Ketogenic diet in children and adolescents: The effects on growth and nutritional status.

The ketogenic diet is known to be a possible adjuvant treatment in several medical conditions, such as in patients with severe or drug-resistant forms of epilepsy. Its use has recently been increasing among adolescents and young adults due to its supposed weight-loss effect, mediated by lipolysis and lowered insulin levels. However, there are still no precise indications on the possible use of ketogenic diets in pediatric age for weight loss. This approach has also recently been proposed for other types of disorder such as inherited metabolic disorders, Prader-Willi syndrome, and some specific types of cancers. Due to its unbalanced ratio of lipids, carbohydrates and proteins, a clinical evaluation of possible side effects with a strict evaluation of growth and nutritional status is essential in all patients following a long-term restrictive diet such as the ketogenic one. The prophylactic use of micronutrients supplementation should be considered before starting any ketogenic diet. Lastly, while there is sufficient literature on possible short-term side effects of ketogenic diets, their possible long-term impact on growth and nutritional status is not yet fully understood, especially when started in pediatric age.

Eosinophilic esophagitis in children: current knowledge to open new horizons.

Eosinophilic Esophagitis (EoE) is a chronic immune/antigen-mediated condition which is also driven by genetic and environmental factors. It has been deeply investigated over the last years and its incidence is widely increasing in childhood. Although atopic diseases are closely linked with EoE, it does not recognize a classical IgE-mediate immune pathogenesis but it is rather a T helper type 2 inflammatory process. Familial clustering supports genetic predisposition in EoE and recent advances in understanding the genetic basis for EoE may eventually translate into targeted management of the disease. EoE diagnosis is based on clinical symptoms, micro, and macroscopic findings along with exclusion of gastroesophageal reflux disease (GERD) evidence. Management of the disease encompasses both dietary and pharmacological solutions that need to be specifically targeted on patients' history, clinical symptoms, and diagnostic evaluations. New therapies, currently not available in children, may represent the basis for future therapeutic options in the next years.

Fighting Fatty Liver Diseases with Nutritional Interventions, Probiotics, Symbiotics, and Fecal Microbiota Transplantation (FMT).

Pediatric obesity is rising worldwide leading the worrying phenomenon of nonalcoholic fatty liver disease (NAFLD) to shift into one of the most frequent causes of chronic liver illness in childhood. Occurrence of NAFLD depends on several factors such as the geographical area and the diagnostic modalities used; overall it ranges between 3% and 10% of pediatric population, increasing up to 70% in patients with metabolic comorbidities (Manco M, Bottazzo G, DeVito R et al, J Am Coll Nutr 27:667-676, 2008).Recent findings have related the intestinal microbiota to a plethora of pathological conditions, including type 2 diabetes (T2D), obesity, and nonalcoholic steatohepatitis (NASH). One of the emerging areas of the study is the link between liver diseases and gut microbiome, which has added new information to the understanding of the so-called gut-liver axis.In order to address the role of gut microbiome in NAFLD onset and progression, it is necessary to "decipher" operational codes for microbiome investigation within the context of advanced laboratory medicine to capture microbiome features and, hence, to address the function of the intestinal microbiome within the gut microbiota-liver axis.Results of these investigations have allowed the beginning of implementing the usage of probiotics and symbiotics in the medical approach of obesity and NAFLD in adults and children. Several randomized clinical trials (RCTs) have been already published on fecal microbiota transplantation (FMT), T2D, NASH, and inflammatory bowel disease (IBD).This review proposes to describe the current state of knowledge on the ways fatty liver diseases can be addressed with nutritional interventions, probiotics, symbiotics, and FMT.

Liver Transplantation and Gut Microbiota Profiling in a Child Colonized by a Multi-Drug Resistant Klebsiella pneumoniae: A New Approach to Move from Antibiotic to "Eubiotic" Control of Microbial Resistance.

