In-hospital systolic blood pressure lowering patterns and risk of rehospitalization for angina in patients with hypertension and coronary artery disease.

This study aimed to examine the association between in-hospital systolic blood pressure (SBP) lowering patterns and rehospitalization for angina in patients with hypertension and coronary artery disease (HT-CAD). This prospective cohort study was conducted in Chinese PLA General Hospital, Beijing, China. We included 730 patients with HT-CAD, who were hospitalized between August 2020 and September 2022. The in-hospital SBP lowering patterns were identified according to SBP level at admission, SBP level at discharge, and the difference between them: normal-stable SBP, more-intensive SBP reduction, less-intensive SBP reduction, and non-reduced SBP. We used Cox proportional hazards regression to estimate the risk of rehospitalization for angina according to SBP lowering patterns. We identified 121 cases of rehospitalization for angina in a median follow-up of 28.2 months. Patients with more-intensive SBP reduction had the lowest incidence rate of rehospitalization for angina, followed by those with normal-stable SBP, less-intensive SBP reduction, and non-reduced SBP. After adjusting for potential confounders, we found that compared with patients with more-intensive SBP reduction, the hazard ratios and 95% confidence intervals of rehospitalization for angina were 1.35 (0.78-2.35) for patients with normal-stable SBP, 2.17 (1.14-4.14) for patients with less-intensive SBP reduction, and 2.99 (1.57-5.68) for patients with non-reduced SBP. This association was more pronounced in patients with multi-vessel stenosis than in patients with single-vessel stenosis. In conclusion, in-hospital SBP lowering patterns were associated with risk of rehospitalization for angina. These results highlighted the importance of intensive in-hospital SBP control in patients with HT-CAD.

A national cross-sectional survey on community spinal cord injury individuals profiles, health-related quality of life and support services in China: implications for healthcare and rehabilitation.

BACKGROUND: Spinal cord injury (SCI) results in severe, permanent functional changes and has become a global health priority due to its high incidence, cost, and disability rate. Current national epidemiological data on SCI in China are limited and outdated. This study aimed to provide a comprehensive, national cross-sectional investigation of SCI epidemiology in China. METHODS: This cross-sectional study included 3055 SCI participants aged 8 to 78 years, conducted from May to September 2023. Data collected encompassed demographic characteristics, employment status, etiology, years lived with disability (YLD), family structure, caregiving status, income, health insurance, paralysis type, and health-related quality of life (HRQoL). Descriptive statistics analyses were used to assess demographic and injury characteristics. Group differences were assessed using t-tests, one-way ANOVA and Chi-square tests. Significant factors were examined using multivariate regression analysis. RESULTS: The majority (88.9%) of respondents were aged 15 to 59 years, with a male-to-female ratio of 2.36:1. Car accidents caused 45.4% of tetraplegia cases, falls caused 35.9% of paraplegia cases, and myelitis was the leading cause of non-traumatic SCI. Among paraplegia participants, 65.5% had complete SCI, while 53.1% of tetraplegia participants had incomplete SCI. Functional improvement was reported by 9.58% of participants. Half (50.3%) of the respondents were unemployed, and 75% had incomes below the national average. HRQoL was significantly lower in the SCI population compared to controls, mainly influenced by injury site, income, age and etiology (p < 0.05). CONCLUSIONS: SCI participants in China exhibit low HRQoL and reemployment rates. Accessible community and vocational rehabilitation programs, alongside robust public medical services, are essential for enhancing reemployment and HRQoL among SCI participants, reducing the overall disease burden.

Parkinson's disease is characterized by vitamin B6-dependent inflammatory kynurenine pathway dysfunction.

