Safety and efficacy of HK-660S in patients with primary sclerosing cholangitis: A randomized double-blind phase 2a trial.

Background/Aims: A clinical unmet need persists for medications capable of modulating the progression of primary sclerosing cholangitis (PSC). This study aimed to assess the clinical feasibility of HK-660S (beta-lapachone) in PSC. Methods: In this multicenter, randomized, double-blind, placebo-controlled, parallel-group phase 2 trial, participants were assigned in a 2:1 ratio to receive either 100 mg of HK-660S or a placebo twice daily for 12 weeks. The primary outcomes were the reduction in serum alkaline phosphatase (ALP) levels and the percentage of participants showing improvements in PSC severity, as determined by magnetic resonance cholangiopancreatography (MRCP) with the Anali score. Secondary endpoints included changes in liver stiffness and adverse events. Results: The analysis included 21 patients, 15 receiving HK-660S, and six receiving a placebo. Improvements in the Anali score were observed in 13.3% of the HK-660S group, with no improvements in the placebo group. HK-660S treatment resulted in a 15.2% reduction in mean ALP levels, compared to a 6.6% reduction in the placebo group. A stratified ad-hoc analysis based on baseline ALP levels showed a statistically significant response in the HK-660S group among those with ALP levels greater than twice the upper limit of normal, with a 50% responder rate (p = 0.05). Additionally, 26.7% of the HK-660S group showed improvements in the enhanced liver fibrosis score, with no improvements in the placebo group. HK-660S was generally well-tolerated. Conclusions: HK-660S is well-tolerated among patients with PSC and may improve bile duct strictures, decrease serum ALP levels, and reduce liver fibrosis. (cris.nih.go.kr, Number KCT0006590).

Ethnic differences in the prevalence of amyloid positivity and cognitive trajectories.

INTRODUCTION: We investigated the prevalence of amyloid beta (Aß) positivity (+) and cognitive trajectories in Koreans and non-Hispanic Whites (NHWs). METHODS: We included 5121 Koreans from multiple centers across South Korea and 929 NHWs from the Alzheimer's Disease Neuroimaging Initiative (ADNI). Participants underwent Aß positron emission tomography and were categorized into cognitively unimpaired (CU), mild cognitive impairment (MCI), and dementia stages. Age, sex, education, and apolipoprotein E. genotype were adjusted using multivariable logistic regression and stabilized inverse probability of treatment weights based on the propensity scores to mitigate imbalances in these variables. RESULTS: The prevalence of Aß+ was lower in CU Koreans than in CU NHWs (adjusted odds ratio 0.60). Aß+ Koreans showed a faster cognitive decline than Aß+ NHWs in the CU (B = -0.314, p = .004) and MCI stages (B = -0.385, p < .001). DISCUSSION: Ethnic characteristics of Aß biomarkers should be considered in research and clinical application of Aß-targeted therapies in diverse populations. HIGHLIGHTS: Koreans have a lower prevalence of Aß positivity compared to NHWs in the CU stage. The effects of Alzheimer's risk factors on Aß positivity differ between Koreans and NHWs. Aß-positive (Aß+) Koreans show faster cognitive decline than Aß+ NHWs in the CU and MCI stages.

Association Between Influenza Vaccination and Acute Kidney Injury Among the Elderly: A Self-Controlled Case Series.

BACKGROUND: Several cases of renal complications, including acute kidney injury (AKI), after influenza vaccination have been reported, but the association remains unproven. We evaluated the association between influenza vaccination and AKI occurrence among the Korean elderly in the 2018-2019 and 2019-2020 seasons. METHODS: We used a large database combining vaccination registration data from the Korea Disease Control and Prevention Agency and claims data from the National Health Insurance Service. The study subjects were patients hospitalized with AKI for the first-time following vaccination among those who received one influenza vaccine in the 2018-2019 or 2019-2020 season. Only those aged 65 or older at the date of vaccination were included. We performed a self-controlled case series study, designating the risk period as 1 to 28 days post-vaccination and the observation period as each influenza season. The adjusted incidence rate ratio (aIRR) was calculated by adjusting for nephrotoxic drug use and influenza infection that may influence AKI occurrence using a conditional Poisson regression model. RESULTS: A total of 16 713 and 16 272 AKI events were identified during the 2018-2019 and 2019-2020 seasons, respectively. The aIRR for AKI was 0.83 (95% confidence interval [CI] = 0.79-0.87) in the 2018-2019 season. The aIRR for the 2019-2020 influenza season was similar to the 2018-2019 season (aIRR = 0.86; 95% CI = 0.82-0.90). CONCLUSIONS: Influenza vaccination is associated with a lower risk of AKI in the elderly over 65. This evidence supports the recommendation of annual influenza vaccination for the elderly. Further studies are needed to determine the biological mechanisms linking the influenza vaccine and AKI.

