TikTok and Bilibili as health information sources on gastroesophageal reflux disease: an assessment of content and its quality.

Gastroesophageal reflux disease (GERD) is a global chronic disease. Short video platforms make it easy for patients with GERD to obtain medical information. However, the quality of information from these videos remains uncertain. This study aimed to systematically assess videos related to GERD on TikTok and Bilibili. We conducted a search and gathered 241 Chinese videos related to GERD and recorded the essential information. Two independent evaluators assessed each video based on the completeness of six components of the GERD guidelines, and assessed the quality and reliability of the information in the videos using recognition tools. Finally, videos from different sources were compared. The uploaders of most videos were medical professionals (86.7%, n = 209). The content was mainly about symptoms and treatment. The quality of the videos information varied depending on the sources. Among videos posted on Bilibili, those posted by medical professionals had a lower content score for definition (P < 0.001). Videos produced by non-gastroenterologists had the highest mean modified DISCERN. (The DISCERN scoring tool was initially created for assessing written publications, but nowadays, it is frequently leveraged for appraising various health-related videos. Details can be found in the text) quality of the videos information was acceptable; however, the content varied significantly depending on the type of source used. Videos with broad content should be carefully screened to meet more needs.

Partial Transfer Learning Method Based on Inter-Class Feature Transfer for Rolling Bearing Fault Diagnosis.

Rolling bearing fault diagnosis methods based on transfer learning always assume that the sample classes in the target domain are consistent with those in the source domain during the training phase. However, it is difficult to collect all fault classes in the early stage of mechanical application. The more likely situation is that the training data in the target domain only contain a subset of the entire health state, which will lead to the problem of label imbalance compared with the source domain. The outlier classes in the source domain that do not have corresponding target domain samples for feature alignment will interfere with the feature transfer of other classes. To address this specific challenge, this study introduces an innovative inter-class feature transfer fault diagnosis approach. By leveraging label information, the method distinctively computes the distribution discrepancies among shared classes, thereby circumventing the deleterious influence of outlier classes on the transfer procedure. Empirical evaluations on two rolling bearing datasets, encompassing multiple partial transfer tasks, substantiate that the proposed method surpasses other approaches, offering a novel and efficacious solution for the realm of intelligent bearing fault diagnosis.

A case of Abernethy malformation type I combined with hepatoblastoma.

This article encountered an extremely rare case of a 2-year-old male with Abernethy malformation Type I combined with hepatoblastoma. Furthermore, the medical history was characterized by several other abnormalities: gross facial asymmetry and cardiac defects，thus, diagnosis of Goldenhar syndrome in the setting of Abernethy type I was made. In this article, we exhibit the typical clinical presentation and Pathology imaging features of this disease.

Complex phenotype in Fanconi renotubular syndrome type 1: Hypophosphatemic rickets as the predominant presentation.

GATM-related Fanconi renotubular syndrome 1 (FRTS1) is a form of renal Fanconi syndrome (RFS), which is a disorder of solute and water reabsorption caused by defects in the function of the entire proximal tubule. Recent findings reveal the molecular basis of FRTS1: Intramitochondrial fiber aggregation triggered by mutant GATM provides a starting point for proximal tubule damage and drives disease progression. As a rare and newly recognized inherited kidney disease, the complex manifestations of FRTS1 are easily underdiagnosed or misdiagnosed. We discuss the complex phenotype of a 26-year-old woman with onset in infancy and a long history of hypophosphatemic rickets. We also identified a novel heterozygous missense variant in the GATM gene in this patient. The novel variant and phenotype we report expand the disease spectrum of FRTS1. We recommend screening for GATM in children with RFS, especially in patients with resistant rickets who have previously had negative genetic testing. In addition, we found pathological deposition of mutant GATM proteins within mitochondria in the patient's urinary sediment cells by a combination of electron microscopy and immunofluorescence. This unique urine cytology experiment has the potential to be a valuable tool for identifying patients with RRTS1.

Three exonic variants in the PHEX gene cause aberrant splicing in a minigene assay.