Parkinson's disease (PD) is a complex multisystem disorder clinically characterized by motor, non-motor, and premotor manifestations. Pathologically, PD involves neuronal loss in the substantia nigra, striatal dopamine deficiency, and accumulation of intracellular inclusions containing aggregates of α-synuclein. Recent studies demonstrate that PD is associated with dysregulated metabolic flux through the kynurenine pathway (KP), in which tryptophan is converted to kynurenine (KYN), and KYN is subsequently metabolized to neuroactive compounds quinolinic acid (QA) and kynurenic acid (KA). This multicenter study used highly sensitive liquid chromatography-tandem mass-spectrometry to compare blood and cerebral spinal fluid (CSF) KP metabolites between 158 unimpaired older adults and 177 participants with PD. Results indicate that increased neuroexcitatory QA/KA ratio in both plasma and CSF of PD participants associated with peripheral and cerebral inflammation and vitamin B6 deficiency. Furthermore, increased QA tracked with CSF tau and severity of both motor and non-motor PD clinical dysfunction. Importantly, plasma and CSF kynurenine metabolites classified PD participants with a high degree of accuracy (AUC = 0.897). Finally, analysis of metabolite data revealed subgroups with distinct KP profiles, and these were subsequently found to display distinct PD clinical features. Together, these data further support the hypothesis that the KP serves as a site of brain and periphery crosstalk, integrating B-vitamin status, inflammation and metabolism to ultimately influence PD clinical manifestation.

Strategies for Non-Covalent Attachment of Antibodies to PEGylated Nanoparticles for Targeted Drug Delivery.

Polyethylene glycol (PEG)-modified nanoparticles (NPs) often struggle with reduced effectiveness against metastasis and liquid tumors due to limited tumor cell uptake and therapeutic efficacy. To address this, actively targeted liposomes with enhanced tumor selectivity and internalization are being developed to improve uptake and treatment outcomes. Using bi-functional proteins to functionalize PEGylated NPs and enhance targeted drug delivery through non-covalent attachment methods has emerged as a promising approach. Among these, the one-step and two-step targeting strategies stand out for their simplicity, efficiency, and versatility. The one-step strategy integrates streptavidin-tagged antibodies or bispecific antibodies (bsAbs: PEG/DIG × marker) directly into PEGylated NPs. This method uses the natural interactions between antibodies and PEG for stable, specific binding, allowing the modification of biotin/Fc-binding molecules like protein A, G, or anti-Fc peptide. Simply mixing bsAbs with PEGylated NPs improves tumor targeting and internalization. The two-step strategy involves first accumulating bsAbs (PEG/biotin × tumor marker) on the tumor cell surface, triggering an initial attack via antibody-dependent and complement-dependent cytotoxicity. These bsAbs then capture PEGylated NPs, initiating a second wave of internalization and cytotoxicity. Both strategies aim to enhance the targeting capabilities of PEGylated NPs by enabling specific recognition and binding to disease-specific markers or receptors. This review provides potential pathways for accelerating clinical translation in the development of targeted nanomedicine.

Meta-analysis of the diagnostic value of functional magnetic resonance imaging for distinguishing unresponsive wakefulness syndrome/vegetative state and minimally conscious state.

Objective: Unresponsive wakefulness syndrome/vegetative state (UWS/VS) and minimally conscious state (MCS) are considered different clinical entities, but their differential diagnosis remains challenging. As a potential clinical tool, functional magnetic resonance imaging (fMRI) could detect residual awareness without the need for the patients' actual motor responses. This study aimed to investigate the diagnostic value of fMRI for distinguishing between UWS/VS and MCS through a meta-analysis of the existing studies. Methods: We conducted a comprehensive search (from the database creation date to November. 2023) for relevant English articles on fMRI for the differential diagnosis of UWS/VS and MCS. The pooled sensitivity, specificity, positive likelihood ratio (LR+), negative likelihood ratio (LR-), summary receiver operating characteristic (SROC) curve, and area under the curve (AUC) were calculated to assess the diagnostic value of fMRI in distinguishing between UWS/VS and MCS. The statistical I 2 test was used to assess heterogeneity, and the source of heterogeneity was investigated by performing a meta-regression analysis. Publication bias was assessed using the Deeks funnel plot asymmetry test. Results: Ten studies were included in the meta-analysis. The pooled sensitivity and specificity were 0.71 (95% CI 0.62-0.79) and 0.71 (95% CI 0.54-0.84), respectively. The fMRI for the differential diagnosis of UWS/VS and MCS has a moderate positive likelihood ratio (2.5) and a relatively low negative likelihood ratio (0.40). Additionally, SROC curves showed that the AUC was 0.76 (95% CI 0.72-0.80). Conclusion: Functional magnetic resonance imaging has a good performance in the differential diagnosis of UWS/VS and MCS, and may provide a potential tool for evaluating the prognosis and guiding the rehabilitation therapy in patients with disorders of consciousness.

