Keep calm and carry H3K27me1 off.

In this issue of Neuron, Torres-Berrío et al.1 show that stress-susceptible mice exhibit elevated H3K27me1 levels in nucleus accumbens neurons due to the action of the SUZ12 VEFS domain, strengthening the link between specific epigenetic changes and long-lasting stress-induced social, emotional, and cognitive alterations.

Unveiling Transcriptional and Epigenetic Mechanisms Within Engram Cells: Insights into Memory Formation and Stability.

Memory traces for behavioral experiences, such as fear conditioning or taste aversion, are believed to be stored through biophysical and molecular changes in distributed neuronal ensembles across various brain regions. These ensembles are known as engrams, and the cells that constitute them are referred to as engram cells. Recent advancements in techniques for labeling and manipulating neural activity have facilitated the study of engram cells throughout different memory phases, including acquisition, allocation, long-term storage, retrieval, and erasure. In this chapter, we will explore the application of next-generation sequencing methods to engram research, shedding new light on the contribution of transcriptional and epigenetic mechanisms to engram formation and stability.

Targeting epigenetic dysregulation in autism spectrum disorders.

Autism spectrum disorders (ASD) comprise a range of neurodevelopmental pathologies characterized by deficits in social interaction and repetitive behaviors, collectively affecting almost 1% of the worldwide population. Deciphering the etiology of ASD has proven challenging due to the intricate interplay of genetic and environmental factors and the variety of molecular pathways affected. Epigenomic alterations have emerged as key players in ASD etiology. Their research has led to the identification of biomarkers for diagnosis and pinpointed specific gene targets for therapeutic interventions. This review examines the role of epigenetic alterations, resulting from both genetic and environmental influences, as a central causative factor in ASD, delving into its contribution to pathogenesis and treatment strategies.

Kdm1a safeguards the topological boundaries of PRC2-repressed genes and prevents aging-related euchromatinization in neurons.

Kdm1a is a histone demethylase linked to intellectual disability with essential roles during gastrulation and the terminal differentiation of specialized cell types, including neurons, that remains highly expressed in the adult brain. To explore Kdm1a's function in adult neurons, we develop inducible and forebrain-restricted Kdm1a knockouts. By applying multi-omic transcriptome, epigenome and chromatin conformation data, combined with super-resolution microscopy, we find that Kdm1a elimination causes the neuronal activation of nonneuronal genes that are silenced by the polycomb repressor complex and interspersed with active genes. Functional assays demonstrate that the N-terminus of Kdm1a contains an intrinsically disordered region that is essential to segregate Kdm1a-repressed genes from the neighboring active chromatin environment. Finally, we show that the segregation of Kdm1a-target genes is weakened in neurons during natural aging, underscoring the role of Kdm1a safeguarding neuronal genome organization and gene silencing throughout life.

Health service and psychotropic medication use for mental health conditions among healthcare workers active during the Spain Covid-19 Pandemic - A prospective cohort study using web-based surveys.

Little is known about healthcare workers' (HCW) use of healthcare services for mental disorders. This study presents data from a 16-month prospective cohort study of Spanish HCW (n = 4,809), recruited shortly after the COVID-19 pandemic onset, and assessed at four timepoints using web-based surveys. Use of health services among HCW with mental health conditions (i.e., those having a positive screen for mental disorders and/or suicidal thoughts and behaviours [STB]) was initially low (i.e., 18.2 %) but increased to 29.6 % at 16-month follow-up. Service use was positively associated with pre-pandemic mental health treatment (OR=1.99), a positive screen for major depressive disorder (OR=1.50), panic attacks (OR=1.74), suicidal thoughts and behaviours (OR=1.22), and experiencing severe role impairment (OR=1.33), and negatively associated with being female (OR = 0.69) and a higher daily number of work hours (OR=0.95). Around 30 % of HCW with mental health conditions used anxiolytics (benzodiazepines), especially medical doctors. Four out of ten HCW (39.0 %) with mental health conditions indicated a need for (additional) help, with most important barriers for service use being too ashamed, long waiting lists, and professional treatment not being available. Our findings delineate a clear mental health treatment gap among Spanish HCW.

Diagnosis of Endometriosis Based on Comorbidities: A Machine Learning Approach.

