The type of DNA damage response after decitabine treatment depends on the level of DNMT activity.

Decitabine and azacytidine are considered as epigenetic drugs that induce DNA methyltransferase (DNMT)-DNA crosslinks, resulting in DNA hypomethylation and damage. Although they are already applied against myeloid cancers, important aspects of their mode of action remain unknown, highly limiting their clinical potential. Using a combinatorial approach, we reveal that the efficacy profile of both compounds primarily depends on the level of induced DNA damage. Under low DNMT activity, only decitabine has a substantial impact. Conversely, when DNMT activity is high, toxicity and cellular response to both compounds are dramatically increased, but do not primarily depend on DNA hypomethylation or RNA-associated processes. By investigating proteome dynamics on chromatin, we show that decitabine induces a strictly DNMT-dependent multifaceted DNA damage response based on chromatin recruitment, but not expression-level changes of repair-associated proteins. The choice of DNA repair pathway hereby depends on the severity of decitabine-induced DNA lesions. Although under moderate DNMT activity, mismatch (MMR), base excision (BER), and Fanconi anaemia-dependent DNA repair combined with homologous recombination are activated in response to decitabine, high DNMT activity and therefore immense replication stress induce activation of MMR and BER followed by non-homologous end joining.

Incorporating uncertainty within dynamic interoceptive learning.

Introduction: Interoception, the perception of the internal state of the body, has been shown to be closely linked to emotions and mental health. Of particular interest are interoceptive learning processes that capture associations between environmental cues and body signals as a basis for making homeostatically relevant predictions about the future. One method of measuring respiratory interoceptive learning that has shown promising results is the Breathing Learning Task (BLT). While the original BLT required binary predictions regarding the presence or absence of an upcoming inspiratory resistance, here we extended this paradigm to capture continuous measures of prediction (un)certainty. Methods: Sixteen healthy participants completed the continuous version of the BLT, where they were asked to predict the likelihood of breathing resistances on a continuous scale from 0.0 to 10.0. In order to explain participants' responses, a Rescorla-Wagner model of associative learning was combined with suitable observation models for continuous or binary predictions, respectively. For validation, we compared both models against corresponding null models and examined the correlation between observed and modeled predictions. The model was additionally extended to test whether learning rates differed according to stimuli valence. Finally, summary measures of prediction certainty as well as model estimates for learning rates were considered against interoceptive and mental health questionnaire measures. Results: Our results demonstrated that the continuous model fits closely captured participant behavior using empirical data, and the binarised predictions showed excellent replicability compared to previously collected data. However, the model extension indicated that there were no significant differences between learning rates for negative (i.e. breathing resistance) and positive (i.e. no breathing resistance) stimuli. Finally, significant correlations were found between fatigue severity and both prediction certainty and learning rate, as well as between anxiety sensitivity and prediction certainty. Discussion: These results demonstrate the utility of gathering enriched continuous prediction data in interoceptive learning tasks, and suggest that the updated BLT is a promising paradigm for future investigations into interoceptive learning and potential links to mental health.

Repeated ecological and life cycle transitions make salamanders an ideal model for evolution and development.

Observations on the ontogeny and diversity of salamanders provided some of the earliest evidence that shifts in developmental trajectories have made a substantial contribution to the evolution of animal forms. Since the dawn of evo-devo there have been major advances in understanding developmental mechanisms, phylogenetic relationships, evolutionary models, and an appreciation for the impact of ecology on patterns of development (eco-evo-devo). Molecular phylogenetic analyses have converged on strong support for the majority of branches in the Salamander Tree of Life, which includes 764 described species. Ancestral reconstructions reveal repeated transitions between life cycle modes and ecologies. The salamander fossil record is scant, but key Mesozoic species support the antiquity of life cycle transitions in some families. Colonization of diverse habitats has promoted phenotypic diversification and sometimes convergence when similar environments have been independently invaded. However, unrelated lineages may follow different developmental pathways to arrive at convergent phenotypes. This article summarizes ecological and endocrine-based causes of life cycle transitions in salamanders, as well as consequences to body size, genome size, and skeletal structure. Salamanders offer a rich source of comparisons for understanding how the evolution of developmental patterns has led to phenotypic diversification following shifts to new adaptive zones.

