, and infarction. Median age at presentation was 6 years, and 30 (57%) were female. Masses were initially noticed on imaging studies performed for unrelated symptoms in 33 children (62%), laboratory abnormalities consistent with liver disease in 11 (21%), and palpable abdominal masses in 9 (17%). Diagnosis was made based on characteristic radiographic findings in 31 (58%), but histopathological examination was required for the remaining 22 (42%). Of the 53 children, 27 (51%) were under observation while 17 (32%) had masses resected. Medications targeting masses were used in 9 (17%) and liver transplantation was performed in 4 (8%). The only death (2%) occurred in a child with multifocal IHH unresponsive to medical management and prior to liver transplant availability. CONCLUSIONS: IHH and focal nodular hyperplasia were the most common lesions. The majority of benign lesions were found incidentally and diagnosed radiologically. Expectant management was sufficient in most children after diagnosis, although surgical intervention including liver transplant was occasionally necessary.

Dimensions of the fetal facial profile in normal pregnancy.

OBJECTIVE: To determine sonographic dimensions of the fetal facial profile in normal pregnancy. METHODS: This was a prospective, cross-sectional study of 397 normal healthy fetuses at 14-33 weeks of gestation. After exclusion of the small numbers of patients at the upper GAs, 379 patients between 14.0 and 26.9 weeks of gestation were included in the analyses. The sagittal plane of the fetal facial profile was evaluated using transvaginal and transabdominal ultrasound. Distances from the tip of the nose to the mouth (the line between the lips), from the mouth to the gnathion (lower chin), from the upper philtrum to the mouth, and from the mouth to the upper concavity of the chin were measured and are presented according to gestational age (GA). RESULTS: There was a significant linear correlation between GA and the distance from the tip of the nose to the mouth (r = 0.943; P < 0.00001; y = -37.98 + 7.54 x GA), from the mouth to the gnathion (r = 0.946; P < 0.00001; y = -46.34 + 7.95 x GA), from the upper level of the philtrum to the mouth (r = 0.71; P < 0.00001; y = 0.22 + 3.33 x GA) and from the mouth to the upper concavity of the chin (r = 0.665; P < 0.00001; y = 1.65 + 2.95 x GA). The ratio between the distance from the tip of the nose to the mouth and that from the mouth to the gnathion was also almost constant throughout gestation, as was the ratio between the distance from the upper philtrum to the mouth and that from the mouth to the upper concavity of the chin. CONCLUSIONS: We provide normative data of the fetal facial profile across GA. Our data offer a potential tool for the prenatal diagnosis of abnormal fetal facial profile.

Surgical outcomes in Alagille syndrome and PFIC: A single institution's 20-year experience.

BACKGROUND: Alagille Syndrome (AGS) and Progressive Familial Intrahepatic Cholestasis (PFIC) are rare pediatric biliary disorders that lead to progressive liver disease. This study reviews our experience with the surgical management of these disorders over the last 20years. METHODS: We retrospectively reviewed the records of children diagnosed with AGS or PFIC from January 1996 to December 2016. Data collected included demographics, surgical intervention (liver transplant or biliary diversion), and complications. RESULTS: Of 37 patients identified with these disorders, 17 patients (8 AGS,9 PFIC) underwent surgical intervention. Mean postsurgical follow-up was 6.9±4.7years. Liver transplantation was the most common procedure (n=14). Two patients who were initially thought to have biliary atresia underwent hepatoportoenterostomy, but were subsequently shown to have Alagille syndrome. Biliary diversion procedures were performed in 3 patients (external n=1, internal n=2). PFIC patients tended to be older at the time of liver transplant compared to AGS (4.3±3.9years vs. 2.4±1.1years, p=0.25). The AGS patient with external diversion had resolution of symptoms and no complications (follow-up: 12.5years). Both PFIC patients with internal diversion (conduit between gallbladder and transverse colon) had resolution of pruritus and no progression of liver disease (follow-up: 3.8 and 4.5years). CONCLUSIONS: AGS and PFIC are rare biliary disorders in children which result in pruritus and progressive liver failure. Three patients in this series (8%) benefited from biliary diversion for control of pruritus and have not to date required transplantation for progressive liver disease. 38% underwent transplantation owing to pruritus and severe liver dysfunction. LEVEL OF EVIDENCE: 2b.

Fli-1, an Ets-related transcription factor, regulates erythropoietin-induced erythroid proliferation and differentiation: evidence for direct transcriptional repression of the Rb gene during differentiation.

Erythropoietin (Epo) is a major regulator of erythropoiesis that alters the survival, proliferation, and differentiation of erythroid progenitor cells. The mechanism by which these events are regulated has not yet been determined. Using HB60, a newly established erythroblastic cell line, we show here that Epo-induced terminal erythroid differentiation is associated with a transient downregulation in the expression of the Ets-related transcription factor Fli-1. Constitutive expression of Fli-1 in HB60 cells, similar to retroviral insertional activation of Fli-1 observed in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia, blocks Epo-induced differentiation while promoting Epo-induced proliferation. These results suggest that Fli-1 modulates the response of erythroid cells to Epo. To understand the mechanism by which Fli-1 regulates erythropoiesis, we searched for downstream target genes whose expression is regulated by this transcription factor. Here we show that the retinoblastoma (Rb) gene, which was previously shown to be involved in the development of mature erythrocytes, contains a Fli-1 consensus binding site within its promoter. Fli-1 binds to this cryptic Ets consensus site within the Rb promoter and transcriptionally represses Rb expression. Both the expression level and the phosphorylation status of Rb are consistent with the response of HB60 cells to Epo-induced terminal differentiation. We suggest that the negative regulation of Rb by Fli-1 could be one of the critical determinants in erythroid progenitor cell differentiation that is specifically deregulated during F-MuLV-induced erythroleukemia.

Three-dimensional power Doppler in the evaluation of painful leiomyomas and focal uterine thickening in pregnancy.

OBJECTIVE: To determine the usefulness of 3-dimensional (3D) power Doppler ultrasound in distinguishing painful leiomyomas from focal myometrial contractions or nonpainful leiomyomas in pregnancy. METHODS: A 2D section of the area of interest in the uterine wall was first obtained in 29 patients to determine whether the pain originated from a myoma or a uterine contraction. Then, volume acquisition was activated and Doppler indices (vascularization index, flow index, and vascularization-flow index) were calculated for thickened and normal uterine wall. RESULTS: Of 15 patients found to have uterine myomas, 11 had multiple tumors and 4 had single tumors. In the remaining 14 patients the painful uterine thickening proved to be a focal contraction. Painful segments of uterine thickening were associated with lower Doppler indices. Painful myomas were found to have significantly lower indices than normal uterine wall (P=0.008, 0.03, and 0.01 for VI, FI, and VFI, respectively, vs. 0.001, 0.003, and 0.01). However, the differences in indices between nonpainful myomas and uterine wall on the one hand, and nonpainful myomas and focal uterine contractions on the other, were not statistically significant. CONCLUSION: Three-dimensional power Doppler ultrasound may be a sensitive and reliable tool for distinguishing painful uterine myomas from focal myometrial contractions and nonpainful myomas.