Persons with reconstructed ACL exhibit altered knee mechanics during high-speed maneuvers.

Anterior cruciate ligament (ACL) injury is a sports trauma that causes long-term disability. The function of the knee during dynamic activities can be severely limited even after successful surgical reconstruction. This study examined the effects of approach velocity during side-step cutting on knee joint mechanics in persons with reconstructed ACL (ACLR). 22 participants (11 with unilateral ACLR, 11 matched-controls) participated. Knee joint mechanics were tested in 3 approach conditions: counter-movement, one-step, and running. Dependent variables, including peak knee flexion, extension, valgus, varus, internal rotation, external rotation angles and corresponding peak joint moments, were assessed during the stance phase of cutting. Two 2×3 ("group" by "approach condition") mixed MANOVA tests were used to examine the effects of ACLR and approach velocity on knee mechanics. ACLR participants exhibited higher knee internal rotator moment (0.22 vs. 0.13 Nm/kg, p=0.003). Inter-group comparisons revealed that the ACLR participants exhibited significantly higher abductor and internal rotator moments only in the running condition (1.86 vs. 1.16 Nm/kg, p=0.018; 0.28 vs. 0.17 Nm/kg, p=0.010, respectively). Our findings suggested that patients with ACLR may be at increased risk of re-injury when participating in high-demand physical activities. Task demand should be considered when prescribing progressive therapeutic interventions to ACLR patients.

Cord blood T cells retain early differentiation phenotype suitable for immunotherapy after TCR gene transfer to confer EBV specificity.

Adoptive T cell therapy can be effective for Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease and melanoma. Transducing high-affinity TCR genes into T lymphocytes is an emerging method to improve potency and specificity of tumor-specific T cells. However, both methods necessitate in vitro lymphocyte proliferation, generating highly differentiated effector cells that display reduced survival and antitumor efficacy postinfusion. TCR-transduction of naive lymphocytes isolated from peripheral blood is reported to provide superior in vivo survival and function. We utilized cord blood (CB) lymphocytes, which comprise mainly naive cells, for transducing EBV-specific TCR. Comparable TCR expression was achieved in adult and CB cells, but the latter expressed an earlier differentiation profile. Further antigen-driven stimulation skewed adult lymphocytes to a late differentiation phenotype associated with immune exhaustion. In contrast, CB T cells retained a less differentiated phenotype after antigen stimulation, remaining CD57-negative but were still capable of antigen-specific polyfunctional cytokine expression and cytotoxicity in response to EBV antigen. CB T cells also retained longer telomeres and in general possessed higher telomerase activity indicative of greater proliferative potential. CB lymphocytes therefore have qualities indicating prolonged survival and effector function favorable to immunotherapy, especially in settings where donor lymphocytes are unavailable such as in solid organ and CB transplantation.

Three-dimensional analysis of deciduous maxillary anterior teeth using cone-beam computed tomography.

The recent introduction of cone-beam computed tomography (CBCT) into the medical field has allowed the nondestructive investigation of internal structures at relatively low cost and radiation exposure. The accuracy of CBCT in both two and three dimensions has been demonstrated, and CBCT has been used successfully for craniofacial anatomy. Knowing the anatomical structure of deciduous teeth is essential for clinical dentistry. However, the root structure of deciduous teeth is rarely reported because of the scarcity of intact deciduous teeth without root resorption. The aim of this study was to evaluate the intact root form of deciduous teeth using CBCT. Data from 38 young children was analyzed using an image-analyzing program. The degree of buccal dilacerations was 26.3° for deciduous maxillary central incisors (DMA), 16.5° for deciduous maxillary lateral incisors (DMB), and 17.5° for deciduous maxillary canines (DMC) in about half of the root length. The crown-to-root ratios were 0.52 for DMA, 0.48 for DMB, and 0.52 for DMC. These data will be helpful for understanding the development of dentition, and for clinical dentistry.

A FFAR1 full agonist restores islet function in models of impaired glucose-stimulated insulin secretion and diabetic non-human primates.