Interoception of breathing and its relationship with anxiety.

Interoception, the perception of internal bodily states, is thought to be inextricably linked to affective qualities such as anxiety. Although interoception spans sensory to metacognitive processing, it is not clear whether anxiety is differentially related to these processing levels. Here we investigated this question in the domain of breathing, using computational modeling and high-field (7 T) fMRI to assess brain activity relating to dynamic changes in inspiratory resistance of varying predictability. Notably, the anterior insula was associated with both breathing-related prediction certainty and prediction errors, suggesting an important role in representing and updating models of the body. Individuals with low versus moderate anxiety traits showed differential anterior insula activity for prediction certainty. Multi-modal analyses of data from fMRI, computational assessments of breathing-related metacognition, and questionnaires demonstrated that anxiety-interoception links span all levels from perceptual sensitivity to metacognition, with strong effects seen at higher levels of interoceptive processes.

Long-term Effectiveness of mHealth Physical Activity Interventions: Systematic Review and Meta-analysis of Randomized Controlled Trials.

BACKGROUND: Mobile health (mHealth) interventions can increase physical activity (PA); however, their long-term impact is not well understood. OBJECTIVE: The primary aim of this study is to understand the immediate and long-term effects of mHealth interventions on PA. The secondary aim is to explore potential effect moderators. METHODS: We performed this study according to the Cochrane and PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. We searched PubMed, the Cochrane Library, SCOPUS, and PsycINFO in July 2020. Eligible studies included randomized controlled trials of mHealth interventions targeting PA as a primary outcome in adults. Eligible outcome measures were walking, moderate-to-vigorous physical activity (MVPA), total physical activity (TPA), and energy expenditure. Where reported, we extracted data for 3 time points (ie, end of intervention, follow-up ≤6 months, and follow-up >6 months). To explore effect moderators, we performed subgroup analyses by population, intervention design, and control group type. Results were summarized using random effects meta-analysis. Risk of bias was assessed using the Cochrane Collaboration tool. RESULTS: Of the 2828 identified studies, 117 were included. These studies reported on 21,118 participants with a mean age of 52.03 (SD 14.14) years, of whom 58.99% (n=12,459) were female. mHealth interventions significantly increased PA across all the 4 outcome measures at the end of intervention (walking standardized mean difference [SMD] 0.46, 95% CI 0.36-0.55; P<.001; MVPA SMD 0.28, 95% CI 0.21-0.35; P<.001; TPA SMD 0.34, 95% CI 0.20-0.47; P<.001; energy expenditure SMD 0.44, 95% CI 0.13-0.75; P=.01). Only 33 studies reported short-term follow-up measurements, and 8 studies reported long-term follow-up measurements in addition to end-of-intervention results. In the short term, effects were sustained for walking (SMD 0.26, 95% CI 0.09-0.42; P=.002), MVPA (SMD 0.20, 95% CI 0.05-0.35; P=.008), and TPA (SMD 0.53, 95% CI 0.13-0.93; P=.009). In the long term, effects were also sustained for walking (SMD 0.25, 95% CI 0.10-0.39; P=.001) and MVPA (SMD 0.19, 95% CI 0.11-0.27; P<.001). We found the study population to be an effect moderator, with higher effect scores in sick and at-risk populations. PA was increased both in scalable and nonscalable mHealth intervention designs and regardless of the control group type. The risk of bias was rated high in 80.3% (94/117) of the studies. Heterogeneity was significant, resulting in low to very low quality of evidence. CONCLUSIONS: mHealth interventions can foster small to moderate increases in PA. The effects are maintained long term; however, the effect size decreases over time. The results encourage using mHealth interventions in at-risk and sick populations and support the use of scalable mHealth intervention designs to affordably reach large populations. However, given the low evidence quality, further methodologically rigorous studies are warranted to evaluate the long-term effects.

Additive Lithography-Organic Monolayer Patterning Coupled with an Area-Selective Deposition.

