In Vivo Human Lacrimal Gland Imaging Using an Ultrasound Biomicroscopy.

Purpose: In the present study, we introduce human lacrimal gland imaging using an ultrasound biomicroscopy (UBM) with a soft cover and show their findings. Methods: The representative UBM findings of palpebral lobes in seven subjects (4 with non-Sjögren dry eye syndrome, 1 with Sjögren syndrome, and 2 healthy subjects) were described in this study. To prolapse the palpebral lobe, the examiner pulled the temporal part of the upper eyelid in the superotemporal direction and directed the subject to look in the inferonasal direction. We scanned the palpebral lobes longitudinally and transversely using UBM. We used an Aviso UBM (Quantel Medical, Clermont-Ferrand, France) with a 50 MHz linear probe and ClearScan. Results: In UBM of two healthy subjects, the echogenicity of the lacrimal gland was lower than that of the sclera and homogeneous. But, the parenchyma of a patient with Sjögren dry eye syndrome was quite inhomogeneous compared to the healthy subjects. In two patients with dry eye syndrome, we were able to observe some lobules in the parenchyma. We could find excretory ducts running parallel at the surface of the longitudinal section in some subjects. In the longitudinal UBM scan of a subject, we observed a tubular structure at a depth of 1500 µm that was considered a blood vessel. It ran from the superonasal to the inferotemporal direction. In a subject, we observed a large cyst beneath the conjunctiva. Conclusions: Lacrimal gland imaging using UBM has both advantages of OCT and sonography, and could be useful for evaluating dry eye syndrome.

Impact of essential and optional ingredients on microbial and metabolic profiles of kimchi.

This study aimed to examine the impacts of essential and optional ingredients on the microbial and metabolic profiles of kimchi during 100 days of fermentation, using a mix-omics approach. Kimchi manufactured without essential ingredients (e.g., red pepper, garlic, ginger, green onion, and radish) had lower lactic acid content. The absence of garlic was associated with a higher proportion of Latilactobacillus and Lactococcus, while the absence of red pepper was associated with a greater proportion of Leuconostoc than the control group. In addition, red pepper and garlic served as primary determinants of the levels of organic acids and biogenic amines. Sugar was positively correlated with the levels of melibiose, and anchovy sauce was positively correlated with the levels of amino acids such as methionine, leucine, and glycine. These findings contribute to a fundamental understanding of how ingredients influence kimchi fermentation, offering valuable insights for optimizing kimchi production to meet various preferences.

Mobile monitoring system detects the disease activity pattern and shows the association with clinical outcomes in patients with newly diagnosed Crohn's disease.

We aimed to determine whether Crohn's disease (CD) activity patterns assessed via a web-based symptom diary can help predict clinical outcomes in patients with newly diagnosed CD. Patients diagnosed with CD within the preceding 3 months were prospectively enrolled at four tertiary centers. All patients recorded their symptoms on a website using a smartphone at least once a week. The index outcomes were disease-related admission and surgery during follow-up. The disease activity from enrollment to outcome or last follow-up was reviewed for pattern analysis. Cox regression analysis was used to identify the predictors of disease outcomes. A total of 102 patients were enrolled. During a median follow-up period of 42 months, 25 (24.5%) and 6 (5.9%) patients required admission and surgery, respectively. Poor activity pattern was an independent predictor of disease-related hospitalization (adjusted hazard ratio [aHR], 3.96; 95% confidence interval [CI] 1.5-10.45; p = 0.005). A poor activity pattern (aHR, 19.48; 95% CI 1.86-203.95; p = 0.013) and female sex (aHR, 11.28; 95% CI 1.49-85.01; p = 0.018) were found to be independent predictors of bowel resection. CD disease activity patterns monitored through the mobile monitoring system may help predict clinical outcomes, such as disease-related hospitalization and surgery, in patients with newly diagnosed CD.

Long non-coding RNA SOX2OT in tamoxifen-resistant breast cancer.

