Gastrodin Ameliorates Post-Stroke Depressive-Like Behaviors Through Cannabinoid-1 Receptor-Dependent PKA/RhoA Signaling Pathway.

Post-stroke depression (PSD) is a significant complication in stroke patients, increases long-term mortality, and exaggerates ischemia-induced brain injury. However, the underlying molecular mechanisms and effective therapeutic targets related to PSD have remained elusive. Here, we employed an animal behavioral model of PSD by combining the use of middle cerebral artery occlusion (MCAO) followed by spatial restraint stress to study the molecular underpinnings and potential therapies of PSD. Interestingly, we found that sub-chronic application of gastrodin (Gas), a traditional Chinese medicinal herb Gastrodia elata extraction, relieved depression-related behavioral deficits, increased the impaired expression of synaptic transmission-associated proteins, and restored the altered spine density in hippocampal CA1 of PSD animals. Furthermore, our results indicated that the anti-PSD effect of Gas was dependent on membrane cannabinoid-1 receptor (CB1R) expression. The contents of phosphorated protein kinase A (p-PKA) and phosphorated Ras homolog gene family member A (p(ser188)-RhoA) were decreased in the hippocampus of PSD-mice, which was reversed by Gas treatment, and CB1R depletion caused a diminished efficacy of Gas on p-PKA and p-RhoA expression. In addition, the anti-PSD effect of Gas was partially blocked by PKA inhibition or RhoA activation, indicating that the anti-PSD effect of Gas is associated with the CB1R-mediated PKA/RhoA signaling pathway. Together, our findings revealed that Gas treatment possesses protective effects against the post-stroke depressive-like state; the CB1R-involved PKA/RhoA signaling pathway is critical in mediating Gas's anti-PSD potency, suggesting that Gas application may be beneficial in the prevention and adjunctive treatment of PSD.

Dysfunction of astrocytic glycophagy exacerbates reperfusion injury in ischemic stroke.

Glycophagy has evolved from an alternative glycogen degradation pathway into a multifaceted pivot to regulate cellular metabolic hemostasis in peripheral tissues. However, the pattern of glycophagy in the brain and its potential therapeutic impact on ischemic stroke remain unknown. Here, we observed that the dysfunction of astrocytic glycophagy was caused by the downregulation of the GABA type A receptor-associated protein like 1 (GABARAPL1) during reperfusion in ischemic stroke patients and mice. PI3K-Akt pathway activation is involved in driving GABARAPL1 downregulation during cerebral reperfusion. Moreover, glycophagy dysfunction-induced glucosamine deficiency suppresses the nuclear translocation of specificity protein 1 and TATA binding protein, the transcription factors for GABARAPL1, by decreasing their O-GlcNAcylation levels, and accordingly feedback inhibits GABARAPL1 in astrocytes during reperfusion. Restoring astrocytic glycophagy by overexpressing GABARAPL1 decreases DNA damage and oxidative injury in astrocytes and improves the survival of surrounding neurons during reperfusion. In addition, a hypocaloric diet in the acute phase after cerebral reperfusion can enhance astrocytic glycophagic flux and accelerate neurological recovery. In summary, glycophagy in the brain links autophagy, metabolism, and epigenetics together, and glycophagy dysfunction exacerbates reperfusion injury after ischemic stroke.

Effectiveness of eHealth interventions for HIV prevention, testing and management: An umbrella review.

BACKGROUND: Human immunodeficiency virus (HIV) infection has become a major contributor to the global burden of disease. Globally, the number of cases of HIV continues to increase. Electronic health (eHealth) interventions have emerged as promising tools to support disease self-management among people living with HIV. The purpose of this umbrella review is to systematically evaluate and summarize the evidence and results of published systematic reviews and meta-analyses on the effectiveness of eHealth interventions for HIV prevention, testing and management. METHODS: PubMed, Embase and the Cochrane Library were searched for reviews. The methodological quality of the included studies was assessed using AMSTAR-2. RESULTS: A total of 22 systematic reviews were included. The methodological quality of the reviews was low or critically low. EHealth interventions range from Internet, computer, or mobile interventions to websites, programs, applications, email, video, games, telemedicine, texting, and social media, or a combination of them. The majority of the reviews showed evidence of effectiveness (including increased participation in HIV management behaviours, successfully changed HIV testing behaviours, and reduced risk behaviours). EHealth interventions were effective in the short term. CONCLUSIONS: Ehealth interventions have the potential to improve HIV prevention, HIV testing and disease management. Due to the limitations of the low methodological quality of the currently available systematic reviews, more high-quality evidence is needed to develop clear and robust recommendations.

