Tsuyoshi Nakai

ABSTRACT Objective Cerebrospinal fluid (CSF) cell‐free mitochondrial DNA (cf‐mtDNA) is a potential biomarker for Parkinson's disease (PD), but its clinical relevance remains unclear. We investigated associations between CSF cf‐mtDNA levels, body composition, nutritional status, and metabolic biomarkers in PD. Methods CSF cf‐mtDNA levels, defined as the copy numbers of two regions of the mtDNA circular molecule (mt64‐ND1 and mt96‐ND5), were quantified in 44 PD patients and 43 controls using multiplex digital PCR. The mt96‐ND5/mt64‐ND1 ratio was calculated to estimate mtDNA deletion burden. Associations with clinical features, body composition, serum nutritional markers, and plasma energy metabolism‐related organic acids were examined. Generalized linear models (GLMs) were performed to adjust for confounders. Results CSF mt64‐ND1 and mt96‐ND5 levels were lower in PD patients than controls (p = 0.002, p = 0.001), while the mt96‐ND5/mt64‐ND1 ratio showed no group difference. GLM analysis identified body composition indices and serum albumin as key determinants of cf‐mtDNA levels. Subgroup analysis showed lower cf‐mtDNA levels in PD patients with preserved body composition and nutritional status. The mt96‐ND5/mt64‐ND1 ratio displayed a biphasic association with body composition and an inverse correlation with plasma 2‐ketoglutaric acid, suggesting a link to energy metabolism. Interpretation CSF cf‐mtDNA levels are reduced in PD and influenced by body composition and nutritional status, supporting their role as a metabolic biomarker. While the cf‐mtDNA deletion ratio remained unchanged, its association with body composition suggests a complex interplay between mitochondrial integrity and metabolism. These findings highlight the relevance of cf‐mtDNA in PD pathophysiology and the need for further study.

Recombinant tissue-type plasminogen activators (rtPA) effectively dissolve blood clots and improve symptoms in patients with acute ischemic stroke and myocardial infraction. Although rtPA are used in patients taking antiplatelets or anticoagulants to improve clinical outcomes, combination therapy may increase the risk of hemorrhagic transformation (HT) and intracerebral hemorrhage (ICH). However, few studies have investigated the risk of HT and ICH associated with these combination therapies. This study aimed to investigate the adverse-event and drug-drug interaction signals for HT and ICH under combination therapy with alteplase and various antiplatelets or anticoagulants, using the Japanese Adverse Drug Event Report database. Adverse-event signals were evaluated using the reporting odds ratio and information components, and drug-drug interaction signals were studied using the Ω shrinkage measure, additive, multiplicative, and Chi-square statistics models. We also investigated predictors of HT and ICH, time-to-onset, and outcomes in patients receiving alteplase. HT and/or ICH signals were detected in patients receiving alteplase in combination with aspirin, P2Y₁₂ inhibitors, cilostazol, ozagrel sodium, direct oral anticoagulants, warfarin potassium, heparin group, or argatroban. Hypertension and diabetes mellitus were significant risk factors for alteplase-induced HT. Most HT and ICH events occurred within 1 day after alteplase administration, and more than 60% of affected patients were not in recovery. In conclusion, continued monitoring is required in patients receiving alteplase in combination with any of the eight types of antiplatelets or the aforementioned anticoagulants. Additionally, the occurrence of HT or ICH within 1 day post-alteplase administration should be considered in patients with hypertension or diabetes mellitus. The findings from this study may help in understanding the risk of HT and ICH induced by rtPA in patients taking antiplatelet or anticoagulant medications, as well as in promoting the appropriate use of rtPA. Further prospective observational studies and randomized controlled trials are needed to assess these finding.

