Glycation Gap Is Associated With Macroproteinuria but Not With Other Complications in Patients With Type 2 Diabetes

OBJECTIVE

We investigated whether glycation gap (G-Gap), an index of intracellular glycation of proteins, was associated with diabetes complications.

RESEARCH DESIGN AND METHODS

We measured concomitantly HbA_1c and fructosamine in 925 patients with type 2 diabetes to calculate the G-Gap, defined as the difference between measured HbA_1c, and fructosamine-based predicted HbA_1c. Patients were explored for retinopathy, nephropathy, peripheral neuropathy, cardiac autonomic neuropathy (n = 512), and silent myocardial ischemia (n = 506).

RESULTS

Macroproteinuria was the only complication that was associated with G-Gap (prevalence in the first, second, and third tertile of G-Gap: 2.9, 6.2, and 11.0%, respectively; P < 0.001). The G-Gap was higher in patients with macroproteinuria than in those without (1.06 ± 1.62 vs. 0.03 ± 1.30%; P < 0.0001). Because HbA_1c was associated with both G-Gap (HbA_1c 7.0 ± 1.4, 7.9 ± 1.4, and 10.1 ± 1.8% in the first, second, and third G-Gap tertile, respectively; P < 0.0001) and macroproteinuria (HbA_1c 8.8 ± 2.2% if macroproteinuria, 8.3 ± 2.0% if none; P < 0.05), and because it could have been a confounder, we matched 54 patients with macroproteinuria and 200 patients without for HbA_1c. Because macroproteinuria was associated with lower serum albumin and fructosamine levels, which might account for higher G-Gap, we calculated in this subpopulation albumin-indexed fructosamine and G-Gap; macroproteinuria was independently associated with male sex (odds ratio [OR] 3.2 [95% CI 1.5–6.7]; P < 0.01), hypertension (2.9 [1.1–7.5]; P < 0.05), and the third tertile of albumin-indexed G-Gap (2.3 [1.1–4.4]; P < 0.05) in multivariate analysis.

CONCLUSIONS

In type 2 diabetic patients, G-Gap was associated with macroproteinuria, independently of HbA_1c, albumin levels, and confounding factors, suggesting a specific role of intracellular glycation susceptibility on kidney glomerular changes.Protein glycation is involved in diabetes complications, and glycated hemoglobin (HbA_1c) level is associated with diabetes complications. Because glycation starts with glucose, it has been assumed that mean blood glucose is at the beginning of this association. However, even if there has been a close correlation between HbA_1c and mean blood glucose level over the previous 3 months, one-fifth (1) to one-third (2) of HbA_1c variance cannot be explained by mean blood glucose. Nonglycemic determinants of HbA_1c actually also could account for diabetes complications. One of the involved mechanisms might be an interindividual variation in the intracellular glycation of proteins, independently of glucose levels: the higher the protein glycation in target tissues (such as retina, kidney, neuronal tissues, and vessels), the more prevalent the tissue damages would be.Some indexes have been developed to estimate nonglycemic determinants of HbA_1c: hemoglobin glycation index is the difference between observed HbA_1c and the value calculated from its regression with mean plasma glucose (3), and glycation gap (G-Gap) (previously called glycosylation gap) is the difference between observed HbA_1c and the value calculated from its regression with fructosamine (4). There are several advantages to consider fructosamine rather than mean blood glucose to evaluate nonglycemic determinants of HbA_1c. First, fructosamine level is more stable than glucose itself. Second, fructosamine represents the 2-week blood glucose exposure, whereas frequent 7-point blood glucose profiles or continuous blood glucose monitoring is required to evaluate mean glucose. Moreover, fructosamine, unlike mean blood glucose, can be used to compare protein glycation in the extracellular space (fructosamine) and in the intracellular space (HbA_1c in red cells and, by assumption, in target tissues).G-Gap has been shown to be consistent over time in type 2 (5,6) and type 1 diabetic patients (4,6). In a study including 40 patients with type 1 diabetes for >15 years, a 1% increase in G-Gap was associated with a 2.9-fold greater frequency of progression in the nephropathy stage. The data demonstrated that nephropathy correlated better with G-Gap than with HbA_1c or fructosamine alone (4). Furthermore, it recently has been shown that G-Gap predicted the progression of nephropathy in type 2 diabetic patients independently of fructosamine, even after adjustment for HbA_1c (5). Nevertheless, in type 1 diabetes, controversial data have been published on G-Gap and retinopathy (7), whereas in type 2 diabetes there has been no report on the relations between G-Gap and complications outside of nephropathy.Thus, the aim of our study was to evaluate, in a large series of type 2 diabetic patients, the potential association between G-Gap and the presence of nephropathy, retinopathy, neuropathy, or silent myocardial ischemia independently of glycemic control.     