The ankle-brachial index, gastrocnemius mitochondrial respirometry, and walking performance in people with and without peripheral artery disease.

Background: Mitochondrial abnormalities exist in lower-extremity peripheral artery disease (PAD), yet the association of the ankle-brachial index (ABI) with mitochondrial respiration in gastrocnemius muscle is unknown. The association of gastrocnemius mitochondrial respiration with 6-minute walk distance in PAD is unknown. Objective: To describe associations of the ABI with mitochondrial respiratory function in gastrocnemius muscle biopsies and associations of gastrocnemius mitochondrial respirometry with 6-minute walk distance in people with and without PAD. Methods: People with (ABI ⩽ 0.90) and without (ABI 1.00-1.40) PAD were enrolled. ABI and 6-minute walk distance were measured. Mitochondrial function of permeabilized myofibers from gastrocnemius biopsies was measured with high-resolution respirometry. Results: A total of 30 people with PAD (71.7 years, mean ABI: 0.64) and 68 without PAD (71.8 years, ABI: 1.17) participated. In non-PAD participants, higher ABI values were associated significantly with better mitochondrial respiration (Pearson correlation for maximal oxidative phosphorylation PCI+II: +0.29, p = 0.016). In PAD, the ABI correlated negatively and not significantly with mitochondrial respiration (Pearson correlation for PCI+II: -0.17, p = 0.38). In people without PAD, better mitochondrial respiration was associated with better 6-minute walk distance (Pearson correlation: +0.51, p < 0.001), but this association was not present in PAD (Pearson correlation: +0.10, p = 0.59). Conclusions: Major differences exist between people with and without PAD in the association of gastrocnemius mitochondrial respiration with ABI and 6-minute walk distance. Among people without PAD, ABI and walking performance were positively associated with mitochondrial respiratory function. These associations were not observed in PAD.

Periplaneta americana extract CII-3 triggers cell senescence through activating ROS-p38 MAPK-p53 signaling pathway in SKOV3 cells.

This study aimed to investigate effect of Periplaneta americana extract CII-3 (CII-3) in senescence of SKOV3 cells. Proliferation, colony forming and cell senescence of SKOV3 cells were determined. ROS production was evaluated by flow cytometry. Transcription of telomerase (TERT), p38 MAPK and p53 gene and protein expression of p-p38 MAPK and p-p53, were identified. CII-3 at different concentrations significantly inhibited SKOV3 proliferation, and 80 µg/ml demonstrated the highest inhibitory effect. CII-3 significantly blocked cell cycle in G0/G1 phase (P<0.01) and reduced colony forming efficiency (P<0.001) of SKOV3 cells compared to those in Control group. CII-3 significantly increased SA-ß-Gal positive staining SKOV3 cells (P<0.001) and reduced mitochondrial membrane potential (P<0.01) compared to those in Control group. CII-3 markedly decreased TERT gene transcription of SKOV3 cells compared to that in Control group (P<0.001). CII-3 also triggered significantly higher ROS levels in SKOV3 cells compared to that in Control group (P<0.001). CII-3 significantly increased p-p38 MAPK (P<0.001), p-p53 (P<0.001) and p21 (P<0.001) expressions of SKOV3 cells compared to those in Control group. In conclusion, CII-3 triggered cell senescence of SKOV3 cells through activating ROS-p38 MAPK-p53 signaling pathway. This study would provide a promising strategy for inhibiting cancer cell proliferation by including cell senescence.

Predicting Long-Term Outcome of Prolonged Disorder of Consciousness in Children Through Machine Learning Based on Conventional Structural Magnetic Resonance Imaging.