Transcriptome Analysis Reveals the Mechanisms of Accumulation and Conversion of Folate Derivatives during Germination of Quinoa (Chenopodium quinoa Willd.) Seeds.

Folate was enriched during quinoa germination, while molecular mechanisms were not well understood. In this study, three quinoa varieties were selected for germination, and changes in substrate content and enzyme activity of the folate biosynthesis pathway were monitored. 5-Methyltetrahydrofolate (5-CH3-THF) and 5-formyltetrahydrofolate (5-CHO-THF) were significantly enriched in quinoa sprouts. Among the selected varieties, QL-2 exhibited the lowest content of the oxidation product MeFox and the highest total folate content. Based on transcriptome analysis, the p-ABA branch was found to be crucial for folate accumulation, while the pterin branch served as a key control point for the one carbon pool by folate pathway, which limited further folate biosynthesis. In the one carbon pool by folate pathway, genes CqMTHFR and CqAMT significantly contributed to the enrichment of 5-CH3-THF and 5-CHO-THF. Findings gained here would facilitate the potential application of quinoa sprouts as an alternative strategy for folate supplementation.

Effects of aerobic exercise or Tai Chi Chuan interventions on problematic mobile phone use and the potential role of intestinal flora: A multi-arm randomized controlled trial.

BACKGROUND: Problematic use of mobile phones (PMPU) has been described as a serious public health issue. METHODS: This study was a parallel three-arm randomized controlled trial and has completed registration (ClinicalTrials.gov Identifier: NCT05843591). Ninety college students with PMPU were randomly assigned to the aerobic exercise group (AE group, n = 30), the Tai Chi Chuan group (TCC group, n = 30), or the wait-list control group (WLC group, n = 30). At the end of the intervention, stool samples from the study participants were collected for biological analysis based on 16 S rDNA amplicon sequencing technology. The primary outcome was addiction symptoms assessed by the Smartphone Addiction Scale-Short Version (SAS-SV). The secondary outcomes are emotional symptoms, physical symptoms, and flora species. RESULTS: Compared with the WLC group, the AE and TCC groups showed reductions in PMPU levels, physical and mental fatigue, but there was no difference between the two groups. Moreover, the effect of increasing self-esteem embodied in the TCC group was not present in the AE group. Compared to the WLC group, the relative abundance of Bacteroidaceae and Bacteroides were lower in the AE group, while the relative abundance of Erysipelotrichaceae and Alistipes were lower in the TCC group. And the relative abundance of Bacteroidaceae, Bacteroides, and Alistipes were significantly and negatively correlated with the decline in PMPU scores. CONCLUSION: AE or TCC is an effective, safe and efficient intervention for college students with PMPU, providing some physiological and psychological benefits and having some impact on their intestinal flora.

Lactate: a prospective target for therapeutic intervention in psychiatric disease.

ABSTRACT: Although antipsychotics that act via monoaminergic neurotransmitter modulation have considerable therapeutic effect, they cannot completely relieve clinical symptoms in patients suffering from psychiatric disorders. This may be attributed to the limited range of neurotransmitters that are regulated by psychotropic drugs. Recent findings indicate the need for investigation of psychotropic medications that target less-studied neurotransmitters. Among these candidate neurotransmitters, lactate is developing from being a waste metabolite to a glial-neuronal signaling molecule in recent years. Previous studies have suggested that cerebral lactate levels change considerably in numerous psychiatric illnesses; animal experiments have also shown that the supply of exogenous lactate exerts an antidepressant effect. In this review, we have described how medications targeting newer neurotransmitters offer promise in psychiatric diseases; we have also summarized the advances in the use of lactate (and its corresponding signaling pathways) as a signaling molecule. In addition, we have described the alterations in brain lactate levels in depression, anxiety, bipolar disorder, and schizophrenia and have indicated the challenges that need to be overcome before brain lactate can be used as a therapeutic target in psychopharmacology.

