The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Cezmi A Akdis - One of the best experts on this subject based on the ideXlab platform.
-
Endotypes of allergic diseases and asthma an important step in building blocks for the future of precision medicine
Allergology International, 2016Co-Authors: Ioana Agache, Cezmi A AkdisAbstract:Discoveries from basic science research in the last decade have brought significant progress in knowledge of pathophysiologic processes of allergic diseases, with a compelling impact on understanding of the natural history, risk prediction, treatment selection or mechanism-specific prevention strategies. The view of the pathophysiology of allergic diseases developed from a mechanistic approach, with a focus on symptoms and organ function, to the recognition of a complex network of immunological pathways. Several subtypes of inflammation and complex immune-regulatory networks and the reasons for their failure are now described, that open the way for the development of new diagnostic tools and innovative targeted-treatments. An Endotype is a subtype of a disease condition, which is defined by a distinct pathophysiological mechanism, whereas a disease phenotype defines any observable characteristic of a disease without any implication of a mechanism. Another key word linked to disease endotyping is biomarker that is measured and evaluated to examine any biological or pathogenic processes, including response to a therapeutic intervention. These three keywords will be discussed more and more in the future with the upcoming efforts to revolutionize patient care in the direction of precision medicine and precision health. The understanding of disease Endotypes based on pathophysiological principles and their validation across clinically meaningful outcomes in asthma, allergic rhinitis, chronic rhinosinusitis, atopic dermatitis and food allergy will be crucial for the success of precision medicine as a new approach to patient management.
-
phenotypes and emerging Endotypes of chronic rhinosinusitis
The Journal of Allergy and Clinical Immunology: In Practice, 2016Co-Authors: Claus Bachert, Cezmi A AkdisAbstract:Chronic rhinosinusitis can be differentiated into several phenotypes based on clinical criteria; however, these phenotypes do not teach us much about the underlying inflammatory mechanisms. Thus, the use of nasal endoscopy and CT scanning, and eventually taking a swab or a biopsy, may not be sufficient to fully appreciate the individual patient's pathology. Endotyping of chronic rhinosinusitis on the basis of pathomechanisms, functionally and pathologically different from others by the involvement of specific molecules or cells, may in contrast provide us with information on the risk of disease progression or recurrence and on the best available treatment, and also helps us identifying innovative therapeutic targets for treatment. Endotyping may best be structured around T helper cells and their downstream events, such as tissue eosinophilia or neutrophilia; this approach involves the cytokines and chemokines related to specific T helper cell populations, and related markers such as IgE. Endotyping is of specific interest at the time of the arrival of new biologicals, confronting us with the challenge of the selection of eligible patients for treatment and predicting their therapeutic response; defining suitable biomarkers is therefore an urgent task. Failure to appreciate the underlying mechanisms and Endotypes of chronic rhinosinusitis may limit progress in the management of the disease at present.
-
precision medicine in patients with allergic diseases airway diseases and atopic dermatitis practall document of the european academy of allergy and clinical immunology and the american academy of allergy asthma immunology
The Journal of Allergy and Clinical Immunology, 2016Co-Authors: Antonella Muraro, Robert F. Lemanske, Lars K. Poulsen, Cezmi A Akdis, T Bieber, Thomas B Casale, Marek Jutel, Peter Hellings, Peck Y Ong, Peter SchmidgrendelmeierAbstract:In this consensus document we summarize the current knowledge on major asthma, rhinitis, and atopic dermatitis Endotypes under the auspices of the PRACTALL collaboration platform. PRACTALL is an initiative of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma & Immunology aiming to harmonize the European and American approaches to best allergy practice and science. Precision medicine is of broad relevance for the management of asthma, rhinitis, and atopic dermatitis in the context of a better selection of treatment responders, risk prediction, and design of disease-modifying strategies. Progress has been made in profiling the type 2 immune response-driven asthma. The Endotype driven approach for non-type 2 immune response asthma, rhinitis, and atopic dermatitis is lagging behind. Validation and qualification of biomarkers are needed to facilitate their translation into pathway-specific diagnostic tests. Wide consensus between academia, governmental regulators, and industry for further development and application of precision medicine in management of allergic diseases is of utmost importance. Improved knowledge of disease pathogenesis together with defining validated and qualified biomarkers are key approaches to precision medicine.