Increased Trapezius Pain Sensitivity Is Not Associated With Increased Tissue Hardness

Fatiguing exercise can affect muscle pain sensitivity and muscle hardness, as seen with work-related neck and shoulder pain. Objective methods to assess muscle pain sensitivity are important because the reliability of manual assessment is generally poor. The aim of this study was (1) to compare coexistence of tender points identified by manual palpation and pressure algometry or hardness assessments and (2) to examine the influence of exercise on muscle pain sensitivity and hardness. Fourteen sites in the upper trapezius muscle were selected for assessments in 12 healthy subjects. Pressure pain thresholds and muscle hardness were examined by computer-controlled pressure algometry at baseline, immediately after static or dynamic exercise, and 20 minutes after static or dynamic exercise. Before recording of pressure pain thresholds, the trapezius muscle was examined for tender points by manual palpation. Two sites with low pressure pain thresholds were typical locations for tender points, and these were the least hard sites. However, manually detected tender points were often (29%) not colocalized with most sensitive sites according to the pressure algometry. A heterogeneous distribution of pressure pain sensitivity and muscle hardness was found in the upper trapezius. The short duration of exercise until exhaustion did not change muscle sensitivity or muscle hardness in asymptomatic muscles.PerspectiveThis study confirms clinical findings with heterogeniosity in pain sensitivity and hardness across the upper trapezius muscle. Developments of new techniques that objectively can identify tender points are important, but thus far, manual palpation is best clinical practice.     

Pain Adaptability in Individuals With Chronic Musculoskeletal Pain Is Not Associated With Conditioned Pain Modulation

Healthy humans can be divided into the pain adaptive (PA) and the pain nonadaptive (PNA) groups; PA showed a greater decrease in pain rating to a cold pressor test (CPT) than PNA. This study examined if the dichotomy of pain adaptability existed in individuals with chronic musculoskeletal pain. CPTs at 2°C and 7°C were used to assess the status of pain adaptability in participants with either chronic nonspecific low back pain or knee osteoarthritis. The participants' potency of conditioned pain modulation (CPM) and local inhibition were measured. The strengths of pain adaptability at both CPTs were highly correlated. PA and PNA did not differ in their demographic characteristics, pain thresholds from thermal and pressure stimuli, or potency of local inhibition or CPM. PA reached their maximum pain faster than PNA (t₄₁ = ?2.76, P?<?.01), and had a gradual reduction of pain unpleasantness over 7 days whereas PNA did not (F_6,246?=?3.01, P?=?.01). The dichotomy of pain adaptability exists in musculoskeletal pain patients. Consistent with the healthy human study, the strength of pain adaptability and potency of CPM are not related. Pain adaptability could be another form of endogenous pain inhibition of which clinical implication is yet to be understood.

Cognitive Function Is Not Associated With Recurrent Foot Ulcers in Patients With Diabetes and Neuropathy

OBJECTIVE

To study whether there is an association between cognitive impairment and the relapse rate of foot ulcers in diabetic patients and those with previous foot ulcers.

RESEARCH DESIGN AND METHODS

This single-center prospective study assessed the association of cognitive function and risk for ulcer relapse in 59 patients with diabetes (mean age 65.1 years, diabetes duration 16.5 years, and A1C 7.4%), peripheral neuropathy, and a history of foot ulceration. Premorbid and current cognitive functions were measured (multiple-choice vocabulary test [Lehrl], number-symbol test, mosaic test [HAWIE-R], and trail-making tests A and B [Reitan]). Prevalence of depression was evaluated retrospectively (diagnoses in patient files or use of antidepressive medication). Patients were re-examined after 1 year.