Hormone receptor (HR)-positive breast cancer can become aggressive after developing hormone-treatment resistance. This study elucidated the role of long non-coding RNA (lncRNA) SOX2OT in tamoxifen-resistant (TAMR) breast cancer and its potential interplay with the tumor microenvironment (TME). TAMR breast cancer cell lines TAMR-V and TAMR-H were compared with the luminal type A cell line (MCF-7). LncRNA expression was assessed via next-generation sequencing, RNA extraction, lncRNA profiling, and quantitative RT-qPCR. SOX2OT overexpression effects on cell proliferation, migration, and invasion were evaluated using various assays. SOX2OT was consistently downregulated in TAMR cell lines and TAMR breast cancer tissue. Overexpression of SOX2OT in TAMR cells increased cell proliferation and cell invasion. However, SOX2OT overexpression did not significantly alter SOX2 levels, suggesting an independent interaction within TAMR cells. Kaplan-Meier plot analysis revealed an inverse relationship between SOX2OT expression and prognosis in luminal A and B breast cancers. Our findings highlight the potential role of SOX2OT in TAMR breast cancer progression. The downregulation of SOX2OT in TAMR breast cancer indicates its involvement in resistance mechanisms. Further studies should explore the intricate interactions between SOX2OT, SOX2, and TME in breast cancer subtypes.

Screening mammography performance according to breast density: a comparison between radiologists versus standalone intelligence detection.

BACKGROUND: Artificial intelligence (AI) algorithms for the independent assessment of screening mammograms have not been well established in a large screening cohort of Asian women. We compared the performance of screening digital mammography considering breast density, between radiologists and AI standalone detection among Korean women. METHODS: We retrospectively included 89,855 Korean women who underwent their initial screening digital mammography from 2009 to 2020. Breast cancer within 12 months of the screening mammography was the reference standard, according to the National Cancer Registry. Lunit software was used to determine the probability of malignancy scores, with a cutoff of 10% for breast cancer detection. The AI's performance was compared with that of the final Breast Imaging Reporting and Data System category, as recorded by breast radiologists. Breast density was classified into four categories (A-D) based on the radiologist and AI-based assessments. The performance metrics (cancer detection rate [CDR], sensitivity, specificity, positive predictive value [PPV], recall rate, and area under the receiver operating characteristic curve [AUC]) were compared across breast density categories. RESULTS: Mean participant age was 43.5 ± 8.7 years; 143 breast cancer cases were identified within 12 months. The CDRs (1.1/1000 examination) and sensitivity values showed no significant differences between radiologist and AI-based results (69.9% [95% confidence interval [CI], 61.7-77.3] vs. 67.1% [95% CI, 58.8-74.8]). However, the AI algorithm showed better specificity (93.0% [95% CI, 92.9-93.2] vs. 77.6% [95% CI, 61.7-77.9]), PPV (1.5% [95% CI, 1.2-1.9] vs. 0.5% [95% CI, 0.4-0.6]), recall rate (7.1% [95% CI, 6.9-7.2] vs. 22.5% [95% CI, 22.2-22.7]), and AUC values (0.8 [95% CI, 0.76-0.84] vs. 0.74 [95% CI, 0.7-0.78]) (all P < 0.05). Radiologist and AI-based results showed the best performance in the non-dense category; the CDR and sensitivity were higher for radiologists in the heterogeneously dense category (P = 0.059). However, the specificity, PPV, and recall rate consistently favored AI-based results across all categories, including the extremely dense category. CONCLUSIONS: AI-based software showed slightly lower sensitivity, although the difference was not statistically significant. However, it outperformed radiologists in recall rate, specificity, PPV, and AUC, with disparities most prominent in extremely dense breast tissue.

Comparative Pharmacokinetics of Gentamicin C1, C1a and C2 in Healthy and Infected Piglets.

Gentamicin, an aminoglycoside antibiotic, is a mixture of therapeutically active C1, C1a, C2 and other minor components. Despite its decades-long use in pigs and other species, its intramuscular (IM) pharmacokinetics/pharmacodynamics (PKs/PDs) are unknown in piglets. Furthermore, the PKs of many drugs differ between healthy and sick animals. Therefore, we investigated the PKs of gentamicin after a single IM dose (10 mg/kg) in healthy piglets and piglets that were intranasally co-infected with Actinobacillus pleuropneumoniae and Pasteurella multocida (PM). The plasma concentrations were measured using validated liquid chromatography/mass spectrometry. The gentamicin exposure was 36% lower based on the area under the plasma concentration-time curve and 16% lower based on the maximum plasma concentration (Cmax) in the infected piglets compared to the healthy piglets, while it was eliminated faster (shorter half-life and larger clearance) in the infected piglets compared to the healthy piglets. The clearance and volume of distribution were the highest for the C1 component. C1, C1a and C2 accounted for 22-25%, 33-37% and 40-42% of the total gentamicin exposure, respectively. The PK/PD target for the efficacy of aminoglycosides (Cmax/minimum inhibitory concentration (MIC) > 10) could be exceeded for PM, with a greater magnitude in the healthy piglets. We suggest integrating this PK information with antibiotic susceptibility data for other bacteria to make informed antibiotic and dosage regimen selections against piglet infections.