The increase of microorganisms multi-drug resistant (MDR) to antibiotics (ATBs) is becoming a global emergency, especially in frail subjects. In chronic liver disease (LD) with indications for liver transplantation (LT), MDR colonization can significantly affect the LT outcome. However, no clear guidelines for microbial management are available. A novel approach toward MDR-colonized patients undergoing LT was developed at our Center refraining from ATBs use during the transplant waiting list, and use of an intensive perioperative prophylaxis cycle. This study aimed to couple clinical evaluation with monitoring of gut microbiota in a pediatric LD patient colonized with MDR Klebsiella pneumoniae (KP) who underwent LT. No peri-transplant complications were reported, and a decontamination from the MDR bacteria occurred during follow-up. Significant changes in gut microbiota, especially during ATB treatment, were reported by microbiota profiling. Patterns of Klebsiella predominance and microbiota diversity revealed opposite temporal trends, with Klebsiella ecological microbiota niches linked to ATB-driven selection. Our infection control program appeared to control complications following LT in an MDR-KP-colonized patient. The perioperative ATB regimen, acting as LT prophylaxis, triggered MDR-KP overgrowth and gut dysbiosis, but buffered infectious processes. Mechanisms modulating the gut ecosystem should be taken into account in MDR colonization clinical management.

Autoimmune liver diseases and inflammatory bowel diseases in children: current issues and future perspectives.

Inflammatory bowel diseases (IBDs) represent a group of intestinal disorders with a chronic and relapsing inflammation of the gut, and with a potential risk of systemic involvement of other organs and systems. Over the pediatric age, an incidence higher than 20% of developing extraintestinal manifestation during follow-up has been reported. The liver and the biliary system are frequently involved, and primary sclerosing cholangitis (PSC) is the most predominant entity with an incidence rate of 6.4-7.8% in children. PSC recognizes a multifactorial pathogenesis, and so far a not fully known mechanism for this association. The peculiar phenotype and the distinct clinical course of patients with IBD and PSC-associated make this 'linkage' an attractive study model to better understand mechanisms underlying these diseases. Approaching to these patients is complex and multidisciplinary, and a unique therapeutic strategy has not been standardized yet. New medications are being studied; however, further studies are needed to fully understand the pathogenesis and to improve the care of these patients. The aim of this paper is to review the recent literature regarding hepatobiliary involvement in IBD patients, with particular attention to PSC, and to provide the latest information for a correct diagnosis and appropriate management.

Twelve Novel Mutations in the SLC26A3 Gene in 17 Sporadic Cases of Congenital Chloride Diarrhea.

OBJECTIVES: We aimed to improve the knowledge of pathogenic mutations in sporadic cases of congenital chloride diarrhea (CCD) and emphasize the importance of functional studies to define the effect of novel mutations. METHODS: All member 3 of solute carrier family 26 (SLC26A3) coding regions were sequenced in 17 sporadic patients with CCD. Moreover, the minigene system was used to analyze the effect of 2 novel splicing mutations. RESULTS: We defined the SLC26A3 genotype of all 17 patients with CCD and identified 12 novel mutations. Using the minigene system, we confirmed the in silico prediction of a complete disruption of splicing pattern caused by 2 of these novel mutations: the c.971+3_971+4delAA and c.735+4_c.735+7delAGTA. Moreover, several prediction tools and a structure-function prediction defined the pathogenic role of 6 novel missense mutations. CONCLUSIONS: We confirm the molecular heterogeneity of sporadic CCD adding 12 novel mutations to the list of known pathogenic mutations. Moreover, we underline the importance, for laboratories that offer molecular diagnosis and genetic counseling, to perform fast functional analysis of novel mutations.

Functional Dyspepsia: An Enigma in a Conundrum.