Endometriosis is defined as the presence of estrogen-dependent endometrial-like tissue outside the uterine cavity. Despite extensive research, endometriosis is still an enigmatic disease and is challenging to diagnose and treat. A common clinical finding is the association of endometriosis with multiple diseases. We use a total of 627,566 clinically collected data from cases of endometriosis (0.82%) and controls (99.18%) to construct and evaluate predictive models. We develop a machine learning platform to construct diagnostic tools for endometriosis. The platform consists of logistic regression, decision tree, random forest, AdaBoost, and XGBoost for prediction, and uses Shapley Additive Explanation (SHAP) values to quantify the importance of features. In the model selection phase, the constructed XGBoost model performs better than other algorithms while achieving an area under the curve (AUC) of 0.725 on the test set during the evaluation phase, resulting in a specificity of 62.9% and a sensitivity of 68.6%. The model leads to a quite low positive predictive value of 1.5%, but a quite satisfactory negative predictive value of 99.58%. Moreover, the feature importance analysis points to age, infertility, uterine fibroids, anxiety, and allergic rhinitis as the top five most important features for predicting endometriosis. Although these results show the feasibility of using machine learning to improve the diagnosis of endometriosis, more research is required to improve the performance of predictive models for the diagnosis of endometriosis. This state of affairs is in part attributed to the complex nature of the condition and, at the same time, the administrative nature of our features. Should more informative features be used, we could possibly achieve a higher AUC for predicting endometriosis. As a result, we merely perceive the constructed predictive model as a tool to provide auxiliary information in clinical practice.

A Novel Classification of Endometriosis Based on Clusters of Comorbidities.

Endometriosis is a heterogeneous, complex, and still challenging disease, due to its epidemiological, etiological and pathogenic, diagnostic, therapeutic, and prognosis characteristics. The classification of endometriosis is contentious, and existing therapies show significant variability in their effectiveness. This study aims to capture and describe clusters of women with endometriosis based on their comorbidity. With data extracted from electronic records of primary care, this study performs a hierarchical clustering with the Ward method of women with endometriosis with a subsequent analysis of the distribution of comorbidities. Data were available for 4055 women with endometriosis, and six clusters of women were identified: cluster 1 (less comorbidity), cluster 2 (anxiety and musculoskeletal disorders), cluster 3 (type 1 allergy or immediate hypersensitivity); cluster 4 (multiple morbidities); cluster 5 (anemia and infertility); and cluster 6 (headache and migraine). Clustering aggregates similar units into similar clusters, partitioning dissimilar objects into other clusters at a progressively finer granularity-in this case, groups of women with similarities in their comorbidities. Clusters may provide a deeper insight into the multidimensionality of endometriosis and may represent diverse "endometriosis trajectories" which may be associated with specific molecular and biochemical mechanisms. Comorbidity-based clusters may be important to the scientific study of endometriosis, contributing to the clarification of its clinical complexity and variability. An awareness of those comorbidities may help elucidate the etiopathogenesis and facilitate the accurate earlier diagnosis and initiation of treatments targeted toward particular subgroups.

Extra centrosomes induce PIDD1-mediated inflammation and immunosurveillance.

Unscheduled increases in ploidy underlie defects in tissue function, premature aging, and malignancy. A concomitant event to polyploidization is the amplification of centrosomes, the main microtubule organization centers in animal cells. Supernumerary centrosomes are frequent in tumors, correlating with higher aggressiveness and poor prognosis. However, extra centrosomes initially also exert an onco-protective effect by activating p53-induced cell cycle arrest. If additional signaling events initiated by centrosomes help prevent pathology is unknown. Here, we report that extra centrosomes, arising during unscheduled polyploidization or aberrant centriole biogenesis, induce activation of NF-κB signaling and sterile inflammation. This signaling requires the NEMO-PIDDosome, a multi-protein complex composed of PIDD1, RIPK1, and NEMO/IKKγ. Remarkably, the presence of supernumerary centrosomes suffices to induce a paracrine chemokine and cytokine profile, able to polarize macrophages into a pro-inflammatory phenotype. Furthermore, extra centrosomes increase the immunogenicity of cancer cells and render them more susceptible to NK-cell attack. Hence, the PIDDosome acts as a dual effector, able to engage not only the p53 network for cell cycle control but also NF-κB signaling to instruct innate immunity.