Background/Objectives: The risk of fractures associated with immune checkpoint inhibitors (ICIs) is increasing; however, the relationship between fracture risk and potential factors, such as osteoporosis and hyperthyroidism, remains unclear. Methods: Using VigiBase, the World Health Organization's global pharmacovigilance database, we investigated the signals for osteoporosis, hyperthyroidism, and fractures associated with ICIs (nivolumab, pembrolizumab, atezolizumab, durvalumab, ipilimumab, and tremelimumab) by calculating information components (ICs) and their 95% confidence intervals (CIs). Furthermore, we estimated the association between the occurrence of fractures in patients receiving ICIs and osteoporosis or hyperthyroidism. Results: Signals of hyperthyroidism (IC = 4.66, 95% CI: 4.58–4.73), but not osteoporosis (IC = −1.79, 95% CI: −2.22 to −1.36) or fractures (IC = −0.21, 95% CI: −0.36 to −0.06), were detected in patients using ICIs. Osteoporosis (odds ratio: 118.00, 95% CI: 61.00–230.00) was associated with an increased reporting frequency of fractures related to ICIs, whereas hyperthyroidism (odds ratio: 0.60, 95% CI: 0.19–1.87) was not associated with such an increase. Conclusions: The VigiBase analysis indicates that the use of ICIs does not increase the reporting frequency of osteoporosis or fractures. Additionally, hyperthyroidism did not increase the reporting frequency of fractures associated with ICIs.

Abstract Purpose Tranexamic acid (TXA) is widely used as an antifibrinolytic drug. However, studies to determine the optimal blood concentration of TXA have produced inconsistent results. During cardiac surgery, cardiopulmonary bypass (CPB) has serious effects on drug distribution, elimination, and plasma concentration. Therefore, we aimed to establish a population pharmacokinetics model of TXA in patients undergoing cardiac surgery with CPB that considers renal function as a covariate, thereby facilitating personalized treatment. Methods In total, 453 TXA plasma samples were prospectively collected from 77 patients who underwent cardiac surgery with CPB. Plasma concentrations were determined by ultra-performance liquid chromatography-tandem mass spectrometry. The population pharmacokinetic model of TXA was analyzed using nonlinear mixed-effects modeling. Results The two-compartment–based model with combined errors was determined as the best. The final model included the effect of bodyweight and CL_cr may be summarized as V ₁ (L) = 12.77 × (bodyweight / 61.4)^0.911, V ₂ (L) = 6.857, CL₁ (L/h) = 3.263 × [CL_cr (L/h) / 61.0]^0.752, CL₂ (L/h) = 2.859. Conclusion Patients who undergo cardiac surgery with CPB may require an adjusted dose of TXA tailored to CPB due to lower CL₁ and increased V ₁. Our TXA population pharmacokinetic model may be useful for developing individualized dosing designs for TXA in patients who undergo cardiac surgery with CPB.

The budding yeast Saccharomyces cerevisiae is commonly used as an expression platform for the production of valuable compounds. Yeast‐based genetic research can uniquely utilize auxotrophy in transformant selection: auxotrophic complementation by an auxotrophic marker gene on exogenous DNA (such as plasmids). However, the number of required auxotrophic nutrients restricts the number of plasmids maintained by the cells. We, therefore, developed novel Δ10 strains that are auxotrophic for 10 different nutrients and new plasmids with two multiple cloning sites and auxotrophic markers for use in Δ10 strains. We confirmed that Δ10 strains were able to maintain 10 types of plasmids. Using plasmids encoding model proteins, we detected the co‐expression of 17 different genes in Δ10 cell lines. We also constructed Δ9 strains that exhibited auxotrophy for nine nutrients and increased growth compared to Δ10. This study opens a new avenue for the co‐expression of a large number of genes in eukaryotic cells.

Abstract Hypertension is a major cause of cardiovascular diseases. Several recent studies reported that pharmacists’ remote follow-up reduced hypertension patients’ blood pressure (BP). This meta-analysis aims to verify whether remote follow-up by pharmacists improves BP levels and reveal the factors that make the intervention effective. The search, conducted using PubMed/Medline, Embase, and Cochrane Library from June to July 2023, targeted articles published between October 1982 and June 2023, using terms including “pharmacist”, “hypertension”, and “randomized controlled trial (RCT)”. The inclusion criteria were: (a) RCTs involving hypertension patients with or without comorbidities, (b) pharmacists using remote communication tools to conduct follow-up encounter during the intervention period, (c) reporting systolic blood pressure (SBP) at baseline and during intervention. SBP was the primary outcome for the meta-analysis. Thirteen studies (3969 participants) were included in this meta-analysis. The mean difference of SBP between intervention group and control group was − 7.35 mmHg (P < 0.0001). Subgroup analyses showed the greater reduction of SBP in the “regularly scheduled follow-up cohort” (− 8.89 mmHg) compared with the “as needed follow-up cohort” (− 3.23 mmHg, P < 0.0001). The results revealed that remote follow-up by pharmacists reduced SBP levels in hypertension patients and scheduled remote follow-up may contribute to the effectiveness.