Enhancing Long-Term Memory in Carbon-Nanotube-Based Optoelectronic Synaptic Devices for Neuromorphic Computing.

This study investigates the impact of spin-coating speed on the performance of carbon nanotube (CNT)-based optoelectronic synaptic devices, focusing on their long-term memory properties. CNT films fabricated at lower spin speeds exhibited a greater thickness and density compared to those at higher speeds. These denser films showed enhanced persistent photoconductivity, resulting in higher excitatory postsynaptic currents (EPSCs) and the prolonged retention of memory states after UV stimulation. Devices coated at a lower spin-coating speed of 2000 RPM maintained EPSCs above 70% for 3600 s, outperforming their higher-speed counterparts in long-term memory retention. Additionally, the study demonstrated that the learning efficiency improved with repeated UV stimulation, with fewer pulses needed to achieve the maximum EPSC in successive learning cycles. These findings highlight that optimizing spin-coating speeds can significantly enhance the performance of CNT-based synaptic devices, making them suitable for applications in neuromorphic computing and artificial neural networks requiring robust memory retention and efficient learning.

De Novo Occurrence of Hb Chile [ß28(B10) Leu→Met] in a Korean Boy with Methemoglobinemia.

Hemoglobin (Hb) Chile, a variant of Hb M, is produced by a point mutation of CTG→ATG on codon 29 (legacy codon 28) of the Hb ß locus gene, which results in an amino acid substitution of Leu→Met. It has been identified in two families worldwide and is inherited in an autosomal dominant manner. Here, we report a case of Hb Chile in which a de novo mutation was detected in the proband. A 17-year-old male presented to the outpatient clinic with a pale appearance. There was cyanosis on his lips and fingers. Blood tests indicated the existence of hemolysis, but complete blood counts revealed no anemia. Peripheral arterial oxygen saturation on pulse oximetry was 80% on room air and did not improve with oxygen supplementation. The level of methemoglobin was 15.4%. Targeted next-generation sequencing identified a heterozygous NM_000518.4(HBB):c.85C > A mutation, indicating Hb Chile. The Hb Chile mutation, on the other hand, was not discovered in his parents, implying that it arose as a result of a de novo mutation. This case highlights the necessity of suspecting Hb gene mutations in patients with unexplained chronic methemoglobinemia, even if there is no family history.

Effects of perceived sleep quality on creative behavior via work engagement: the moderating role of gender.

BACKGROUND: Sleep quality significantly impacts employees' attitudes and behaviors. Using ego depletion theory, we examined the influence of sleep quality on work engagement and creative behavior, also investigating gender differences in these effects. METHODS: A multi-wave survey approach was employed with a six-week interval between waves for data collection. Participants were recruited online across two waves, totaling 322 employees from the United Kingdom and the United States. RESULTS: Regression analyses revealed a statistically significant positive relationship between sleep quality and creative behavior, mediated by work engagement. Additionally, gender moderated both the direct and indirect effects of sleep quality. CONCLUSION: The study found a positive relationship between sleep quality and creative behavior, mediated by work engagement, with notable gender differences. Sleep quality had a stronger impact on work engagement for men than women, and a stronger indirect effect on creative behavior through work engagement. These findings add to the existing literature on the influence of sleep quality on creative behavior.

Difference in trajectories according to early amyloid accumulation in cognitively unimpaired elderly.