BACKGROUND: The prognosis of prolonged disorders of consciousness (pDoC) in children has consistently posed a formidable challenge in clinical decision-making. OBJECTIVE: This study aimed to develop a machine learning (ML) model based on conventional structural magnetic resonance imaging (csMRI) to predict outcomes in children with pDoC. METHODS: A total of 196 children with pDoC were included in this study. Based on the consciousness states 1 year after brain injury, the children were categorized into either the favorable prognosis group or the poor prognosis group. They were then randomly assigned to the training set (n = 138) or the test set (n = 58). Semi-quantitative visual assessments of brain csMRI were conducted and Least Absolute Shrinkage and Selection Operator regression was used to identify significant features predicting outcomes. Based on the selected features, support vector machine (SVM), random forests (RF), and logistic regression (LR) were used to develop csMRI, clinical, and csMRI-clinical-merge models, respectively. Finally, the performances of all models were evaluated. RESULTS: Seven csMRI features and 4 clinical features were identified as important predictors of consciousness recovery. All models achieved satisfactory prognostic performances (all areas under the curve [AUCs] >0.70). Notably, the csMRI model developed using the SVM exhibited the best performance, with an AUC, accuracy, sensitivity, and specificity of 0.851, 0.845, 0.844, and 0.846, respectively. CONCLUSIONS: A csMRI-based prediction model for the prognosis of children with pDoC was developed, showing potential to predict recovery of consciousness 1 year after brain injury and is worth popularizing in clinical practice.

Disparity landscapes of viral-induced structural variations in hepatocellular carcinoma: Mechanistic characterization and functional implications.

Oncoviruses can integrate into the host genome and cause tumorigenesis. In particular, hepatitis B virus (HBV) infection accounts for more than 50% of hepatocellular carcinoma (HCC) worldwide. We revealed the global geographical disparity of HBV integration that the landscape of HBV integration between HCC tumor and non-tumorous liver varied in regional cohorts, suggesting the different degrees of clonal enrichment. Most HBV integrations were positionally enriched at telomeres and centromeres (T&C) and they highlighted the novel co-involvement of HBV integration, which likely introduces genomic instability in HCC development. This was confirmed by phospho-H2AX staining. We constructed a large meta-cohort of multiple ethnicities to refine the landscape of HBV integration. This enables the gene set/family level exploration. As TERT is the most frequently integrated gene, we further investigated the underlying mechanistic modulation of TERT transcription activation and revealed the concurrent influence by the orientation and relative distance of HBV integration. Additionally, clonal disparity of HBV integration was observed among patients and the higher level of clonal disparity score can indicate poor patients' prognostication. Taken together, our study uncovered the different levels of clonal enrichment of HBV integration, mechanistic insights, and prognostic biomarker signature, to strengthen our understanding in HBV-associated hepatocarcinogenesis.

The role of intestinal microbiota and metabolites in intestinal inflammation.

With the imbalance of intestinal microbiota, the body will then face an inflammatory response, which has serious implications for human health. Bodily allergies, injury or pathogens infections can trigger or promote inflammation and alter the intestinal environment. Meanwhile, excessive changes in the intestinal environment cause the imbalance of microbial homeostasis, which leads to the proliferation and colonization of opportunistic pathogens, invasion of the body's immune system, and the intensification of inflammation. Some natural compounds and gut microbiota and metabolites can reduce inflammation; however, the details of how they interact with the gut immune system and reduce the gut inflammatory response still need to be fully understood. The review focuses on inflammation and intestinal microbiota imbalance caused by pathogens. The body reacts differently to different types of pathogenic bacteria, and the ingestion of pathogens leads to inflamed gastrointestinal tract disorders or intestinal inflammation. In this paper, unraveling the interactions between the inflammation, pathogenic bacteria, and intestinal microbiota based on inflammation caused by several common pathogens. Finally, we summarize the effects of intestinal metabolites and natural anti-inflammatory substances on inflammation to provide help for related research of intestinal inflammation caused by pathogenic bacteria.

Effects of High-altitude Hypoxia on Drug Metabolism and Pharmacokinetics of Sedative-hypnotic Drugs and Regulatory Mechanism.

Sedative hypnotics effectively improve sleep quality under high-altitude hypoxia by reducing central nervous system excitability. High-altitude hypoxia causes sleep disorders and modifies the metabolism and mechanisms of drug action, impacting medication therapy's effectiveness. This review aims to provide a theoretical basis for the treatment of central nervous system diseases in high-altitude areas by summarizing the progress and mechanism of sedative-hypnotics in hypoxic environments, as well as the impact of high-altitude hypoxia on sleep.

Blood-Brain Barrier Permeability is Affected by Changes in Tight Junction Protein Expression at High-Altitude Hypoxic Conditions-this may have Implications for Brain Drug Transport.