PURPOSE: Minimally invasive surgery (MIS) using a thoraco- or laparoscope is becoming a more common surgical technique. In MIS, a magnified view from a thoracoscope helps surgeons conduct precise operations. However, there is a risk of the visible area becoming narrow. To confirm that the operation field is safe, the surgeon will draw the thoracoscope back to check the marginal area of the target and insert it again many times during MIS. To reduce the surgeon's load, we aim to visualize the entire thoracic cavity using a newly developed device called "panorama vision ring" (PVR). METHOD: The PVR is used instead of a wound retractor or a trocar. It is a ring-type socket with one big hole for the thoracoscope and four small holes for tiny cameras placed around the big hole. The views from the tiny cameras are fused into one wider view that visualizes the entire thoracic cavity. A surgeon can proceed with an operation by checking what exists outside of the thoracoscopic view. Also, she/he can check whether or not bleeding has occurred from the image of the entire cavity. RESULTS: We evaluated the view-expansion ability of the PVR by using a three-dimensional full-scale thoracic model. The experimental results showed that the entire thoracic cavity could be visible in a panoramic view generated by the PVR. We also demonstrated pulmonary lobectomy in virtual MIS using the PVR. Surgeons could perform a pulmonary lobectomy while checking the entire cavity. CONCLUSION: We developed the PVR, which uses tiny auxiliary cameras to create a panoramic view of the entire thoracic cavity during MIS. We aim to make MIS safer for patients and more comfortable for surgeons through the development of the PVR.

<jats:title>Abstract</jats:title><jats:p>Teicoplanin is an important antimicrobial agent for methicillin-resistant<jats:italic>Staphylococcus aureus</jats:italic>infections. To enhance its clinical effectiveness while preventing adverse effects, therapeutic drug monitoring (TDM) of teicoplanin trough concentration is recommended. Given the importance of the early achievement of therapeutic concentrations for treatment success, initial dosing regimens are deliberately designed based on patient information.</jats:p><jats:p>Considerable effort has been dedicated to developing an optimal initial dose plan for specific populations; however, comprehensive strategies for tailoring teicoplanin dosing have not been successfully implemented. The initial dose planning of teicoplanin is conducted at the clinician’s discretion and is thus strongly dependent on the clinician’s experience and expertise.</jats:p><jats:p>The present study aimed to use a machine learning (ML) approach to integrate clinicians’ knowledge into a predictive model for initial teicoplanin dose planning. We first confirmed that dose planning by pharmacists dedicated to TDM (hereafter TDM pharmacists) significantly improved early therapeutic target attainment for patients without an intensive care unit or high care unit stay, providing the first evidence that dose planning of teicoplanin by experienced clinicians enhances early teicoplanin therapeutic exposure. Next, we used a dataset of teicoplanin initial dose planning by TDM pharmacists to train and implement the model, yielding a model that emulated TDM pharmacists’ decision-making for dosing. We further applied ML to cases without TDM pharmacist dose planning and found that the target attainment rate of the initial teicoplanin concentration markedly increased. Our study opens a new avenue for tailoring the initial dosing regimens of teicoplanin using a TDM pharmacist-trained ML system.</jats:p><jats:sec><jats:title>Importance</jats:title><jats:p>Teicoplanin is used for treating methicillin-resistant<jats:italic>Staphylococcus aureus</jats:italic>infections. Given the importance of early adequate teicoplanin exposure, initial dosing regimens are adjusted for patient characteristics. However, tailoring teicoplanin dosing is challenging for most clinicians. In this study, we first showed that initial dosing regimens by pharmacists dedicated to therapeutic drug monitoring significantly improved early achievement of targeted concentration. In addition, we leveraged machine learning approach to develop the predictive model that tailors initial dosing regimens at the levels of experienced pharmacists. The target attainment rate of patients without experienced pharmacists’ dose planning was significantly increased by applying the model. Therefore, machine learning approach may provide new avenues for tailoring initial teicoplanin dosing.</jats:p></jats:sec>