BACKGROUND AND PURPOSE: Amyloid ß (Aß), a major biomarker of Alzheimer's disease, leads to tau accumulation, neurodegeneration and cognitive decline. Modelling the trajectory of Aß accumulation in cognitively unimpaired (CU) individuals is crucial, as treatments targeting Aß are anticipated. The evolution of Aß levels was investigated to determine whether it could lead to classification into different groups by studying longitudinal Aß changes in older CU individuals, and differences between the groups were compared. METHODS: A total of 297 CU participants were included from the Alzheimer's Disease Neuroimaging Initiative database, and these participants underwent apolipoprotein E (APOE) genotyping, neuropsychological testing, brain magnetic resonance imaging, and an average of 3.03 follow-up 18F-florbetapir positron emission tomography scans. Distinct Aß trajectory patterns were classified using latent class growth analysis, and longitudinal cognitive performances across these patterns were assessed with a linear mixed effects model. RESULTS: The optimal model consisted of three classes, with a high entropy value of 0.947. The classes were designated as follows: class 1, non-accumulation group (n = 197); class 2, late accumulation group (n = 70); and class 3, early accumulation group (n = 30). The late accumulation and early accumulation groups had more APOE ε4 carriers than the non-accumulation group. The longitudinal analysis of cognitive performance revealed that the early accumulation group showed the steepest decline (modified Preclinical Alzheimer's Cognitive Composite with digit symbol substitution [mPACCdigit], p < 0.001; modified Preclinical Alzheimer's Cognitive Composite with trails B [mPACCtrailsB], p < 0.001) and the late accumulation group showed a steeper decline (mPACCdigit, p = 0.014; mPACCtrailsB, p = 0.007) compared to the non-accumulation group. CONCLUSIONS: Our study showed the heterogeneity of Aß accumulation trajectories in CU older individuals. The prognoses for cognitive decline differ according to the Aß trajectory patterns.

Rare Non-Cryptic NUP98 Rearrangements Associated With Myeloid Neoplasms and Their Poor Prognostic Impact.

Background: NUP98 rearrangements (NUP98r), associated with various hematologic malignancies, involve more than 30 partner genes. Despite their clinical significance, reports on the clinicopathological characteristics of rare NUP98r remain limited. We investigated the characteristics of patients with myeloid neoplasms harboring NUP98r among those identified as having 11p15 translocation in chromosomal analysis. Methods: We retrospectively reviewed results from bone marrow chromosomal analyses conducted between 2011 and 2023 and identified 15 patients with 11p15 translocation. Subsequently, NUP98r were evaluated using FISH and/or reverse transcription PCR, and clinical and laboratory data of the patients were analyzed. Results: NUP98r were identified in 11 patients initially diagnosed as having AML (N=8), myelodysplastic syndrome (N=2), or chronic myelomonocytic leukemia (N=1), with a median age of 44 yrs (range, 4-77 yrs). Three patients had a history of chemotherapy. In total, five NUP98 fusions were identified: NUP98::DDX10 (N=3), NUP98::HOXA9 (N=2), NUP98::PSIP1 (N=2), NUP98::PRRX1 (N=1), and NUP98::HOXC11 (N=1). Patients with NUP98r exhibited a poor prognosis, with a median overall survival of 12.0 months (95% confidence interval [CI], 3.4-29.6 months) and a 5-yr overall survival rate of 18.2% (95% CI, 5.2%-63.7%). Conclusions: Our study revealed the clinical and genetic characteristics of patients with myeloid neoplasms harboring rare and non-cryptic NUP98r. Given its association with poor prognosis, a comprehensive evaluation is crucial for identifying previously underdiagnosed NUP98r in patients with myeloid neoplasms.

Acute Emotional Impact of Peer Suicide and Student-Related Factors.

OBJECTIVE: This study explored the negative emotional impact of peer suicide on adolescent students during the early stages of becoming aware of suicide and examined the adverse effects of related environmental and mental health factors. METHODS: This study was conducted from March 1, 2020 to December 31, 2021, targeting students enrolled in middle and high schools where student suicide occurred. Emotional impact was assessed using the Korean version of the Impact of Event Scale-Revised (IES-R-K), State-Trait Anxiety Inventory-X, Center for Epidemiologic Studies-Depression Scale (CES-D), and Beck's Scale for Suicide Ideation (SSI). χ2 test and Pearson's correlation analysis were performed to analyze subgroup differences and explore relationships between scale scores, respectively. RESULTS: Of the 2,382 participants, 25.1% belonged to the post-traumatic stress disorder (PTSD) or PTSD tendency group on the IES-R-K, and 22.2% showed abnormalities in the State-Trait Anxiety Inventory-State. Students in the same class as the deceased student and those in the same grade but in different classes had higher risk rates than those in different grades. Boarding school students had 1.9 times higher odds of experiencing emotional impacts than non-boarding students. On the CES-D, 10.4% of the students showed potential depression, with 3.7 times higher odds of experiencing emotional impact than those with normal scores. Moreover, 4.5% of the students reported suicidal ideation on the SSI, with those experiencing mild suicidal ideation having 1.9 times higher odds of experiencing emotional impact than those with normal scores. CONCLUSION: A significant proportion of students experienced negative impacts of peer suicide. Students in close-knit environments, such as the same class, grade, and boarding school as the deceased, or those with depression or anxiety experienced a more pronounced negative impact.

