Cytokine Gene Polymorphisms in Patients with Chronic Inflammatory Demyelinating Polyneuropathy.

Innate and adaptive immune responses exert their role in CIDP pathogenesis through cytokine production. Single-nucleotide polymorphisms (SNPs) may alter cytokine gene expression, with a potential influence on the pathogenesis of autoimmune diseases. However, cytokine gene SNPs have not been assessed in CIDP patients yet. We assessed functional SNPs in the genes encoding IL-10 (rs1800896, rs1800871, rs1800872 and rs3024505), IL-6 (rs1800795), TNF (rs1800629 and rs361525), IL-12B (rs3212227), IFN-γ (rs2430561), GM-CSF (rs25882) and IL-17F (rs11465553) in a cohort of 88 CIDP patients and 486 healthy controls (HCs) via qPCR. We found an association of SNP in the IL10 promotor and CIDP occurrence. Major homozygotes (AA) were more frequent in the HCs compared to CIDP patients (p = 0.049), but the GA genotype prevailed among the patients (p = 0.032). A lower frequency of the C allele was observed for rs1800871 and rs1800872 in CIDP patients compared to the HCs (p = 0.048). A higher proportion of A carriers at position -1082 (rs1800896) (presumed to be a low IL-10 producer) was noted in patients with milder disability (low INCAT). All mild-INCAT patients were C carriers for rs1800871 and rs1800872 in IL10 (p = 0.038). Furthermore, the IL6 rs1800795 GG genotype was more frequent in patients (p = 0.049) and the CG heterozygote in the HCs (p = 0.013). Among the CIDP patients, being a G carrier for this SNP was associated with a higher frequency of type 2 diabetes (T2D) compared to being a non-carrier (p = 0.032). Our data indicate a possible association of the IL10 and IL6 SNPs with CIDP, but also with disease severity and T2D occurrence. Given the paucity of CIDP patients, multicentric studies are necessary to draw definite conclusions on these associations.

Reliability and validity of the Stroke and Aphasia Quality of Life Scale-39 (SAQOL-39) for a Serbian population.

Purpose: Aphasia has a negative impact on a person's quality of life (QOL). The Stroke Aphasia Quality of Life-39 scale (SAQOL-39) is a widely-used measure of health-related quality of life (HRQOL) developed for people with aphasia that has been translated into several languages. Its psychometric properties have been examined not only in English, but also in other languages. This study examined the reliability and validity of a translation and adaptation of the SAQOL-39 into Serbian in Serbian-speaking people with aphasia.Method: Using forward and backward translation, the SAQOL-39 was translated and adapted from English into Serbian and its psychometric properties were examined in 90 Serbian-speaking people with a broad range of times post-onset of aphasia. Internal consistency, test-retest reliability and other analyses were conducted.Result: Internal consistency and test-retest reliability of the Serbian version was high (Cronbach's α > 0.9; ICC ≥0.87), which is similar to versions of the scale in other languages.Conclusion: The Serbian translation and adaptation of the SAQOL-39 was shown to be a valid and reliable measure of QOL in people with aphasia with reliable psychometric properties and is suitable for the assessment of Serbian people with aphasia.

Downregulation of LKB1/AMPK Signaling in Blood Mononuclear Cells Is Associated with the Severity of Guillain-Barre Syndrome.

AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolic and immune functions mainly through the inhibition of the mechanistic target of rapamycin (mTOR)-dependent anabolic pathways and the activation of catabolic processes such as autophagy. The AMPK/mTOR signaling pathway and autophagy markers were analyzed by immunoblotting in blood mononuclear cells of 20 healthy control subjects and 23 patients with an acute demyelinating form of Guillain-Barré syndrome (GBS). The activation of the liver kinase B1 (LKB1)/AMPK/Raptor signaling axis was significantly reduced in GBS compared to control subjects. In contrast, the phosphorylated forms of mTOR activator AKT and mTOR substrate 4EBP1, as well as the levels of autophagy markers LC3-II, beclin-1, ATG5, p62/sequestosome 1, and NBR1 were similar between the two groups. The downregulation of LKB1/AMPK signaling, but not the activation status of the AKT/mTOR/4EBP1 pathway or the levels of autophagy markers, correlated with higher clinical activity and worse outcomes of GBS. A retrospective study in a diabetic cohort of GBS patients demonstrated that treatment with AMPK activator metformin was associated with milder GBS compared to insulin/sulphonylurea therapy. In conclusion, the impairment of the LKB1/AMPK pathway might contribute to the development/progression of GBS, thus representing a potential therapeutic target in this immune-mediated peripheral polyneuropathy.