The free fatty acid receptor 1 (FFAR1/GPR40) mediates fatty acid-induced insulin secretion from pancreatic ß-cells. At least 3 distinct binding sites exist on the FFAR1 receptor and numerous synthetic ligands have been investigated for their anti-diabetic actions. Fasiglifam, binds to site-1 and stimulates intra-cellular calcium release and improves glycemic control in diabetic patients. Recently, small molecule FFAR1 agonists were discovered which bind to site-3, stimulating both intra-cellular calcium and cAMP, resulting in insulin and glucagon-like peptide-1 (GLP-1) secretion. The ability of our site-3 FFAR1 agonist (compound A) to control blood glucose was evaluated in spontaneously diabetic cynomolgus monkeys during an oral glucose tolerance test. In type-2 diabetic (T2D) animals, significant reductions in blood glucose and insulin were noted. To better understand the mechanism of these in vivo findings, we evaluated the effect of compound A in islets under several conditions of dysfunction. First, healthy human and non-human primate islets were treated with compound A and showed potentiation of insulin and glucagon secretion from both species. Next, we determined glucose-responsive insulin secretion under gluco-lipotoxic conditions and from islets isolated from type-2 diabetic humans. Despite a dysfunctional phenotype that failed to secrete insulin in response to glucose, site-3 FFAR1 agonism not only enhanced insulin secretion, but restored glucose responsiveness across a range of glucose concentrations. Lastly, we treated ex vivo human islets chronically with a sulfonylurea to induce secondary beta-cell failure. Again, this model showed reduced glucose-responsive insulin secretion that was restored and potentiated by site-3 FFAR1 agonism. Together these data suggest a mechanism for FFAR1 where agonists have direct effects on islet hormone secretion that can overcome a dysfunctional T2D phenotype. These unique characteristics of FFAR1 site-3 agonists make them an appealing potential therapy to treat type-2 diabetes.

Lower levels of glutathione in the brains of secondary progressive multiple sclerosis patients measured by 1H magnetic resonance chemical shift imaging at 3 T.

BACKGROUND: Disability levels for patients with secondary progressive multiple sclerosis (SPMS) often worsen despite a stable MRI T(2) lesion burden. The presence of oxidative stress in the absence of measurable inflammation could help explain this phenomenon. In this study, the assessment of an in vivo marker of oxidative stress, cerebral glutathione (GSH), using magnetic resonance chemical shift imaging (CSI) is described, and GSH levels were compared in patients with SPMS and healthy controls. OBJECTIVE: To assess whether GSH, a key antioxidant in the brain, is lower in the SPMS patients compared to matched controls. METHODS: Seventeen patients with SPMS (Expanded Disability Status Scale=4.0-7.0; length of MS diagnosis=19.4 ± 7 years) and 17 age- and gender-matched healthy controls were studied. GSH levels were measured in the fronto-parietal regions of the brain using a specially designed magnetic resonance spectroscopy technique, CSI of GSH, at 3T. RESULTS: The levels of GSH were lower for SPMS patients than for controls, the largest reduction (18.5%) being in the frontal region (p=0.001). CONCLUSION: The lower GSH levels in these patients indicate the presence of oxidative stress in SPMS. This process could be at least partially responsible for ongoing functional decline in SPMS.

Epitope focusing in the primary cytotoxic T cell response to Epstein-Barr virus and its relationship to T cell memory.