-
The Complex Type 2 Endotype in Allergy and Asthma: From Laboratory to Bedside
Current Allergy and Asthma Reports, 2015Co-Authors: Ioana Agache, Kazunari Sugita, Hideaki Morita, Mübeccel Akdis, Cezmi A AkdisAbstract:Better management of allergic diseases needs a sharpened understanding of disease heterogeneity and mechanisms in relation to clinically significant outcomes. Phenotypes describing observable clinical and morphologic characteristics and unique responses to treatment have been developed; however, they do not relate to disease mechanisms. Recently, extended heterogeneous and disease-related metabolic, inflammatory, immunological, and remodeling pathways have been described, and reproducible patterns are defined as disease Endotypes. An Endotype might consist of several intricated mechanisms that cannot be clearly separated into “pure single molecular mechanism” thus being a “complex Endotype.” The description of an Endotype may rely on biomarkers, which can be the signature of a complex underlying pathway or a key molecule associated with or directly playing a role in a particular disease Endotype. The Th2 type inflammation can be defined as a complex Endotype in asthma and linked to mechanisms of disease development and response to treatment and to disease outcomes such as exacerbations and remodeling. The type 2 complex Endotype in allergies and asthma includes innate lymphoid cells, T helper 2 cells, tissue eosinophilia, and IgE production. Currently, emerging Endotype-driven strategies in asthma, particularly the development of biologicals that target a single molecular pathway, are being focused for solving individualized clinical problems on disease outcomes. Progress is also being made for endotyping rhinitis, chronic rhinosinusitis, and atopic dermatitis.
-
Endotypes and phenotypes of chronic rhinosinusitis a practall document of the european academy of allergy and clinical immunology and the american academy of allergy asthma immunology
The Journal of Allergy and Clinical Immunology, 2013Co-Authors: Cezmi A Akdis, Peter Hellings, Claus Bachert, Cemal Cingi, Mark S Dykewicz, Robert M Naclerio, Robert P Schleimer, Dennis K LedfordAbstract:Chronic rhinosinusitis (CRS) is a complex disease consisting of several disease variants with different underlying pathophysiologies. Limited knowledge of the mechanisms of these disease subgroups is possibly the greatest obstacle in understanding the causes of CRS and improving treatment. It is generally agreed that there are clinically relevant CRS phenotypes defined by an observable characteristic or trait, such as the presence or absence of nasal polyps. Defining the phenotype of the patient is useful in making therapeutic decisions. However, clinical phenotypes do not provide full insight into all underlying cellular and molecular pathophysiologic mechanisms of CRS. Recognition of the heterogeneity of CRS has promoted the concept that CRS consists of multiple groups of biological subtypes, or “Endotypes,” which are defined by distinct pathophysiologic mechanisms that might be identified by corresponding biomarkers. Different CRS Endotypes can be characterized by differences in responsiveness to different treatments, including topical intranasal corticosteroids and biological agents, such as anti–IL-5 and anti-IgE mAb, and can be based on different biomarkers that are linked to underlying mechanisms. CRS has been regarded as a single disease entity in clinical and genetic studies in the past, which can explain the failure to identify consistent genetic and environmental correlations. In addition, better identification of Endotypes might permit individualization of therapy that can be targeted against the pathophysiologic processes of a patient's Endotype, with potential for more effective treatment and better patient outcomes.
Kousaku Murata - One of the best experts on this subject based on the ideXlab platform.
