Chronic glucocorticoid management in neuromuscular disease: A survey of neuromuscular neurologists.

INTRODUCTION/AIMS: Glucocorticoids (GC) are first-line therapy for many neuromuscular diseases. There is a lack of guidelines regarding the prevention and management of GC complications in the context of neuromuscular disease, introducing the potential for practice variation, that may compromise quality of care. Our aim was to evaluate the practice patterns among Canadian adult neuromuscular neurologists on the screening, management, and treatment of GC-related complications and to identify variances in practice. METHODS: A web-based anonymous questionnaire was disseminated to 99 Canadian adult neuromuscular neurologists. Questions addressed patterns of screening, prevention, monitoring, and treatment of GC-induced adverse events, including infection prophylaxis, vaccination, bone health, hyperglycemia, and other complications. RESULTS: Seventy-one percent completed the survey. Of those, 52% perform screening blood work prior to initiating GC, 56% screen for infections, and 18% for osteoporosis. The majority monitor glycemic control and blood pressure (>85%). Thirty-two (46%) reported that they do not primarily monitor GC complications, but rather provide recommendations to the primary care physician. Pneumocystis jiroveci pneumonia prophylaxis was never used by 29%, and 29% recommend vaccinations prior to GC initiation. Calcium supplementation was recommended by 80% to prevent osteoporosis. Only 36% were aware of any existing guidelines for preventing GC complications, and 91% endorsed a need for neurology-specific guidelines. DISCUSSION: There is substantial variability in the management of GC adverse effects among neuromuscular neurologists, often not corresponding to limited published literature. Our results support the need for improved education and neurology-specific guidelines to help standardize practice and improve and prevent complications.

A Real-World Study of Nusinersen Effects in Adults with Spinal Muscular Atrophy Type 2 and 3.

BACKGROUND: Spinal muscular atrophy (SMA) is a progressive genetic disorder characterized by muscle weakness ultimately leading to pulmonary impairments that can be fatal. The recent approval of nusinersen, a disease-modifying therapy, substantially changed the prognosis for patients, particularly in children. However, real-world evidence about its long-term effectiveness in adults remains limited. This study aimed to document longitudinal data on motor function, pulmonary function and patient-reported outcome measures of Canadian adults with SMA type 2 and 3 treated with nusinersen. METHODS: Outcomes from 17 patients were collected at the Institut de réadaptation en déficience physique de Québec during routine clinical visits over 36 months post nusinersen treatment, using the Hammersmith Functional Motor Scale Expanded for SMA (HFMSE), Revised Upper Limb Module (RULM), 6-Minute Walk Test (6MWT), Children's Hospital of Philadelphia Adult Test of Neuromuscular Disorders (CHOP-ATEND), SMA functional rating scale (SMAFRS), pulmonary function testing and subjective changes reported by patients. RESULTS: After 36 months, 9 patients showed motor function improvement. Changes beyond the minimal clinically important difference were seen for four patients on the HFMSE, four patients on the RULM and five patients on the 6MWT. Pulmonary function remained stable for most subjects. Subjective positive changes were reported in 88% of patients and five patients showed improvement in the SMAFRS. CONCLUSION: This real-world study demonstrates the positive effects of nusinersen in adults with SMA types 2 and 3. Although stabilizing the patient's condition is considered therapeutic success, this study shows an improvement in motor function and subjective gains in several patients.

A Study on the Incidence and Prevalence of 5q Spinal Muscular Atrophy in Canada Using Multiple Data Sources.

OBJECTIVES: Spinal muscular atrophy (SMA) is a leading genetic cause of infant death and represents a significant burden of care. An improved understanding of the epidemiology of SMA in Canada may help inform strategies to improve the standard of care for individuals living with SMA. METHODS: We employed a multisource approach to estimate the minimal incidence and prevalence of 5q SMA and to gain greater insight into recent clinical practices and treatment trends for the Canadian SMA population. Data sources included the Canadian Paediatric Surveillance Program (CPSP), Canadian Neuromuscular Disease Registry (CNDR), and molecular genetics laboratories in Canada. RESULTS: The estimated annual minimum incidence of 5q SMA was 4.38, 3.44, and 7.99 cases per 100,000 live births in 2020 and 2021, based on CPSP, CNDR, and molecular genetics laboratories data, respectively, representing approximately 1 in 21,472 births (range 12,516-29,070) in Canada. SMA prevalence was estimated to be 0.85 per 100,000 persons aged 0-79 years. Delay in diagnosis exists across all SMA subtypes. Most common presenting symptoms were delayed milestones, hypotonia, and muscle weakness. Nusinersen was the most common disease-modifying treatment received. Most patients utilized multidisciplinary clinics for management of SMA. CONCLUSION: This study provides data on the annual minimum incidence of pediatric 5q SMA in Canada. Recent therapeutic advances and newborn screening have the potential to drastically alter the natural history of SMA. Findings underline the importance of ongoing surveillance of the epidemiology and long-term health outcomes of SMA in the Canadian population.