The relationship between primary and memory cytotoxic T lymphocyte (CTL) responses, and the factors influencing entry into memory, are poorly understood. Here we address this in the context of Epstein-Barr virus (EBV), a persistent human herpesvirus in which memory CTL responses in long-term virus carriers are highly focused on epitopes preferentially drawn from just three of the eight available virus latent proteins, EBNAs 3A, 3B, and 3C. To determine whether this unusual level of focusing is a consequence of long-term virus challenge, we carried out a detailed analysis of EBV antigen/epitope specificities in the primary virus-induced CTL response in 10 infectious mononucleosis (IM) patients of different HLA types. Primary effectors, studied in ex vivo assays and by limiting dilution cloning in vitro, were again highly skewed toward a small number of viral epitopes, almost all derived from the EBNA3 proteins, with CTL to the immunodominant epitope accounting for at least 1% of the circulating CD8+ IM T cell pool. This is the first unequivocal demonstration of an EBV-specific CD8+ CTL response in IM. Prospective studies on individual patients showed that, whereas all of the EBV reactivities found in CTL memory had been detectable earlier during primary infection, the memory population was not simply a scaled down version of the primary response. In particular (a) differences in the relative frequencies of CTL to immunodominant versus subdominant epitopes appeared to be much less marked in memory than in primary populations, and (b) we found at least one clear example in which a significant virus-specific reactivity within the primary response was never detectable in memory.

T cell responses and virus evolution: loss of HLA A11-restricted CTL epitopes in Epstein-Barr virus isolates from highly A11-positive populations by selective mutation of anchor residues.

Epstein-Barr virus (EBV) is a B lymphotropic herpesvirus of humans that elicits strong HLA class I-restricted cytotoxic T lymphocyte (CTL) responses. An influence of such responses on virus evolution was first suggested by our finding that EBV isolates from the highly HLA A11-positive Papua New Guinea (PNG) population carried a lys-thr mutation at residue 424 of the nuclear antigen EBV-encoded nuclear antigen (EBNA4) that destroyed the immunodominant target epitope for A11-restricted CTL recognition. Here we turn to a much larger population, Southern Chinese, where the A11 allele is again present in over 50% of the individuals. Each of 23 EBV isolates analyzed from this population were also mutated in the EBNA4 416-424 epitope, the mutations selectively involving one of the two anchor residues in positions 2 (417 val-leu) or 9 (424 lys-asp, -arg or -thr) that are critical for A11-peptide interaction. The majority of the Chinese isolates and all 10 PNG isolates also carried mutations affecting positions 1 and 2 of the next most immunodominant A11-restricted epitope, EBNA4 residues 399-408. These changes clearly affected antigenicity since A11-positive lymphoblastoid cell lines (LCLs) carrying these mutant EBV strains were not recognized by A11-restricted CTLs raised against the prototype B95.8 virus. Furthermore, Chinese donors naturally infected with these mutant viruses did not mount detectable A11-restricted CTL responses on in vitro stimulation with autologous LCL cells carrying either the B95.8 or their endogenous EBV strain. In two different highly A11-positive populations, therefore, immune pressure appears to have selected for resident EBV strains lacking immunodominant A11-restricted CTL epitopes.

Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines against which they were raised.

We have raised CD8+ cytotoxic T lymphocytes (CTL) from three Epstein-Barr virus-seropositive donors by incubating peripheral blood lymphocytes with irradiated autologous B95.8-strain EBV-transformed B lymphoblastoid cells (LCL). However, to detect lysis in a standard 51Cr release assay of the LCL against which these CTL were raised, superinfection with recombinant vaccinia expressing the appropriate EBV protein or incubation with the peptide epitope was necessary. The untreated LCL were not lysed, even though Western blotting demonstrated that they expressed the EBV antigens containing the CTL epitopes. We have found CTL of this phenotype that are restricted by human leukocyte antigen-A2, -A3, -B7, or -B39, and which recognize the EBV latent proteins, EBV nuclear antigen (EBNA)-3A, EBNA-3C, or terminal protein. During these experiments, we identified a new human leukocyte antigen-A3-restricted EBNA-3A epitope, residues 603-611, RLRAEAGVK. We raised a spontaneous LCL, transformed by endogenous EBV, from one donor, but this was also not lysed. For at least one of the epitopes, CTL from another donor lysed the LCL without superinfection or addition of peptides. We conclude that the CTL were unable to achieve a high enough avidity of interaction with untreated LCL to trigger effector function, although the LCL were able to stimulate them to grow in vitro for up to 4 mo. To assess whether a small percentage of the LCL might possess a higher antigen density, we used an assay of tumor necrosis factor release from a CTL clone, which was able to detect antigen-bearing cells representing only 1% of a stimulating LCL population. Nevertheless, the untreated autologous LCL line failed to stimulate tumor necrosis factor release.