Changes to blood-brain barrier structure and function may affect the delivery of drugs into the brain. It is worthwhile to exploring more study on how the blood-brain barrier changes in structure and function and how that affects drug transport in high-altitude hypoxic environment. The DIA high-throughput sequencing technique indicate that the rats blood-brain barrier has been identified to have 7252 proteins overall and 8 tight junction proteins, among which Claudin-7 was a plateau-specific tight junction protein under high-altitude hypoxia, and based on the interaction network study, 2421 proteins are found to interact with one another, with ZO-1 being the primary target. The results of the projected gene function analysis demonstrated that changes in tight junction proteins are related to the control of TRP channels by inflammatory mediators, the wnt signaling pathway, the ABC transporter system, and drug metabolism-CYP450 enzyme regulation. Additionally, the electron microscopy, the Evans blue combination with confocal laser scanning microscopy, and the Western Blot and RT-qPCR revealed that high-altitude hypoxic environment induces blood-brain barrier tight junctions to open, blood-brain barrier permeability increases, ZO-1, Occludin, Claudin-5 protein and mRNA expression decreased. Our research implies that structural and functional alterations in the blood-brain barrier induced by high altitude hypoxia may impact drug transport inside the central nervous system, and that drug transporters and drug-metabolizing enzymes may be key players in this process.

Icariin ameliorates TNF-α/IFN-γ-induced oxidative stress, inflammatory response and apoptosis of human immortalized epidermal cells through the WTAP/SERPINB4 axis.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by increased sensitivity to environmental allergens and irritants. Icariin, a natural compound extracted from the herb Epimedium, has been traditionally used for its potential anti-inflammatory and antioxidant properties. This study aimed to investigate the regulatory effects of icariin on AD-like symptoms and to elucidate its underlying mechanisms. The effects of icariin on TNF-α/IFN-γ-induced HaCaT cell injury were assessed using various assays, including cell counting kit-8 for cell viability, flow cytometry for reactive oxygen species (ROS) levels, and colorimetric assays for malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity. In addition, the study performed enzyme-linked immunosorbent assays to assess cytokines (IL-1ß, IL-6, and IL-8) and chemokines (MDC, TARC, and RANTES) levels. Flow cytometry was used to quantify apoptotic rate, while a wound-healing assay was conducted to assess cell migration. The expression of WT1 associated protein (WTAP) and serpin family B member 4 (SERPINB4) at the mRNA and protein levels was determined using qRT-PCR and western blotting, respectively. The associations between WTAP and SERPINB4 were analyzed using RNA immunoprecipitation assay and m6A RNA immunoprecipitation assay. Icariin treatment significantly mitigated TNF-α/IFN-γ-induced oxidative stress, inflammatory response, and apoptosis in HaCaT cells, while also reversing the inhibitory effect on cell migration. Icariin reduced the expression of WTAP in TNF-α/IFN-γ-stimulated HaCaT cells. Overexpression of WTAP reversed the effects of icariin in TNF-α/IFN-γ-stimulated HaCaT cells. WTAP silencing inhibited the mRNA stability of SERPINB4 through the m6A modification. SERPINB4 overexpression attenuated the effects of WTAP silencing on oxidative stress, inflammatory response, apoptosis, and migration of TNF-α/IFN-γ-stimulated HaCaT cells. Icariin treatment downregulated SERPINB4 expression by regulating WTAP in TNF-α/IFN-γ-stimulated HaCaT cells. Icariin ameliorated TNF-α/IFN-γ-induced human immortalized epidermal cell injury through the WTAP/SERPINB4 axis, highlighting the potential for targeted interventions in AD pathogenesis.

The superior mesenteric artery angle in diagnosis of nutcracker syndrome: a systematic review and meta-analysis.

