Objective: To evaluate the efficacy and safety of ruxolitinib combined with low-dose hormone for patients with acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: Thirty patients with aGVHD after allo-HSCT admitted to the Department of Hematology of the General Hospital of Western Theater Command from November 2021 to November 2024 were retrospectively analyzed. All patients were treated with low-dose hormone (methylprednisolone 0.3-1 mg kg -d ) combined with ruxolitinib 5-10 mg d . The efficacy and adverse reactions were observed during the follow-up period to analyze the survival outcomes of the patients. Results: A total of 30 patients with aGVHD after allo-HSCT were included in this study, consisting of 15 (50%) males and 15 (50%) females with a median age of 34 year-old (ranging from 14 to 62). Classification by disease type: there were 18 cases of acute myeloid leukemia, 4 cases of acute lymphoblastic leukemia, 4 cases of aplastic anemia, and 4 cases of myelodysplastic syndrome. Classification by aGVHD severity: there were 27 cases (90%) of Ⅱ-Ⅳ degree aGVHD and 11 cases (36.7%) of Ⅲ-Ⅳ degree aGVHD. Ruxolitinib in combination with low-dose glucocorticoid treatment yield responses in 28 (93.3%) patients, of which 27 (90%) achieved complete remission (CR), while 1 (3.3%) showed partial remission (PR). One patient (3.3%) had no response (NR), and 1 patient (3.3%) exhibited progressed disease (PD). Overall survival (OS) at 1 year of transplantation was 73.9% (95%CI 49.5% to 87.7%), progression-free survival (PFS) was 93.3% (95%CI 75.9% to 98.3%), non-relapse mortality (NRM) was 20.6% (95%CI 7.9% to 47.4%), and median survival time was 27.6 months. Conclusion: Ruxolitinib combined with low-dose hormones is safe and effective in the treatment of aGVHD after allo-HSCT.

Consciousness, which underpins human perception, cognition, and behavioural responses, has long been a major focus of neuroscience and medicine. Alterations in consciousness occur not only during reversible processes such as sleep and anaesthesia, but also in pathological conditions, including disorders of consciousness (DOC) and brain injury. With recent advances in the synchronized acquisition and analysis of multimodal physiological signals, such as electroencephalography (EEG) and electrocardiography (ECG), the dynamic interactions between the heart and brain have become increasingly apparent. A growing body of evidence indicates that heart-brain interactions are closely associated with the maintenance and transition of consciousness states, offering a novel perspective for elucidating their underlying mechanisms and supporting relevant clinical assessments. Current approaches to heart-brain interaction analysis for consciousness-state assessment can be broadly grouped into three categories: ECG-based methods, EEG-based methods, and heart-brain coupling analysis. These approaches characterize heart-brain interactions during changes in consciousness from the complementary perspectives of autonomic regulation, cortical interoceptive processing, and cross-system information transfer, respectively. Nevertheless, this field remains at an early stage. Most studies have focused on a single experimental context or a fixed analytical method, with insufficient systematic consideration of the applicability and limitations of different approaches. Moreover, the mechanisms underlying the emergence and alteration of consciousness remain incompletely understood. Accordingly, this review examines heart-brain interaction methods employed in recent consciousness-state assessment studies in accordance with progressively deeper analytical perspectives, and systematically evaluates the advantages and limitations of these methods across distinct consciousness-related conditions. Heart rate variability (HRV) is one of the classical ECG-based indices used to investigate heart-brain interactions. It refers to fluctuations in consecutive interbeat intervals over a specified time period and is regarded as a physiological measure of autonomic nervous system regulation that may indirectly reflect the strength of heart-brain interactions. Depending on the analytical dimension, HRV analysis is commonly divided into time-domain, frequency-domain, and complexity-index analyses. These approaches characterize autonomic activity in terms of temporal fluctuations, the distribution of frequency components, and nonlinear dynamical properties, respectively. Although extensive research supports their physiological interpretation, they cannot characterize directional interactions from the heart to the brain. Among EEG-based methods for heart-brain interaction analysis, EEG activity directly related to cardiac fluctuations has received particular attention. One of the most widely used measures is the heartbeat-evoked potential (HEP). HEP-based approaches are considered to directly reflect the capacity of the cerebral cortex to process cardiac afferent signals and may be especially advantageous for characterizing rapidly changing consciousness levels, such as those observed during sleep. However, they do not comprehensively capture dynamic bidirectional information exchange between the brain and heart and are susceptible to cardiac artefacts, respiration, and variation in preprocessing procedures. Heart-brain coupling analysis quantifies functional associations between ECG and EEG through cross-system interaction measures, thereby assessing dynamic interactions between the heart and brain. It emphasizes information transfer, feedback regulation, and cross-timescale coordination between the two systems, offering a more comprehensive perspective for investigating changes in consciousness during sleep, anaesthesia, and DOC. However, the neurophysiological mechanisms and physiological interpretation of individual computational indices have not yet been fully established, and the robustness of their results and their potential for clinical generalization require further validation. Finally, this review summarizes the commonalities and differences among the three classes of methods across research domains involving different consciousness states. It further argues that heart-brain interaction research requires deeper investigation at both the mechanistic level of consciousness and the level of clinical application. Current challenges and key issues are identified, and future directions and feasible pathways for heart-brain interaction research are discussed, with the aim of providing new perspectives for researchers in related fields.