Cornulin, a marker of late epidermal differentiation, is down-regulated in eczema.

BACKGROUND: Eczema is a common chronic inflammatory skin disorder which shows strong genetic predisposition. To identify new potential molecular determinants of the disease pathogenesis, we performed a gene expression study in an eczema mouse model. This analysis identified a marked down regulation of the cornulin gene (CRNN), a member of the epidermal differentiation complex, in the eczema-like skin. We then investigated CRNN as an eczema candidate gene and studied its polymorphism and the expression in the skin of eczema patients. METHODS: An eczema-like phenotype was induced in mice by allergen (Der p2) patching. Gene expression analysis was performed with the subtractive suppression hybridization method and validated by real time PCR and the transmission disequilibrium test was used to test for genetic associations in 406 multiplex eczema families. RESULTS: Der p 2 patched mice developed a localized eczema and a Th 2 skewed systemic response. Real time PCR analysis confirmed a down regulation of CRNN mRNA in eczema-like skin in the mouse model and in human eczema. The CRNN polymorphism rs941934 was significantly associated with atopic eczema in the genetic analysis (P = 0.006), though only as part of an extended haplotype including a known associated variant (2282del4) in the filaggrin gene. CONCLUSIONS: CRNN mRNA expression is decreased in eczematous skin. Further studies are needed to verify whether the associated cornulin polymorphism contribute to the genetic susceptibility in eczema.

Aspirin prevention of cholesterol gallstone formation in prairie dogs.

When prairie dogs (Cynomys ludovicianus) are fed a diet containing cholesterol, a marked increase in gallbladder mucin secretion parallels the evolution of cholesterol supersaturated bile. Gelation of mucin precedes the precipitation of cholesterol liquid and solid crystals and the development of gallstones. Aspirin given to prairie dogs inhibited mucin hypersecretion and gel accumulation and prevented gallstone formation without influencing the cholesterol content of supersaturated bile. This suggests that gallbladder mucin is a nucleation matrix for cholesterol gallstones.

A novel statistical model for mandibular helical axis analysis.

The purpose of this study was to establish a new statistical method for the analysis of noisy mandibular helical axis parameters, especially the position vector of the finite helical axis (FHA). The subjects were children with anterior cross-bite who had received orthodontic treatment. Maximum mouth-opening was measured by means of an opto-electronic motion analysis system. These movements were compared with similar movement in the same group after treatment of their anterior cross-bite. Each curve of FHA position vectors was modelled as a spline function with random coefficients. To determine the optimal number of knots, two criteria were used: deviance information criteria (DIC) and mean squared prediction error (MSE). We were interested in estimating a typical curve for a population. Self-modelling regression (SEMOR) was extended to three dimensions to model groups of three-dimensional curves. Each curve was modelled as a spline function using nine knots. Population average curves were created using SEMOR. This study provided detailed information about jaw movement for comparing cross-bite to normal occlusion by calculating the population mean curves of the position vector of the FHA. Our results suggested that the two population mean curves for the position vector of the FHA were significantly different in the closing phase. The combination of a spline function with random coefficients and SEMOR extended to three dimensions can be used not only for FHA analysis but also for the analysis of other jaw movements.

Retroviral transfer of a dominant TCR prevents surface expression of a large proportion of the endogenous TCR repertoire in human T cells.