Gold Coast criteria expand clinical trial eligibility in amyotrophic lateral sclerosis.

INTRODUCTION/AIMS: Consensus criteria to formalize the diagnosis of amyotrophic lateral sclerosis (ALS) and refine clinical trial populations have evolved. The recently proposed Gold Coast consensus criteria are intended to simplify use and increase sensitivity. We aimed to evaluate the potential impact of these criteria on clinical trial eligibility. METHODS: We performed a single-center, retrospective study of people diagnosed with ALS between 2016 and 2021 to determine the numbers of those meeting Gold Coast, revised El Escorial (rEEC) criteria, and Awaji criteria. We identified the proportion of those who would have been eligible for participation in three major ALS clinical trials if Gold Coast were used in place of rEEC definite/probable criteria. (rEEC D/P). RESULTS: Two hundred six people with ALS were included in our study. 48.5% met Gold Coast criteria but not rEEC D/P. Using the Gold Coast criteria would result in higher rates of clinical trial eligibility after other inclusion criteria were met: 95.2% vs 42.5% (P < .001) in a phase III study of riluzole; 100% vs 31.0% (P = .002) in a phase III study of edaravone; and 95.6% vs 45.3% (P < .001) in an ongoing phase III study of sodium phenylbutyrate and taurursodiol. The sensitivity of the Gold Coast criteria (96.1%; 95% confidence interval [CI], 92.2%-98.2%) was significantly higher than that of rEEC D/P (47.6%; 95% CI, 40.6%-54.6%; for difference, χ2  = 117.6; P < .001). DISCUSSION: Until robust biomarkers are available to diagnose ALS, consensus diagnostic criteria remain necessary. Gold Coast criteria would expand research and clinical trial eligibility and improve external validity of clinical trial results.

A National Spinal Muscular Atrophy Registry for Real-World Evidence.

BACKGROUND: Spinal muscular atrophy (SMA) is a devastating rare disease that affects individuals regardless of ethnicity, gender, and age. The first-approved disease-modifying therapy for SMA, nusinursen, was approved by Health Canada, as well as by American and European regulatory agencies following positive clinical trial outcomes. The trials were conducted in a narrow pediatric population defined by age, severity, and genotype. Broad approval of therapy necessitates close follow-up of potential rare adverse events and effectiveness in the larger real-world population. METHODS: The Canadian Neuromuscular Disease Registry (CNDR) undertook an iterative multi-stakeholder process to expand the existing SMA dataset to capture items relevant to patient outcomes in a post-marketing environment. The CNDR SMA expanded registry is a longitudinal, prospective, observational study of patients with SMA in Canada designed to evaluate the safety and effectiveness of novel therapies and provide practical information unattainable in trials. RESULTS: The consensus expanded dataset includes items that address therapy effectiveness and safety and is collected in a multicenter, prospective, observational study, including SMA patients regardless of therapeutic status. The expanded dataset is aligned with global datasets to facilitate collaboration. Additionally, consensus dataset development aimed to standardize appropriate outcome measures across the network and broader Canadian community. Prospective outcome studies, data use, and analyses are independent of the funding partner. CONCLUSION: Prospective outcome data collected will provide results on safety and effectiveness in a post-therapy approval era. These data are essential to inform improvements in care and access to therapy for all SMA patients.

Two placebo-controlled crossover studies in healthy subjects to evaluate gastric acid neutralization by an alginate-antacid formulation (Gaviscon Double Action).