-
structural determinants responsible for substrate recognition and mode of action in family 11 polysaccharide lyases
Journal of Biological Chemistry, 2009Co-Authors: Akihito Ochiai, Bunzo Mikami, Wataru Hashimoto, Takafumi Itoh, Kousaku MurataAbstract:A saprophytic Bacillus subtilis secretes two types of rhamnogalacturonan (RG) lyases, Endotype YesW and exotype YesX, which are responsible for an initial cleavage of the RG type I (RG-I) region of plant cell wall pectin. Polysaccharide lyase family 11 YesW and YesX with a significant sequence identity (67.8%) cleave glycoside bonds between rhamnose and galacturonic acid residues in RG-I through a β-elimination reaction. Here we show the structural determinants for substrate recognition and the mode of action in polysaccharide lyase family 11 lyases. The crystal structures of YesW in complex with rhamnose and ligand-free YesX were determined at 1.32 and 1.65 A resolution, respectively. The YesW amino acid residues such as Asn152, Asp172, Asn532, Gly533, Thr534, and Tyr595 in the active cleft bind to rhamnose molecules through hydrogen bonds and van der Waals contacts. Other rhamnose molecules are accommodated at the noncatalytic domain far from the active cleft, revealing that the domain possibly functions as a novel carbohydrate-binding module. A structural comparison between YesW and YesX indicates that a specific loop in YesX for recognizing the terminal saccharide molecule sterically inhibits penetration of the polymer over the active cleft. The loop-deficient YesX mutant exhibits YesW-like Endotype activity, demonstrating that molecular conversion regarding the mode of action is achieved by the addition/removal of the loop for recognizing the terminal saccharide. This is the first report on a structural insight into RG-I recognition and molecular conversion of exotype to Endotype in polysaccharide lyases.
-
Molecular identification of Sphingomonas sp. A1 alginate lyase (A1-IV') as a member of novel polysaccharide lyase family 15 and implications in alginate lyase evolution.
Journal of Bioscience and Bioengineering, 2005Co-Authors: Wataru Hashimoto, Osamu Miyake, Akihito Ochiai, Kousaku MurataAbstract:Abstract Sphingomonas sp. A1 (strain A1) produces three Endotypes (A1-I [65 kDa], A1-II [25 kDa], and A1-III [40 kDa]) and an exotype (A1-IV [86 kDa]) alginate lyases in cytoplasm. These four enzymes cooperatively depolymerize alginate into constituent monosaccharides. In addition to the genes for these lyases, novel genes encoding hypothetical proteins homologous with A1-IV were found in the genomes of many bacteria including strain A1. One such protein, A1-IV′ (90 kDa) of strain A1, was overexpressed in Escherichia coli cells, purified, and characterized. A1-IV′ catalyzed the cleavage of glycosidic bonds in alginate through a β-elimination reaction and released unsaturated di- and trisaccharides as main products, thus indicating that the enzyme is an Endotype alginate lyase. A1-IV′, which differed from A1-IV in some enzymatic properties, was not expressed in strain A1, suggesting that A1-IV′ has no significant role in alginate metabolism. A1-IV′ and other A1-IV homologs facilitate the creation of novel polysaccharide lyase family 15 based on their primary structures, implying the evolution route of alginate lyases in family PL-15.
-
origin and diversity of alginate lyases of families pl 5 and 7 in sphingomonas sp strain a1
Journal of Bacteriology, 2004Co-Authors: Osamu Miyake, Wataru Hashimoto, Akihito Ochiai, Kousaku MurataAbstract:ABSTRACT Sphingomonas sp. strain A1 has three Endotype alginate lyases (A1-I, A1-II [family PL-7], and A1-III [family PL-5]), each of which is encoded by a single gene. In addition to those of these lyases, a gene (the A1-II′ gene) showing significant identity with the A1-II gene was present in the bacterial genome and coded for an alginate lyase with broad substrate specificity. Since no expression of A1-II′ was observed even in bacterial cells grown on alginate, the A1-II′ gene was thought to be a silent gene derived from the A1-II gene, presumably through duplication, modification, and translocation.
Claus Bachert - One of the best experts on this subject based on the ideXlab platform.
-
Endotype driven approach for chronic rhinosinusitis
Implementing precision medicine in best practices of chronic airway diseases, 2018Co-Authors: Claus Bachert, Peter TomassenAbstract:Abstract Chronic rhinosinusitis (CRS) can be differentiated into several phenotypes based on clinical criteria, using nasal endoscopy and computed tomography scanning; in contrast, endotyping of CRS aims to clarify the pathomechanisms, to provide information on the risk of disease progression or recurrence and on the possible use of innovative biologic treatment. Endotyping may best be structured around T-helper cells and their downstream events, such as tissue eosinophilia or neutrophilia; this approach involves the cytokines and chemokines related to specific T-helper cell populations and related markers such as IgE, ECP, and MPO. Endotyping is of specific interest for the development of new biologicals and prediction of the therapeutic response to them. Defining suitable biomarkers is, therefore, an urgent task.