The combination of area-selective deposition (ASD) with a patternable organic monolayer provides a versatile additive lithography platform, enabling the generation of a variety of nanoscale feature geometries. Stearate hydroxamic acid self-assembled monolayers (SAMs) were patterned with extreme ultraviolet (λ = 13.5 nm) or electron beam irradiation and developed with ASD to achieve line space patterns as small as 50 nm. Density functional theory was employed to aid in the synthesis of hydroxamic acid derivatives with optimized packing density to enhance the imaging contrast and improve dose sensitivity. Near-edge X-ray absorption fine structure spectroscopy and infrared spectroscopy reveal that the imaging mechanism is based on improved deposition inhibition provided by the cross-linking of the SAM to produce a more effective barrier during a subsequent deposition step. With patterned substrates composed of coplanar copper lines and silicon spacers, hydroxamic acids selectively formed monolayers on the metal portions and could undergo a pattern-wise exposure followed by ASD in the first combination of a patternable monolayer with ASD. This material system presents an additional capability compared to traditional ASD approaches that generally reflect a starting patterned surface. Furthermore, this bottoms-up additive approach to lithography may be a viable alternative to subtractive nanoscale feature generation.

Graft Dilatation May Cause Secondary Regurgitation in Aortic Valve-Sparing Operations.

Aortic valve-sparing operations are meant to stabilize the root in order to provide stable aortic valve function. The possibility of graft dilatation has been known in other forms of aortic replacement but is largely ignored in valve-preserving surgery. We describe graft dilatation as a cause of failure late after aortic valve reimplantation. It was corrected by applying the concepts of aortic valve repair.

Unicuspid aortic valve repair with bicuspidization in the paediatric population.

OBJECTIVES: Aortic stenosis or regurgitation that requires operations in children often results from unicuspid valve morphology. In all paediatric patients with this anomaly, we have performed unicuspid valve repair by bicuspidization, creating a new commissure via adding patch material. This study reviewed our experience with this procedure. METHODS: All patients with a unicuspid aortic valve who underwent bicuspidization at ≤18 years of age between 2003 and 2018 were evaluated. Autologous pericardium had initially been used for cusp augmentation. Since 2014, decellularized xenogeneic tissue or expanded polytetrafluoroethylene membrane has been applied. RESULTS: There were 60 consecutive patients. The median operative age was 13 (1-18) years. Thirty patients had prior surgical or catheter valvuloplasties. Aortic regurgitation, aortic stenosis and their combination were present in 22, 11 and 27 patients, respectively. Autologous pericardium decellularized tissue and expanded polytetrafluoroethylene were used in 45, 11 and 4 patients, respectively, without intraoperative conversion to valve replacement. The overall survival was 96% at 10 years with 2 late deaths. Twenty patients underwent aortic valve reoperation due to patch degeneration (n = 11), suture dehiscence of the patch (n = 3), subaortic stenosis (n = 3) or other reasons (n = 3). Freedom from aortic valve reoperation was 73% and 50% at 5 and 10 years, respectively. Fifteen patients (25%) required valve replacement with pulmonary autograft (n = 14) or prosthesis (n = 1) 5.2 (0.2-13) years after bicuspidization at the age of 19 (10-32) years. CONCLUSIONS: Bicuspidization is a safe and reproducible alternative to valve replacement with acceptable freedom from reoperation. It can serve as a bridge to valve replacement providing superior outcomes in adults.

Surface Initiated Polymer Thin Films for the Area Selective Deposition and Etching of Metal Oxides.

The area selective growth of polymers and their use as inhibiting layers for inorganic film depositions may provide a valuable self-aligned process for fabrication. Polynorbornene (PNB) thin films were grown from surface-bound initiators and show inhibitory properties against the atomic layer deposition (ALD) of ZnO and TiO2. Area selective control of the polymerization was achieved through the synthesis of initiators that incorporate surface-binding ligands, enabling their selective attachment to metal oxide features versus silicon dielectrics, which were then used to initiate surface polymerizations. The subsequent use of these films in an ALD process enabled the area selective deposition (ASD) of up to 39 nm of ZnO. In addition, polymer thickness was found to play a key role, where films that underwent longer polymerization times were more effective at inhibiting higher numbers of ALD cycles. Finally, while the ASD of a TiO2 film was not achieved despite blanket studies showing inhibition, the ALD deposition on polymer regions of a patterned film produced a different quality metal oxide and therefore altered its etch resistance. This property was exploited in the area selective etch of a metal feature. This demonstration of an area selective surface-grown polymer to enable ASD and selective etch has implications for the fabrication of both micro- and nanoscale features and surfaces.