Traumatic stress symptoms among Spanish healthcare workers during the COVID-19 pandemic: a prospective study.

AIM: To investigate the occurrence of traumatic stress symptoms (TSS) among healthcare workers active during the COVID-19 pandemic and to obtain insight as to which pandemic-related stressful experiences are associated with onset and persistence of traumatic stress. METHODS: This is a multicenter prospective cohort study. Spanish healthcare workers (N = 4,809) participated at an initial assessment (i.e., just after the first wave of the Spain COVID-19 pandemic) and at a 4-month follow-up assessment using web-based surveys. Logistic regression investigated associations of 19 pandemic-related stressful experiences across four domains (infection-related, work-related, health-related and financial) with TSS prevalence, incidence and persistence, including simulations of population attributable risk proportions (PARP). RESULTS: Thirty-day TSS prevalence at T1 was 22.1%. Four-month incidence and persistence were 11.6% and 54.2%, respectively. Auxiliary nurses had highest rates of TSS prevalence (35.1%) and incidence (16.1%). All 19 pandemic-related stressful experiences under study were associated with TSS prevalence or incidence, especially experiences from the domains of health-related (PARP range 88.4-95.6%) and work-related stressful experiences (PARP range 76.8-86.5%). Nine stressful experiences were also associated with TSS persistence, of which having patient(s) in care who died from COVID-19 had the strongest association. This association remained significant after adjusting for co-occurring depression and anxiety. CONCLUSIONS: TSSs among Spanish healthcare workers active during the COVID-19 pandemic are common and associated with various pandemic-related stressful experiences. Future research should investigate if these stressful experiences represent truly traumatic experiences and carry risk for the development of post-traumatic stress disorder.

CBP and p300 Jointly Maintain Neural Progenitor Viability but Play Unique Roles in the Differentiation of Neural Lineages.

The paralogous lysine acetyltransferases 3 (KAT3), CBP and P300, play critical roles during neurodevelopment, but their specific roles in neural precursors maintenance and differentiation remain obscure. In fact, it is still unclear whether these proteins are individually or jointly essential in processes such as proliferation of neural precursors, differentiation to specific neural cell types, or both. Here, we use subventricular zone-derived neurospheres as a potential ex vivo developmental model to analyze the proliferation and differentiation of neural stem cells (NSCs) lacking CBP, p300, or both proteins. The results showed that CBP and p300 are not individually essential for maintenance and proliferation of NSCs, although their combined ablation seriously compromised cell division. In turn, the absence of either of the two proteins compromised the differentiation of NSC into the neuronal and astrocytic lineages. Single-nucleus RNA sequencing analysis of neural cell cultures derived from CBP or p300 mutant neurospheres revealed divergent trajectories of neural differentiation upon CBP or p300 ablation, confirming unique functions and nonredundant roles in neural development. These findings contribute to a better understanding of the shared and individual roles of KAT3 proteins in neural differentiation and the etiology of neurodevelopmental disorders caused by their deficiency.

CBP Is Required for Establishing Adaptive Gene Programs in the Adult Mouse Brain.

Environmental factors and life experiences impinge on brain circuits triggering adaptive changes. Epigenetic regulators contribute to this neuroadaptation by enhancing or suppressing specific gene programs. The paralogous transcriptional coactivators and lysine acetyltransferases CREB binding protein (CBP) and p300 are involved in brain plasticity and stimulus-dependent transcription, but their specific roles in neuroadaptation are not fully understood. Here we investigated the impact of eliminating either CBP or p300 in excitatory neurons of the adult forebrain of mice from both sexes using inducible and cell type-restricted knock-out strains. The elimination of CBP, but not p300, reduced the expression and chromatin acetylation of plasticity genes, dampened activity-driven transcription, and caused memory deficits. The defects became more prominent in elderly mice and in paradigms that involved enduring changes in transcription, such as kindling and environmental enrichment, in which CBP loss interfered with the establishment of activity-induced transcriptional and epigenetic changes in response to stimulus or experience. These findings further strengthen the link between CBP deficiency in excitatory neurons and etiopathology in the nervous system.SIGNIFICANCE STATEMENT How environmental conditions and life experiences impinge on mature brain circuits to elicit adaptive responses that favor the survival of the organism remains an outstanding question in neurosciences. Epigenetic regulators are thought to contribute to neuroadaptation by initiating or enhancing adaptive gene programs. In this article, we examined the role of CREB binding protein (CBP) and p300, two paralogous transcriptional coactivators and histone acetyltransferases involved in cognitive processes and intellectual disability, in neuroadaptation in adult hippocampal circuits. Our experiments demonstrate that CBP, but not its paralog p300, plays a highly specific role in the epigenetic regulation of neuronal plasticity gene programs in response to stimulus and provide unprecedented insight into the molecular mechanisms underlying neuroadaptation.