OBJECTIVE: To investigate the intragastric acid neutralization activity of a combined alginate-antacid formulation. SIGNIFICANCE: Published studies have investigated the reflux-suppressing alginate component of Gaviscon Double Action (Gaviscon DA; RB, UK) but intragastric acid neutralization activity of the antacid component has not been evaluated in vivo. METHODS: Intragastric pH monitoring, using a custom-made 10-electrode catheter, was evaluated in a two-part exploratory study in healthy subjects; Part I (n = 6) tested suitability of the catheter using antacid tablets (Rennie; Bayer, Germany); Part II (n = 12) evaluated gastric acid neutralization activity of Gaviscon DA liquid (20 ml) versus placebo in fasted subjects using a randomized, open-label, crossover design. The primary endpoint was the percentage of time that intragastric pH ≥4 was measured during 30 min post-treatment. A confirmatory study of identical design was subsequently conducted (n = 20). RESULTS: Monitoring pH using the multielectrode catheter was a viable approach, directly detecting changes in intragastric pH following a single dose of antacid tablets. In the exploratory study, the percentage of time that pH ≥4 during 30 minutes post-treatment was 46.8% with Gaviscon DA liquid versus 4.7% with placebo (p = 0.0004). These findings were supported by the confirmatory study, where pH ≥4 was recorded 50.8% of the time with Gaviscon DA versus 3.5% with placebo (p = 0.0051). In this study, Gaviscon DA was safe and well tolerated. CONCLUSIONS: These studies demonstrate the effective acid neutralizing capacity of Gaviscon DA versus placebo in healthy, fasted subjects. This adds to the evidence base for the combination of alginates and antacids.

Provincial Differences in the Diagnosis and Care of Amyotrophic Lateral Sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease resulting in muscle weakness, dysarthria and dysphagia, and ultimately respiratory failure leading to death. Half of the ALS patients survive less than 3 years, and 80% of the patients survive less than 5 years. Riluzole is the only approved medication in Canada with randomized controlled clinical trial evidence to slow the progression of ALS, albeit only to a modest degree. The Canadian Neuromuscular Disease Registry (CNDR) collects data on over 140 different neuromuscular diseases including ALS across ten academic institutions and 28 clinics including ten multidisciplinary ALS clinics. METHODS: In this study, CNDR registry data were analyzed to examine potential differences in ALS care among provinces in time to diagnosis, riluzole and feeding tube use. RESULTS: Significant differences were found among provinces, in time to diagnosis from symptom onset, in the use of riluzole and in feeding tube use. CONCLUSIONS: Future investigations should be undertaken to identify factors contributing to such differences, and to propose potential interventions to address the provincial differences reported.

The Canadian Neuromuscular Disease Registry: Connecting patients to national and international research opportunities.

INTRODUCTION: Patient registries serve an important role in rare disease research, particularly for the recruitment and planning of clinical trials. The Canadian Neuromuscular Disease Registry was established with the primary objective of improving the future for neuromuscular (NM) patients through the enablement and support of research into potential treatments. METHODS: In this report, we discuss design and utilization of the Canadian Neuromuscular Disease Registry with special reference to the paediatric cohort currently enrolled in the registry. RESULTS: As of July 25, 2017, there are 658 paediatric participants enrolled in the registry, 249 are dystrophinopathies (229 are Duchenne muscular dystrophy), 57 are myotonic dystrophy participants, 98 spinal muscular atrophy participants and 65 are limb girdle muscular dystrophy. A total of 175 patients have another NM diagnosis. The registry has facilitated 20 clinical trial inquiries, 5 mail-out survey studies and 5 other studies in the paediatric population. DISCUSSION: The strengths of the registry are discussed. The registry has proven to be an invaluable tool to NM disease research and has increased Canada's visibility as a competitive location for the conduct of clinical trials for NM therapies.

X-linked spinal muscular atrophy in mice caused by autonomous loss of ATP7A in the motor neuron.