As defined by Rome III, there are 4 abdominal pain-related functional gastrointestinal disorders in children: irritable bowel syndrome, functional dyspepsia (FD), abdominal migraine, and functional abdominal pain. Dyspepsia is a constellation of symptoms referable to the gastroduodenal region of the upper gastrointestinal tract. FD refers to dyspeptic symptoms that cannot currently be explained by an organic cause, and affects 25% to 40% of the adult population over a lifetime. In children, this condition results in increased specialist consultations, with reported prevalence between 3% and 27%. The Rome III criteria for pediatric FD include the presence or persistence of recurrent pain or discomfort centered in the upper abdomen, without evidence of organic disease or change in frequency of stools. Symptoms must be chronic, occurring at least weekly and over a period of at least 6 months. The goal of this article is to provide a narrative review of diagnosis and management of the FD in the pediatric population. A comprehensive search of published literature using the PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) database was carried out to identify all articles published in English from 1998 to November 2015, using 3 key terms; "FD," "functional gastrointestinal disorders," and "children."

Clinical Features and Risk Factors of Autoimmune Liver Involvement in Pediatric Inflammatory Bowel Disease.

OBJECTIVES: Autoimmune liver disease is reported in up to 7.8% of children with inflammatory bowel disease. A distinct inflammatory bowel disease phenotype has been suggested in adults and in small pediatric cohorts. The aim of the study was to evaluate the features of inflammatory bowel disease associated with autoimmune liver diseases and to analyze the characteristics of the liver disease. METHODS: Information on patients was obtained from the Italian Pediatric Inflammatory Bowel Disease Registry. Data of patients with and without autoimmune liver disease were compared. RESULTS: Autoimmune liver disease was detected in 6.8% of the 677 patients enrolled and was significantly associated with the diagnosis of ulcerative colitis (83%), with pancolonic involvement (84%), and with perinuclear antineutrophil cytoplasmic antibody positivity (41%) (all Ps < 0.05). Autoimmune liver disease was defined as sclerosing cholangitis in 61% of the patients and as an overlap syndrome in 33%. Concomitant intra- and extrahepatic biliary involvement was reported in 61% of cases, whereas exclusive extrahepatic lesions were reported in 21%. Hepatobiliary complications were observed in 9% of the patients during follow-up. CONCLUSIONS: Autoimmune liver disease, especially sclerosing cholangitis, was significantly more common in patients with extensive ulcerative colitis. Although complications are relatively rare in the pediatric age, monitoring is recommended.

Role of prebiotics and probiotics in pediatric diseases.

The increasing knowledge about the composition and activities of the microflora has shown the close link between the bacteria and the health of the human organism. For this reason it has focused attention on the possibility of modulating the gut flora. The use of probiotics and prebiotics has increased enormously in recent years, more for real beneficial effects demonstrated in patients than for their safety profiles. However, it is recorded an indiscriminate use also in conditions in which there are no scientific evidence to support. Their use in case of immunocompromised patients or with severe and debilitating chronic diseases should be very prudent, because of the risk of complications including sepsis. The use of a probiotic cannot ignore the knowledge of the genus and species of the strain and in pediatric patients has been demonstrated their role for treating acute viral gastroenteritis and preventing antibiotic-associated diarrhea in healthy children. Moreover probiotics are considered as an option for recurrent and relapsing antibiotic sensitive pouchitis and in select patients with mild ulcerative colitis. Further studies are needed to evaluate clinical conditions that may require their use and to define the optimal doses and intake durations.