Augmentation of intracranial self-stimulation induced by amphetamine-like drugs in Period circadian regulator 2 knockout mice is associated with intracellular Ca2+ levels.

Over the past decade, new psychoactive substances (NPS) have emerged in the illegal drug market and have continued to attract attention from the international community. Among these, amphetamine-like NPS, classified as stimulants, constitute a significant proportion. However, the pharmacological characteristics and mechanisms underlying addiction to amphetamine-like NPS remain poorly understood. Given that circadian rhythms are linked to the brain stimulation effects of methamphetamine (METH) and amphetamine, we investigated the effects of METH, 1-(4-methoxyphenyl)-N-methylpropan-2-amine (PMMA), and 1-(benzofuran-5-yl)-N-ethylpropan-2-amine (5-EAPB) on intracranial self-stimulation (ICSS) in wild-type (WT) or Period circadian regulator 2 knockout mice. Amphetamine-like drugs increase intracellular Ca2+ levels to provoke dopamine release, so we examined the impact of Per2 knockdown on intracellular Ca2+ levels in PC12 cells to elucidate a potential mechanism underlying NPS-induced ICSS enhancement. Our ICSS results showed that METH and PMMA significantly increased brain stimulation in Per2 knockout mice compared to WT mice. Similarly, METH and PMMA induced higher Ca2+ fluorescence intensity in Per2 knockdown PC12 cells than in control cells. In contrast, 5-EAPB did not produce significant changes in either ICSS or Ca2+ signaling. These findings suggest that Per2 plays a crucial role in the brain stimulation effects of amphetamine-like drugs through the regulation of intracellular Ca2+.

Effect of polycyclic aromatic hydrocarbon exposure on amnestic mild cognitive impairment and Alzheimer's disease: A matched case-control study.

There is an emerging body of evidence concerning the neurological effect of air pollutants on cognitive function and increased risk of neurodegeneration. Although previous studies have suggested that polycyclic aromatic hydrocarbons (PAHs) are neurotoxic, the effect of PAHs exposure on neurodegeneration remains unclear. This study aimed to investigate the association between PAH exposure and the risk of developing amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). For this matched case-control cross-sectional study, we recruited patients aged ≥50 years diagnosed with aMCI and AD from the Samsung Medical Center, Seoul, Korea, between 2014 and 2019. For each patient, we randomly selected four cognitively healthy controls through frequency matching based on sex, age group, and education level. Urinary levels of four PAH metabolites, 1-hydroxypyrene (1-OHP), 1-hydroxyphenanthrene (1-OHPhe), 2-hydroxyfluorene (2-OHFlu), and 2-naphthol (2-NAP), were measured. A conditional logistic regression model was used to evaluate the association, adjusting for potential confounders. A total of 212 patients with aMCI with 848 matched controls, and 267 patients with AD with 1068 matched controls were included in the analyses to estimate the risk of PAH exposure. We found that elevated urinary levels of PAH metabolites (specifically, 1-OHP and 2-NAP) were significantly associated with an increased risk of aMCI and AD. An increase of one unit in log-transformed level of urinary 1-OHP was associated with a 1.15- and 1.16-times higher risk of aMCI and AD, respectively. An increase of one unit in log-transformed level of urinary 2-NAP was associated with a 1.11- and 1.13-times higher risk of aMCI and AD, respectively. These findings indicate that PAH exposure may increase the risk of aMCI and AD, especially for the elderly population. Considering the widespread distribution of PAHs in the environment, reducing PAH exposure may be an effective strategy for the prevention of neurodegenerative diseases.