Reelin is an extracellular matrix protein that is mainly produced in Cajal-Retzius cells and controls neuronal migration, which is important for the proper formation of cortical layers in the developmental stage of the brain. In the adult brain, Reelin plays a crucial role in the regulation of N-methyl-D-aspartate receptor-dependent synaptic function, and its expression decreases postnatally. Clinical studies showed reductions in Reelin protein and mRNA expression levels in patients with psychiatric disorders; however, the causal relationship remains unclear. Reelin-deficient mice exhibit an abnormal neuronal morphology and behavior, while Reelin supplementation ameliorates learning deficits, synaptic dysfunctions, and spine loss in animal models with Reelin deficiency. These findings suggest that the neuronal deficits and brain dysfunctions associated with the down-regulated expression of Reelin are attenuated by enhancements in its expression and functions in the brain. In this review, we summarize findings on the role of Reelin in neuropsychiatric disorders and discuss potential therapeutic approaches for neuropsychiatric disorders associated with Reelin dysfunctions.

Alzheimer’s disease (AD) is an age-related and progressive neurodegenerative disorder. It is widely accepted that AD is mainly caused by the accumulation of extracellular amyloid β (Aβ) and intracellular neurofibrillary tau tangles. Aβ begins to accumulate years before the onset of cognitive impairment, suggesting that the benefit of currently available interventions would be greater if they were initiated in the early phases of AD. To understand the mechanisms of AD pathogenesis, various transgenic mouse models with an accelerated accumulation of Aβ and tau tangles have been developed. However, none of these models exhibit all pathologies present in human AD. To overcome these undesirable phenotypes, APP knock-in mice, which were presented with touchscreen-based tasks, were developed to better evaluate the efficacy of candidate therapeutics in mouse models of early-stage AD. This review assesses several AD mouse models from the aspect of biomarkers and cognitive impairment and discusses their potential as tools to provide novel AD therapeutic approaches.

S-allylcysteine (SAC), a major thioallyl compound contained in mature garlic extract (MGE), is known to be a neuroactive compound. This study was designed to investigate the effects of SAC on primary cultured hippocampal neurons and cognitively impaired senescence-accelerated mice prone 10 (SAMP10). Treatment of these neurons with MGE or SAC significantly increased the total neurite length and number of dendrites. SAMP10 mice fed MGE or SAC showed a significant improvement in memory dysfunction in pharmacological behavioral analyses. The decrease of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, N-methyl-d-aspartate (NMDA) receptor, and phosphorylated α-calcium/calmodulin-dependent protein kinase II (CaMKII) in the hippocampal tissue of SAMP10 mice fed MGE or SAC was significantly suppressed, especially in the MGE-fed group. These findings suggest that SAC positively contributes to learning and memory formation, having a beneficial effect on brain function. In addition, multiple components (aside from SAC) contained in MGE could be useful for improving cognitive function by acting as neurotrophic factors.

BACKGROUND: Polyriboinosinic-polyribocytidylic acid (polyI:C) triggers a strong innate immune response that mimics immune activation by viral infections. Induction of interferon-induced transmembrane protein 3 (Ifitm3) in astrocytes has a crucial role in polyI:C-induced neurodevelopmental abnormalities. Through a quantitative proteomic screen, we previously identified candidate astroglial factors, such as matrix metalloproteinase-3 (Mmp3) and follistatin-like 1 (Fstl1), in polyl:C-induced neurodevelopmental impairment. Here, we characterized the Ifitm3-dependent inflammatory processes focusing on astrocyte-derived Fstl1 following polyI:C treatment to assess the neuropathologic role of Fstl1. METHODS: Astrocytes were treated with PBS (control) or polyI:C (10 μg/mL). The conditioned medium was collected 24 h after the polyI:C treatment and used as astrocyte condition medium (ACM). The expression of Fstl1 mRNA and extracellular Fstl1 protein levels were analyzed by quantitative PCR and western blotting, respectively. For functional studies, neurons were treated with ACM and the effects of ACM on dendritic elongation were assayed. To examine the role of Fstl1, recombinant Fstl1 protein and siRNA for Fstl1 were used. To investigate the expression of Fstl1 in vivo, neonatal mice were treated with vehicle or polyI:C on postnatal day 2 to 6. RESULTS: ACM prepared with polyI:C (polyI:C ACM) contained significantly higher Fstl1 protein than control ACM, but no increase in Fstl1 was observed in polyI:C ACM derived from Ifitm3-deficient astrocytes. We found that the production of Fstl1 involves the inflammatory responsive molecule Ifitm3 in astrocytes and influences neuronal differentiation. In agreement, the levels of Fstl1 increased in the hippocampus of polyI:C-treated neonatal mice. COS7 cells co-transfected with both Fstl1 and Ifitm3 had higher extracellular levels of Fstl1 than the cells transfected with Fstl1 alone. Treatment of primary cultured hippocampal neurons with recombinant Fstl1 impaired dendritic elongation, and the deleterious effect of polyI:C ACM on dendritic elongation was attenuated by knockdown of Fstl1 in astrocytes. CONCLUSIONS: The extracellular level of Fstl1 is regulated by Ifitm3 in astrocytes, which could be involved in polyI:C-induced neurodevelopmental impairment.