Background: X-linked hypophosphatemia (XLH, OMIM 307800) is a rare phosphorus metabolism disorder caused by PHEX gene variants. Many variants simply classified as missense or nonsense variants were only analyzed at the DNA level. However, growing evidence indicates that some of these variants may alter pre-mRNA splicing, causing diseases. Therefore, this study aimed to use bioinformatics tools and a minigene assay to ascertain the effects of PHEX variations on pre-mRNA splicing. Methods: We analyzed 174 variants in the PHEX gene described as missense or nonsense variants. Finally, we selected eight candidate variants using bioinformatics tools to evaluate their effects on pre-mRNA splicing using a minigene assay system. The complementary DNA (cDNA) sequence for the PHEX gene (RefSeq NM_000444.6) serves as the basis for DNA variant numbering. Results: Of the eight candidate variants, three were found to cause abnormal splicing. Variants c.617T>G p.(Leu206Trp) and c.621T>A p.(Tyr207*) in exon 5 altered the splicing of pre-mRNA, owing to the activation of a cryptic splice site in exon 5, which produced an aberrant transcript lacking a part of exon 5, whereas variant c.1700G>C p.(Arg567Pro) in exon 16 led to the activation of a cryptic splice site in intron 16, resulting in a partial inclusion of intron 16. Conclusion: Our study employed a minigene system, which has a great degree of flexibility to assess abnormal splicing patterns under the circumstances of patient mRNA samples that are not available, to explore the impact of the exonic variants on pre-mRNA splicing. Based on the aforementioned experimental findings, we demonstrated the importance of analyzing exonic variants at the mRNA level.

CARD11-BCL10-MALT1 Complex-Dependent MALT1 Activation Facilitates Myocardial Oxidative Stress in Doxorubicin-Treated Mice via Enhancing k48-Linked Ubiquitination of Nrf2.

Aims: Downregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) contributes to doxorubicin (DOX)-induced myocardial oxidative stress, and inhibition of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) increased Nrf2 protein level in rat heart suffering ischemia/reperfusion, indicating a connection between MALT1 and Nrf2. This study aims to explore the role of MALT1 in DOX-induced myocardial oxidative stress and the underlying mechanisms. Results: The mice received a single injection of DOX (15 mg/kg, i.p.) to induce myocardial oxidative stress, evidenced by increases in the levels of reactive oxidative species as well as decreases in the activities of antioxidative enzymes, concomitant with a downregulation of Nrf2; these phenomena were reversed by MALT1 inhibitor. Similar phenomena were observed in DOX-induced oxidative stress in cardiomyocytes. Mechanistically, knockdown or inhibition of MALT1 notably attenuated the interaction between Nrf2 and MALT1 and decreased the k48-linked ubiquitination of Nrf2. Furthermore, inhibition or knockdown of calcium/calmodulin-dependent protein kinase II (CaMKII-δ) reduced the phosphorylation of caspase recruitment domain-containing protein 11 (CARD11), subsequently disrupted the assembly of CARD11, B cell lymphoma 10 (BCL10), and MALT1 (CBM) complex, and reduced the MALT1-dependent k48-linked ubiquitination of Nrf2 in DOX-treated mice or cardiomyocytes. Innovation and Conclusion: The E3 ubiquitin ligase function of MALT1 accounts for the downregulation of Nrf2 and aggravation of myocardial oxidative stress in DOX-treated mice, and CaMKII-δ-dependent phosphorylation of CARD11 triggered the assembly of CBM complex and the subsequent activation of MALT1.

Identification of seven variants in the col4a1 gene that alter RNA splicing by minigene assay.

Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.

A novel heterozygous variant of the SALL1 gene with atypical Townes-Brocks syndrome phenotypes in Chinese family.