Deep learning-based quantification of brain atrophy using 2D T1-weighted MRI for Alzheimer's disease classification.

Background: Determining brain atrophy is crucial for the diagnosis of neurodegenerative diseases. Despite detailed brain atrophy assessments using three-dimensional (3D) T1-weighted magnetic resonance imaging, their practical utility is limited by cost and time. This study introduces deep learning algorithms for quantifying brain atrophy using a more accessible two-dimensional (2D) T1, aiming to achieve cost-effective differentiation of dementia of the Alzheimer's type (DAT) from cognitively unimpaired (CU), while maintaining or exceeding the performance obtained with T1-3D individuals and to accurately predict AD-specific atrophy similarity and atrophic changes [W-scores and Brain Age Index (BAI)]. Methods: Involving 924 participants (478 CU and 446 DAT), our deep learning models were trained on cerebrospinal fluid (CSF) volumes from 2D T1 images and compared with 3D T1 images. The performance of the models in differentiating DAT from CU was assessed using receiver operating characteristic analysis. Pearson's correlation analyses were used to evaluate the relations between 3D T1 and 2D T1 measurements of cortical thickness and CSF volumes, AD-specific atrophy similarity, W-scores, and BAIs. Results: Our deep learning models demonstrated strong correlations between 2D and 3D T1-derived CSF volumes, with correlation coefficients r ranging from 0.805 to 0.971. The algorithms based on 2D T1 accurately distinguished DAT from CU with high accuracy (area under the curve values of 0.873), which were comparable to those of algorithms based on 3D T1. Algorithms based on 2D T1 image-derived CSF volumes showed high correlations in AD-specific atrophy similarity (r = 0.915), W-scores for brain atrophy (0.732 ≤ r ≤ 0.976), and BAIs (r = 0.821) compared with those based on 3D T1 images. Conclusion: Deep learning-based analysis of 2D T1 images is a feasible and accurate alternative for assessing brain atrophy, offering diagnostic precision comparable to that of 3D T1 imaging. This approach offers the advantage of the availability of T1-2D imaging, as well as reduced time and cost, while maintaining diagnostic precision comparable to T1-3D.

Different associations between body mass index and Alzheimer's markers depending on metabolic health.

BACKGROUND: Increasing evidence supports the association between body mass index (BMI), Alzheimer's disease, and vascular markers. Recently, metabolically unhealthy conditions have been reported to affect the expression of these markers. We aimed to investigate the effects of BMI status on Alzheimer's and vascular markers in relation to metabolic health status. METHODS: We recruited 1,736 Asians without dementia (71.6 ± 8.0 years). Participants were categorized into underweight, normal weight, or obese groups based on their BMI. Each group was further divided into metabolically healthy (MH) and unhealthy (MU) groups based on the International Diabetes Foundation definition of metabolic syndrome. The main outcome was Aß positivity, defined as a Centiloid value of 20.0 or above and the presence of vascular markers, defined as severe white matter hyperintensities (WMH). Logistic regression analyses were performed for Aß positivity and severe WMH with BMI status or interaction terms between BMI and metabolic health status as predictors. Mediation analyses were performed with hippocampal volume (HV) and baseline Mini-Mental State Examination (MMSE) scores as the outcomes, and linear mixed models were performed for longitudinal change in MMSE scores. RESULTS: Being underweight increased the risk of Aß positivity (odds ratio [OR] = 2.37, 95% confidence interval [CI] 1.13-4.98), whereas obesity decreased Aß positivity risk (OR = 0.63, 95% CI 0.50-0.80). Especially, obesity decreased the risk of Aß positivity (OR = 0.38, 95% CI 0.26-0.56) in the MH group, but not in the MU group. Obesity increased the risk of severe WMH (OR = 1.69, 1.16-2.47). Decreased Aß positivity mediate the relationship between obesity and higher HV and MMSE scores, particularly in the MH group. Obesity demonstrated a slower decline in MMSE (ß = 1.423, p = 0.037) compared to being normal weight, especially in the MH group. CONCLUSIONS: Our findings provide new evidence that metabolic health has a significant effect on the relationship between obesity and Alzheimer's markers, which, in turn, lead to better clinical outcomes.

Extracts of Prunella vulgaris Enhanced Pentobarbital-Induced Sleeping Behavior in Mice Potentially via Adenosine A2A Receptor Activity.