Pre-Exposure Prophylaxis Care Continuum for HIV Risk Populations: An Umbrella Review of Systematic Reviews and Meta-Analyses.

Pre-exposure prophylaxis (PrEP) is an effective biomedical strategy for HIV prevention. This umbrella review is aimed at providing a comprehensive summary of the current status of each stage of the PrEP care cascade. A systematic literature search was conducted in PubMed, Embase, and Cochrane. Additionally, a Measurement Tool to Assess Systematic Reviews 2 (AMSTAR-2) tool and Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) checklist were used to evaluate their methodological and reporting quality, respectively. A total of 30 systematic reviews met the inclusion criteria. According to the results of methodological quality assessment, 3 reviews were rated as low, while 27 as critically low. Furthermore, the results of the reporting quality evaluation revealed a mean score of 23.03 for the included reviews. Across all the reviews, awareness of PrEP was generally moderate in all populations, and the acceptability was even higher compared with awareness. Unfortunately, the PrEP uptake among different groups was even less optimal, although the adherence was almost above moderate, and several barriers that hindered the utilization of PrEP were identified, and the most common are as follows: cost, stigma, lack of knowledge, mistrust, low risk perception, and more. Although PrEP has proven to be an effective prevention method to date, the promotion of PrEP failed to achieve the anticipated outcome. To reinforce the generalization of and use of PrEP, and effectively control HIV transmission, it is urgent to identify the underlying causes of low uptake rates so that efficient interventions can be implemented.

Age-dependent changes in the gut microbiota and serum metabolome correlate with renal function and human aging.

Human aging is invariably accompanied by a decline in renal function, a process potentially exacerbated by uremic toxins originating from gut microbes. Based on a registered household Chinese Guangxi longevity cohort (n = 151), we conducted comprehensive profiling of the gut microbiota and serum metabolome of individuals from 22 to 111 years of age and validated the findings in two independent East Asian aging cohorts (Japan aging cohort n = 330, Yunnan aging cohort n = 80), identifying unique age-dependent differences in the microbiota and serum metabolome. We discovered that the influence of the gut microbiota on serum metabolites intensifies with advancing age. Furthermore, mediation analyses unveiled putative causal relationships between the gut microbiota (Escherichia coli, Odoribacter splanchnicus, and Desulfovibrio piger) and serum metabolite markers related to impaired renal function (p-cresol, N-phenylacetylglutamine, 2-oxindole, and 4-aminohippuric acid) and aging. The fecal microbiota transplantation experiment demonstrated that the feces of elderly individuals could influence markers related to impaired renal function in the serum. Our findings reveal novel links between age-dependent alterations in the gut microbiota and serum metabolite markers of impaired renal function, providing novel insights into the effects of microbiota-metabolite interplay on renal function and healthy aging.

Research Progress of Palladium-Plated Copper Bonding Wire in Microelectronic Packaging.

Wire-bonding technology is the most commonly used chip interconnection technology in microelectronic packaging. Metal bonding wire is the key material for wire bonding and plays an important role in the reliability of electronic devices. In recent years, palladium-plated copper (PdCu) bonding wire has been widely used because of its low cost, good electrical and thermal conductivity, the fact that it is not easy to oxidize, and its high reliability. Therefore, it is necessary to review its research progress. In this paper, the preparation and application of palladium-plated copper bonding wire are reviewed. Firstly, the preparation methods of electroplating, electroless plating, and direct plating are introduced. Secondly, the factors affecting the distribution of Pd in free air balls and bonding interfaces, the effect of Pd on the formation and growth of intermetallic compounds in PdCu wire, stitch bond, and reliability of PdCu wire are summarized and analyzed in the application process. Finally, its development prospect is prospected. Hopefully, this review can help readers to have a comprehensive understanding of the preparation and application of palladium-plated copper bonding wires, and can accelerate the promotion of its application in more fields in the future.

AAformer: Auto-Aligned Transformer for Person Re-Identification.