ATP7A is a copper-transporting P-type ATPase that is essential for cellular copper homeostasis. Loss-of-function mutations in the ATP7A gene result in Menkes disease, a fatal neurodegenerative disorder resulting in seizures, hypotonia and failure to thrive, due to systemic copper deficiency. Most recently, rare missense mutations in ATP7A that do not impact systemic copper homeostasis have been shown to cause X-linked spinal muscular atrophy type 3 (SMAX3), a distal hereditary motor neuropathy. An understanding of the mechanistic and pathophysiological basis of SMAX3 is currently lacking, in part because the disease-causing mutations have been shown to confer both loss- and gain-of-function properties to ATP7A, and because there is currently no animal model of the disease. In this study, the Atp7a gene was specifically deleted in the motor neurons of mice, resulting in a degenerative phenotype consistent with the clinical features in affected patients with SMAX3, including the progressive deterioration of gait, age-dependent muscle atrophy, denervation of neuromuscular junctions and a loss of motor neuron cell bodies. Taken together, these data reveal autonomous requirements for ATP7A that reveal essential roles for copper in the maintenance and function of the motor neuron, and suggest that SMAX3 is caused by a loss of ATP7A function that specifically impacts the spinal motor neuron.

Autonomous requirements of the Menkes disease protein in the nervous system.

Menkes disease is a fatal neurodegenerative disorder arising from a systemic copper deficiency caused by loss-of-function mutations in a ubiquitously expressed copper transporter, ATP7A. Although this disorder reveals an essential role for copper in the developing human nervous system, the role of ATP7A in the pathogenesis of signs and symptoms in affected patients, including severe mental retardation, ataxia, and excitotoxic seizures, remains unknown. To directly examine the role of ATP7A within the central nervous system, we generated Atp7a(Nes) mice, in which the Atp7a gene was specifically deleted within neural and glial cell precursors without impairing systemic copper homeostasis, and compared these mice with the mottled brindle (mo-br) mutant, a murine model of Menkes disease in which Atp7a is defective in all cells. Whereas mo-br mice displayed neurodegeneration, demyelination, and 100% mortality prior to weaning, the Atp7a(Nes) mice showed none of these phenotypes, exhibiting only mild sensorimotor deficits, increased anxiety, and susceptibility to NMDA-induced seizure. Our results indicate that the pathophysiology of severe neurological signs and symptoms in Menkes disease is the result of copper deficiency within the central nervous system secondary to impaired systemic copper homeostasis and does not arise from an intrinsic lack of ATP7A within the developing brain. Furthermore, the sensorimotor deficits, hypophagia, anxiety, and sensitivity to NMDA-induced seizure in the Atp7a(Nes) mice reveal unique autonomous requirements for ATP7A in the nervous system. Taken together, these data reveal essential roles for copper acquisition in the central nervous system in early development and suggest novel therapeutic approaches in affected patients.

Copper homeostasis at the host-pathogen interface.

The trace element copper is indispensable for all aerobic life forms. Its ability to cycle between two oxidation states, Cu(1+) and Cu(2+), has been harnessed by a wide array of metalloenzymes that catalyze electron transfer reactions. The metabolic needs for copper are sustained by a complex series of transporters and carrier proteins that regulate its intracellular accumulation and distribution in both pathogenic microbes and their animal hosts. However, copper is also potentially toxic due in part to its ability to generate reactive oxygen species. Recent studies suggest that the macrophage phagosome accumulates copper during bacterial infection, which may constitute an important mechanism of killing. Bacterial countermeasures include the up-regulation of copper export and detoxification genes during infection, which studies suggest are important determinants of virulence. In this minireview, we summarize recent developments that suggest an emerging role for copper as an unexpected component in determining the outcome of host-pathogen interactions.

Maternofetal and neonatal copper requirements revealed by enterocyte-specific deletion of the Menkes disease protein.

The essential requirement for copper in early development is dramatically illustrated by Menkes disease, a fatal neurodegenerative disorder of early childhood caused by loss-of-function mutations in the gene encoding the copper transporting ATPase ATP7A. In this study, we generated mice with enterocyte-specific knockout of the murine ATP7A gene (Atp7a) to test its importance in dietary copper acquisition. Although mice lacking Atp7a protein within intestinal enterocytes appeared normal at birth, they exhibited profound growth impairment and neurological deterioration as a consequence of copper deficiency, resulting in excessive mortality prior to weaning. Copper supplementation of lactating females or parenteral copper injection of the affected offspring markedly attenuated this rapid demise. Enterocyte-specific deletion of Atp7a in rescued pregnant females did not restrict embryogenesis; however, copper accumulation in the late-term fetus was severely reduced, resulting in early postnatal mortality. Taken together, these data demonstrate unique and specific requirements for enterocyte Atp7a in neonatal and maternofetal copper acquisition that are dependent on dietary copper availability, thus providing new insights into the mechanisms of gene-nutrient interaction essential for early human development.