Minimum Information Variability in Linear Langevin Systems via Model Predictive Control.

Controlling the time evolution of a probability distribution that describes the dynamics of a given complex system is a challenging problem. Achieving success in this endeavour will benefit multiple practical scenarios, e.g., controlling mesoscopic systems. Here, we propose a control approach blending the model predictive control technique with insights from information geometry theory. Focusing on linear Langevin systems, we use model predictive control online optimisation capabilities to determine the system inputs that minimise deviations from the geodesic of the information length over time, ensuring dynamics with minimum "geometric information variability". We validate our methodology through numerical experimentation on the Ornstein-Uhlenbeck process and Kramers equation, demonstrating its feasibility. Furthermore, in the context of the Ornstein-Uhlenbeck process, we analyse the impact on the entropy production and entropy rate, providing a physical understanding of the effects of minimum information variability control.

Genomic and Metabolomic Analyses of Nocardiopsis maritima YSL2 as the Mycorrhizosphere Bacterium of Suaeda maritima (L.) Dumort.

Nine bacteria were isolated from the episphere of Suaeda maritima (L.) Dumort. Among them, the bacterial strain YSL2 displayed the highest antimicrobial activity on agar plates and exhibited significant novelty compared with other bacteria based on 16S rRNA analysis. Consequently, Nocardiopsis maritima YSL2T was subjected to phenotypic characterization and whole-genome sequencing. Phylogenetic analysis revealed its close association with Nocardiopsis aegyptia SNG49T. Furthermore, genomic analysis of strain YSL2T revealed the presence of various gene clusters, indicating its potential for producing antimicrobial secondary metabolites. Upon cultivation on a large scale, maritiamides A and B (1 and 2) were isolated and characterized as cyclic hexapeptides based on nuclear magnetic resonance, ultraviolet, infrared, and mass spectrometric data. The absolute configurations of the amino acid residues in the maritiamides were determined through chiral derivatization, utilizing FDAA and GITC. Maritiamides 1 and 2 exhibited promising antibacterial activities against Staphylococcus epidermidis and weakly inhibited the growth of Escherichia coli and Pseudomonas fluorescens.

Mutational analysis of 23 autosomal short tandem repeats based on trio paternity testing in the Korean population.

This study aimed to estimate A-STR mutation rates in 2,317 Korean parent-child trios by examining 20 Combined DNA Index System (CODIS) core loci (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045) and three non-CODIS loci (Penta E, Penta D, and SE33). Locus-specific mutation rate estimates varied from 0.00 to 8.63 × 10-3 per generation, with an average mutation rate of 1.62 × 10-3 (95 % CI, 1.39-1.88 × 10-3). We also combined data from previous studies to obtain comprehensive genetic values for the Korean population, and the average mutation rate was 1.59 × 10-3 (95 % CI, 1.38-1.82 × 10-3). Single-step mutations (95.69 %) and double-step mutations (3.35 %) were observed in the mutation pattern analysis, and cases expected to have multi-step mutations (0.96 %) were also observed. Large-sized alleles exhibited more loss mutations than gain mutations, and paternal mutations (62.68 %) were more frequently observed than maternal mutations (19.62 %). The calculated values and features of the 23 A-STRs explored in this study are expected to play a crucial role in establishing criteria for forensic genetic interpretation.

Impact of leisure physical activity and resistance exercise on the prevalence of depressive symptoms in Korean adults: Analysis of the Korean National Health and Nutrition Examination Survey.