In person re-identification (re-ID), extracting part-level features from person images has been verified to be crucial to offer fine-grained information. Most of the existing CNN-based methods only locate the human parts coarsely, or rely on pretrained human parsing models and fail in locating the identifiable nonhuman parts (e.g., knapsack). In this article, we introduce an alignment scheme in transformer architecture for the first time and propose the auto-aligned transformer (AAformer) to automatically locate both the human parts and nonhuman ones at patch level. We introduce the "Part tokens (PARTs)", which are learnable vectors, to extract part features in the transformer. A PART only interacts with a local subset of patches in self-attention and learns to be the part representation. To adaptively group the image patches into different subsets, we design the auto-alignment. Auto-alignment employs a fast variant of optimal transport (OT) algorithm to online cluster the patch embeddings into several groups with the PARTs as their prototypes. AAformer integrates the part alignment into the self-attention and the output PARTs can be directly used as part features for retrieval. Extensive experiments validate the effectiveness of PARTs and the superiority of AAformer over various state-of-the-art methods.

NeuWS: Neural wavefront shaping for guidestar-free imaging through static and dynamic scattering media.

Diffraction-limited optical imaging through scattering media has the potential to transform many applications such as airborne and space-based imaging (through the atmosphere), bioimaging (through skin and human tissue), and fiber-based imaging (through fiber bundles). Existing wavefront shaping methods can image through scattering media and other obscurants by optically correcting wavefront aberrations using high-resolution spatial light modulators-but these methods generally require (i) guidestars, (ii) controlled illumination, (iii) point scanning, and/or (iv) statics scenes and aberrations. We propose neural wavefront shaping (NeuWS), a scanning-free wavefront shaping technique that integrates maximum likelihood estimation, measurement modulation, and neural signal representations to reconstruct diffraction-limited images through strong static and dynamic scattering media without guidestars, sparse targets, controlled illumination, nor specialized image sensors. We experimentally demonstrate guidestar-free, wide field-of-view, high-resolution, diffraction-limited imaging of extended, nonsparse, and static/dynamic scenes captured through static/dynamic aberrations.

Longitudinal association between problematic smartphone use and sleep disorder among Chinese college students during the COVID-19 pandemic.

BACKGROUND: Problematic smartphone use (PSU) and sleep disorders (SD) are common public health problems among college students. While previous cross-sectional studies have found a relationship between PSU and SD, the causal direction of this relationship remains unclear. This study aims to examine the longitudinal changes of PSU and SD during the COVID-19 pandemic, determine the causal relationship between them, and identify confounding factors that affect this association. METHODS: The study sample consisted of 1186 Chinese college students (47.7% male) with a mean age of 18.08 years. Participants completed the Smartphone Addiction Scale - Short Version (SAS-SV) and the Pittsburgh Sleep Quality Index (PSQI) at both baseline and follow-up surveys, conducted one year apart. The cross-lagged panel model (CLPM) was used to examine the causal relationship between PSU and SD, stratified by gender and duration of daily physical activity. The fixed effect panel regression was used to confirm the findings of CLPM. RESULTS: The results of the CLPM analysis showed a significant bidirectional relationship between PSU and SD for the overall sample, which was consistent with the fixed effects model findings. However, subgroup analyses revealed that the bidirectional association disappeared among males or those who engaged in daily physical activity for more than 1 h. CONCLUSIONS: Our study shows a significant bidirectional association between PSU and SD, with variations across gender and daily physical activity levels. Encouraging physical activity may serve as a potential intervention to disrupt the bidirectional association between PSU and SD, which has important implications for public health strategies aimed at reducing the negative consequences of PSU and SD.

Effects of exercise or tai chi on Internet addiction in college students and the potential role of gut microbiota: A randomized controlled trial.

OBJECTIVE: This study aimed to explore the effect of exercise or tai chi on Internet addiction disorder (IAD) among college students and clarified the abundance and population changes of gut microbiota in different groups. Thus explore the potential role of gut microbiota between exercise and IAD. METHODS: A total of 93 subjects diagnosed with mild IAD were randomly assigned to the exercise group, the tai chi group, and the control group. The intervention groups received exercise or tai chi for 8 weeks and the control group was evaluated without any intervention. Fecal samples were collected after the intervention. RESULTS: 1) Analysis found a significant intervention effect with the exercise group showing an average decrease of 8.84 points on the Internet addiction test (IAT) compared with the control group (95%CI -15.41 to-2.27, P = 0.004). But there was no significant difference between the control group and the tai chi group. 2) Both exercise (P = 0.018) and tai chi (P = 0.026) could significantly relieve fatigue symptoms. 3) The relative abundance of the Betaproteobacteria, Porphyromonadaceae, Sutterellaceae, and Alistipes were significantly decreased in the exercise group compared with the control group, and the relative abundance of Escherichia was significantly increased in the exercise group. 4) The relative abundance of Betaproteobacteria, Sutterellaceae, and Escherichia had significant differences between the improved group and the no-improved group. CONCLUSION: Exercise intervention has a considerable effect on treating IAD. Exercise and tai chi might have effectiveness in relieving the symptoms of fatigue. Exercise intervention regulates the gut microflora and changes the abundance of microflora to improve IAD. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, identifier NCT05529368.