Graphene quantum dot antioxidant and proautophagic actions protect SH-SY5Y neuroblastoma cells from oxidative stress-mediated apoptotic death.

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical (•OH), superoxide anion (O2•-), and lipid peroxidation. Nonselective antioxidants, •OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing •OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy-limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proautophagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NH4Cl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both •OH/NO scavenging and induction of cytoprotective autophagy.

Different eplet software programs give discordant and incorrect results: An analysis of HLAMatchmaker vs Fusion Matchmaker Eplet calling software.

HLA eplet matching is a novel approach to define acceptable HLA mismatches for transplant recipients. We performed an eplet analysis of three different transplant case-series to determine if the available software programs gave accurate results. Eplet analysis was performed for three different transplant case-series typed by NGS for all HLA class I and II loci. The three different HLA datasets were entered into both the HLAMatchmaker program (v2.1) and OLI Fusion MatchMaker (v4.2) software tools. Eplet results which were discordant were cross referenced against eplet registry and published HLA allele sequence data to determine the correct assignments. The comparison reveals that there was poor concordance between the two eplet programs. Analysis of the same donor/recipient pair often gave rise to different total eplet scores, incorrect eplet mismatches and antibody verification status, and both programs have eplets assigned to incorrect HLA alleles. Overall, the OLI Fusion MatchMaker eplet tool gave more accurate and useful eplet results. Eplet matching is still primarily a research tool. Before eplet matching can enter routine clinical practice further work is required to validate the accuracy of available eplet software programs. Incorrect eplet assignment could have serious adverse consequences in the clinical transplant setting.

Extending knowledge of the public awareness of aphasia in the Balkans: Serbia and Montenegro.

PURPOSE: Public awareness of aphasia has been surveyed in a number of countries revealing that it is universally low. We report results of surveys in the Balkan countries Serbia and Montenegro and compare results with data from Croatia and Slovenia. METHODS: Convenience surveys of the general public were conducted in public places like shopping centers/malls and parks in Serbia (N = 400) and Montenegro (N = 500) using an adapted version of the public awareness of aphasia survey questionnaire. Respondents were asked whether they have heard of aphasia and tested with questions about aphasia. Information on gender, age, occupation and education was recorded. OUTCOMES: Twelve percent (Serbia) and 11% (Montenegro) had heard of aphasia, but just 4% (Serbia) and 3.2% (Montenegro) had a basic knowledge of aphasia. Age, gender and occupation interacted variably with awareness. Between 16% (Slovenia) and 60% (Croatia) said they had heard of aphasia (10.5% overall mean for the four countries) and basic knowledge of aphasia across the four countries ranged between 3.2 and 7%. CONCLUSIONS: Levels of awareness of aphasia in the Balkans are low and variably associated with age, gender, socio-economic and educational levels. Respondents with some knowledge of aphasia gained it through personal or professional interaction with aphasia or the media. The data provide a basis for awareness raising in Balkan countries to reduce stigmatization, improve community access and understanding. Implications for rehabilitation Awareness of aphasia is low universally, even among healthcare workers. Low public awareness of a condition, like aphasia, results in under-funded research and service provision. In order to raise public awareness of aphasia we need to know how many members of the general public know about it. Improvements in public awareness could positively affect funding, the quality of services, and the public understanding and acceptance of individuals with aphasia in the community. Improving awareness of aphasia in those who come into contact with aphasic people, like healthcare workers, could significantly improve the healthcare experience of people with aphasia and their families.