Townes-Brocks syndrome (TBS) is an autosomal dominant disorder characterised by the triad of anorectal, thumb, and ear malformations. It may also be accompanied by defects in kidney, heart, eyes, hearing, and feet. TBS has been demonstrated to result from heterozygous variants in the SALL1 gene, which encodes zinc finger protein believed to function as a transcriptional repressor. The clinical characteristics of an atypical TBS phenotype patient from a Chinese family are described, with predominant manifestations including external ear dysplasia, unilateral renal hypoplasia with mild renal dysfunction, and hearing impairment. A novel heterozygous variant c.3060T>A (p.Tyr1020*) in exon 2 of the SALL1 gene was identified in this proband. Pyrosequencing of the complementary DNA of the proband revealed that the variant transcript accounted for 48% of the total transcripts in peripheral leukocytes, indicating that this variant transcript has not undergone nonsense-mediated mRNA decay. This variant c.3060T > A is located at the terminal end of exon 2, proximal to the 3' end of the SALL1 gene, and exerts a relatively minor impact on protein function. We suggest that the atypical TBS phenotype observed in the proband may be attributed to the truncated protein retaining partial SALL1 function.

Health information in short videos about metabolic dysfunction-associated steatotic liver disease: Analysing quality and reliability.

BACKGROUND & AIMS: Short videos, crucial for disseminating health information on metabolic dysfunction-associated steatotic liver disease (MASLD), lack a clear evaluation of quality and reliability. This study aimed to assess the quality and reliability of MASLD-related videos on Chinese platforms. METHODS: Video samples were collected from three platforms (TikTok, Kwai and Bilibili) during the period from November 2019 to July 2023. Two independent reviewers evaluated the integrity of the information contained therein by scoring six key aspects of its content: definition, epidemiology, risk factors, outcomes, diagnosis and treatment. The quality and reliability of the videos were assessed using the Journal of the American Medical Association (JAMA) criteria, the Global Quality Score (GQS) and the modified DISCERN score. RESULTS: A total of 198 videos were included. The video content exhibited an overall unsatisfactory quality, with a primary emphasis on risk factors and treatment, while diagnosis and epidemiology were seldom addressed. Regarding the sources of the videos, the GQS and modified DISCERN scores varied significantly between the platforms (p = .003), although they had generally similar JAMA scores (p = .251). Videos created by medical professionals differed significantly in terms of JAMA scores (p = .046) compared to those created by nonmedical professionals, but there were no statistically significant differences in GQS (p = .923) or modified DISCERN scores (p = .317). CONCLUSIONS: The overall quality and reliability of the videos were poor and varied between platforms and uploaders. Platforms and healthcare professionals should strive to provide more reliable health-related information regarding MASLD.

Three exonic variants in the COL4A5 gene alter RNA splicing in a minigene assay.

BACKGROUND: X-linked Alport syndrome (XLAS) is an inherited renal disease caused by rare variants of COL4A5 on chromosome Xq22. Many studies have indicated that single nucleotide variants (SNVs) in exons can disrupt normal splicing process of the pre-mRNA by altering various splicing regulatory signals. The male patients with XLAS have a strong genotype-phenotype correlation. Confirming the effect of variants on splicing can help to predict kidney prognosis. This study aimed to investigate whether single nucleotide substitutions, located within three bases at the 5' end of the exons or internal position of the exons in COL4A5 gene, cause aberrant splicing process. METHODS: We analyzed 401 SNVs previously presumed missense and nonsense variants in COL4A5 gene by bioinformatics programs and identified candidate variants that may affect the splicing of pre-mRNA via minigene assays. RESULTS: Our study indicated three of eight candidate variants induced complete or partial exon skipping. Variants c.2678G>C and c.2918G>A probably disturb classic splice sites leading to corresponding exon skipping. Variant c.3700C>T may disrupt splicing enhancer motifs accompanying with generation of splicing silencer sequences resulting in the skipping of exon 41. CONCLUSION: Our study revealed that two missense variants positioned the first nucleotides of the 5' end of COL4A5 exons and one internal exonic nonsense variant caused aberrant splicing. Importantly, this study emphasized the necessity of assessing the effects of SNVs at the mRNA level.

A novel discrete linkage-type electrode for radiofrequency-induced intestinal anastomosis.