PURPOSE: Nutcracker syndrome (NCS) can be caused by narrowness of the superior mesenteric artery (SMA) angle. Nevertheless, the cut-off value of the SMA angle is controversial and variable. Therefore, the present study evaluated the optimal SMA angle to maximize diagnostic performance for NCS diagnosis by conducting a meta-analysis. METHODS: We comprehensively searched the English literature related to the diagnosis of NCS from the perspective of SMA (from the date of database inception to June 2022). The accuracy of an SMA angle less than 41° in the diagnosis of NCS was evaluated by calculating the pooled sensitivity (SEN), pooled specificity (SPE), positive likelihood ratio (LR+), negative likelihood ratio (LR-), summary receiver operating characteristic (SROC) curve and area under the curve (AUC) value. The I2 test and meta-regression analysis were used to assess heterogeneity and sources of heterogeneity, respectively. Publication bias was assessed using Deeks' funnel plot asymmetry test. RESULTS: Six studies (526 patients) met the inclusion criteria. SEN and SPE were 0.94 (95% confidence interval (CI) 0.80-0.99) and 0.85 (95% CI 0.65-0.94), respectively. The LR + value was 6.0, and the LR- value was 0.07, revealing that SMA angles less than 41° exhibited an excellent ability to help confirm or exclude NCS. Additionally, SROC curves showed that the AUC of SMA angles less than 41° for the diagnosis of NCS was 0.96, indicating that SMA angles less than 41° have good efficacy for helping to diagnose NCS. CONCLUSION: This study explored the diagnostic efficacy of the cut-off value of the SMA angle by meta-analysis. According to the high SPE and SEN results, SMA angles less than 41° have good efficacy in facilitating NCS diagnosis.

Grandparenting and Physical Activity.

Objective: The aim of this study was to examine a continuum of grandparenting intensity and its association with physical activity using three perspectives: grandparents are active, the more constraints perspective, and the selection bias perspective. Method: We use 2014 data from the Health and Retirement Study (HRS), a nationally-representative panel study of the US population over the age of 50 and their spouses (n = 17,851). Results: We found that greater grandparenting intensity was inversely associated with physical activity, providing support for both the more constraints perspective and the selection bias perspective. Discussion: We discuss the implications of inequality in which the most advantaged with physical activity are those who were either not grandparents, or grandparents who provided less care.

Liposomes with Low Levels of Grafted Poly(ethylene glycol) Remain Susceptible to Destabilization by Anti-Poly(ethylene glycol) Antibodies.

Binding of anti-PEG antibodies to poly(ethylene glycol) (PEG) on the surface of PEGylated liposomal doxorubicin (PLD) in vitro and in rats can activate complement and cause the rapid release of doxorubicin from the liposome interior. Here, we find that irinotecan liposomes (IL) and L-PLD, which have 16-fold lower levels of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG2000 in their liposome membrane as compared to PLD, generate less complement activation but remain sensitive to destabilization and drug release by anti-PEG antibodies. Complement activation and liposome destabilization correlated with the theoretically estimated number of antibody molecules bound per liposome. Drug release from liposomes proceeded through the alternative complement pathway but was accelerated by the classical complement pathway. In contrast to PLD destabilization by anti-PEG immunoglobulin G (IgG), which proceeded by the insertion of membrane attack complexes in the lipid bilayer of otherwise intact PLD, anti-PEG IgG promoted the fusion of L-PLD, and IL to form unilamellar and oligo-vesicular liposomes. Anti-PEG immunoglobulin M (IgM) induced drug release from all liposomes (PLD, L-PLD, and IL) via the formation of unilamellar and oligo-vesicular liposomes. Anti-PEG IgG destabilized both PLD and L-PLD in rats, indicating that the reduction of PEG levels on liposomes is not an effective approach to prevent liposome destabilization by anti-PEG antibodies.

Inferences for the distribution of the duration of response in a comparative clinical study.

Duration of response is an important endpoint used in drug development. Prolonged duration for response is often viewed as an early indication of treatment efficacy. However, there are numerous difficulties in studying the distribution of duration of response based on observed data subject to right censoring in practice. The most important obstacle is that the distribution of the duration of response is in general not identifiable in the presence of censoring due to the simple fact that there is no information on the joint distribution of time to response and time to progression beyond the largest follow-up time. In this article, we introduce the restricted duration of response as a replacement of the conventional duration of response. The distribution of restricted duration of response is estimable and we have proposed several nonparametric estimators in this article. The corresponding inference procedure and additional downstream analysis have been developed. Extensive numerical simulations have been conducted to examine the finite sample performance of the proposed estimators. It appears that a new regression-based two-step estimator for the survival function of the restricted duration of response tends to have a robust and superior performance, and we recommend its use in practice. A real data example from oncology has been used to illustrate the analysis for restricted duration of response.