Characterization of the Gut Microbiota and Mycobiota in Italian Pediatric Patients With Primary Sclerosing Cholangitis and Ulcerative Colitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory, cholestatic liver disease of unknown etiopathogenesis, often associated with inflammatory bowel diseases. Recent evidence ascribes, together with immunologic and environmental components, a significant role to the intestinal microbiota or its molecules in the PSC pathogenesis. METHODS: By metagenomic sequencing of 16S rRNA and ITS2 loci, we describe the fecal microbiota and mycobiota of 26 pediatric patients affected by PSC and concomitant ulcerative colitis (PSC-UC), 27 patients without PSC but with UC (UC), and 26 healthy subjects (CTRLs). RESULTS: Compared with CTRL, the bacterial and fungal gut dysbiosis was evident for both PSC-UC and UC groups; in particular, Streptococcus, Saccharomyces, Sporobolomyces, Tilletiopsis, and Debaryomyces appeared increased in PSC-UC, whereas Klebsiella, Haemophilus, Enterococcus Collinsella, Piptoporus, Candida, and Hyphodontia in UC. In both patient groups, Akkermansia, Bacteroides, Parabacteroides, Oscillospira, Meyerozyma and Malassezia were decreased. Co-occurrence analysis evidenced the lowest number of nodes and edges for fungi networks compared with bacteria. Finally, we identified a specific patient profile, based on liver function tests, bacterial and fungal signatures, that is able to distinguish PSC-UC from UC patients. CONCLUSIONS: We describe the gut microbiota and mycobiota dysbiosis associated to PSC-UC disease. Our results evidenced a gut imbalance, with the reduction of gut commensal microorganisms with stated anti-inflammatory properties (ie, Akkermansia, Bacteroides, Parabacteroides, Oscillospira, Meyerozyma, and Malassezia) and the increase of pathobionts (ie, Streptococcus, Saccharomyces, and Debaryomyces) that could be involved in PSC progression. Altogether, these events may concur in the pathophysiology of PSC in the framework of UC.

In this study, we report the gut microbiota and mycobiota dysbiosis in pediatric patients affected by primary sclerosing cholangitis (PSC) associated with ulcerative colitis (UC), with an increase in pro-inflammatory pathobionts and a reduction in anti-inflammatory commensals.

DNA methylation of the TPMT gene and azathioprine pharmacokinetics in children with very early onset inflammatory bowel disease.

BACKGROUND: Thiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). AIMS: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. METHODS: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients' erythrocytes by HPLC and associated with patients' age group and TPMT DNA methylation. RESULTS: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10-5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. CONCLUSION: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.

Impact of Two Antibiotic Therapies on Clinical Outcome and Gut Microbiota Profile in Liver Transplant Paediatric Candidates Colonized by Carbapenem-Resistant Klebsiella pneumoniae CR-KP.

Colonization by multidrug-resistant (MDR) organisms in liver transplant (LT) candidates significantly affects the LT outcome. To date, consensus about patient management is lacking, including microbiological screening indications. This pilot study aimed to evaluate the impact of carbapenem-resistant Klebsiella pneumoniae (CR-KP) colonization in LT paediatric candidates to enable optimal prevention and therapeutic strategies that exploit both clinical and microbiological approaches. Seven paediatric patients colonized by CR-KP were evaluated before and until one-year post LT. At the time of the transplant, patients were stratified based on antibiotic (ATB) prophylaxis into two groups: 'standard ATB' (standard ATB prophylaxis), and 'targeted ATB' (MDR antibiogram-based ATB prophylaxis). Twenty-eight faecal samples were collected during follow-up and used for MDR screening and gut microbiota 16S rRNA-based profiling. Post-transplant hospitalization duration was comparable for both groups. With the exception of one patient, no serious infections and/or complications, nor deaths were recorded. A progressive MDR decontamination was registered. In the 'standard ATB' group, overall bacterial richness increased. Moreover, 6 months after LT, Lactobacillus and Bulleidia were increased and Enterobacteriaceae and Klebsiella spp. were reduced. In the 'targeted ATB' group Klebsiella spp., Ruminococcus gnavus, Erysipelotrichaceae, and Bifidobacterium spp. were increased 12 months after LT. In conclusion, both antibiotics prophylaxis do not affect nor LT outcomes or the risk of intestinal bacterial translocation. However, in the 'standard ATB' group, gut microbiota richness after LT was increased, with an increase of beneficial lactic acid- and short-chain fatty acids (SCFA)-producing bacteria and the reduction of harmful Enterobacteriaceae and Klebsiella spp. It could therefore be appropriate to administer standard prophylaxis, reserving the use of ATB-based molecules only in case of complications.

Phenotype and Natural History of Children With Coexistent Inflammatory Bowel Disease and Celiac Disease.