-
Endotype-driven care pathways in patients with chronic rhinosinusitis
Journal of Allergy and Clinical Immunology, 2018Co-Authors: Claus Bachert, Nan Zhang, Peter W. Hellings, Jean BousquetAbstract:Chronic rhinosinusitis (CRS) has been differentiated clinically into CRS without nasal polyps and CRS with nasal polyps, with both forms subjected to glucocorticosteroid and antibiotic treatments and, if not successful, to nasal and sinus surgery tailored to endoscopic and computed tomographic scan findings. The elaboration of Endotypes based on pathomechanisms involving different immune responses offers new possibilities in terms of prediction of prognosis and risks and sophisticated guidance in personalized pharmacotherapy, surgical approaches, and innovative treatment approaches in the CRS field with various biologics. Surgical approaches can vary from classical functional endoscopic sinus surgery to extended and "reboot" approaches, with the idea to completely remove the dysfunctional and inflamed mucosa and replace it with a newly grown healthy mucosa. Biologics in this field are targeting the type 2 cytokines IL-4, IL-5, and IL-13, as well as IgE. Phase I and II study results are promising, and phase III studies are currently being performed. The development of Endotype-driven integrated care pathways appreciating these innovations are now needed for the management of CRS.
-
phenotypes and emerging Endotypes of chronic rhinosinusitis
The Journal of Allergy and Clinical Immunology: In Practice, 2016Co-Authors: Claus Bachert, Cezmi A AkdisAbstract:Chronic rhinosinusitis can be differentiated into several phenotypes based on clinical criteria; however, these phenotypes do not teach us much about the underlying inflammatory mechanisms. Thus, the use of nasal endoscopy and CT scanning, and eventually taking a swab or a biopsy, may not be sufficient to fully appreciate the individual patient's pathology. Endotyping of chronic rhinosinusitis on the basis of pathomechanisms, functionally and pathologically different from others by the involvement of specific molecules or cells, may in contrast provide us with information on the risk of disease progression or recurrence and on the best available treatment, and also helps us identifying innovative therapeutic targets for treatment. Endotyping may best be structured around T helper cells and their downstream events, such as tissue eosinophilia or neutrophilia; this approach involves the cytokines and chemokines related to specific T helper cell populations, and related markers such as IgE. Endotyping is of specific interest at the time of the arrival of new biologicals, confronting us with the challenge of the selection of eligible patients for treatment and predicting their therapeutic response; defining suitable biomarkers is therefore an urgent task. Failure to appreciate the underlying mechanisms and Endotypes of chronic rhinosinusitis may limit progress in the management of the disease at present.
-
current and future treatment options for adult chronic rhinosinusitis focus on nasal polyposis
The Journal of Allergy and Clinical Immunology, 2015Co-Authors: Luo Zhang, Claus Bachert, Phillippe GevaertAbstract:Chronic rhinosinusitis (CRS) affects more than 10% of the population in the United States and Europe. Recent findings point to a considerable variation of inflammatory subtypes in patients with CRS with nasal polyps and patients with CRS without nasal polyps. According to current guidelines, glucocorticosteroids and antibiotics are the principle pharmacotherapeutic approaches; however, they fail in a group of patients who share common clinical and laboratory markers. Several clinical phenotypes often leading to uncontrolled disease, including adult nasal polyposis, aspirin-exacerbated respiratory disease, and allergic fungal rhinosinusitis, are characterized by a common Endotype: a TH2 bias is associated with a higher likelihood of comorbid asthma and recurrence after surgical treatment. As a consequence, several innovative approaches targeting the TH2 bias with humanized mAbs have been subjected to proof-of-concept studies in patients with CRS with nasal polyps with or without comorbid asthma: omalizumab, reslizumab, mepolizumab, and recently dupilumab. Future concepts using upstream targets, such as GATA-3, also focus on this Endotype. This current development might result in advantages in the treatment of patients with the most severe CRS.