RESULTS

Three patients (5%) died during follow-up (one of sepsis and two of heart problems). The remaining 56 patients (48%) developed 27 new foot ulcerations (78% superficial ulcerations [Wagner stage 1]). Characteristics of patients with and without ulcer relapse were not different. In a binary logistic regression analysis, cognitive function is not predictive of foot reulceration.

CONCLUSIONS

Cognitive function is not an important determinant of foot reulceration.Diabetic patients and those with a history of foot ulcers are at risk for foot reulceration (1,2). Although cognitive function is known to be impaired in patients with diabetes compared with that in nondiabetic control subjects (3), no studies have examined the potential role of cognitive impairment, an important factor for educational success (4), in the development or recurrence of diabetic foot ulcers. Our hypothesis is that cognitive function is associated with the relapse rate of foot ulcers in patients with diabetes and previous foot ulcers.     

Diabetes Distress but Not Clinical Depression or Depressive Symptoms Is Associated With Glycemic Control in Both Cross-Sectional and Longitudinal Analyses

OBJECTIVE

To determine the concurrent, prospective, and time-concordant relationships among major depressive disorder (MDD), depressive symptoms, and diabetes distress with glycemic control.

RESEARCH DESIGN AND METHODS

In a noninterventional study, we assessed 506 type 2 diabetic patients for MDD (Composite International Diagnostic Interview), for depressive symptoms (Center for Epidemiological Studies-Depression), and for diabetes distress (Diabetes Distress Scale), along with self-management, stress, demographics, and diabetes status, at baseline and 9 and 18 months later. Using multilevel modeling (MLM), we explored the cross-sectional relationships of the three affective variables with A1C, the prospective relationships of baseline variables with change in A1C over time, and the time-concordant relationships with A1C.

RESULTS

All three affective variables were moderately intercorrelated, although the relationship between depressive symptoms and diabetes distress was greater than the relationship of either with MDD. In the cross-sectional MLM, only diabetes distress but not MDD or depressive symptoms was significantly associated with A1C. None of the three affective variables were linked with A1C in prospective analyses. Only diabetes distress displayed significant time-concordant relationships with A1C.

CONCLUSIONS

We found no concurrent or longitudinal association between MDD or depressive symptoms with A1C, whereas both concurrent and time-concordant relationships were found between diabetes distress and A1C. What has been called “depression” among type 2 diabetic patients may really be two conditions, MDD and diabetes distress, with only the latter displaying significant associations with A1C. Ongoing evaluation of both diabetes distress and MDD may be helpful in clinical settings.Clinical depression, depressive affect, and diabetes distress are prevalent emotional states found among patients with diabetes (1). These states are associated with high morbidity and mortality (2,3). One line of research has explored whether depression is a risk factor for diabetes or whether diabetes is a risk factor for depression. There are substantive data to suggest that depression is indeed a risk factor for subsequent diabetes (4) and that there may be a bidirectional relationship between depression and diabetes over time (5). A second line of research has explored the linkages between depression and glycemic control among patients who already have diabetes. Here the findings are less clear. In a landmark study published in 2000, Lustman et al. (6) presented a meta-analysis of the literature on depression and glucose control among patients who already have diabetes and reported a modest but significant effect size (d = 0.19). They raised several cautions about interpreting their results, however, because of concerns that some previous studies mixed type 1 and type 2 diabetic patients, used symptom measures that were not tied to defined diagnoses, were primarily cross-sectional, and lacked appropriate demographic and lifestyle controls. Subsequent studies of depression and glycemic control among patients who already have diabetes also have yielded mixed findings, and Georgiades et al. (7) recently listed 7 studies that demonstrated a significant relationship and 10 that did not. Furthermore, intervention trials to reduce depression among patients with diabetes have not consistently led to corresponding reductions in A1C or to improvements in self-care behavior (8,9), and trials to improve diabetes self-care and glycemic control have not consistently led to a reduction in depression (10). Consequently, the causal linkages and pathways between depression and glycemic control among patients who already have diabetes are well studied but unclear.Two major factors that contribute to this lack of clarity concern problems of definition and related measurement. Depression among patients with diabetes has been defined and measured in three ways in clinical research: 1) as a syndrome that meets DSM-IV criteria for major depressive disorder (MDD) usually assessed by a well-standardized, semistructured interview (e.g., Composite International Diagnostic Interview [11]); 2) as depressive symptoms assessed by general symptom inventories (e.g., Beck Depression Inventory [12] or Center for Epidemiological Studies-Depression Scale [CES-D] [13]) (counts of the number and/or severity of depressive symptoms as assessed by an instrument that documents mood states but does not link or associate each with particular events or life circumstances, such as diabetes); and 3) as distress linked specifically to diabetes and its management assessed by diabetes-specific distress questionnaires (e.g., Problem Areas in Diabetes Scale [14] or Diabetes Distress Scale [15]). Unfortunately, distinctions among these three potentially different affective conditions often have not been made clear across studies; the term “depression” has often been used to refer to all three, and a large number of scales and measures have been used inconsistently to measure each. Consequently, a lack of clarity regarding what was being assessed and differences in the types of measures used have exacerbated the problems of exploring the relationship between depression and glycemic control.In a three-wave, longitudinal, observational study of 506 type 2 diabetic patients, we sought to clarify the differences and similarities among these three approaches to defining and measuring depression and their interrelationships with glycemic control by examining the systematic covariation of all three with glycemic control in the same cross-sectional, prospective, and time-varying analyses. Using well-established measures of each, our goals were to clarify issues of definition to provide clearer targets for the development of appropriate interventions. Three research questions were posed: First, what is the concurrent, independent relationship between each of these three affective constructs and A1C (cross-sectional analysis)? Second, does the level or occurrence of any or all of these three at initial assessment significantly predict changes in A1C over subsequent study waves (prospective analysis). Third, do fluctuations in any or all of these three correspond with fluctuations in A1C over study waves (time-varying analysis)? In addition, we explored the impact of patient demographics, diabetes status, medications, self-management behaviors, and extradisease stressors in each analysis.     