The origin and differentiation potential of smooth muscle cells in coronary atherosclerosis.

OBJECTIVES: To determine the phenotypical state of smooth muscle cells during the pathogenesis of an atherosclerotic lesion, and to determine the morphological state of the endothelium and the origin of foam cells. METHODS: Twenty-one samples of atherosclerotically changed right coronary arteries, which were divided into six subgroups based on the stage of atherosclerosis, were analyzed. The tissues were fixed in formalin and embedded in paraffin. Sections of 5 mum thickness were stained immunocytochemically using a labelled streptavidin-biotin/horse radish peroxidase kit (Dako, Denmark) for the identification of vimentin, alpha-smooth muscle actin, myosin heavy chains, desmin, S-100 protein, CD3, CD31, CD34, CD45, CD68 and proliferating cell nuclear antigen protein. RESULTS: The present study showed that there is first functional and then morphological damage of the endothelium in the late stages of atherosclerosis. The preatheroma stage revealed the presence of intimal changes of smooth muscle cells, with expression of vimentin and alpha-smooth muscle actin and a lack of expression of desmin, which led to a switch to a synthetic phenotype. The described changes progressed into the later stages of atherosclerosis. Along with these changes, a large number of foam cells of variant origin were observed; some of the foam cells developed from monocyte-macrophage lineage (CD68-immunoreactive) and others originated from smooth muscle cells (vimentin- and S-100-immunoreactive). The late stages of atherosclerosis development, such as the atheroma stage, include intimal changes with the formation of a lipid core (S-100-immunoreactive cells and cell necrosis), while fibrosis in the lipid core and the accumulation of collagen fibres with extreme hypocellularity are characteristics of the fibroatheroma stage.

Acquired dyslexia in Serbian speakers with Broca's and Wernicke's aphasia.

This study examined patterns of acquired dyslexia in Serbian aphasic speakers, comparing profiles of groups with Broca's versus Wernicke's aphasia. The study also looked at the relationship of reading and auditory comprehension and between reading comprehension and reading aloud in these groups. Participants were 20 people with Broca's and 20 with Wernicke's aphasia. They were asked to read aloud and to understand written material from the Serbian adaptation of the Boston Diagnostic Aphasia Examination. A Serbian Word Reading Aloud Test was also used. The people with Broca's aphasia achieved better results in reading aloud and in reading comprehension than those with Wernicke's aphasia. Those with Wernicke's aphasia showed significantly more semantic errors than those with Broca's aphasia who had significantly more morphological and phonological errors. From the data we inferred that lesion sites accorded with previous work on networks associated with Broca's and Wernicke's aphasia and with a posterior-anterior axis for reading processes centred on (left) parietal-temporal-frontal lobes.

Analysis of voice impairment in aphasia after stroke-underlying neuroanatomical substrates.

Phonation is a fundamental feature of human communication. Control of phonation in the context of speech-language disturbances has traditionally been considered a characteristic of lesions to subcortical structures and pathways. Evidence suggests however, that cortical lesions may also implicate phonation. We carried out acoustic and perceptual analyses of the phonation of /a/ in 60 males with aphasia (20 Wernicke's, 20 Broca's, 20 subcortical aphasia) and 20 males matched in age with no neurological or speech-language disturbances. All groups with aphasia were significantly more impaired on the majority of acoustic and perceptual measures as compared with the control speakers. Within the subjects with aphasia, subjects with subcortical aphasia were more impaired on most measures compared to subjects with Broca's aphasia, and they, in turn, more impaired than those with Wernicke's aphasia. Lesions in regions involved in sound production-perception result in dysfunction of the entire neurocognitive system of articulation-phonological language processing.

Investigation of language and motor skills in Serbian speaking children with specific language impairment and in typically developing children.