-
Endotypes and phenotypes of chronic rhinosinusitis a practall document of the european academy of allergy and clinical immunology and the american academy of allergy asthma immunology
The Journal of Allergy and Clinical Immunology, 2013Co-Authors: Cezmi A Akdis, Peter Hellings, Claus Bachert, Cemal Cingi, Mark S Dykewicz, Robert M Naclerio, Robert P Schleimer, Dennis K LedfordAbstract:Chronic rhinosinusitis (CRS) is a complex disease consisting of several disease variants with different underlying pathophysiologies. Limited knowledge of the mechanisms of these disease subgroups is possibly the greatest obstacle in understanding the causes of CRS and improving treatment. It is generally agreed that there are clinically relevant CRS phenotypes defined by an observable characteristic or trait, such as the presence or absence of nasal polyps. Defining the phenotype of the patient is useful in making therapeutic decisions. However, clinical phenotypes do not provide full insight into all underlying cellular and molecular pathophysiologic mechanisms of CRS. Recognition of the heterogeneity of CRS has promoted the concept that CRS consists of multiple groups of biological subtypes, or “Endotypes,” which are defined by distinct pathophysiologic mechanisms that might be identified by corresponding biomarkers. Different CRS Endotypes can be characterized by differences in responsiveness to different treatments, including topical intranasal corticosteroids and biological agents, such as anti–IL-5 and anti-IgE mAb, and can be based on different biomarkers that are linked to underlying mechanisms. CRS has been regarded as a single disease entity in clinical and genetic studies in the past, which can explain the failure to identify consistent genetic and environmental correlations. In addition, better identification of Endotypes might permit individualization of therapy that can be targeted against the pathophysiologic processes of a patient's Endotype, with potential for more effective treatment and better patient outcomes.
Robert F. Lemanske - One of the best experts on this subject based on the ideXlab platform.
-
precision medicine in allergic disease food allergy drug allergy and anaphylaxis practall document of the european academy of allergy and clinical immunology and the american academy of allergy asthma and immunology
Allergy, 2017Co-Authors: Antonella Muraro, Wesley Burks, Robert F. Lemanske, Hansuwe Simon, Carsten Bindslevjensen, David A. Khan, Lars K. Poulsen, Maria J Torres, Mariana Castells, Hugh A. SampsonAbstract:This consensus document summarizes the current knowledge on the potential for precision medicine in food allergy, drug allergy, and anaphylaxis under the auspices of the PRACTALL collaboration platform. PRACTALL is a joint effort of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma and Immunology, which aims to synchronize the European and American approaches to allergy care. Precision medicine is an emerging approach for disease treatment based on disease Endotypes, which are phenotypic subclasses associated with specific mechanisms underlying the disease. Although significant progress has been made in defining Endotypes for asthma, definitions of Endotypes for food and drug allergy or for anaphylaxis lag behind. Progress has been made in discovery of biomarkers to guide a precision medicine approach to treatment of food and drug allergy, but further validation and quantification of these biomarkers are needed to allow their translation into practice in the clinical management of allergic disease.
-
precision medicine in patients with allergic diseases airway diseases and atopic dermatitis practall document of the european academy of allergy and clinical immunology and the american academy of allergy asthma immunology
The Journal of Allergy and Clinical Immunology, 2016Co-Authors: Antonella Muraro, Robert F. Lemanske, Lars K. Poulsen, Cezmi A Akdis, T Bieber, Thomas B Casale, Marek Jutel, Peter Hellings, Peck Y Ong, Peter SchmidgrendelmeierAbstract:In this consensus document we summarize the current knowledge on major asthma, rhinitis, and atopic dermatitis Endotypes under the auspices of the PRACTALL collaboration platform. PRACTALL is an initiative of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma & Immunology aiming to harmonize the European and American approaches to best allergy practice and science. Precision medicine is of broad relevance for the management of asthma, rhinitis, and atopic dermatitis in the context of a better selection of treatment responders, risk prediction, and design of disease-modifying strategies. Progress has been made in profiling the type 2 immune response-driven asthma. The Endotype driven approach for non-type 2 immune response asthma, rhinitis, and atopic dermatitis is lagging behind. Validation and qualification of biomarkers are needed to facilitate their translation into pathway-specific diagnostic tests. Wide consensus between academia, governmental regulators, and industry for further development and application of precision medicine in management of allergic diseases is of utmost importance. Improved knowledge of disease pathogenesis together with defining validated and qualified biomarkers are key approaches to precision medicine.