Synaptic plasticity in hippocampal neurons has been thought to represent a variety of memories. Although accumulating evidence indicates a crucial role of BDNF/TrkB/Akt signaling in the synaptic plasticity of the hippocampus, the mechanism by which Akt, a serine/threonine kinase, controls activity-dependent neuronal plasticity remains unclear. Girdin (also known as APE, GIV, and HkRP1), an actin-binding protein involved both in the remodeling of the actin cytoskeleton and in cell migration, has been identified as a substrate of Akt. Previous studies have demonstrated that deficit of neuronal migration in the hippocampus of Girdin-deficient (Girdin(-/-)) mice is independent on serine phosphorylation of Girdin at S1416 (Girdin S1416) by Akt. In the present study, we focused on the role of Girdin S1416 phosphorylation in BDNF/TrkB/Akt signaling associated with synaptic plasticity. We found that Girdin in the hippocampus was phosphorylated at S1416 in an activity-dependent manner. Phosphorylation-deficient knock-in mice (Girdin(SA/SA) mice), in which S1416 is replaced with alanine, exhibited shrinkage of spines, deficit of hippocampal long-term potentiation, and memory impairment. These phenotypes of Girdin(SA/SA) mice resembled those of Girdin(+/-) mice, which have 50% loss of Girdin expression. Furthermore, Girdin interacted with Src kinase and NR2B subunit of NMDA receptor, leading to phosphorylation of the NR2B subunit and NMDA receptor activation. Our findings suggest that Girdin has two different functions in the hippocampus: Akt-independent neuronal migration and Akt-dependent NR2B phosphorylation through the interaction with Src, which is associated with synaptic plasticity in the hippocampus underlying memory formation.

Disrupted-in-schizophrenia-1 (DISC1) has been widely associated with several psychiatric disorders, including schizophrenia, mood disorders and autism. We previously reported that a deficiency of DISC1 may induce low anxiety and/or high impulsivity in mice with disruption of exons 2 and 3 of the Disc1 gene (Disc1(Δ2-3/Δ2-3)). It remains unclear, however, if deficiency of DISC1 leads to specific alterations in distinct neuronal systems. In the present study, to understand the role of DISC1 in γ-aminobutyric acid (GABA) interneurons and mesocorticolimbic dopaminergic (DAergic) neurons, we investigated the number of parvalbumin (PV)-positive interneurons, methamphetamine (METH)-induced DA release and the expression levels of GABAA, DA transporter (DAT) and DA receptors in wild-type (Disc1(+/+)) and Disc1(Δ2-3/Δ2-3) mice. Female Disc1(Δ2-3/Δ2-3) mice showed a significant reduction of PV-positive interneurons in the hippocampus, while no apparent changes were observed in mRNA expression levels of GABAA receptor subunits. METH-induced DA release was significantly potentiated in the nucleus accumbens (NAc) of female Disc1(Δ2-3/Δ2-3) mice, although there were no significant differences in the expression levels of DAT. Furthermore, the expression levels of DA receptor mRNA were upregulated in the NAc of female Disc1(Δ2-3/Δ2-3) mice. Male Disc1(Δ2-3/Δ2-3) mice showed no apparent differences in all experiments. DISC1 may play a critical role in gender-specific developmental alteration in GABAergic inhibitory interneurons and DAergic neurons.