Multiomic Analysis of Neurons with Divergent Projection Patterns Identifies Novel Regulators of Axon Pathfinding.

Axon pathfinding is a key step in neural circuits formation. However, the transcriptional mechanisms regulating its progression remain poorly understood. The binary decision of crossing or avoiding the midline taken by some neuronal axons during development represents a robust model to investigate the mechanisms that control the selection of axonal trajectories. Here, to identify novel regulators of axon guidance, this work compares the transcriptome and chromatin occupancy profiles of two neuronal subpopulations, ipsilateral (iRGC) and contralateral retinal ganglion cells (cRGC), with similar functions but divergent axon trajectories. These analyses retrieved a number of genes encoding for proteins not previously implicated in axon pathfinding. In vivo functional experiments confirm the implication of some of these candidates in axonal navigation. Among the candidate genes, γ-synuclein is identified as essential for inducing midline crossing. Footprint and luciferase assays demonstrate that this small-sized protein is regulated by the transcription factor (TF) Pou4f1 in cRGCs. It is also shown that Lhx2/9 are specifically expressed in iRGCs and control a program that partially overlaps with that regulated by Zic2, previously described as essential for iRGC specification. Overall, the analyses identify dozens of new molecules potentially involved in axon guidance and reveal the regulatory logic behind the selection of axonal trajectories.

Evaluation of Physical Therapy Interventions for Improving Musculoskeletal Pain and Quality of Life in Older Adults.

Background: The ageing process may lead to functional limitations, musculoskeletal pain, and worsened quality of life. The aim of this paper is to evaluate two physical therapy interventions for reducing musculoskeletal pain and improving quality of life in older adults. Methods: A cohort study was carried out with older people (60−75 years old). The Geriatric Physical Therapy group (n = 70) received massage therapy, therapeutic exercise, and therapeutic education program for 5 weeks; the Standardized Therapeutic Exercise group (n = 140) received a standardized therapeutic exercise and therapeutic education program for 3 weeks. Health-related quality of life (SF-36v2) and musculoskeletal pain intensity (VAS) were collected at baseline (A0), post-intervention (A1), and 12 weeks after baseline (A2). Results: There was pain intensity reduction in both groups (p < 0.05) and health-related quality of life improvement, except for Emotional Role (p = 0.34); Physical Function (p = 0.07), Bodily Pain (p = 0.02), and General Health (p = 0.09). At A2 there was a difference (p < 0.05) for neck pain in favor of the Geriatric Physical Therapy Group. Conclusions: Within the limitations of the study, it was possible to conclude that both physical therapy interventions showed a positive effect for reducing non-specific neck pain and low back pain in older adults, which may contribute to health-related quality of life improvement.

Direct and Indirect Costs Related to Physical Activity Levels in Patients with Diabetes Mellitus in Spain: A Cross-Sectional Study.

Diabetes mellitus (DM) is a global public health concern. DM is importantly linked to the modern lifestyle. Lifestyle-based interventions currently represent a critical preventive and therapeutic approach for patients with DM. Increasing physical activity has proven multiple benefits to prevent this condition; however, there is still room for further progress in this field, especially in terms of the effect of exercise in patients with already established DM. This study intends to examine the economic relationship between physical activity and direct/indirect costs in patients with DM. We analyze a national representative sample (n = 1496) of the general population of Spain, using available data from the National Health Survey of 2017 (NHS 2017). Our results show that 63.7% of the sample engaged in some degree of physical activity, being more frequent in men (67.5%), younger individuals (80.0%), and those with higher educational levels (69.7%). Conversely, lower levels of physical activity were associated with female sex, older subjects, and various comorbidities. Our study estimates that 2151 € per (51% in direct costs) patient may be saved if a minimum level of physical activity is implemented, primarily, due to a decrease in indirect costs (absenteeism and presenteeism). This study shows that physical activity will bring notable savings in terms of direct and indirect costs in patients with DM, particularly in some vulnerable groups.