BACKGROUND: Adult patients with both inflammatory bowel disease (IBD) and celiac disease (CeD) have peculiar phenotypic features. This study aimed at describing the characteristics and natural history of children with both IBD and CeD. METHODS: This was a case-control study based on a national registry. Cases included children diagnosed with both IBD and CeD. Two matched IBD controls without CeD, and 2 matched CeD controls were selected for each case. Inflammatory bowel disease phenotype and natural history, comprising growth and pubertal development, were compared between groups. RESULTS: Forty-nine (1.75%) patients with IBD and CeD were identified out of 2800 patients with IBD. Compared with patients with IBD alone, patients with IBD and CeD presented more frequently with autoimmune diseases (odds ratio, 2.81; 95% CI, 0.97-8.37; P = 0.04). Ileocolonic localization (46.1% vs 73.1%), treatment with azathioprine (46.2% vs 71.2%), and anti-TNF biologics (46.2% vs 69.2%) were less common in patients with Crohn's disease and CeD than in patients with Crohn's disease alone. Patients with ulcerative colitis and CeD had an increased risk of colectomy despite similar medical treatments compared with patients with ulcerative colitis alone (13.0% vs 0%). Pubertal delay was more common in patients with IBD and CeD compared with patients with IBD alone (14.9% vs 3.2%; odds artio, 5.24; 95% CI, 1.13-33.0; P = 0.02) and CeD alone (14.9% vs 1.1%; P = 0.002). CONCLUSIONS: Children with IBD and CeD may have peculiar features with a higher risk for autoimmune diseases, colectomy, and pubertal delay compared with IBD alone.

Fat soluble vitamins deficiency in pediatric chronic liver disease: The impact of liver transplantation.

BACKGROUND: Children affected with chronic liver disease are at risk for fat-soluble vitamins (FSV) deficiency, in this scenario the role of liver transplant has been only partially explored. AIMS: This study aimed to evaluate the prevalence of FSV deficiency in a cohort of paediatric patients awaiting liver transplant, analyze relationships between plasma vitamin concentrations and risk of acute rejections and liver fibrosis and assess the impact of the transplant on vitamin status. METHODS: 166 children candidates for liver transplant were retrospectively evaluated. Vitamin concentrations were measured before and 12 months after transplantation. RESULTS: Before transplant vitamin A, vitamin E and vitamin D deficiency was found in 66.6%, 40.6% and 36.3% of patients, respectively. 12 months after surgery, the prevalence of deficiency decreased to 29,5% and 2,6% for vitamin A and E while remained the same for vitamin D (36.3%). No association was found between vitamin status and the risk of acute rejections or the severity of liver fibrosis. CONCLUSION: Liver transplant was effective to improve vitamin A and E, but it did not affect vitamin D. A consensus is needed to define optimal nutritional management of these patients in order to prevent deficiencies.

WITHDRAWN: Fat soluble vitamins deficiency in pediatric chronic liver disease: The impact of liver transplantation.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.dld.2019.10.005. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Standard Polymeric Formula Tube Feeding in Neurologically Impaired Children: A Five-Year Retrospective Study.

Malnutrition is frequent in neurologically impaired (NI) children. Enteral feeding via gastrostomy tube is increasingly being used to provide adequate nutrition. Our aim was to assess the outcomes of exclusive gastrostomy tube feeding with standard polymeric formula in children with NI, severe oro-motor dysfunction, and malnutrition, and to investigate the role of the underlying NI-associated disease. A five-year retrospective study from January 2013 to November 2017 was conducted. The primary aim was to assess the nutritional outcomes of exclusive gastrostomy tube feeding with standard polymeric formula in malnourished NI children. The secondary aim was to investigate gastrostomy complications and the impact of the underlying NI-associated disease on the nutritional outcomes. We enrolled 110 consecutive children with NI. Of these patients, 34.5% (N = 38) were categorized as malnourished and started exclusive enteral feeding with a standard (1.0 kcal/mL) polymeric formula (Nutrini, Nutricia) after percutaneous endoscopic gastrostomy (PEG) placement. Seventy-three percent of patients (N = 28) had cerebral palsy (CP); other diagnoses included metabolic (13%, N = 5) and genetic (13%, N = 5) diseases. Tricep skinfold thickness had significantly improved in all patients at 12-months follow-up, while body weight and body mass index showed significant increases mainly in children with CP. No serious complications occurred. We found that standard polymeric formula via gastrostomy tube represents a safe and efficient nutritional intervention in children with NI and malnutrition.