Specific language impairment (SLI) is usually defined as a developmental language disorder which does not result from a hearing loss, autism, neurological and emotional difficulties, severe social deprivation, low non-verbal abilities. Children affected with SLI typically have difficulties with the acquisition of different aspects of language and by definition, their impairment is specific to language and no other skills are affected. However, there has been a growing body of literature to suggest that children with SLI also have non-linguistic deficits, including impaired motor abilities. The aim of the current study is to investigate language and motor abilities of a group of thirty children with SLI (aged between 4 and 7) in comparison to a group of 30 typically developing children matched for chronological age. The results showed that the group of children with SLI had significantly more difficulties on the language and motor assessments compared to the control group. The SLI group also showed delayed onset in the development of all motor skills under investigation in comparison to the typically developing group. More interestingly, the two groups differed with respect to which language abilities were correlated with motor abilities, however Imitation of Complex Movements was the unique skill which reliably predicted expressive vocabulary in both typically developing children and in children with SLI.

Comparison of the recovery patterns of language and cognitive functions in patients with post-traumatic language processing deficits and in patients with aphasia following a stroke.

UNLABELLED: In this study we investigated the recovery patterns of language and cognitive functions in patients with post-traumatic language processing deficits and in patients with aphasia following a stroke. The correlation of specific language functions and cognitive functions was analyzed in the acute phase and 6 months later. Significant recovery of the tested functions was observed in both groups. However, in patients with post-traumatic language processing deficits the degree of recovery of most language functions and some cognitive functions was higher. A significantly greater correlation was revealed within language and cognitive functions, as well as between language functions and other aspects of cognition in patients with post-traumatic language processing deficits than in patients with aphasia following a stroke. Our results show that patients with post-traumatic language processing deficits have a different recovery pattern and a different pattern of correlation between language and cognitive functions compared to patients with aphasia following a stroke. LEARNING OUTCOMES: (1) Better understanding of the differences in recovery of language and cognitive functions in patients who have suffered strokes and those who have experienced traumatic brain injury. (2) Better understanding of the relationship between language and cognitive functions in patients with post-traumatic language processing deficits and in patients with aphasia following a stroke. (3) Better understanding of the factors influencing recovery.

[Cytohistological and immunohistochemical characteristics of vascular remodelling in diseases of the blood vessels].

INTRODUCTION: Vascular remodelling is an adaptive process involving the adjustment of the structure and function of blood vessels to long-term changes in haemodynamic conditions. This process leads to structural alterations within vessel walls in different cardiovascular diseases, such as hypertension, atherosclerosis, and coarctation of the aorta. OBJECTIVE: We investigated the histochemical and immunocytochemical characteristics of morphological lesions in coronary atherosclerosis and coarctation of the aorta. METHOD: Twenty-one samples of atherosclerotically modified right coronary arteries, divided into 6 segments, were analysed. We also examined 10 samples of coarctation segments, excised during surgery. The segments were stained histochemically (using orcein and alcian blue-PAS), immunocytochemically (using alpha-smooth muscle actin-alpha-SMA, vimentin, desmin, myosin heavy chains-MHC, CD3, CD45, S-100, and Proliferating Cell Nuclear Antigen-PCNA), and for electron microscopy. RESULTS: The results of our study of morphological lesions in coronary atherosclerosis demonstrated initial functional and then, in the later stages of atherosclerosis, morphological, damage to the endothelium. The preatheroma stage revealed the presence of intimal dedifferentiation of smooth muscle cells, with the expression of vimentin and alpha-SMA, and the lack of expression of desmin. Along with these changes, a huge number of foam cells of variant origin were noticed. Some of them were CD68-immunoreactive while others were both vimentin- and S-100-immunoreactive. All examined samples of the coarctation of the aorta demonstrated the presence of dedifferentiated smooth muscle cells as well as a diminution in cell numbers, followed by apoptotic smooth muscle cells, and the absence of inflammatory cells. CONCLUSION: Some foam cells develop from monocyte-macrophage lineage (CD68-immunoreactive), while others originate from smooth muscle cells (vimentin and S-100-immunoreactive). Coarctation of the aorta is characterised by a diminution in cell numbers (apoptosis) as well as their dedifferentiation from contractile to synthetic phenotype.

