Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of microtubule-associated protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach, we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into three modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wild type and phosphomimetic MAP2 reduced protein synthesis in vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz-that a large proportion of individuals have a "MAP2opathy"-in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

The context-dependence of extinction learning has been well studied and requires the hippocampus. However, the underlying neural mechanisms are still poorly understood. Using memory-driven reinforcement learning and deep neural networks, we developed a model that learns to navigate autonomously in biologically realistic virtual reality environments based on raw camera inputs alone. Neither is context represented explicitly in our model, nor is context change signaled. We find that memory-intact agents learn distinct context representations, and develop ABA renewal, whereas memory-impaired agents do not. These findings reproduce the behavior of control and hippocampal animals, respectively. We therefore propose that the role of the hippocampus in the context-dependence of extinction learning might stem from its function in episodic-like memory and not in context-representation per se. We conclude that context-dependence can emerge from raw visual inputs.

Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the "gatekeeper" residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application.

The transcription factor eomesodermin (EOMES) promotes interleukin (IL)-10 expression in CD4+ T cells, which has been linked to immunosuppressive and cytotoxic activities. We detected cytotoxic, programmed cell death protein-1 (PD-1) and EOMES co-expressing CD4+ T cells in lymph nodes (LNs) of patients with chronic lymphocytic leukemia (CLL) or diffuse large B-cell lymphoma. Transcriptome and flow cytometry analyses revealed that EOMES does not only drive IL-10 expression, but rather controls a unique transcriptional signature in CD4+ T cells, that is enriched in genes typical for T regulatory type 1 (TR1) cells. The TR1 cell identity of these CD4+ T cells was supported by their expression of interferon gamma and IL-10, as well as inhibitory receptors including PD-1. TR1 cells with cytotoxic capacity accumulate also in Eµ-TCL1 mice that develop CLL-like disease. Whereas wild-type CD4+ T cells control TCL1 leukemia development after adoptive transfer in leukopenic Rag2-/- mice, EOMES-deficient CD4+ T cells failed to do so. We further show that TR1 cell-mediated control of TCL1 leukemia requires IL-10 receptor (IL-10R) signaling, as Il10rb-deficient CD4+ T cells showed impaired antileukemia activity. Altogether, our data demonstrate that EOMES is indispensable for the development of IL-10-expressing, cytotoxic TR1 cells, which accumulate in LNs of CLL patients and control TCL1 leukemia in mice in an IL-10R-dependent manner.

During drug development, evaluation of drug and its metabolite is an essential process to understand drug activity, stability, toxicity and distribution. Liquid chromatography (LC) coupled with mass spectrometry (MS) has become the standard analytical tool for screening and identifying drug metabolites. Unlike LC/MS approach requiring liquifying the biological samples, we showed that spectral imaging (or spectral microscopy) could provide high-resolution images of doxorubicin (dox) and its metabolite doxorubicinol (dox'ol) in single living cells. Using this new method, we performed measurements without destroying the biological samples. We calculated the rate constant of dox translocating from extracellular moiety into the cell and the metabolism rate of dox to dox'ol in living cells. The translocation rate of dox into a single cell for spectral microscopy and LC/MS approaches was similar (~ 1.5 pM min-1 cell-1). When compared to spectral microscopy, the metabolism rate of dox was underestimated for about every 500 cells using LC/MS. The microscopy approach further showed that dox and dox'ol translocated to the nucleus at different rates of 0.8 and 0.3 pM min-1, respectively. LC/MS is not a practical approach to determine drug translocation from cytosol to nucleus. Using various methods, we confirmed that when combined with a high-resolution imaging, spectral characteristics of a molecule could be used as a powerful approach to analyze drug metabolism. We propose that spectral microscopy is a new method to study drug localization, translocation, transformation and identification with a resolution at a single cell level, while LC/MS is more appropriate for drug screening at an organ or tissue level.