-
asthma Endotypes a new approach to classification of disease entities within the asthma syndrome
The Journal of Allergy and Clinical Immunology, 2011Co-Authors: Jan Lotvall, Robert F. Lemanske, Cezmi A Akdis, Thomas B Casale, Leonard B Bacharier, Leif Bjermer, Adnan Custovic, A J Wardlaw, Sally E Wenzel, Paul A GreenbergerAbstract:It is increasingly clear that asthma is a complex disease made up of number of disease variants with different underlying pathophysiologies. Limited knowledge of the mechanisms of these disease subgroups is possibly the greatest obstacle in understanding the causes of asthma and improving treatment and can explain the failure to identify consistent genetic and environmental correlations to asthma. Here we describe a hypothesis whereby the asthma syndrome is divided into distinct disease entities with specific mechanisms, which we have called "asthma Endotypes." An "Endotype" is proposed to be a subtype of a condition defined by a distinct pathophysiological mechanism. Criteria for defining asthma Endotypes on the basis of their phenotypes and putative pathophysiology are suggested. Using these criteria, we identify several proposed asthma Endotypes and propose how these new definitions can be used in clinical study design and drug development to target existing and novel therapies to patients most likely to benefit. This PRACTALL (PRACtical ALLergy) consensus report was produced by experts from the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma & Immunology.
Antonella Muraro - One of the best experts on this subject based on the ideXlab platform.
-
precision medicine in allergic disease food allergy drug allergy and anaphylaxis practall document of the european academy of allergy and clinical immunology and the american academy of allergy asthma and immunology
Allergy, 2017Co-Authors: Antonella Muraro, Wesley Burks, Robert F. Lemanske, Hansuwe Simon, Carsten Bindslevjensen, David A. Khan, Lars K. Poulsen, Maria J Torres, Mariana Castells, Hugh A. SampsonAbstract:This consensus document summarizes the current knowledge on the potential for precision medicine in food allergy, drug allergy, and anaphylaxis under the auspices of the PRACTALL collaboration platform. PRACTALL is a joint effort of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma and Immunology, which aims to synchronize the European and American approaches to allergy care. Precision medicine is an emerging approach for disease treatment based on disease Endotypes, which are phenotypic subclasses associated with specific mechanisms underlying the disease. Although significant progress has been made in defining Endotypes for asthma, definitions of Endotypes for food and drug allergy or for anaphylaxis lag behind. Progress has been made in discovery of biomarkers to guide a precision medicine approach to treatment of food and drug allergy, but further validation and quantification of these biomarkers are needed to allow their translation into practice in the clinical management of allergic disease.
-
precision medicine in patients with allergic diseases airway diseases and atopic dermatitis practall document of the european academy of allergy and clinical immunology and the american academy of allergy asthma immunology
The Journal of Allergy and Clinical Immunology, 2016Co-Authors: Antonella Muraro, Robert F. Lemanske, Lars K. Poulsen, Cezmi A Akdis, T Bieber, Thomas B Casale, Marek Jutel, Peter Hellings, Peck Y Ong, Peter SchmidgrendelmeierAbstract:In this consensus document we summarize the current knowledge on major asthma, rhinitis, and atopic dermatitis Endotypes under the auspices of the PRACTALL collaboration platform. PRACTALL is an initiative of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma & Immunology aiming to harmonize the European and American approaches to best allergy practice and science. Precision medicine is of broad relevance for the management of asthma, rhinitis, and atopic dermatitis in the context of a better selection of treatment responders, risk prediction, and design of disease-modifying strategies. Progress has been made in profiling the type 2 immune response-driven asthma. The Endotype driven approach for non-type 2 immune response asthma, rhinitis, and atopic dermatitis is lagging behind. Validation and qualification of biomarkers are needed to facilitate their translation into pathway-specific diagnostic tests. Wide consensus between academia, governmental regulators, and industry for further development and application of precision medicine in management of allergic diseases is of utmost importance. Improved knowledge of disease pathogenesis together with defining validated and qualified biomarkers are key approaches to precision medicine.