A bidirectional association between smartphone addiction and depression among college students: A cross-lagged panel model.

Background: Smartphone addiction (SA) is associated with adverse consequences, especially for freshmen. Evidence indicates that SA is associated with depression, and it is necessary to conduct a longitudinal study to explore the association further. Methods: SA (measured by the Smartphone Addiction Scale-Short Version) and depression (measured by the Zung's Self-Rating Depression Scale) among 1,186 freshmen were surveyed at baseline and a respective 12-month follow-up for each participant. The application of a cross-lagged panel model approach (CLPM) revealed an association between SA and depression after adjusting for demographic variables. Results: The CLPM results showed a significant path from baseline SA to follow-up depression (ß = 0.08, P < 0.001) and a significant path from baseline depression to follow-up SA (ß = 0.08, P < 0.001). Compared with the overall cross-lagged model, the cross-lagged coefficient of the path from baseline SA to follow-up depression increased in the female group (ß = 0.10, P = 0.015), and the cross-lagged coefficient of the path from baseline depression to follow-up SA also increased significantly (ß = 0.15, P < 0.001). In contrast, the cross-lagged model in the male group showed no predictive effect between SA and depression (P > 0.05). Conclusions: The current study showed a significant bidirectional association between smartphone addiction and depression among freshmen, but only in the female population.

Learning Semantics-Consistent Stripes With Self-Refinement for Person Re-Identification.

Aligning human parts automatically is one of the most challenging problems for person re-identification (re-ID). Recently, the stripe-based methods, which equally partition the person images into the fixed stripes for aligned representation learning, have achieved great success. However, the stripes with fixed height and position cannot well handle the misalignment problems caused by inaccurate detection and occlusion and may introduce much background noise. In this article, we aim at learning adaptive stripes with foreground refinement to achieve pixel-level part alignment by only using person identity labels for person re-ID and make two contributions. 1) A semantics-consistent stripe learning method (SCS). Given an image, SCS partitions it into adaptive horizontal stripes and each stripe is corresponding to a specific semantic part. Specifically, SCS iterates between two processes: i) clustering the rows to human parts or background to generate the pseudo-part labels of rows and ii) learning a row classifier to partition a person image, which is supervised by the latest pseudo-labels. This iterative scheme guarantees the accuracy of the learned image partition. 2) A self-refinement method (SCS+) to remove the background noise in stripes. We employ the above row classifier to generate the probabilities of pixels belonging to human parts (foreground) or background, which is called the class activation map (CAM). Only the most confident areas from the CAM are assigned with foreground/background labels to guide the human part refinement. Finally, by intersecting the semantics-consistent stripes with the foreground areas, SCS+ locates the human parts at pixel-level, obtaining a more robust part-aligned representation. Extensive experiments validate that SCS+ sets the new state-of-the-art performance on three widely used datasets including Market-1501, DukeMTMC-reID, and CUHK03-NP.

Association between mobile phone addiction, sleep disorder and the gut microbiota: a short-term prospective observational study.