The herbivore digestive tract is home to a complex community of anaerobic microbes that work together to break down lignocellulose. These microbiota are an untapped resource of strains, pathways and enzymes that could be applied to convert plant waste into sugar substrates for green biotechnology. We carried out more than 400 parallel enrichment experiments from goat faeces to determine how substrate and antibiotic selection influence membership, activity, stability and chemical productivity of herbivore gut communities. We assembled 719 high-quality metagenome-assembled genomes (MAGs) that are unique at the species level. More than 90% of these MAGs are from previously unidentified herbivore gut microorganisms. Microbial consortia dominated by anaerobic fungi outperformed bacterially dominated consortia in terms of both methane production and extent of cellulose degradation, which indicates that fungi have an important role in methane release. Metabolic pathway reconstructions from MAGs of 737 bacteria, archaea and fungi suggest that cross-domain partnerships between fungi and methanogens enabled production of acetate, formate and methane, whereas bacterially dominated consortia mainly produced short-chain fatty acids, including propionate and butyrate. Analyses of carbohydrate-active enzyme domains present in each anaerobic consortium suggest that anaerobic bacteria and fungi employ mostly complementary hydrolytic strategies. The division of labour among herbivore anaerobes to degrade plant biomass could be harnessed for industrial bioprocessing.

Background: Osteogenesis imperfecta (OI) is a rare disease characterized by increased bone fragility and susceptibility for fractures. Only few studies have compared the management for femoral fractures in children with OI. Nevertheless, no cohort studies have described the treatment for femoral fractures in adults with OI in Taiwan. This study aimed to investigate and compare the incidence of union and non-union femoral fractures and the best treatment options to avoid non-union fractures. Methods: We enrolled 72 patients with OI who were older than 18 years at MacKay Memorial Hospital between January 2010 and December 2018. Femoral fracture incidence, non-union rate, and treatment modality were analyzed. Results: Of 72 patients with OI, 11 patients had femoral fractures and 4 patients of them had >1 femoral fracture. The incidence for all types of femoral fractures was 651 fractures per 100,000 person-years annually. In 15 total fractures, 4 fractures resulted in non-union, and patients with type 4 OI mostly had shaft fractures. The best outcomes for non-union shaft fracture is achieved by surgical treatment. Conclusion: Adults with OI tended to develop femoral fractures and non-unions. Adults with type 4 OI were particularly at high risk for non-unions in shaft fractures with conservative treatment.

Di-(2-ethylhexyl) phthalate (DEHP) that is one of the most commonly used phthalates in manufacturing plastic wares regulates tumorigenesis. Thymosin beta-4 (TB4), an actin-sequestering protein, has been reported as a novel regulator to form primary cilia that are antenna-like organelles playing a role in various physiological homeostasis and pathological development including tumorigenesis. Here, we investigated whether DEHP affects tumor growth via primary cilium (PC) formation via the axis of TB4 gene expression and the production of reactive oxygen species (ROS). Tumor growth was increased by DEHP treatment that enhanced TB4 expression, PC formation and ROS production. The number of cells with primary cilia was enhanced time-dependently higher in HeLa cells incubated in the culture medium with 0.1% fetal bovine serum (FBS). The number of cells with primary cilia was decreased by the inhibition of TB4 expression. The incubation of cells with 0.1% FBS enhanced ROS production and the transcriptional activity of TB4 that was reduced by ciliobrevin A (CilioA), the inhibitor of ciliogenesis. ROS production was decreased by catalase treatment but not by mito-TEMPO, which affected to PC formation with the same trend. H2O2 production was reduced by siRNA-based inhibition of TB4 expression. H2O2 also increased the number of ciliated cells, which was reduced by siRNA-TB4 or the co-incubation with CilioA. Tumor cell viability was maintained by ciliogenesis, which was correlated with the changes of intracellular ATP amount rather than a simple mitochondrial enzyme activity. TB4 overexpression enhanced PC formation and DEHP-induced tumor growth. Taken together, data demonstrate that DEHP-induced tumor growth might be controlled by PC formation via TB4-H2O2 axis. Therefore, it suggests that TB4 could be a novel bio-marker to expect the risk of DEHP on tumor growth.