BACKGROUND: Various physical activity (PA) guidelines recommend leisure PA and resistance exercise (RE). However, the impact of PA and RE on the prevalence of depressive symptoms remains unclear. We investigated whether meeting PA and RE guidelines is associated with the prevalence of depressive symptoms using nationally representative samples. METHODS: We analyzed data from 11,829 (5111 male and 6718 female) participants aged 19+ years in the Korean National Health and Nutrition Examination Survey (2016 and 2018). Those with doctor's diagnosis of depression or with a cut-off score of ≥11 on the Patient Health Questionnaire-9 were defined as having depressive symptoms. PA and RE were measured using a validated Global PA Questionnaire. Logistic regression analysis was used to examine the association between participating in leisure PA, simultaneously meeting RE guidelines, and experiencing depressive symptoms. RESULTS: Those who participated in leisure PA and met RE guidelines had a lower prevalence of depressive symptoms than those who did not (p for trend <0.001). After adjusting for covariates, those who participated in leisure PA and met RE guideline were significantly less likely to have depressive symptoms in male (OR = 0.52, 95 % CI 0.32-0.84) and female (OR = 0.71, 95 % CI 0.48-1.06), compared with those who did not participate in leisure PA but also did not meet RE guideline. LIMITATIONS: The cross-sectional nature of the data prevented causal claims. CONCLUSIONS: Participation in leisure PA and RE was associated with a lower prevalence of depressive symptoms. This suggests both leisure PA and RE should be encouraged as effective means for preventing depression.

Impact of AI for Digital Breast Tomosynthesis on Breast Cancer Detection and Interpretation Time.

"Just Accepted" papers have undergone full peer review and have been accepted for publication in Radiology: Artificial Intelligence. This article will undergo copyediting, layout, and proof review before it is published in its final version. Please note that during production of the final copyedited article, errors may be discovered which could affect the content. Purpose To develop an artificial intelligence (AI) for diagnosis of breast cancer in digital breast tomosynthesis (DBT) and investigate whether it could improve diagnostic accuracy and reduce reading time of radiologists. Materials and methods A deep learning AI algorithm was developed and validated for DBT with retrospectively collected examinations (January 2010 to December 2021) from 14 institutions in the United States and South Korea. A multicenter, reader study was performed to compare the performance of 15 radiologists (7 breast specialists, 8 general radiologists) in interpreting DBT examinations from 258 women (mean, 56 years ± 13.41 [SD]), including 65 cancer cases, with and without the use of AI. Area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and reading time were evaluated. Results The AUC for standalone AI performance was 0.93 (95% CI: 0.92,0.94). With AI, radiologists' AUC improved from 0.90 (0.86, 0.93) to 0.92 (0.88, 0.96; P = .003) in the reader study. AI showed higher specificity (89.64% (85.34, 93.94)) than radiologists (77.34% (75.82, 78.87; P < .001)). When reading with AI, radiologists' sensitivity increased from 85.44% (83.22, 87.65) to 87.69% (85.63, 89.75; P = .04), with no evidence of a difference in specificity. Reading time decreased from 54.41 seconds (52.56, 56.27) without AI to 48.52 seconds (46.79, 50.25) with AI (P < .001). Interreader agreement measured by Fleiss kappa increased from 0.59 to 0.62, respectively. Conclusion The AI model showed better diagnostic accuracy than radiologists in breast cancer detection and reduced reading times. The concurrent use of AI in DBT interpretation could improve both accuracy and efficiency. ©RSNA, 2024.

Sex Difference in the Effect of Bifidobacterium longum on Repeated Water Avoidance Stress-induced Gut Dysbiosis in Wistar Rats.

Dysbiosis in gut microbiota is known to contribute to development of irritable bowel syndrome. We tried to investigate the effect of Bifidobacterium longum on repeated water avoidance stress (WAS) in a Wistar rat model. The three groups (no-stress, WAS, and WAS with B. longum) of rats were allocated to sham or WAS for 1 hour daily for 10 days, and B. longum was administered through gavage for 10 days. Fecal pellet numbers were counted at the end of each 1-hour session of WAS. After 10 days of repeated WAS, the rats were eutanized, and the feces were collected. WAS increased fecal pellet output (FPO) significantly in both sexes (P < 0.001), while the female B. longum group showed significantly decreased FPO (P = 0.005). However, there was no consistent change of myeloperoxidase activity and mRNA expression of interleukin-1ß and TNF-α. Mast cell infiltration at colonic submucosa increased in the female WAS group (P = 0.016). In terms of fecal microbiota, the repeated WAS groups in both sexes showed different beta-diversity compared to control and WAS with B. longum groups. WAS-induced mast cell infiltration was reduced by the administration of B. longum in female rats. Moreover, administration of B. longum relieved WAS-caused dysbiosis, especially in female rats. In conclusion, B. longum was beneficial for WAS-induced stress in rats, especially in females.