Bidirectional communication between the gut microbiota and the brain has sparked interest in exploring the link between mobile phone addiction (MPA) and sleep disorders (SD) in microbiome research. However, investigating the role of gut microbiota in this relationship using animal models presents challenges due to the unique nature of MPA, and human research in this area is scarce. We recruited 99 healthy college students to evaluate the gut microbiome using 16S rRNA gene amplicon sequencing and assess MPA and SD at baseline and after a two-month follow-up. Multiple covariate-adjusted statistical models, including linear regression, permutational multivariate analysis of variance and so on, were employed to determine microbiome associations with MPA at baseline and changes in SD at follow-up. Our findings revealed negative associations between MPA and three alpha diversity metrics, along with alterations in bacterial composition. MPA showed negative associations with the relative abundance of Bacteroidetes, while displaying positive associations with Actinobacteria and Bifidobacteriales. Conversely, Actinobacteria exhibited a negative association with increased SD. This study has established a significant link between MPA and a decrease in the alpha diversity of the gut microbiota. Actinobacteria was associated with MPA and SD, respectively. Additional investigation is needed to fully comprehend the relationship between comorbid behavioral disorders and the gut microbiota.

Neuronal GPER Participates in Genistein-Mediated Neuroprotection in Ischemic Stroke by Inhibiting NLRP3 Inflammasome Activation in Ovariectomized Female Mice.

Estrogen replacement therapy (ERT) is potentially beneficial for the prevention and treatment of postmenopausal cerebral ischemia but inevitably increases the risk of cerebral hemorrhage and breast cancer when used for a long period of time. Genistein, a natural phytoestrogen, has been reported to contribute to the recovery of postmenopausal ischemic stroke with reduced risks. However, the underlying mechanism of genistein-mediated neuroprotection remains unclear. We reported that genistein exerted significant neuroprotective effects by enhancing the expression of neuronal G protein-coupled estrogen receptor (GPER) in the ischemic penumbra after cerebral reperfusion in ovariectomized (OVX) mice, and this effect was achieved through GPER-mediated inhibition of nod-like receptor protein 3 (NLRP3) inflammasome activation. In addition, we found that peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) was the pivotal molecule that participated in GPER-mediated inhibition of NLRP3 inflammasome activation in OVX mice after ischemia/reperfusion (I/R) injury. Our data suggest that the neuronal GPER/PGC-1α pathway plays an important role in genistein-mediated neuroprotection against I/R injury in OVX mice.

Effects of different interventions on internet addiction: A meta-analysis of random controlled trials.

OBJECTIVE: To evaluate the effects of different interventions on Internet addiction (IA), a meta-analysis and network meta-analyses were performed. METHODS: We searched PubMed, Cochrane, Embase, Web of Science, PsycINFO, ProQuest, CNKI, WanFang, VIP database, and CBM from their inception to August 2021 to identify randomized controlled trials (RCTs) where the effects of interventions on IA were assessed. The risk of bias was evaluated according to the Revised Cochrane risk-of-bias tool for randomized trials (RoB2). The R studio Software and Stata 14.0 were used to perform traditional meta-analysis and network meta-analyses. RESULTS: A total of 59 RCTs including 3832 participants were incorporated into meta-analysis. The results of the traditional meta-analysis of 24 studies showed that CBT, group counseling, sports intervention, and Internet-based intervention could significantly reduce IA levels (SMD = -1.90, 95%CI: -2.26 to -1.55, P < 0.01, I2 = 85.9%) as compared to no-treatment control groups. Network meta-analyses based on different scales showed that combined interventions had the highest probability of being the best interventions for IA (SUCRA = 91.0 % based on IAT; SUCRA = 89.0 % based on CIAS). DISCUSSION: Most interventions have significant effects on the treatment of IA. Compared with single interventions, combined interventions showed a more pronounced improvement in Internet addiction symptoms.

The Component and Functional Pathways of Gut Microbiota Are Altered in Populations with Poor Sleep Quality - A Preliminary Report.

With the development of genome sequencing, many researchers have investigated the mechanism by which the intestinal microbiota influences sleep across the brain-gut axis. However, the relationship between gut microbiota and sleep disorder remains unclear. Thus, we studied the difference in gut microbiota composition between poor sleep quality- and normal populations, which helps set the ground for future research. The recruited college students provided baseline information and stool samples and completed the Pittsburgh Sleep Quality Index (PSQI). We compared the two groups' gut microbiota composition and functional differentiation by using the 16S rRNA gene sequencing analysis. The main bacterial difference and the most critical effect were mainly concentrated within Tenericutes and Elusimicrobia. Compared with the healthy control group, some functions of the gut microbiota were impaired in the poor sleep quality group, such as butanoate metabolism and propanoate metabolism. Bacterial taxa with significant differences raised the possibility for future diagnosis and treatment of sleep problems.