Reexamining remodelling in children.

OBJECTIVES: Some studies have suggested that root remodelling is unsuitable as valve-sparing aortic root replacement in children because of the increased risk of valve failure. This study reviewed our experience with root remodelling in children. METHODS: All patients who underwent root remodelling at the age of ≤18 years between 1999 and 2016 were evaluated. In 2004, cusp effective height was measured intraoperatively and prolapse was corrected with central plication on the cusp. Suture annuloplasty was introduced after 2009 for annular dilatation. RESULTS: There were 17 consecutive patients. The median age at operation was 14 (2.8-18) years. Aortic valve morphology was tricuspid in 10 patients, bicuspid in 5 patients, unicuspid in 1 patient and a pulmonary autograft in 1 patient. Marfan syndrome, Loeys-Dietz syndrome and other connective tissue diseases were present in 11, 1 and 2 patients, respectively. Five patients had more than moderate aortic regurgitation. The median graft size used for root remodelling was 24 (18-26) mm. Cusp repair and annuloplasty were performed in 15 and 14 patients, respectively. The mean follow-up time was 6.5 ± 4.3 years. One patient with preoperatively severely depressed ventricular function died in the hospital from persistent heart failure. One patient (operated on before 2004) required aortic valve reoperation due to cusp prolapse. One patient with a unicuspid valve had developed moderate aortic regurgitation, and the other 14 patients had mild or less regurgitation. The median diameter of the sinus of Valsalva at the last follow-up was 36 (30-43) mm, Z-score of 1.5 (-3.5 to 3.9). CONCLUSIONS: Root remodelling can be performed in children with favourable results by appropriate cusp repair and annuloplasty.

Insulin-like growth factor 1 stimulates the angiogenic activity of adipose tissue-derived microvascular fragments.

Angiogenesis in adipose tissue is promoted by insulin-like growth factor 1 signaling. We analyzed whether this regulatory mechanism also improves the angiogenic activity of adipose tissue-derived microvascular fragments. Murine adipose tissue-derived microvascular fragments were cultivated for 24 h in the University of Wisconsin (UW) solution supplemented with vehicle, insulin-like growth factor 1, or a combination of insulin-like growth factor 1 and insulin-like growth factor-binding protein 4. Subsequently, we assessed their cellular composition, viability, proliferation, and growth factor expression. Moreover, cultivated adipose tissue-derived microvascular fragments were seeded onto collagen-glycosaminoglycan scaffolds, which were implanted into dorsal skinfold chambers to study their vascularization and incorporation. Insulin-like growth factor 1 increased the viability and growth factor expression of adipose tissue-derived microvascular fragments without affecting their cellular composition and proliferation. Accordingly, scaffolds containing insulin-like growth factor 1-stimulated adipose tissue-derived microvascular fragments exhibited an enhanced in vivo vascularization and incorporation. These positive insulin-like growth factor 1 effects were reversed by additional exposure of adipose tissue-derived microvascular fragments to insulin-like growth factor-binding protein 4. Our findings indicate that insulin-like growth factor 1 stimulation of adipose tissue-derived microvascular fragments is suitable to improve their vascularization capacity.

Seizure detection based on heart rate variability using a wearable electrocardiography device.

OBJECTIVE: To assess the feasibility and accuracy of seizure detection based on heart rate variability (HRV) using a wearable electrocardiography (ECG) device. Noninvasive devices for detection of convulsive seizures (generalized tonic-clonic and focal to bilateral tonic-clonic seizures) have been validated in phase 2 and 3 studies. However, detection of nonconvulsive seizures still needs further research, since currently available methods have either low sensitivity or an extremely high false alarm rate (FAR). METHODS: In this phase 2 study, we prospectively recruited patients admitted to long-term video-EEG monitoring (LTM). ECG was recorded using a dedicated wearable device. Seizures were automatically detected using HRV parameters computed off-line, blinded to all other data. We compared the performance of 26 automated algorithms with the seizure time-points marked by experts who reviewed the LTM recording. Patients were classified as responders if >66% of their seizures were detected. RESULTS: We recruited 100 consecutive patients and analyzed 126 seizures (108 nonconvulsive and 18 convulsive) from 43 patients who had seizures during monitoring. The best-performing HRV algorithm combined a measure of sympathetic activity with a measure of how quickly HR changes occurred. The algorithm identified 53.5% of the patients with seizures as responders. Among responders, detection sensitivity was 93.1% (95% CI: 86.6%-99.6%) for all seizures and 90.5% (95% CI: 77.4%-97.3%) for nonconvulsive seizures. FAR was 1.0/24 h (0.11/night). Median seizure detection latency was 30 s. Typically, patients with prominent autonomic nervous system changes were responders: An ictal change of >50 heartbeats per minute predicted who would be responder with a positive predictive value of 87% and a negative predictive value of 90%. SIGNIFICANCE: The automated HRV algorithm, using ECG recorded with a wearable device, has high sensitivity for detecting seizures, including the nonconvulsive ones. FAR was low during the night. This approach is feasible in patients with prominent ictal autonomic changes.