The latent membrane protein-2 (LMP2) of Epstein-Barr virus is a potential target for T-cell receptor (TCR) gene therapy of Hodgkin lymphoma and nasopharyngeal carcinoma. Here, we modified a human leukocyte antigen-A2-restricted, LMP2-specific TCR to achieve efficient expression following retroviral TCR gene transfer. The unmodified TCR was poorly expressed in primary human T cells, suggesting that it competed inefficiently with endogenous TCR chains for cell surface expression. In order to improve this TCR, we replaced the human constant region with murine sequences, linked the two TCR genes using a self-cleaving 2A sequence and finally, codon optimized the TCR-alpha-2A-beta cassette for efficient translation in human cells. Retroviral transfer of the modified TCR resulted in efficient surface expression and HLA-A2/LMP2 pentamer binding. The transduced cells showed peptide-specific interferon-gamma and interleukin-2 production and killed target cells displaying the LMP2 peptide. Importantly, the introduced LMP2-TCR suppressed the cell surface expression of a large proportion of endogenous TCR combinations present in primary human T cells. The design of dominant TCR is likely to improve TCR gene therapy by reducing the risk of potential autoreactivity of endogenous and mispaired TCR combinations.

Thymol and related alkyl phenols activate the hTRPA1 channel.

BACKGROUND AND PURPOSE: Thymol, a major component of thyme and oregano, has medical uses in oral care products as an astringent and antibiotic. Its distinctive sharp odour and pungent flavour are considered aversive properties. The molecular basis of these aversive properties is not well understood. EXPERIMENTAL APPROACH: The ability of thymol to activate human transient receptor potential channel A1 (hTRPA1) expressed in stably transfected human embryonic kidney 293 (HEK293) cells was measured by membrane potential and calcium-sensitive dyes in a fluorescence-imaging plate reader (FLIPR) assay. Direct activation of hTRPA1 currents was measured by whole-cell voltage clamp recording. Intracellular calcium changes were measured using fura-2 dye. The FLIPR assay was also used to measure membrane potential changes elicited by thymol after pretreatment with camphor, a known TRPA1 inhibitor. The ability of related alkyl phenols to activate hTRPA1 was also determined. KEY RESULTS: Thymol potently activated a membrane potential response and intracellular calcium increase in hTRPA1-expressing HEK293 cells in a concentration-dependent manner. Activation by thymol desensitized hTRPA1 to further exposure to thymol or the known ligand allyl isothiocyanate (AITC). The related phenols 2-tert-butyl-5-methylphenol, 2,6-diisopropylphenol (propofol) and carvacrol also activated hTRPA1. Phenols with less bulky carbon substitutions and lower logP values were less potent in general. The response to thymol was blocked by camphor. CONCLUSIONS AND IMPLICATIONS: These results suggest a role for hTRPA1 activation in the reported pungent and aversive properties of some of these pharmaceutically important phenols.

The Singaporean public beliefs about the causes of mental illness: results from a multi-ethnic population-based study.

AIMS: To identify the common causal beliefs of mental illness in a multi-ethnic Southeast Asian community and describe the sociodemographic associations to said beliefs. The factor structure to the causal beliefs scale is explored. The causal beliefs relating to five different mental illnesses (alcohol abuse, depression, obsessive-compulsive disorder (OCD), dementia and schizophrenia) and desire for social distance are also investigated. METHODS: Data from 3006 participants from a nationwide vignette-based study on mental health literacy were analysed using factor analysis and multiple logistic regression to address the aims. Participants answered questions related to sociodemographic information, causal beliefs of mental illness and their desire for social distance towards those with mental illness. RESULTS: Physical causes, psychosocial causes and personality causes were endorsed by the sample. Sociodemographic differences including ethnic, gender and age differences in causal beliefs were found in the sample. Differences in causal beliefs were shown across different mental illness vignettes though psychosocial causes was the most highly attributed cause across vignettes (endorsed by 97.9% of respondents), followed by personality causes (83.5%) and last, physical causes (37%). Physical causes were more likely to be endorsed for OCD, depression and schizophrenia. Psychosocial causes were less often endorsed for OCD. Personality causes were less endorsed for dementia but more associated with depression. CONCLUSIONS: The factor structure of the causal beliefs scale is not entirely the same as that found in previous research. Further research on the causal beliefs endorsed by Southeast Asian communities should be conducted to investigate other potential causes such as biogenetic factors and spiritual/supernatural causes. Mental health awareness campaigns should address causes of mental illness as a topic. Lay beliefs in the different causes must be acknowledged and it would be beneficial for the public to be informed of the causes of some of the most common mental illnesses in order to encourage help-seeking and treatment compliance.