Chronic Venous Disease in Pregnant Women Causes an Increase in ILK in the Placental Villi Associated with a Decrease in E-Cadherin.

Chronic venous disease (CVD) is a multifactorial vascular disorder frequently manifested in lower limbs in the form of varicose veins (VVs). Women are a vulnerable population for suffering from CVD, especially during pregnancy, when a plethora of changes occur in their cardiovascular system. Previous studies have indicated a worrisome association between CVD in pregnancy with the placental structure and function. Findings include an altered cellular behavior and extracellular matrix (ECM) composition. Integrin-linked kinase (ILK) is a critical molecule involved in multiple physiological and pathological conditions, and together with cadherins, is essential to mediate cell to ECM and cell to cell interplay, respectively. Thus, the aim of this study was to evaluate the implication of ILK and a set of cadherins (e-cadherin, cadherin-6 and cadherin-17) in placentas of women with CVD in order to unravel the possible pathophysiological role of these components. Gene expression (RT-qPCR) and protein expression (immunohistochemistry) studies were performed. Our results show a significant increase in the gene and protein expression of ILK, cadherin-6 and cadherin-17 and a decrease of e-cadherin in the placenta of women with CVD. Overall, this work shows that an abnormal expression of ILK, e-cadherin, cadherin-6 and cadherin-17 may be implicated in the pathological changes occurring in the placental tissue. Further studies should be conducted to determine the possible associations of these changes with maternal and fetal well-being.

Control of protein synthesis and memory by GluN3A-NMDA receptors through inhibition of GIT1/mTORC1 assembly.

De novo protein synthesis is required for synapse modifications underlying stable memory encoding. Yet neurons are highly compartmentalized cells and how protein synthesis can be regulated at the synapse level is unknown. Here, we characterize neuronal signaling complexes formed by the postsynaptic scaffold GIT1, the mechanistic target of rapamycin (mTOR) kinase, and Raptor that couple synaptic stimuli to mTOR-dependent protein synthesis; and identify NMDA receptors containing GluN3A subunits as key negative regulators of GIT1 binding to mTOR. Disruption of GIT1/mTOR complexes by enhancing GluN3A expression or silencing GIT1 inhibits synaptic mTOR activation and restricts the mTOR-dependent translation of specific activity-regulated mRNAs. Conversely, GluN3A removal enables complex formation, potentiates mTOR-dependent protein synthesis, and facilitates the consolidation of associative and spatial memories in mice. The memory enhancement becomes evident with light or spaced training, can be achieved by selectively deleting GluN3A from excitatory neurons during adulthood, and does not compromise other aspects of cognition such as memory flexibility or extinction. Our findings provide mechanistic insight into synaptic translational control and reveal a potentially selective target for cognitive enhancement.

Areas of Interest and Attitudes Toward Antiobesity Drugs: Thematic and Quantitative Analysis Using Twitter.

BACKGROUND: Antiobesity drugs are prescribed for the treatment of obesity in conjunction with healthy eating, physical activity, and behavior modification. However, poor adherence rates have been reported. Attitudes or beliefs toward medications are important to ascertain because they may be associated with patient behavior. The analysis of tweets has become a tool for health research. OBJECTIVE: The aim of this study is to investigate the content and key metrics of tweets referring to antiobesity drugs. METHODS: In this observational quantitative and qualitative study, we focused on tweets containing hashtags related to antiobesity drugs between September 20, 2019, and October 31, 2019. Tweets were first classified according to whether they described medical issues or not. Tweets with medical content were classified according to the topic they referred to: side effects, efficacy, or adherence. We additionally rated it as positive or negative. Furthermore, we classified any links included within a tweet as either scientific or nonscientific. Finally, the number of retweets generated as well as the dissemination and sentiment score obtained by the antiobesity drugs analyzed were also measured. RESULTS: We analyzed a total of 2045 tweets, 945 of which were excluded according to the criteria of the study. Finally, 320 out of the 1,100 remaining tweets were also excluded because their content, although related to drugs for obesity treatment, did not address the efficacy, side effects, or adherence to medication. Liraglutide and semaglutide accumulated the majority of tweets (682/780, 87.4%). Notably, the content that generated the highest frequency of tweets was related to treatment efficacy, with liraglutide-, semaglutide-, and lorcaserin-related tweets accumulating the highest proportion of positive consideration. We found the highest percentages of tweets with scientific links in those posts related to liraglutide and semaglutide. Semaglutide-related tweets obtained the highest probability of likes and were the most disseminated within the Twitter community. CONCLUSIONS: This analysis of posted tweets related to antiobesity drugs shows that the interest, beliefs, and experiences regarding these pharmacological treatments are heterogeneous. The efficacy of the treatment accounts for the majority of interest among Twitter users.