Self-screening questionnaire for perianal fistulizing disease in patients with Crohn's disease.

Background/Aims: A poor prognostic factor for Crohn's disease (CD) includes perianal fistulizing disease, including perianal fistula and/or perianal abscess. Currently, a tool to assess perianal symptoms in patients with CD remains nonexistent. This study aimed to develop a perianal fistulizing disease self-screening questionnaire for patients with CD. Methods: This prospective pilot study was conducted at three tertiary referral centers between January 2019 and May 2020. We formulated questions on perianal symptoms, including tenesmus, anal discharge, bleeding, pain, and heat. A 4-point Likert scale was used to rate each question. Patients with CD completed a questionnaire and underwent pelvic magnetic resonance imaging (MRI). Results: Overall, 93 patients were enrolled, with 51 (54.8%) diagnosed with perianal fistulizing disease, as determined by pelvic MRI. The Spearman correlation findings demonstrated that anal pain (p = 0.450, p < 0.001) and anal discharge (p = 0.556, p < 0.001) were the symptoms that most significantly correlated with perianal disease. For anal pain and discharge, the area under the receiver operating characteristic curve of the scores was significantly higher than that of the combined score for all five symptoms (0.855 vs. 0.794, DeLong's test p = 0.04). For the two symptoms combined, the sensitivity, specificity, and positive predictive and negative predictive values were 88.2, 73.8, 80.4, and 83.8%, respectively, with 81.7% accuracy for detecting perianal fistulizing disease. Conclusions: This study indicates that simple questions regarding anal pain and discharge can help accurately identify the presence of perianal fistulizing disease in patients with CD.

The Critical Role of Intracellular Bacterial Communities in Uncomplicated Recurrent Urinary Cystitis: A Comprehensive Review of Detection Methods and Diagnostic Potential.

Urinary tract infections (UTIs) are among the most common bacterial infections worldwide and are particularly prevalent in women. Recurrent UTIs significantly diminish quality of life due to their symptoms and frequent relapses. Patients often experience immediate relapse following slightly strenuous activities or intense psychological stress. In this review, we explore why infections persist despite the advent of various treatments and suggest strategies to manage recurrent cystitis by targeting the mechanisms of adhesion and infection. Vitamin D levels and the expression of neutrophil gelatinase-associated lipocalin are linked to the recurrence of UTIs. During a UTI, bacteria employ adhesins to invade the urinary tract, adhere to urothelial cells, and then penetrate these cells, where they rapidly multiply to establish intracellular bacterial communities. Bacteria can also form quiescent intracellular reservoirs that escape immune responses and antibiotic treatments, leading to recurrence under certain conditions. The surface proteins of bacteria and D-mannose are crucial in the adhesion of bacteria to the urothelium. Understanding these processes provides valuable insights into potential therapeutic approaches that focus on preventing bacterial attachment and cluster formation. By disrupting the ability of bacteria to adhere to and form clusters on cells, we can better manage recurrent UTIs and improve patient outcomes.

Comparison of the Prognosis of Upper-Third Gastric Cancer With That of Middle and Lower-Third Gastric Cancer.