Mixed Botryoid and Spindle Cell Bladder Rhabdomyosarcoma: An Outstanding Pediatric Case.

We report a case of a 3-year-old North African child, initially assessed for nonspecific urinary symptoms such as haematuria and burning urination. The ultrasound evaluation showed a vegetating mass occupying the lumen with weak vascular signs at the Colour-Doppler evaluation. An explorative cystoscopy was performed and it revealed a nonbleeding lesion, white in colour, pedunculated, projecting into the lumen, and associated with a brown satellite formation. Histological examination showed a mixed Botryoid and Spindle Cell Rhabdomyosarcoma. This mixed histology has not been described before and no statistical data are reported in literature so far. Despite the Embryonal Rhabdomyosarcoma variant being the most common, the association characterized by two histological Rhabdomyosarcoma subtypes such as Botryoid and Spindle Cell is rarely observed and it is important to get an accurate histological diagnosis in order to immediately start the correct treatment protocol.

Pediatric gastrointestinal bleeding: Perspectives from the Italian Society of Pediatric Gastroenterology.

There are many causes of gastrointestinal bleeding (GIB) in children, and this condition is not rare, having a reported incidence of 6.4%. Causes vary with age, but show considerable overlap; moreover, while many of the causes in the pediatric population are similar to those in adults, some lesions are unique to children. The diagnostic approach for pediatric GIB includes definition of the etiology, localization of the bleeding site and determination of the severity of bleeding; timely and accurate diagnosis is necessary to reduce morbidity and mortality. To assist medical care providers in the evaluation and management of children with GIB, the "Gastro-Ped Bleed Team" of the Italian Society of Pediatric Gastroenterology, Hepatology and Nutrition (SIGENP) carried out a systematic search on MEDLINE via PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) to identify all articles published in English from January 1990 to 2016; the following key words were used to conduct the electronic search: "upper GIB" and "pediatric" [all fields]; "lower GIB" and "pediatric" [all fields]; "obscure GIB" and "pediatric" [all fields]; "GIB" and "endoscopy" [all fields]; "GIB" and "therapy" [all fields]. The identified publications included articles describing randomized controlled trials, reviews, case reports, cohort studies, case-control studies and observational studies. References from the pertinent articles were also reviewed. This paper expresses a position statement of SIGENP that can have an immediate impact on clinical practice and for which sufficient evidence is not available in literature. The experts participating in this effort were selected according to their expertise and professional qualifications.

Pediatric inflammatory bowel disease: specificity of very early onset.

INTRODUCTION: The incidence of inflammatory bowel disease (IBD) has increased over the last 50 years. It is now recognized that several genetic defects can express an IBD-like phenotype at very early onset (<6 years). AREAS COVERED: The aim of this review was to update knowledge concerning the specificity of IBD at onset <6 years, which can include conventional/standard IBD as well as monogenic IBD-like diseases. Expert commentary: We found that females are less prone than males to develop monogenic disorders, which have X-linked heritability in several cases. Furthermore, the Crohn's Diseases (CD) subtype seems to be suggestive of monogenic disorders while Unclassified IBD (IBDU) subtype is predominantly found in conventional/standard IBD at onset <6 years. Isolated colonic location is prevalent in both the subsets of IBD at onset <6 years if compared to IBD at later onset. Monogenic disorders require more aggressive medical and surgical treatments and can be complicated by the occurrence of lymphomas.