INTRODUCTION: For decades, radiofrequency (RF)-induced tissue fusion has garnered great attention due to its potential to replace sutures and staples for anastomosis of tissue reconstruction. However, the complexities of achieving high bonding strength and reducing excessive thermal damage present substantial limitations of existing fusion devices. MATERIALS AND METHODS: This study proposed a discrete linkage-type electrode to carry out ex vivo RF-induced intestinal anastomosis experiments. The anastomotic strength was examined by burst pressure and shear strength test. The degree of thermal damage was monitored through an infrared thermal imager. And the anastomotic stoma fused by the electrode was further investigated through histopathological and ultrastructural observation. RESULTS: The burst pressure and shear strength of anastomotic tissue can reach 62.2 ± 3.08 mmHg and 8.73 ± 1.11N, respectively, when the pressure, power and duration are 995 kPa, 160 W and 13 s, and the thermal damage can be controlled within limits. Histopathological and ultrastructural observation indicate that an intact and fully fused stomas with collagenic crosslink can be formed. CONCLUSION: The discrete linkage-type electrode presents favorable efficiency and security in RF-induced tissue fusion, and these results are informative to the design of electrosurgical medical devices with controllable pressure and energy delivery.

High efficiency and stable partial nitration achieved via gel immobilization.

Long-term high efficiency and stable partial nitrification (PN) performance was achieved using gel-immobilized partial nitrifying bacteria. The PN characteristics of the filler under high and low ammonia nitrogen concentrations and low temperature were comprehensively studied and the rapid reactivation was achieved after reactor breakdown or long stagnation period. The results showed that the maximum ammonia oxidation rate was 66.8 mg•(L•h)-1 and the nitrite accumulation rate was above 95 % for the filler. Efficient and stable PN performance depends on the high abundance of ammonia-oxidizing bacteria (AOB) inside the filler and dynamically microbial community. In addition, the oxygen-limited zone and competition between the microorganisms inside the filler effectively inhibited the growth of nitrite oxidizing bacteria, and the sludge outside the filler assisted in this process, which supported the dominant position of AOB in fillers. This study provides a reliable technology for the practical application of the PN nitrogen removal process.

Functional analysis of the CTNS gene exonic variants predicted to affect splicing.

Cystinosis is a severe, monogenic systemic disease caused by variants in CTNS gene. Currently, there is growing evidence that exonic variants in many diseases can affect pre-mRNA splicing. The impact of CTNS gene exonic variants on splicing regulation may be underestimated due to the lack of routine studies at the RNA level. Here, we analyzed 59 exonic variants in the CTNS gene using bioinformatics tools and identified candidate variants that may induce splicing alterations by minigene assays. We identified six exonic variants that induce splicing alterations by disrupting the ratio of exonic splicing enhancers/exonic splicing silencers (ESEs/ESSs) or by interfering with the recognition of classical splice sites, or both. Our results help in the correct molecular characterization of variants in cystinosis and inform emerging therapies. Furthermore, our work suggests that the combination of in silico and in vitro assays facilitates to assess the effects of DNA variants driving rare genetic diseases on splicing regulation and will enhance the clinical utility of variant functional annotation.

Optimization of Pedicle Screw Parameters for Enhancing Implant Stability Based on Finite Element Analysis.

OBJECTIVE: To improve implant stability parameters, including pedicle screw (PS) outer diameter, thread depth, and pitch, by finite element analysis. METHODS: Insertion and pullout of the PS were simulated by finite element analysis, and the precision of simulation was evaluated by comparison with mechanical tests. Influences of the parameters on the maximum insertion torque and maximum pullout force were analyzed by computational simulations, including single-factor analysis and orthogonal experiments. RESULTS: The simulation results agreed with the mechanical test results. The order of parameters influencing insertion torque and pullout force was outer diameter > pitch > thread depth. When the pilot hole diameter is 0.1 mm larger than the inner diameter of the PS, the calculated Pearson correlation coefficient between the maximum insertion torque and maximum pullout force was r = 0.99. The optimized PS had a maximum insertion torque of 485.16 N·mm and a maximum pullout force of 1726.33 N, 23.9% and 9.1% higher, respectively, than the values of standard screws. CONCLUSIONS: The presently used models are feasible for evaluating the implant stability of PSs. The maximum insertion torque and maximum pullout force of PSs are highly correlated and can be improved by increasing the outer diameter and decreasing pitch. Although with the parameters of the PS, pedicle size and bone mineral density are 2 additional factors to consider for better implant stability.