Rationale: To assess the longitudinal changes and relationships of clinical measures and extent of CT lung abnormalities in COVID-19. Methods: 81 patients with COVID-19 were prospectively enrolled and followed until discharge. CT scores were quantified on a basis of a CT scoring system where each lung was divided into 3 zones: upper (above the carina), middle, and lower (below the inferior pulmonary vein) zones; each zone was evaluated for percentage of lung involvement on a scale of 0-4 (0, 0%; 1, 0-24%; 2, 25% - 49%; 3, 50% -74%; 4, >74%).Temporal trends of CT scores and the laboratory parameters characteristic of COVID-19 were analyzed. Correlations between the two were determined at three milestones (initial presentation, worst CT manifestation, and recovery finding before discharge). Their correlations with duration to worst CT manifestation and discharge from symptom onset were evaluated. Results: CT scores peaked during illness days 6-11 (median: 5), and stayed steady. C-reactive protein and lactate dehydrogenase increased, peaked on illness days 6-8 and 8-11 (mean: 23.5 mg/L, 259.9 U/L), and gradually declined. Continual decrease and increase were observed in hemoglobin and lymphocyte count, respectively. Albumin reduced and remained at low levels with a nadir on illness days 12-15 (36.6 g/L). Both initial (r = 0.58, 0.64, p < 0.05) and worst CT scores (r = 0.47, 0.65, p < 0.05) were correlated with C-reactive protein and lactate dehydrogenase; and CT scores before discharge, only with albumin (r = -0.41, p < 0.05). Duration to worst CT manifestation was associated with initial and worst CT scores (r = 0.33, 0.29, p < 0.05). No parameters were related to timespan to discharge. Conclusion: Our results illustrated the temporal changes of characteristic clinical measures and extent of CT lung abnormalities in COVID-19. CT scores correlated with some important laboratory parameters, and might serve as prognostic factors.

Background Women with breast cancer (BC) commonly experience weight gain during chemotherapy, although there is conflicting evidence regarding the contributing factors. This study aimed to evaluate body weight changes among women undergoing chemotherapy for non-metastatic BC during the first year after diagnosis, and to determine whether baseline body weight and/or hemoglobin concentration values were associated with weight changes during chemotherapy. Methods This retrospective study evaluated patients who were treated at the King Abdulaziz University Hospital (Saudi Arabia) during 2010-2019. A total of 228 women were included based on the following criteria: new diagnosed BC, age of 18-80 years, non-metastatic disease, and initial chemotherapy treatment for BC. The patients' baseline characteristics, including body weight during the first chemotherapy cycle, were collected from their electronic medical records. Each patient's weight was then followed at each hospital visit until the last chemotherapy cycle. In addition, data were collected regarding tumor status, menopausal status, chemotherapy regimen, hemoglobin concentration, recurrence status, and death. Results The mean patient age was 53.37±10.9 years and 55.7% of the patients were pre-menopausal. The vast majority of patients underwent surgery (96.9%) and most patients received adjuvant chemotherapy (63.6%) or adjuvant radiotherapy (68.9%). The mean number of chemotherapy cycles was 6.29±1.74 (taxane-based: 1.67±1.36 cycles, anthracycline-based: 2.61±1.81 cycles). At the end of chemotherapy, the body weight changes were classified as increased (41.7% of patients, mean increase: 3.39 kg), decreased (35.5% of patients, mean decrease: -4.12 kg), or stable (22.8%). Factors that predicted weight gain after chemotherapy included younger age at diagnosis (p<0.029), pre-menopausal status (p<0.003), and a high number of taxane-based chemotherapy cycles (p<0.029). Conclusions Chemotherapy for BC did not lead to significant changes in body weight among women in Saudi Arabia. Weight gain in this setting was significantly associated with younger age, pre-menopausal status, and a high number of taxane-based chemotherapy cycles.

While sustained nuclear factor-κB (NF-κB) activation is critical for proinflammatory molecule expression, regulators of NF-κB activity during chronic inflammation are not known. We investigated the role of focal adhesion kinase (FAK) on sustained NF-κB activation in tumor necrosis factor-α (TNF-α)-stimulated endothelial cells (ECs) both in vitro and in vivo. We found that FAK inhibition abolished TNF-α-mediated sustained NF-κB activity in ECs by disrupting formation of TNF-α receptor complex-I (TNFRC-I). Additionally, FAK inhibition diminished recruitment of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and the inhibitor of NF-κB (IκB) kinase (IKK) complex to TNFRC-I, resulting in elevated stability of IκBα protein. In mice given TNF-α, pharmacological and genetic FAK inhibition blocked TNF-α-induced IKK-NF-κB activation in aortic ECs. Mechanistically, TNF-α activated and redistributed FAK from the nucleus to the cytoplasm, causing elevated IKK-NF-κB activation. On the other hand, FAK inhibition trapped FAK in the nucleus of ECs even upon TNF-α stimulation, leading to reduced IKK-NF-κB activity. Together, these findings support a potential use for FAK inhibitors in treating chronic inflammatory diseases.

Patients with established Parkinson's disease (PD) display differences in peripheral blood markers of immune function, including leukocyte differential counts, compared with controls. These differences may be useful biomarkers to predict PD and may shed light on pathogenesis. We sought to identify whether peripheral immune dysregulation was associated with increased risk of subsequent PD diagnosis.