[Cytohistologic and immunohistochemical characteristics of the aortic intima and media in coarctation of the aorta of the adult type]. / Citohistoloske i imunohistohemijske odlike intime i medije aorte u koarktaciji adultnog tipa.

INTRODUCTION: Classically, coarctation of the aorta has been divided into infantile and adult forms. The vascular malformation responsible for coarctation is a defect of the vessel intima and media giving rise to a prominent posterior infolding ("the posterior shelf") which, in some cases, may extend around the entire circumference of the aorta. Histological examination of the coarcted aortic segment discloses intimal and medial lesion consisting of thickened ridges that protrude posteriorly into the aortic lumen. Intimal proliferation and disruption of elastic tissue may occur in adult type. OBJECTIVE: The smooth muscle cells phenotype in the aortic intimal thickening, presence of inflammatory cells and contents of the intimal and medial pseudocysts were investigated. MATERIAL AND METHODS: The samples of coarctation segments excised at surgery from 10 patients aged from 2 to 13 years were examined. For light microscopy, the specimens were dehydrated in graded ethanol (70-100%), cleared in xylol and embedded in paraffin. Sections of 5 microm thick were cut on Leica SM 2000R and Leica Reinhart Austria microtome and stained with orcein and Alcian blue-PAS at pH 1.0 and pH 2.5. Immunocytochemical staining was performed on 5 microm sections from formaldehyde-fixed paraffin-embedded blocks, using a labeled streptavidin-biotin method with an LSAB kit (Dako). Sections were deparaffinized and rehydrated. After microwave treatment of 21 minutes in citrate buffer pH 6.0, endogenous peroxidase activity was blocked with 3% H2O2 for 15 minutes. The sections were first incubated with the primary antibody for 60 minutes (alpha-smooth muscle actin-alpha-SMA, vimentin, desmin, myosin haevy chains-MHC, CD3, CD45, S-100 and Proliferating Cell Nuclear Antigen-PCNA), then with biotinylated link antibody and finally with peroxidase-labeled streptavidin. Slides were counter-stained with hematoxylin, washed in water and mounted. For electron microscopy, the primary fixative consisted of 2.5% glutaraldehyde in 0.1 M sodium cacodylate-HCl buffer (pH 7.4) for 24 h at 4 degrees C. The specimens were postfixed for 1 h at 4 degrees C in 1% osmium tetroxide in 0.1 M cacodylate buffer and 4.8% uranyl acetate for 24 h at 4 degrees C. The samples were dehydrated in graded ethanol (70-100%) and embedded in Epon 812. The samples were cut with a diamond knife on an LKB Ultratome. Ultra-thin sections were stained with 2% uranyl acetate and alkaline lead citrate. RESULTS: All samples had focal intimal thickening on the posterior aortic wall, with accumulation of mucins which were stained with Alcian blue-PAS on pH 1.0, followed by prominent hypocellularity. Rare smooth muscle cells (SMC) showed immunoreactivity on alpha-SMA and vimentin, but not on desmin, MHC or CD3 and CD45. A large number of cells in apoptosis was noticed in the inner media on the posterior wall. On the anteromedial wall, a large number of PCNA- and S-100- positive cells was noted in the inner media while one layer of MHC- and desmin-positive cells was noted in the outer media. The elastic lamellae were focally disrupted by pools which were stained with Alcian blue-PAS at pH 1.0. DISCUSSION: In all examined samples, the immunocytochemical and TEM results revealed the presence of dedifferentiated smooth muscle cells which express alpha-SMA and vimentin, with a lack of expression of desmin and MHC. Results of this study also showed the reduction of cell number in the intima and media, followed by apoptotic smooth muscle cells in the inner media of the posterior wall and the absence of inflammatory cells. Such finding suggests that apoptosis but not necrosis may be the mechanism of reduction of cell number. The presence of smooth muscle cell proliferation in the inner media of the anteromedial wall and one layer of differentiated SMC in the outer part may lead us to suppose that changes of media (including dedifferentiation of the cells and disruption of elastic tissue) appear from inner to outer part and from posterior to anteromedial wall. The presence of pseudocysts which are stained with Alcian blue-PAS at pH 1.0 show large amount of mucins in elastic fibers. CONCLUSION: The intimal thickening on the posterior aortic wall is composed of small number of dedifferentiated smooth muscle cells (SMC). Some of these cells are in apoptosis. On the anteromedial wall, the intima and media are composed of proliferated SMC and small number of SMC which exhibit contractile phenotype. In all parts of the aortic wall, there is a large number of pseudocysts with large amount of mucins, without presence of inflammatory cells.