Optimizing the hydrolysis-acidification stage in municipal wastewater treatment: comparison of immobilized fillers and granular sludge.

The decomposition of organic macromolecules in sewage currently benefits substantially from hydrolysis-acidification. The full use of its qualities can help domestic sewage to biodegrade more quickly, which promotes the subsequent aerobic reactions. This study evaluated the hydrolysis-acidification performance of granular sludge and filler in residential sewage. Both forms were highly effective at producing volatile fatty acids (VFAs) at the beginning of the reaction, but the granular sludge gradually disintegrated over time, particularly at low temperatures. The production of VFAs decreased (68.08 mg/L), and the effluent dissolved organic nitrogen (DON) increased (6.23 mg/L). However, the effluent of fillers remained at a lower level (1.3 mg/L) and produced more VFAs (74.13 mg/L). High-throughput sequencing revealed that the filler included a greater quantity of hydrolytic-acidifying bacteria than the granular sludge, which resulted in higher performance. In this study, the optimal form of utilizing hydrolytic acidifying bacteria was discussed to provide a theoretical basis to improve the full utilization of organic matter in domestic sewage and the removal of as much total nitrogen as possible.

Inhibition of astroglial hemichannels ameliorates infrasonic noise induced short-term learning and memory impairment.

As a kind of environmental noise, infrasonic noise has negative effects on various human organs. To date, research has shown that infrasound impairs cognitive function, especially the ability for learning and memory. Previously, we demonstrated that impaired learning and memory induced by infrasound was closely related with glia activation; however, the underlying mechanisms remain unclear. Connexin 43 hemichannels (Cx43 HCs), which are mainly expressed in hippocampal astrocytes, are activated under pathological conditions, lending support to the hypothesis that Cx43 HCs might function in the impaired learning and memory induced by infrasound. This study revealed that that blocking hippocampal Cx43 HCs or downregulating hippocampal Cx43 expression significantly alleviated impaired learning and memory induced by infrasound. We also observed that infrasound exposure led to the abundant release of glutamate and ATP through Cx43 HCs. In addition, the abundant release of glutamate and ATP depended on proinflammatory cytokines. Our finds suggested that the enhanced release of ATP and glutamate by astroglial Cx43 HCs may be involved in the learning and memory deficits caused by infrasound exposure.

Higher oxidative balance scores are associated with lower nonalcoholic fatty liver disease and not with fibrosis in US adults.

BACKGROUND AND AIMS: Little research have focused on the relationship between systemic oxidative stress status and NAFLD and fibrosis. The Oxidative Balance Score (OBS) is employed to evaluate whole-body lifestyle and diet exposures related to oxidative stress, with higher OBS scores implying exposure to more antioxidants. This study aimed to explore whether OBS is correlated with NAFLD and NAFLD-related fibrosis. METHODS AND RESULTS: 12,223 participants from NHANES 2003-2018 were enrolled in this study. NAFLD was defined as USFLI ≥30 and liver fibrosis was determined as FIB-4 ≥ 2.67. OBS was scored by 20 lifestyle and dietary factors. Weighted logistic regression and restricted cubic splines were used to assess the association between OBS and NAFLD and fibrosis. The prevalence of NAFLD was 29.67%. There was a significant negative correlation between OBS, dietary OBS, lifestyle OBS and NAFLD and no correlation with NAFLD-related fibrosis. Compared to the lowest quartile, the adjusted ORs for the highest quartile of OBS, lifestyle OBS, dietary OBS and NAFLD were 0.55(95%CI:0.35,0.85), 0.12(95%CI:0.08,0.16), 0.70(95%CI:0.52,0.94) respectively. In stratified analyses, lifestyle OBS was negatively associated with NAFLD across gender, dietary OBS was only negatively correlated with NAFLD in men, and any OBS was not observed to be relevant to NAFLD-related fibrosis. CONCLUSIONS: OBS was negatively associated with NAFLD, but not with NAFLD-related fibrosis. The findings underline the significance of adhering to an antioxidant lifestyle and diet, which can help prevent NAFLD but seems to be ineffective in preventing fibrosis in individuals with NAFLD.

Viral infections inhibit saponin biosynthesis and photosynthesis in Panax notoginseng.