BACKGROUND: Treatment with chimeric antigen receptor-T (CAR-T) cells has shown promising effectiveness in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), although the process of preparing for this therapy usually takes a long time. We have recently created CD19 Fast-CAR-T (F-CAR-T) cells, which can be produced within a single day. The objective of this study was to evaluate and contrast the effectiveness and safety of CD19 F-CAR-T cells with those of CD19 conventional CAR-T cells in the management of R/R B-ALL. METHODS: A multicenter, retrospective analysis of the clinical data of 44 patients with R/R B-ALL was conducted. Overall, 23 patients were administered with innovative CD19 F-CAR-T cells (F-CAR-T group), whereas 21 patients were given CD19 conventional CAR-T cells (C-CAR-T group). We compared the rates of complete remission (CR), minimal residual disease (MRD)-negative CR, leukemia-free survival (LFS), overall survival (OS), and the incidence of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) between the two groups. RESULTS: Compared with the C-CAR-T group, the F-CAR-T group had significantly higher CR and MRD-negative rates (95.7% and 91.3%, respectively; 71.4% and 66.7%, respectively; P = 0.036 and P = 0.044). No significant differences were observed in the 1-year or 2-year LFS or OS rates between the two groups: the 1-year and 2-year LFS for the F-CAR-T group vs.C-CAR-T group were 47.8% and 43.5% vs. 38.1% and 23.8% (P = 0.384 and P = 0.216), while the 1-year and 2-year OS rates were 65.2% and 56.5% vs. 52.4% and 47.6% (P = 0.395 and P = 0.540). Additionally, among CR patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) following CAR-T-cell therapy, there were no significant differences in the 1-year or 2-year LFS or OS rates: 57.1% and 50.0% vs. 47.8% and 34.8% (P = 0.506 and P = 0.356), 64.3% and 57.1% vs. 65.2% and 56.5% (P = 0.985 and P = 0.883), respectively. The incidence of CRS was greater in the F-CAR-T group (91.3%) than in the C-CAR-T group (66.7%) (P = 0.044). The incidence of ICANS was also greater in the F-CAR-T group (30.4%) than in the C-CAR-T group (9.5%) (P = 0.085), but no treatment-related deaths occurred in the two groups. CONCLUSION Compared with C-CAR-T-cell therapy, F-CAR-T-cell therapy has a superior remission rate but also leads to a tolerably increased incidence of CRS/ICANS. Further research is needed to explore the function of allo-HSCT as an intermediary therapy after CAR-T-cell therapy.

Guided by the theory of "the kidney storing essence, governing the bones, and producing marrow", this study explored the mechanism of icariin(ICA) in regulating the osteogenic differentiation of rat bone mesenchymal stem cells(BMSCs) through caveolin-1(Cav1) via in vitro and in vivo experiments, aiming to provide a theoretical basis for the prevention and treatment of postmenopausal osteoporosis with traditional Chinese medicine(TCM). Primary cells were obtained from 4-week-old female SD rats using the whole bone marrow adherent method. Flow cytometry was used to detect the expression of surface markers CD29, CD90, CD11b, and CD45. The potential for osteogenic and adipogenic differentiation was assessed. The effect of ICA on cell viability was determined using the CCK-8 assay, and the impact of ICA on the formation of mineralized nodules was verified by alizarin red staining. A stable Cav1-silenced cell line was constructed using lentivirus. The effect of Cav1 silencing on osteogenic differentiation was observed via alizarin red staining. Western blot analysis was conducted to detect the expression of Cav1, Hippo/TAZ, and osteogenic markers such as Runt-related transcription factor 2(RUNX2) and alkaline phosphatase(ALP). The results showed that primary cells were successfully obtained using the whole bone marrow adherent method, positively expressing surface markers of rat BMSCs and possessing the potential for both osteogenic and adipogenic differentiation. The CCK-8 assay and alizarin red staining results indicated that 1×10~(-7) mol·L~(-1) was the optimal concentration of ICA for intervention in this experiment(P<0.05). During osteogenic induction, ICA inhibited Cav1 expression(P<0.05) while promoting TAZ expression(P<0.05). Alizarin red staining demonstrated that Cav1 silencing significantly promoted the osteogenic differentiation of BMSCs. After ICA intervention, TAZ expression was activated, and the expression of osteogenic markers ALP and RUNX2 was increased. In conclusion, Cav1 silencing significantly promotes the osteogenic differentiation of BMSCs, and ICA promotes this differentiation by inhibiting Cav1 and regulating the Hippo/TAZ signaling pathway.