[Surgery for coarctation of the aorta in infants younger than three months]. / Hirursko lecenje koarktacije aorte kod odojceta uzrasta do tri meseca.

INTRODUCTION: Recurrent coarctation is a complication which is seen at a consistent rate following repair for coarctation of the aorta in young infants. OBJECTIVE: This retrospective analysis was carried out to compare the results between resection with end-to-end anastomosis (ETE), and resection with extended end-to-end anastomosis (E-ETE), in this age group during late follow-up period. The role of ductus arteriosus is not clearly defined and the second objective of this study was to analyze intimal thickening in aortic coarctation. MATERIAL AND METHODS: From 1999 to 2003, 45 patients less than 3 months of age underwent repair of aortic coarctation. Mean age was 24 days (2-89 days), average weight was 3.5 +/- 0.6 kg (2.4-5.2 kg). The method of repair was ETE in 14 (31.1%) patients, E-ETE in 29 (64.4%) patients and other techniques were applied in 2 cases. Demographic, morphometric, clinical and operative variables were analyzed for correlation with recurrent arch obstruction. In order to characterize the components of intimal thickening in coarctation, narrowed segments of aorta resected from 16 neonates during surgery were examined immunocytochemically and by electron microscopy. For light microscopy, the specimens were dehydrated in graded ethanol (70-100%), cleared in xylol and embedded in paraffin. Immunocytochemical staining was performed in 5 microm sections from formaldehyde-fixed paraffin-embedded blocks, using a labeled streptavidin-biotin method with an LSAB kit (Dako). RESULTS: Early mortality was 6.7% (CI 95%, 2.9%-10.4%). All early deaths (3 patients) occurred in infants with associated ventricular septal defects (p<0.05). The mean follow-up for all patients was 30 +/- 21 months (range 1.5-63 months). During mean follow-up of 2 months, recurrent arch obstruction was diagnosed in 9 patients (21.4%). Two patients with associated complex heart defects died before re-intervention, one had mild gradient on catheterization (20 mm Hg) and one is waiting for catheterization. Five patients were reoperated and the mean time to re-intervention was 4 months (range 2.6-6 months). Kaplan-Meier freedom from recoarctation was 78.1 +/- 6.4% at 5 years in the whole group. Freedom from recoarctation was 60.6 +/- 15.4% at 25 months in ETE group and 86.2 +/- 6.4% at 60 months in E-ETE group (p=0.062). Factors associated with recoarctation, obtained by univariable Cox regression, included abnormal right subclavian artery (p=0.003), hypoplastic proximal transverse aortic arch (Z < or = -2, p=0.025) and weight at operation < or = 3 kg (p=0.02). Abnormal origin of the right subclavian artery was the only independent predictor of recoarctation obtained by multivariable Cox regression analysis. DISCUSSION: All examined specimens had intimal thickening of the posterior aortic wall, with accumulation of smooth muscle cells (SMC) with alpha smooth muscle actin (alpha-SMA) and vimentin-immunoreactivity (but not desmin and MHC) and also expressed PCNA and S-100. In the inner media of the anteromedial wall of the aorta, all specimens had large number of SMC expressing desmin and MHC. SMC in the inner media exhibit contractile phenotype and their origin could be ductal. CONCLUSION: Both procedures are effective for coarctation repair in young infants. Risk of recoarctation is a function of the complex anatomy of the arch, while residual ductal tissue may play a significant role.