The placenta protects the fetus against excessive stress-associated maternal cortisol during pregnancy. We studied whether exposure to radiofrequency electromagnetic field (RF-EMF) radiation during pregnancy can cause changes in dams and their placentas. Pregnant Sprague-Dawley rats were divided into cage-control, sham-exposed, and RF-exposed groups. They were exposed to RF-EMF signals at a whole-body specific absorption rate of 4 W/kg for 8 h/day from gestational Day 1 to 19. Levels of cortisol in the blood, adrenal gland, and placenta were measured by enzyme-linked immunosorbent assay. Levels of adrenocorticotropic hormone and corticotropin-releasing hormone were monitored in maternal blood. Expression levels of placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) messenger RNA (mRNA) were measured by reverse transcription polymerase chain reaction. Morphological changes in the placenta were analyzed using hematoxylin and eosin staining. Fetal parts of the placenta were measured using Zen 2.3 blue edition software. Maternal cortisol in circulating blood (RF: 230 ± 24.6 ng/ml and Sham: 156 ± 8.3 ng/ml) and the adrenal gland (RF: 58.3 ± 4.5 ng/ml and Sham: 30 ± 3.8 ng/ml) was significantly increased in the RF-exposed group (P < 0.05). Placental cortisol was stably maintained, and the level of placental 11β-HSD2 mRNA expression was not changed in the RF-exposed group. RF-EMF exposure during pregnancy caused a significant elevation of cortisol levels in circulating blood; however, no changes in the placental barrier were observed in pregnant rats. Bioelectromagnetics. © 2021 Bioelectromagnetics Society.

A giant barocaloric effect (BCE) in a molecular material Fe3 (bntrz)6 (tcnset)6 (FBT) is reported, where bntrz = 4-(benzyl)-1,2,4-triazole and tcnset = 1,1,3,3-tetracyano-2-thioethylepropenide. The crystal structure of FBT contains a trinuclear transition metal complex that undergoes an abrupt spin-state switching between the state in which all three FeII centers are in the high-spin (S = 2) electronic configuration and the state in which all of them are in the low-spin (S = 0) configuration. Despite the strongly cooperative nature of the spin transition, it proceeds with a negligible hysteresis and a large volumetric change, suggesting that FBT should be a good candidate for producing a large BCE. Powder X-ray diffraction and calorimetry reveal that the material is highly susceptible to applied pressure, as the transition temperature spans the range from 318 at ambient pressure to 383 K at 2.6 kbar. Despite the large shift in the spin-transition temperature, its nonhysteretic character is maintained under applied pressure. Such behavior leads to a remarkably large and reversible BCE, characterized by an isothermal entropy change of 120 J kg-1 K-1 and an adiabatic temperature change of 35 K, which are among the highest reversible values reported for any caloric material thus far.

Programmed cell death protein 1 (PD-1), an immune checkpoint receptor expressed by activated T, B, and NK cells, is a well-known target for cancer immunotherapy. Tislelizumab (BGB-A317) is an anti-PD-1 antibody that has recently been approved for treatment of Hodgkin's lymphoma and urothelial carcinoma. Here, we show that tislelizumab displayed remarkable antitumor efficacy in a B16F10/GM-CSF mouse model. Structural biology and Surface plasmon resonance (SPR) analyses revealed unique epitopes of tislelizumab, and demonstrated that the CC' loop of PD-1, a region considered to be essential for binding to PD-1 ligand 1 (PD-L1) but not reported as targeted by other therapeutic antibodies, significantly contributes to the binding of tislelizumab. The binding surface of tislelizumab on PD-1 overlaps largely with that of the PD-L1. SPR analysis revealed the extremely slow dissociation rate of tislelizumab from PD-1. Both structural and functional analyses align with the observed ability of tislelizumab to completely block PD-1/PD-L1 interaction, broadening our understanding of the mechanism of action of anti-PD-1 antibodies.