Atogepant Is Not Associated With Clinically Meaningful Alanine Aminotransferase Elevations in Healthy Adults

Atogepant is a potent, selective, oral calcitonin gene–related peptide (CGRP) receptor antagonist in development for migraine prevention. The chemical structure of atogepant is distinct from previous CGRP receptor antagonists, which were associated with elevated serum alanine aminotransferase (ALT) in clinical trials. Here, we report the safety, tolerability, and pharmacokinetics (PKs) of a once‐daily supratherapeutic dose (170 mg) of atogepant for 28 days from a randomized, double‐blind, placebo‐controlled phase I trial in healthy participants. Overall safety, hepatic safety, and plasma PK parameters were evaluated. Thirty‐four participants aged 23–55 years enrolled; 28 (82.4%) completed the study in accordance with the protocol. Multiple doses of 170 mg atogepant for 28 consecutive days were generally well‐tolerated. All adverse events (AEs; reported in 87.0% of the atogepant group; 72.7%, placebo) were mild in severity except one serious AE of subarachnoid hemorrhage due to a bicycle accident and not considered related to treatment. There were two discontinuations due to AEs, both with atogepant, one considered possibly related to treatment. Over 28 days of treatment, no participant receiving atogepant had an ALT elevation above 1.5 × upper limit of normal. Change from baseline in serum ALT levels was not different between atogepant and placebo. Atogepant is rapidly absorbed (median time to maximum plasma concentration, ~ 2 hours) with an apparent terminal half‐life of ~ 11 hours, and no evidence of accumulation after once‐daily dosing. Overall, atogepant at a high oral dose is safe and well‐tolerated in healthy participants with no clinically meaningful elevations in ALT.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

☑ Atogepant is a potent, selective, oral calcitonin gene–related peptide (CGRP) receptor antagonist in development for migraine prevention. The impact of atogepant on changes in alanine aminotransferase (ALT) has not yet been evaluated in a dedicated clinical trial.

• WHAT QUESTION DID THIS STUDY ADDRESS?

☑ The potential impact of once‐daily supratherapeutic doses (170 mg) of atogepant for 28 days on ALT levels in healthy adult participants.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

☑ Once‐daily supratherapeutic doses of atogepant for 28 days were found to be safe and well‐tolerated in healthy participants with no clinically meaningful elevations in ALT.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

☑ These findings will provide clinicians with the knowledge that atogepant, an oral CGRP receptor antagonist, is unlikely to be associated with drug‐induced liver injury.