Differential proteomics in the search for biomarkers of radiotherapy resistance.

The individualization of radiotherapy treatment would be beneficial for cancer patients; however, there are no predictive biomarkers of radiotherapy resistance in routine clinical use. This article describes the body of work in this field where comparative proteomics methods have been used for the discovery of putative biomarkers associated with radiotherapy resistance. A large number of differentially expressed proteins have been reported, mostly from the study of novel radiotherapy-resistant cell lines. Here, we have assessed these putative biomarkers through the discovery, confirmation and validation phases of the biomarker pipeline, and inform the reader on the current status of proteomics-based findings. Suggested avenues for future work are discussed.

A Canadian Adult Spinal Muscular Atrophy Outcome Measures Toolkit: Results of a National Consensus using a Modified Delphi Method.

BACKGROUND: Spinal Muscular Atrophy (SMA) is a rare disease that affects 1 in 11 000 live births. Recent developments in SMA treatments have included new disease-modifying therapies that require high quality data to inform decisions around initiation and continuation of therapy. In Canada, there are no nationally agreed upon outcome measures (OM) used in adult SMA. Standardization of OM is essential to obtain high quality data that is comparable among neuromuscular clinics. OBJECTIVE: To develop a recommended toolkit and timing of OM for assessment of adults with SMA. METHODS: A modified delphi method consisting of 2 virtual voting rounds followed by a virtual conference was utilized with a panel of expert clinicians treating adult SMA across Canada. RESULTS: A consensus-derived toolkit of 8 OM was developed across three domains of function, with an additional 3 optional measures. Optimal assessment frequency is 12 months for most patients regardless of therapeutic access, while patients in their first year of receiving disease-modifying therapy should be assessed more frequently. CONCLUSIONS: The implementation of the consensus-derived OM toolkit will improve monitoring and assessment of adult SMA patients, and enrich the quality of real-world evidence. Regular updates to the toolkit must be considered as new evidence becomes available.

Clinically Meaningful Difference for the Infant Gastroesophageal Questionnaire Revised version (I-GERQ-R): A Quantitative Synthesis.

BACKGROUND: Gastroesophageal reflux disease (GORD) is a common condition affecting 30% of infants aged 0-23 months. The Infant Gastroesophageal Questionnaire Revised version (I-GERQ-R) is an observer-reported outcome measures (ObsRO) developed to evaluate the impact of GORD on young infants. However, evidence regarding the clinically important difference (CID) for the I-GERQ-R is limited. The aim of this study was to determine a CID for the I-GERQ-R. METHODS: A literature review was undertaken (PsycInfo, Embase, MedLine and EconLit databases) for longitudinal studies involving the I-GERQ-R. Articles were not limited by language or publication date. A random effects model was applied to calculate an overall CID, along with I2 and Q statistics. Publication bias was also assessed. RESULTS: The search identified 42 articles; 11 were selected for full-text review and 7 articles were identified for full data extraction. The studies included a total of 661 infants (range: 30 to 313); 424 infants had been diagnosed with GORD (64%). The age range of the infants across the studies was from birth to 7 months. The overall CID was -6.54 (95% confidence interval: -4.35 to -8.74), Q = 17.96, p=0.08 and I2=22.04. CONCLUSION: This study derived a CID for the I-GERQ-R and indicated a threshold around 6 could signify a clinically important difference for this instrument. The lower limit of the 95% confidence interval suggested a threshold of 3 to 4 could represent a minimally important difference. These results may help inform clinical decisions in evaluating meaningful change in symptom severity in children affected by GORD.

Randomized clinical trial: a double-blind, placebo-controlled study to assess the clinical efficacy and safety of alginate-antacid (Gaviscon Double Action) chewable tablets in patients with gastro-oesophageal reflux disease.