Autoimmune diseases such as psoriasis, rheumatoid arthritis, and systemic lupus erythematosus (SLE) have high rates of hypertension and cardiovascular disease. Systemic lupus erythematosus is a prototypic autoimmune disorder that primarily affects women of childbearing age and is associated with a loss of self-tolerance, autoreactive B and T lymphocytes, and the production of autoantibodies, especially to nuclear components. In this study, we hypothesized that the pristane-inducible model of SLE would develop hypertension and vascular dysfunction as the disease progressed. To test this hypothesis, female C57BL/6 mice were administered PBS or pristane. Seven months after pristane administration, mice developed various autoantibodies, including anti-dsDNA IgG, anti-ssDNA IgG, and anti-nRNP IgG, as well as hypergammaglobulinemia. Several other immunological changes, including increased circulating neutrophils and increased CD4- CD8- (double negative) thymocytes were also detected. Mean arterial pressure (MAP) was elevated in pristane-treated mice when compared to PBS-treated mice. In addition, second-order mesenteric arteries from pristine-treated mice had impaired relaxation to the endothelium-dependent vasodilator acetylcholine compared to PBS-treated mice. These data suggest that the immune system dysfunction present in the pristane model of lupus contributes to the development of hypertension and vascular dysfunction.

Salmonellosis in poultry is an important disease that seriously impedes the development of the poultry industry. The increased resistance to antimicrobials against Salmonella has been a major public health concern worldwide. We conducted a study from January to June 2016 in and around the Rajshahi district of Bangladesh on the commercial chicken to isolate, identify and characterize poultry-specific Salmonella, to assess the potential risk factors and to determine the antimicrobial resistance pattern of the isolates. The overall prevalence of Salmonella enterica was 41% (49/120) [95% CI: 31.95%-50.17%] with 41.7% in broiler chicken (25/60) [95% CI: 29.06%-55.12%] and 40% in layer chicken (24/60, 40%) [95% CI: 27.56%-53.46%]. Samples collected from Rajshahi city (OR = 1.37, 95% CI: 0.50-3.73) and Puthia Upazila (OR = 1.51, 95% CI: 0.56-4.12) were more likely to be positive for Salmonella than Charghat Upazila. Salmonella detection was 1.3 times higher in chicken, providing loose feed than those provided ready feed. All the isolates fermented dextrose, maltose and mannitol with the production of acid and gas, but did not ferment sucrose and lactose. The isolates showed catalase, MR, citrate utilization test and TSI agar test positive, but indole and V-P tests negative. Salmonella isolates were sensitive to ciprofloxacin (90%), gentamycin (80%), amoxicillin (75%), streptomycin (70%), ampicillin (45%) and sulfamethoxazole-trimethoprim (45%), whereas highly resistant to penicillin (100%) and nalidixic acid (100%) followed by sulfamethoxazole-trimethoprim (55%), ampicillin (40%) and amoxicillin (25%). Salmonella enterica is endemic in commercial chicken production in Bangladesh with high prevalence. A considerable proportion of Salmonella isolates was found to be resistant to the majority of the common antimicrobial drugs. A good biosecurity system could be effective for the reduction of Salmonella. It is necessary to obtain universal commitments to establish prudent antibiotic use policies.

While mesenchymal stromal cells are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID-19, trauma, or cancers. Here, we tested a novel preclinical approach aimed at improving the safety of mesenchymal stromal cell (MSC) systemic administration by use of a bioreactor. In this system, MSCs are seeded on the exterior of a hollow-fiber filter, sequestering them behind a hemocompatible semipermeable membrane with defined pore-size and permeability to allow for a molecularly defined cross talk between the therapeutic cells and the whole blood environment, including blood cells and signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against directly injected "free" MSCs, a model of systemic administration. Our results showed that restricting MSCs exposure to plasma via a bioreactor extends the time necessary for clot formation to occur when compared with "free" MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Furthermore, application of this technology in two in vivo models did not require additional heparin in fully anticoagulated experimental animals to maintain target activated clotting time levels relative to heparin anticoagulated controls. Taken together the clinical use of bioreactor housed MSCs could offer a novel method to control systemic MSC exposure and prolong clot formation time.

Infographics are pictorial representations of information intended to disseminate information quickly and clearly. Their use has increased in the past decade due to wider and easy access to technology. Infographics are being increasingly used for public advisories, disseminating protocols for healthcare professionals, and post-publication promotion of research. Due to their potential to rapidly reach a vast audience, these have gained larger importance during the coronavirus disease 2019 pandemic. Two key aspects determine the quality of infographics, content and visual appeal. In this brief, the authors attempt to delineate the key aspects of designing an infographic, and the freeware that they may have at their disposal for creating informative, appealing, and useful infographics.

In organ transplantation, the need for immune modulation rather than immune suppression has been emphasized. In this study, we investigated whether combinatorial treatments of with thalidomide (TM) and dexamethasone (DX) might be new approaches to induce systemic immunomodulation on T cells and other immune cells that regulate the expression of co-inhibitory molecules.