Cusp Nadir Relocation by Root Remodeling in Unicuspid Aortic Valve Repair.

We describe a root remodeling technique for the unicuspid and unicommissural aortic valve with a moderately hypoplastic anterior commissure. This technique achieves symmetric bicuspidization without patch insertion to cusps. Partial cusp fusion of the anterior commissure is divided, and two symmetric tongues of a Dacron graft are sutured to each center of cusp insertion lines. The cusp nadirs are relocated posteriorly, and the bicuspidized valve has two sufficiently high commissures from a new angulated virtual basal ring. To optimize valve configuration, annuloplasty and prolapse correction according to effective height concept are added. This technique is a promising option in select patients.

University of Wisconsin solution for the xeno-free storage of adipose tissue-derived microvascular fragments.

Aim: Adipose tissue-derived microvascular fragments (ad-MVF) are vascularization units for regenerative medicine. We investigated whether University of Wisconsin (UW) solution is suitable for their xeno-free storage. Materials & methods: Murine ad-MVF were cultivated for 24 h in 4°C or 20°C UW solution and 20°C endothelial cell growth medium (control). The ad-MVF were seeded onto collagen-glycosaminoglycan scaffolds, which were analyzed in dorsal skinfold chambers by intravital fluorescence microscopy and histology. Results: All implants exhibited microvascular networks on day 14 with the highest functional microvessel density in controls. Ad-MVF cultivation in UW solution at 4°C resulted in an improved scaffold vascularization compared with cultivation at 20°C. Conclusion: UW solution is suitable for the hypothermic storage of ad-MVF.

Early-onset stroke in two siblings with Neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, characterized by cafe-au-lait macules, benign neurofibromas as well as malignant peripheral nerve sheath tumours, freckling in the axillary or inguinal regions, optic glioma and Lisch nodules (iris hamartomas) and further manifestations like bone deformities etc. Additionally, NF1 patients are at increased risk of early-onset cerebrovascular diseases, the pathogenesis of which has not been clarified yet. Here we report the first case of two siblings with NF1 who suffered an acute ischemic stroke. Professionals treating NF1 patients should be aware of the elevated risk of stroke in this population. Large prospective studies are needed to establish optimal guidelines for diagnosis, monitoring and treatment of cerebrovascular disease in patients suffering from NF1, as well as to achieve a consensus on routine vascular screening in NF1.

Cirolanides wassenichae sp. nov., a freshwater, subterranean Cirolanidae (Isopoda, Cymothoida) with additional records of other species from Texas, United States.

Cirolanides wassenichae sp. nov., is described from the phreatic zone of the Edwards Aquifer, Texas, USA where it is sympatric with Cirolanides texensis Benedict, 1896. Its status as a new species is based on both morphological and molecular data. Number of antennula articles (3-5 vs 9-15), size (mean sizes of 9.5 and 8.8 mm vs 11.1 and 10.4 mm for males and females, respectively), morphology of pereopods 1-3 (haptorial to semi-haptorial in 1-3 vs only 1 haptorial), and shape of pleotelson (squared, slightly indented vs rounded) are key morphological characteristics that distinguish C. wassenichae sp. nov. from C. texensis. Phylogenies based on cytochrome oxidase 1 and large ribosomal subunit 28S show that divergent morphologies correspond to reciprocally monophyletic groups for both nuclear and mitochondrial datasets. The genus Cirolanides is in need of revision, as our description of C. wassenichae sp. nov. renders C. texensis paraphyletic.