BACKGROUND: The alginate-antacid Gaviscon Double Action (Gaviscon DA) has a combined acid-neutralizing and reflux-suppressing action. Response to treatment in a symptomatic gastro-oesophageal reflux disease (GERD) population has not yet been tested in a large-scale clinical study. AIM: The aim of this study was to assess the efficacy and safety of Gaviscon DA compared with matched placebo tablets in the reduction of upper gastrointestinal symptoms in patients with GERD. PARTICIPANTS AND METHODS: In this multicentre, randomized, double-blind, placebo-controlled study, adults with GERD symptoms (N=424) received Gaviscon DA or placebo tablets for 7 days. The primary endpoint was a clinically important reduction of at least 1.5 points in the Reflux Disease Questionnaire (RDQ) GERD dimension (combined heartburn/regurgitation) between baseline and the end of the treatment. Secondary endpoints included the change in RDQ score from baseline for individual RDQ dimensions and Overall Treatment Evaluation. RESULTS: A significantly greater proportion of patients treated with Gaviscon DA met the primary endpoint compared with placebo (47.8 vs. 33.2%, respectively, P=0.0031; odds ratio: 1.85, 95% confidence interval: 1.23-2.78). A significant treatment effect was also observed for heartburn, regurgitation and dyspepsia individually. Patients in the Gaviscon DA group rated their overall treatment response greater than patients in the placebo group [mean Overall Treatment Evaluation (SD): 3.2 (3.08) vs. 2.2 (3.34); P<0.001]. No notable differences in the incidence of adverse events were observed between treatments. CONCLUSION: The alginate-antacid combination, Gaviscon DA, is an effective and well-tolerated treatment to reduce reflux symptoms and associated dyspepsia in symptomatic GERD patients.

T-type calcium channels functionally interact with spectrin (α/ß) and ankyrin B.

This study describes the functional interaction between the Cav3.1 and Cav3.2 T-type calcium channels and cytoskeletal spectrin (α/ß) and ankyrin B proteins. The interactions were identified utilizing a proteomic approach to identify proteins that interact with a conserved negatively charged cytosolic region present in the carboxy-terminus of T-type calcium channels. Deletion of this stretch of amino acids decreased binding of Cav3.1 and Cav3.2 calcium channels to spectrin (α/ß) and ankyrin B and notably also reduced T-type whole cell current densities in expression systems. Furthermore, fluorescence recovery after photobleaching analysis of mutant channels lacking the proximal C-terminus region revealed reduced recovery of both Cav3.1 and Cav3.2 mutant channels in hippocampal neurons. Knockdown of spectrin α and ankyrin B decreased the density of endogenous Cav3.2 in hippocampal neurons. These findings reveal spectrin (α/ß) / ankyrin B cytoskeletal and signaling proteins as key regulators of T-type calcium channels expressed in the nervous system.

Multiple di-leucines in the ATP7A copper transporter are required for retrograde trafficking to the trans-Golgi network.

The ATP7A protein is a ubiquitous copper-transporting P-type ATPase that is mutated in the lethal pediatric disorder of copper metabolism, Menkes disease. The steady-state location of ATP7A is within the trans-Golgi network (TGN), where it delivers copper to copper-dependent enzymes within the secretory pathway. However, ATP7A constantly cycles between the TGN and the plasma membrane, and in the presence of high copper concentrations, the exocytic arm of this cycling pathway is enhanced to promote a steady-state distribution of ATP7A to post-Golgi vesicles and the plasma membrane. A single di-leucine endocytic motif within the cytosolic carboxy tail of ATP7A (1487LL) was previously shown to be essential for TGN localization by functioning in retrieval from the plasma membrane, however, the requirement of other di-leucine signals in this region has not been fully investigated. While there has been some success in identifying sequence elements within ATP7A required for trafficking and catalysis, progress has been hampered by the instability of the ATP7A cDNA in high-copy plasmids during replication in Escherichia coli. In this study, we find that the use of DNA synthesis to generate silent mutations across the majority of both mouse and human ATP7A open reading frames was sufficient to stabilize these genes in high-copy plasmids, thus permitting the generation of full-length expression constructs. Using the stabilized mouse Atp7a construct, we identify a second di-leucine motif in the carboxy tail of ATP7A (1459LL) as essential for steady-state localization in the TGN by functioning in endosome-to-TGN trafficking. Taken together, these findings demonstrate that multiple di-leucine signals are required for recycling ATP7A from the plasma membrane to the TGN and illustrate the utility of large-scale codon reassignment as a simple and effective approach to circumvent cDNA instability in high-copy plasmids.