Efficacy of Manual Therapy and Transcutaneous Electrical Nerve Stimulation in Cervical Mobility and Endurance in Subacute and Chronic Neck Pain: A Randomized Clinical Trial.

Neck pain is a frequent health problem. Manual therapy (MT) and transcutaneous electrical nerve stimulation (TENS) are recommended techniques for treatment of mechanical neck disorders (MND) in Spanish Public Primary Care Physiotherapy Services. The aim of this study was to compare the efficacy of MT versus TENS in active mobility and endurance in cervical subacute or chronic neck pain. Ninety patients with MND were randomly allocated to receive ten 30-min sessions of either MT or TENS, in a multi-centered study through 12 Primary Care Physiotherapy Units in the Madrid community. Active cervical range of motion (CD-ROM) and endurance (Palmer and Epler test) were evaluated pre- and post-intervention and at 6-month follow-up. A generalized linear model of repeated measures was constructed for the analysis of differences. Post-intervention MT yielded a significant improvement in active mobility and endurance in patients with subacute or chronic MND, and at 6-month follow-up the differences were only significant in endurance and in sagittal plane active mobility. In the TENS group, no significant improvement was detected. With regard to other variables, MT improved mobility and endurance more effectively than TENS at post-intervention and at 6-month follow-up in the sagittal plane. Only MT generated significant improvements in cervical mobility and endurance in the three movement planes.

Areas of Interest and Attitudes towards the Pharmacological Treatment of Attention Deficit Hyperactivity Disorder: Thematic and Quantitative Analysis Using Twitter.

We focused on tweets containing hashtags related to ADHD pharmacotherapy between 20 September and 31 October 2019. Tweets were classified as to whether they described medical issues or not. Tweets with medical content were classified according to the topic they referred to: side effects, efficacy, or adherence. Furthermore, we classified any links included within a tweet as either scientific or non-scientific. We created a dataset of 6568 tweets: 4949 (75.4%) related to stimulants, 605 (9.2%) to non-stimulants and 1014 (15.4%) to alpha-2 agonists. Next, we manually analyzed 1810 tweets. In the end, 481 (48%) of the tweets in the stimulant group, 218 (71.9%) in the non-stimulant group and 162 (31.9%) in the alpha agonist group were considered classifiable. Stimulants accumulated the majority of tweets. Notably, the content that generated the highest frequency of tweets was that related to treatment efficacy, with alpha-2 agonist-related tweets accumulating the highest proportion of positive consideration. We found the highest percentages of tweets with scientific links in those posts related to alpha-2 agonists. Stimulant-related tweets obtained the highest proportion of likes and were the most disseminated within the Twitter community. Understanding the public view of these medications is necessary to design promotional strategies aimed at the appropriate population.

Transcriptome and epigenome analysis of engram cells: Next-generation sequencing technologies in memory research.

Transcription and epigenetic changes are integral components of the neuronal response to stimulation and have been postulated to be drivers or substrates for enduring changes in animal behavior, including learning and memory. Memories are thought to be deposited in neuronal assemblies called engrams, i.e., groups of cells that undergo persistent physical or chemical changes during learning and are selectively reactivated to retrieve the memory. Despite the research progress made in recent years, the identity of specific epigenetic changes, if any, that occur in these cells and subsequently contribute to the persistence of memory traces remains unknown. The analysis of these changes is challenging due to the difficulty of exploring molecular alterations that only occur in a relatively small percentage of cells embedded in a complex tissue. In this review, we discuss the recent advances in this field and the promise of next-generation sequencing (NGS) and epigenome editing methods for overcoming these challenges and address long-standing questions concerning the role of epigenetic mechanisms in memory encoding, maintenance and expression.