PURPOSE: Gastric cancer is one of the most common cancers in Korea, and the proportion of upper-third gastric cancers has been steadily increasing over the last two decades. This study aimed to evaluate the effect of tumor location on gastric cancer prognosis. MATERIALS AND METHODS: We retrospectively reviewed 2,466 patients who underwent gastrectomy for pathologically proven gastric cancer between January 2011 and December 2016. The patients were divided into an upper-third group (U group; n=419, 17.0%) and a middle- and lower-third group (ML group; n=2,047, 83.0%). Clinicopathological characteristics, overall survival (OS), and recurrence-free survival (RFS) after surgery were compared. RESULTS: The U group had more advanced disease than the ML group and a higher incidence of N3b disease for T3 (12.0% vs. 4.9%, p=0.023) and T4 tumors (33.3% vs. 17.5%, p=0.001). The 5-year RFS rate for stage III disease was marginally lower in the U group than that in the ML group (47.1% vs. 56.7%, p=0.082). The upper third location was an independent prognostic factor for both OS (hazard ratio [HR], 1.350; 95% confidence interval [CI], 1.065-1.711) and RFS (HR, 1.430; 95% CI, 1.080-1.823). CONCLUSIONS: Upper-third gastric cancer shows extensive node metastasis compared to those located more distally in ≥T3 tumors. The upper third location is an independent prognostic factor for both OS and RFS and may have an adverse impact on RFS, particularly in patients with stage III gastric cancer.

Periaqueductal gray activates antipredatory neural responses in the amygdala of foraging rats.

Pavlovian fear conditioning research suggests that the interaction between the dorsal periaqueductal gray (dPAG) and basolateral amygdala (BLA) acts as a prediction error mechanism in the formation of associative fear memories. However, their roles in responding to naturalistic predatory threats, characterized by less explicit cues and the absence of reiterative trial-and-error learning events, remain unexplored. In this study, we conducted single-unit recordings in rats during an 'approach food-avoid predator' task, focusing on the responsiveness of dPAG and BLA neurons to a looming robot predator. Optogenetic stimulation of the dPAG triggered fleeing behaviors and increased BLA activity in naive rats. Notably, BLA neurons activated by dPAG stimulation displayed immediate responses to the robot, demonstrating heightened synchronous activity compared to BLA neurons that did not respond to dPAG stimulation. Additionally, the use of anterograde and retrograde tracer injections into the dPAG and BLA, respectively, coupled with c-Fos activation in response to predatory threats, indicates that the midline thalamus may play an intermediary role in innate antipredatory defensive functioning.

Comparison of Enlarged Perivascular Spaces in Early-Onset and Late-Onset Alzheimer Disease-related Cognitive Impairment: A Single Clinic-based Study in South Korea.

We examined whether there were differences in the presence of centrum semiovale-enlarged perivascular spaces (CSO-ePVS) and basal ganglia-ePVS (BG-ePVS) among patients with Alzheimer disease-related cognitive impairment (ADCI) based on their age of onset. Out of a total of 239 patients with cognitive impairment, 155 with positive amyloid-PET results were included. Among these, 43 had early-onset ADCI (EOADCI) and 112 had late-onset ADCI (LOADCI). Patients with LOADCI exhibited a higher prevalence of hypertension, lacunes, white matter hyperintensities, and BG-ePVS than those with EOADCI. BG-ePVS showed a significant correlation with age at the onset and the number of lacunes, whereas CSO-ePVS did not exhibit any association. The higher prevalence of BG-ePVS in patients with LOADCI might be attributable to vascular risk factors (hypertension) and cerebral small vessel disease (CSVD). These findings support the hypothesis that BG-ePVS is associated with CSVD and vascular risk factors, whereas CSO-ePVS is associated with cerebral amyloid angiopathy.

Dynamic mucus secretion in ventral surfaces of toe pads of the tree frog (Dryophytes japonica).

The tree frog is a prominent amphibian among terrestrial vertebrates known for its ability to adhere to various surfaces through the capillary forces of water in the microchannels between micropillars on its disc-shaped toe pads, a phenomenon known as wet adhesion. However, the secretion pattern of mucus on the attachment surface of living tree frog toe pads and the distribution of active mucus pores (AMPs) have not yet been fully elucidated. In this study, we utilized synchrotron X-ray micro-computed tomography and interference reflection microscopy to obtain the spatial distribution of the entire population of ventral mucus glands on the toe pads of living tree frogs and the real-time mucus secretion patterns from the ventral mucus pores on the contact surface under different environmental conditions. We observed that the number and secretion frequency of AMPs on the toe pad are regulated according to environmental conditions. Such dynamic mucus secretion on the tree frog's toe pad could contribute to the understanding of capillary force regulation for wet adhesion and the development of adhesive surfaces by mimicking the mucus-secreting toe pad.