Migraine is a highly prevalent and burdensome chronic neurological disease. ¹ , ² It is the second largest cause of disability worldwide, ³ and the leading cause of disability during the most productive ages of 15–49 years. ⁴ The disabling effects of migraine can also exert negative changes on many aspects of life, including work productivity, quality of life, and finances. ⁵ , ⁶ , ⁷ Effective and safe preventive treatment options are needed to help reduce the burden of migraine.Inhibition of calcitonin gene−related peptide (CGRP), a potent vasodilatory protein strongly implicated in the pathophysiology of migraine, has emerged as a targeted approach for migraine prevention and treatment. ⁸ Small‐molecule oral CGRP receptor antagonists (called gepants), such as ubrogepant ⁹ and rimegepant, ¹⁰ have demonstrated efficacy in the acute treatment of migraine attacks and were recently approved by the US Food and Drug Administration, ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ and rimegepant is being evaluated for prevention in adult patients with migraine ({"type":"clinical-trial","attrs":{"text":"NCT03732638","term_id":"NCT03732638"}}NCT03732638). Monoclonal antibodies that target CGRP or the CGRP receptor are currently available for adults in the United States and Europe. ⁸ Monoclonal antibodies require parenteral administration (subcutaneous or intravenous injection) and have a long elimination half‐life. ⁸ Therefore, the development of orally administered CGRP receptor antagonists may provide an alternative for people who prefer an oral route of administration over an injection.Atogepant is a potent, selective, small‐molecule antagonist of the CGRP receptor that is currently in development for the prevention of migraine, with a half‐life of ~ 11 hours. Atogepant is chemically distinct from prior oral CGRP receptor antagonists, notably telcagepant and MK‐3207, which were discontinued because of drug‐induced liver injury (DILI). ¹⁶ The efficacy and safety of atogepant in migraine prevention was demonstrated in a phase IIb/III clinical trial conducted subsequent to this trial in which treatment with atogepant, compared with placebo, significantly decreased monthly migraine days over 12 weeks. ¹⁷ Atogepant is in phase III development for migraine prevention (ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03700320","term_id":"NCT03700320"}}NCT03700320, {"type":"clinical-trial","attrs":{"text":"NCT03777059","term_id":"NCT03777059"}}NCT03777059, {"type":"clinical-trial","attrs":{"text":"NCT03855137","term_id":"NCT03855137"}}NCT03855137, and {"type":"clinical-trial","attrs":{"text":"NCT03939312","term_id":"NCT03939312"}}NCT03939312).This study evaluated the safety, tolerability, and pharmacokinetics (PKs) of multiple oral 170 mg doses of atogepant in healthy adult participants. The 170 mg dose is substantially higher than doses being tested in phase III clinical trials (once daily 10, 30, and 60 mg, and twice daily 30 and 60 mg). The primary objectives were to evaluate the safety and tolerability, and the mean fold change from baseline of alanine aminotransferase (ALT) after 28 days of once‐daily atogepant dosing in healthy participants. The secondary objective was to obtain preliminary plasma PK data following multiple‐dose administration of atogepant. The primary safety end point was mean fold change from baseline in serum ALT.     

Mitral Annular Calcification Is Associated with Pulse Wave Velocity but Not with Augmentation Index

Objective

To investigate the possible relationship between mitral annular calcification (MAC) and arterial stiffness.

Subjects and Methods

Forty-two patients (mean age 68 ± 6 years) with MAC and an age-matched control group of 41 individuals (mean age 66 ± 6 years) were studied. Arterial stiffness and wave reflections of the study population were evaluated by using applanation tonometry (SphygmoCor). Aortic pulse wave velocity (PWV) was measured as an index of aortic stiffness. The heart rate-corrected augmentation index (AIx@75) was estimated as a composite marker of wave reflections and arterial stiffness.

Results

Aortic PWV was significantly higher in patients with MAC (12.2 ± 2.3 m/s) than in controls (10.1 ± 1.3 m/s, p = 0.0001). However, AIx@75 was similar between the groups (28 ± 10 vs. 29 ± 10%, p = 0.59). Multivariate analysis involving the whole population revealed that brachial diastolic blood pressure (β = 1.87, p = 0.04) and MAC (β = 0.41, p = 0.0001) were independent determinants of aortic PWV.