PPARG locus haplotype variation and exacerbations in asthma.

The peroxisome proliferator-activated receptor gamma (PPARgamma) regulates inflammation and may play a role in asthma. Using mouthwash-derived DNA and clinical interviews and measurements, we investigated the association of previously characterized single-nucleotide polymorphisms in the PPARG gene (Pro12Ala, C1431T, and C-681G) with asthma exacerbations in patients aged 3-22 years (n=569). The common homozygous haplotype combination of the Pro12 and C1431 alleles was associated with increased risk for asthma exacerbations (ProC, odds ratio (OR) 1.87, 95% confidence interval 1.25-2.79; P=0.002). The ProC genotype was associated with increased school absences (OR 1.82, 95% confidence interval 1.21-2.76; P=0.004) and hospital admissions (OR 2.32, 95% confidence interval 1.18-4.58; P=0.015) over the preceding 6 months. The population-attributable risk of this genotype was 33%. Common genetic variation at the PPARG locus may play an important role in modulating the long-term control of asthma in children and young adults.

Role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones.

Because mucin glycoproteins may be important in the pathophysiology of gallstones, we studied the relationship among biliary lipids, gallbladder mucin secretion, and gallstone formation in cholesterol-fed prairie dogs. Organ culture studies of gallbladder explants revealed that the incorporation of [(3)H]glucosamine into tissue and secretory gallbladder glycoproteins was significantly increased at 3, 5, 8, and 14 d of feeding. Peak secretion of labeled mucin occurred at 5 d, when total tissue and secreted glycoprotein production was fivefold greater than control. Gel filtration of the secreted glycoprotein on Sepharose 4B indicated that the majority of radioactivity was present in a macromolecule of > 1 million molecular weight. The increased secretion of gallbladder mucin was organ specific, in that [(3)H]glucosamine incorporation into glycoproteins of stomach and colon was unaffected by cholesterol feeding. Similarly, the incorporation of [(3)H]mannose into gallbladder membrane glycoproteins was not altered by cholesterol feeding. The rate of glycoprotein synthesis and secretion returned to normal upon withdrawal of the cholesterol diet, and ligation of the cystic duct before cholesterol feeding prevented gallbladder mucin hypersecretion. Both results indicate that the stimulus to mucin secretion was a constituent of bile. Gallbladder bile after 5 d contained cholesterol in micelles, liquid crystals, and crystals, whereas hepatic bile remained a single micellar phase throughout cholesterol feeding. For this reason the cholesterol-saturation indices of gallbladder bile were compared in both homogenized and centrifuged samples. The micellar phase of gallbladder bile was appreciably less saturated than homogenized bile at 5 and 8 d, which reflects the continuous nucleation of cholesterol in the gallbladder. Purified human gallbladder mucin gels were shown to induce nucleation of lecithin-cholesterol liquid crystals from supersaturated hepatic bile. These in turn gave rise to cholesterol monohydrate crystals within 18 h. Control supersaturated hepatic bile could not be nucleated by the addition of other proteins, and was stable for days upon standing. These results suggest that the increase in cholesterol content of bile in cholesterolfed prairie dogs stimulates gallbladder mucus hypersecretion, and that gallbladder mucus gel is a nucleating agent for biliary cholesterol.

Cytotoxic T lymphocyte responses to Epstein-Barr virus.

Epstein-Barr virus induces a potent cytotoxic T lymphocyte response in man that is preferentially directed towards a particular subset of the virus latent cycle antigens; the immunodominance of these proteins, apparent in both primary and memory responses, may reflect some differential access to the HLA class I processing pathway. Effector cells recognizing these immunodominant antigens can reverse the growth of virus-induced lymphoproliferative lesions in immunosuppressed patients; however, responses to some of the subdominant latent proteins will be needed to target other virus-positive tumours.

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.