Virus-infected Panax notoginseng plants with chlorotic, mosaic, and pitted leaves are ubiquitous in the primary P. notoginseng-producing region in Wenshan autonomous prefecture, Yunnan province, China. However, the viruses that infect P. notoginseng and the effects of viral infections on the biosynthesis of secondary metabolites and photosynthesis remain unknown. This study identified a variety of viruses infecting P. notoginseng plants via deep-sequencing of small RNA (sRNA). Of the 10 identified viruses, seven had not previously been detected in P. notoginseng, including Cauliflower mosaic virus and Soybean chlorotic mottle virus. In addition, the simultaneous infection of P. notoginseng by Panax notoginseng virus A (PnVA), Panax cryptic virus 4 (PCV4), and Tomato yellow leaf curl China virus (TYLCCNV) was confirmed by PCR. Moreover, a quantitative PCR analysis showed that the expression levels of key genes related to saponin biosynthesis were generally down-regulated in the virus-infected P. notoginseng. Additionally, high-performance liquid chromatography results indicated the saponin content decreased in the roots of virus-infected P. notoginseng plants. The activities of photosynthesis-related enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase, fructose 1,6-bisphosphatase, and fructose 1,6-biphosphate aldolase, decreased significantly in the virus-infected P. notoginseng plants. The viral infections also induced the expression of antioxidant genes and increased antioxidant enzyme activities. Furthermore, the expression levels of many resistance-related genes were up-regulated in P. notoginseng plants inoculated with a viral suspension. The study results provide the foundation for future research on P. notoginseng viral diseases, which may lead to the development of enhanced disease control measures.

Non-retroareolar male mucinous breast cancer without gynecomastia development in an elderly man: A case report.

BACKGROUND: Male breast cancer (MBC) is an extremely rare condition and accounts for less than 1% of all breast cancers, and malignant tumors occur in less than 1% of the affected men. Mucinous breast cancer is extremely rare and accounts for 2% of all invasive breast cancers. Generally, MBC is accompanied by a retroareolar mass. CASE SUMMARY: Herein, we report a case of male mucinous breast carcinoma (MMBC) without gynecomastia development and with mass localization outside the common retroareolar region, wherein the mass was a painless nodule in the right breast of a 64-year-old man. We also discuss the clinical and pathological characteristics of this unusual tumor. The excised breast specimen showed pure mucinous carcinoma. The patient had strong expression of estrogen and progesterone receptors, a low Ki-67 proliferation index of the tumor cells, and negative pathological axillary lymph nodes. The patient underwent modified radical mastectomy and axillary lymph node dissection, followed by tamoxifen hormone therapy. CONCLUSION: To the best of our knowledge, this is the first case report of MMBC in the non-retroareolar region of the nipple without gynecomastia development. Mucinous tumors are easily missed during diagnosis, and the incidence of axillary lymph node metastases of chest mucinous tumors has increased.

Limited Impact of Soil Microorganisms on the Endophytic Bacteria of Tartary Buckwheat (Fagopyrum tataricum).

Soil has been considered the main microbial reservoir for plants, but the robustness of the plant microbiome when the soil resource is removed has not been greatly considered. In the present study, we tested the robustness of the microbiota recruited by Tartary buckwheat (Fagopyrum tataricum Gaertn.), grown on sterile humus soil and irrigated with sterile water. Our results showed that the microbiomes of the leaf, stem, root and next-generation seeds were comparable between treated (grown in sterile soil) and control plants (grown in non-sterile soil), indicating that the plants had alternative robust ways to shape their microbiome. Seed microbiota contributed greatly to endophyte communities in the phyllosphere, rhizosphere and next-generation seeds. The microbiome originated from the seeds conferred clear benefits to seedling growth because seedling height and the number of leaves were significantly increased when grown in sterilized soil. The overall microbiome of the plant was affected very little by the removal of the soil microbial resource. The microbial co-occurrence network exhibited more interactions, and Proteobacteria was enriched in the root of Tartary buckwheat planted in sterilized soil. Our research broadens the understanding of the general principles governing microbiome assembly and is widely applicable to both microbiome modeling and sustainable agriculture.