Subnormothermic short-term cultivation improves the vascularization capacity of adipose tissue-derived microvascular fragments.

Adipose tissue-derived microvascular fragments (ad-MVFs) are promising vascularization units for tissue engineering. In this study, we analysed the effects of normothermic (37°C) and subnormothermic (20°C) short-term cultivation on their viability and network forming capacity. Ad-MVFs from green fluorescent protein (GFP)+ and GFP- C57BL/6 mice were cultivated for 24 hr at 37°C or 20°C. Freshly isolated, noncultivated ad-MVFs served as controls. Number, length, viability, proliferation, and angiogenic activity of the ad-MVFs were assessed by microscopic analysis and proteome profiling. GFP+ ad-MVFs were seeded onto collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of GFP- mice to analyse their vascularization by means of intravital fluorescence microscopy, histology, and immunohistochemistry. Depending on the temperature, short-term cultivation of ad-MVFs markedly changed their expression of multiple proangiogenic and antiangiogenic factors. Moreover, cultivation at 37°C significantly increased the number of apoptotic cells within ad-MVFs, whereas 20°C preserved the viability of ad-MVFs and even promoted the proliferation of endothelial and perivascular cells. Accordingly, ad-MVFs cultivated at 20°C also exhibited an enhanced in vivo vascularization capacity when compared with normothermically cultivated ad-MVFs and noncultivated controls. This was indicated by an accelerated network formation, an increased microvascular remodelling, and a higher density of GFP+ microvessels within implanted matrices. Thus, if ad-MVFs require short-term storage before in vivo application, subnormothermic cultivation should be preferred to normothermic cultivation.

Effects of cryopreservation on adipose tissue-derived microvascular fragments.

Adipose tissue-derived microvascular fragments (ad-MVF) are effective vascularization units for tissue engineering. They rapidly reassemble into new microvascular networks after seeding on scaffolds and subsequent in vivo implantation. Herein, we analyzed whether the vascularization capacity of ad-MVF is affected by cryopreservation. Ad-MVF were isolated from the epididymal fat pads of C57BL/6 mice and cryopreserved for 7 days to compare their morphology, viability, cellular composition, and protein expression with freshly isolated control ad-MVF. Moreover, cryopreserved and control ad-MVF from green fluorescent protein (GFP)+ donor mice were seeded on collagen-glycosaminoglycan matrices (Integra® ), which were implanted into dorsal skinfold chambers of GFP- recipient animals to study their vascularization and incorporation using intravital fluorescence microscopy, histology, and immunohistochemistry. Cryopreservation of ad-MVF did not affect vessel morphology and cellular composition. However, cryopreservation was associated with an increased rate of necrotic cells and a significantly reduced number of transplantable ad-MVF. This was compensated by a higher angiogenic activity of the remaining ad-MVF, as indicated by significantly elevated expression levels of pro-angiogenic factors when compared to controls. Accordingly, cryopreserved and control ad-MVF induced a comparable vascularization and incorporation of implanted Integra® without differences in microvascular network formation, maturation, and remodeling. Enhanced angiogenic sprouting even resulted in a higher fraction of GFP+ microvessels within the implants of the cryopreservation group. These findings indicate that cryopreservation of ad-MVF is feasible and, thus, offers the exciting opportunity to build up stocks of readily available vascularization units for future tissue engineering applications.

Chelated charge assisted halogen bonding enhanced halide recognition by a pyridinium-iodotriazolium axle containing [2]rotaxane.

Four halogen and hydrogen bonding rotaxane host systems featuring pyridinium bis-amide-iodotriazole/prototriazole and pyridinium bis-amide-iodotriazolium/prototriazolium axle components have been synthesized by CuAAC-mediated mono-stoppering chloride anion templation and post-rotaxanation methylation reactions. In competitive 45 : 45 : 10 CDCl3-CD3OD-D2O aqueous solvent media, the dicationic halogen bonding rotaxane displayed a notable enhanced binding affinity and marked selectivity for Br- over other halides and nitrate and dihydrogen phosphate oxoanions in contrast to an all hydrogen bonding counterpart which is attributed to chelated charge assisted halogen bonding interactions.