Increasing epidemiological evidence indicates that prenatal infection and childhood central nervous system infection with various viral pathogens enhance the risk for several neuropsychiatric disorders. Polyriboinosinic-polyribocytidilic acid (polyI:C) is known to induce strong innate immune responses that mimic immune activation by viral infections. Our previous findings suggested that activation of the innate immune system in astrocytes results in impairments of neurite outgrowth and spine formation, which lead to behavioral abnormalities in adulthood. To identify candidates of astrocyte-derived humoral factors that affect neuronal development, we analyzed astrocyte-conditioned medium (ACM) from murine astrocyte cultures treated with polyI:C (polyI:C-ACM) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Through a quantitative proteomic screen, we found that 13 protein spots were differentially expressed compared with ACM from vehicle-treated astrocytes (control-ACM), and characterized one of the candidates, matrix metalloproteinase-3 (Mmp3). PolyI:C treatment significantly increased the expression levels of Mmp3 mRNA and protein in astrocytes, but not microglia. PolyI:C-ACM was associated with significantly higher Mmp3 protein level and enzyme activity than control-ACM. The addition of recombinant Mmp3 into control-ACM impaired dendritic elongation of primary cultured hippocampal neurons, while the deleterious effect of polyI:C-ACM on neurite elongation was attenuated by knockdown of Mmp3 in astrocytes. These results suggest that Mmp3 is a possible mediator of polyI:C-ACM-induced neurodevelopmental impairment.

Senescence-accelerated mouse prone 8 (SAMP8) is a model of aging characterized by the early onset of learning and memory impairment and various pathological features of Alzheimer's disease (AD). Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from citrus peels, ameliorates learning and memory impairment in olfactory-bulbectomized mice, amyloid precursor protein transgenic mice, and NMDA receptor antagonist-treated mice. Here, we present evidence that this natural compound improves age-related cognitive impairment and reduces oxidative stress and tau phosphorylation in SAMP8 mice. Treatment with nobiletin (10 or 50mg/kg) reversed the impairment of recognition memory and context-dependent fear memory in SAMP8 mice. Treatment with nobiletin also restored the decrease in the GSH/GSSG ratio in the brain of SAMP8 mice. In addition, increases in glutathione peroxidase and manganese-superoxide dismutase activities, as well as a decrease in protein carbonyl level, were observed in the brain of nobiletin-treated SAMP8 mice. Furthermore, nobiletin reduced tau phosphorylation in the hippocampus of SAMP8 mice. Together, the markedly beneficial effects of nobiletin represent a potentially useful treatment for ameliorating the learning and memory deficits, oxidative stress, and hyperphosphorylation of tau in aging as well as age-related neurodegenerative diseases such as AD.

Platinum(IV) [Pt(IV)] complex, satraplatin, is currently in clinical trials for the treatment of various cancers. As a key step of the anti-cancer effect exertion, satraplatin is supposed to be reduced by endogenous reductants to platinum(II) [Pt(II)] complex. In this study, we investigated the interaction of DNA, Pt(IV), and the endogenous reductants such as ascorbic acid (AsA) and glutathione (GSH). As a model Pt(IV) compound, cis-diammine-tetrachloro-Pt(IV) [cis-Pt(IV)], which is a prodrug of cisplatin [cis-diammine-dichloro-Pt(II), cis-Pt(II)], was incubated with calf thymus DNA in the presence of AsA or GSH. In the presence of AsA, cis-Pt(IV) induced oxidative DNA damage. Hydroxyl radical scavengers suppressed the AsA-associated oxidative damage, thereby suggesting that hydroxyl radicals are involved in the DNA oxidation. cis-Pt(II)-like CD spectral change and crosslink formation in calf thymus DNA were also observed during this DNA oxidation, suggesting cis-Pt(IV) reduction by AsA and DNA conformational change induced by the newly formed cis-Pt(II) binding to DNA. GSH did not induce oxidative DNA damage likely due to its own hydroxyl radical scavenging ability. Further, GSH suppressed the Pt(II)-mediated DNA conformational change and crosslink formation, suggesting that GSH sequesters the cis-Pt(II) away from DNA by GSH-cis-Pt(II) complex formation.