Conclusion

The data showed that MAC was significantly associated with increased arterial stiffness and that it was an independent determinant of aortic PWV.Key Words: Mitral annular calcification, Pulse wave velocity, Augmentation index     

Salivary Cortisol Release and Hypothalamic Pituitary Adrenal Axis Feedback Sensitivity in Fibromyalgia Is Associated With Depression But Not With Pain

Results on hypothalamicpituitary-adrenal (HPA) axis function in fibromyalgia are heterogeneous and studies that integrate psychological and biological mechanisms in the search for pathways to fibromyalgia are rare. The goal of the study was to evaluate cortisol release and HPA axis feedback regulation in fibromyalgia and its association with psychopathology and pain. Beneath assessment of pain thresholds and self-report of pain, salivary free cortisol release over the day before and after intake of 0.5 mg of dexamethasone was measured in 21 female patients with fibromyalgia and 26 control women. Depression was assessed by questionnaires and clinical interview. We found reduced feedback sensitivity and slightly enhanced cortisol release in patients with fibromyalgia compared with healthy control subjects. Post hoc analyses showed that these effects are exclusively found in those patients, who also had major depressive disorder. Patients with fibromyalgia had lower pain pressure threshold, whereas heat pain thresholds were comparable with control subjects. Pain pressure and heat pain thresholds were not associated with cortisol release. On the other hand measurements of affective pain experience and depression were positively correlated with salivary cortisol over the day. Our results support the hypotheses that HPA axis related alterations are associated with affective disturbances, for example, depression, in patients with fibromyalgia.     

Functional Status Impairment Is Associated With Unplanned Readmissions

Objective

To determine whether functional status on admission to a Comprehensive Integrated Inpatient Rehabilitation Program (CIIRP) is associated with unplanned readmission to acute care.

Design

Retrospective cohort study.

Setting

Academic hospital-based CIIRP.

Participants

Consecutive patients (N=1515) admitted to a CIIRP between January 2009 and June 2012.

Interventions

Patients' functional status, the primary exposure variable, was assessed using tertiles of the total FIM score at CIIRP admission, with secondary analyses using the FIM motor and cognitive domains. A propensity score, consisting of 25 relevant clinical and demographic variables, was used to adjust for confounding in the analysis.

Main Outcome Measures

Readmission to acute care was categorized as (1) readmission before planned discharge from the CIIRP, (2) readmission within 30 days of discharge from the CIIRP, and (3) total readmissions from both groups, with total readmissions being the a priori primary outcome.

Results

Among the 1515 patients, there were 347 total readmissions. Total readmissions were significantly associated with FIM scores, with adjusted odds ratios (AORs) and 95% confidence intervals (CIs) for the lowest and middle FIM tertiles versus the highest tertile (AOR=2.6; 95% CI, 1.9–3.7; P<.001 and AOR=1.7; 95% CI, 1.2–2.4; P=.002, respectively). There were similar findings for secondary analyses of readmission before planned discharge from the CIIRP (AOR=3.5; 95% CI, 2.2–5.8; P<.001 and AOR=2.1; 95% CI, 1.3–3.5l P=.002, respectively), and a weaker association for readmissions after discharge from the CIIRP (AOR=1.6; 95% CI, 1.0–2.4; P=.047 and AOR=1.3; 95% CI, 0.8–1.9; P=.28, respectively). The FIM motor domain score was more strongly associated with readmissions than the FIM cognitive score.

Conclusions

Functional status on admission to the CIIRP is strongly associated with readmission to acute care, particularly for motor aspects of functional status and readmission before planned discharge from the CIIRP. Efforts to reduce hospital readmissions should consider patient functional status as an important and potentially modifiable risk factor.     

Fear of Movement Is Not Associated With Objective and Subjective Physical Activity Levels in Chronic Nonspecific Low Back Pain

Objectives

To assess the association of physical activity measures, derived with an accelerometer and a self-reported questionnaire, with fear of movement in patients with chronic nonspecific low back pain (LBP) and to investigate the association between disability and fear of movement in this population.