The increasing prevalence of sleep dysregulation cases has prompted the search for effective and safe sleep-enhancing agents. Numerous medications used in the treatment of sleep disorders function by enhancing γ-aminobutyric acid neurotransmitter activity. Unfortunately, these substances may induce significant adverse effects in chronic users, such as dependence and motor behavior impairments. Consequently, there is a growing interest in exploring therapeutic sleep-enhancing agents derived from natural sources, with the anticipation of causing less severe side effects. Prunella vulgaris (PV), a perennial plant indigenous to South Korea, exhibits various pharmacological effects, likely attributed to its chemical composition. Rosmarinic acid, one of its components, has previously demonstrated sleep-potentiating properties, suggesting the potential for PV to exhibit similar pharmacological effects. This study aims to investigate the potential effects of repeated administration of PV extract on the sleep behavior, brainwave activity, sleep-wake cycle, and physiological behavior of mice. Findings indicate that PV extracts exhibit sleep-enhancing effects in mice, characterized by prolonged sleep duration and a reduced onset time of pentobarbital-induced sleep. However, PV extracts only reduced alpha wave powers, with minor alterations in wakefulness and rapid-eye-movement sleep duration. In contrast to diazepam, PV extracts lack adverse effects on locomotor activity, motor coordination, or anxiety in mice. Receptor-binding assay and caffeine treatment support the potential involvement of adenosine A2A receptors in the effects of PV, suggesting distinct mechanisms of action compared to diazepam, despite both exhibiting sleep-altering effects. Overall, our results suggest that PV holds promise as a potential source of sleep-aiding agents.

Sihler's staining of the anterior belly of digastric muscle for botulinum toxin injection.

PURPOSE: The anterior belly of the digastric muscle (ABDM) is the target of botulinum toxin injection; however, anatomical considerations related to the injection point are absent. This study used Sihler's staining to analyze the intramuscular nerve distribution of ABDM to identify the most effective botulinum toxin injection points. METHODS: We used 12 specimens from 6 embalmed cadavers in this study. The specimens were manually dissected to preserve the mylohyoid nerve and subjected to Sihler's staining. From the gnathion to and hyoid bone, the ABDM was divided into three equal parts, distinguishing the anterior, middle, and posterior thirds. RESULTS: Only a branch of the mylohyoid nerve entered the ABDM, and its entry point was located in the middle-third region in all cases. The nerve endings were concentrated in the middle third (100%), followed by the anterior third (58.3%) and were not observed in the posterior third. CONCLUSION: The landmarks used in this study (gnathion and hyoid bone) are easily palpable on the skin surface, allowing clinicians to target the most effective injection site (middle third of ABDM). These results provide scientific and anatomic evidence for injection points, and will aid in the management of ABDM injection procedures in clinical practice.

Unraveling the Link between Perceived ESG and Psychological Well-Being: The Moderated Mediating Roles of Job Meaningfulness and Pay Satisfaction.

Enhancing corporate accountability in areas such as environment, social, and governance (ESG) has solidified its role in the discussion on improving corporate resilience and growth. ESG management activities not only augment corporate sustainability and risk control but also influence the professional roles and personal lives of members through their perceived ESG. Historically, most ESG research has centered on the interrelation of corporate ESG endeavors and outcomes, while studies focusing on the influence of perceived ESG on members have been sparse. In this light, our investigation, rooted in the conservation of resources theory, aimed to delineate the mechanistic link between perceived ESG and members' psychological well-being. This study employed a stratified random sampling technique and collected data across three waves, each spaced four weeks apart. Our sample comprised 325 Korean employees working in administrative, technical, service, and sales roles. This study recruited 325 Korean employees across three time-lagged phases and found that ESG comprehension enhances job meaningfulness, subsequently amplifying psychological wellness. Intriguingly, as pay satisfaction escalates, the mediating role of job meaningfulness between perceived ESG and well-being intensifies. Our study underscores that for organizations to harness perceived ESG to boost psychological well-being via job meaningfulness, managing pay satisfaction is imperative. These findings highlight a crucial policy implication: policymakers must actively promote ESG awareness and incorporate it into employee compensation strategies. This integration is essential to cultivating a healthier, more engaged workforce and driving long-term organizational success.