Animals ; Mesenchymal Stem Cells/metabolism* ; Caveolin 1/genetics* ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; Cell Differentiation/drug effects* ; Female ; Signal Transduction/drug effects* ; Flavonoids/administration & dosage* ; Protein Serine-Threonine Kinases/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Cells, Cultured ; Humans

This study aimed to investigate the effects of caffeoylquinic acids from Erigeron breviscapus(EBCQA) on cognitive impairment and mitochondrial dysfunction in Alzheimer's disease(AD), and to explore its underlying mechanisms. The impacts of EBCQA on paralysis, β-amyloid(Aβ) oligomerization, and mRNA expression of mitophagy-related genes [PTEN-induced putative kinase 1(PINK1) homolog-encoding gene pink-1, Parkin homolog-encoding gene pdr-1, Bcl-2 interacting coiled-coil protein 1(Beclin 1) homolog-encoding gene bec-1, microtubule-associated protein 1 light chain 3(LC3) homolog-encoding gene lgg-1, autophagic adapter protein 62(p62) homolog-encoding gene sqst-1] were examined in the AD Caenorhabditis elegans CL4176 model, along with mitochondrial functions including adenosine triphosphate(ATP) content, enzyme activities of mitochondrial respiratory chain complexes Ⅰ,Ⅲ, and Ⅳ, and mitochondrial membrane potential. Additionally, the effects of EBCQA on the green fluorescent protein(GFP)/red fluorescent protein from Discosoma sp.(DsRed) ratio, the expression of phosphatidylethanolamine-modified and GFP-labeled LGG-1(PE-GFP::LGG-1)/GFP-labeled LGG-1(GFP::LGG-1), and GFP-labeled SQST-1(GFP::SQST-1) proteins were investigated in transgenic C. elegans strains. The effect of EBCQA on paralysis was further evaluated after RNA interference(RNAi)-mediated suppression of the pink-1 and pdr-1 genes in CL4176 strain. An AD rat model was established through intraperitoneal injection of D-galactose and intragastric administration of aluminum trichloride. The effects of β-nicotinamide mononucleotide(NMN) and EBCQA on learning and memory ability, neuronal morphology, mitophagy occurrence, mitophagy-related protein expression(PINK1, Parkin, Beclin 1, LC3-Ⅱ/LC3-Ⅰ, p62), and mitochondrial functions(ATP content; enzyme activities of mitochondrial respiratory chain complexes Ⅰ, Ⅲ, and Ⅳ; mitochondrial membrane potential) were investigated in this AD rat model. The results showed that EBCQA delayed paralysis onset in the CL4176 strain, reduced Aβ oligomer formation, and upregulated the mRNA expression levels of lgg-1, bec-1, pink-1, and pdr-1, while downregulating sqst-1 mRNA expression. EBCQA also enhanced ATP content, mitochondrial membrane potential, and the activities of mitochondrial respiratory chain complexes Ⅰ, Ⅲ, and Ⅳ. Furthermore, EBCQA improved the PE-GFP::LGG-1/GFP::LGG-1 ratio, reduced GFP::SQST-1 expression, and decreased the GFP/DsRed ratio. Notably, the ability of EBCQA to delay paralysis was significantly reduced following RNAi-mediated suppression of pink-1 and pdr-1 in CL4176 strain. In AD rats, the administration of NMN or EBCQA significantly improved learning and memory, restored neuronal morphology in the hippocampus, increased autophagosome numbers, and upregulated the expression of PINK1, Parkin, Beclin 1, and the LC3-Ⅱ/LC3-Ⅰ ratio, while reducing p62 expression. Additionally, the treatment with NMN or EBCQA both elevated ATP content, mitochondrial respiratory chain complex Ⅰ, Ⅲ, and Ⅳ activities, and mitochondrial membrane potential in the hippocampus. The above findings indicate that EBCQA improves cognitive impairment and mitochondrial dysfunction in AD, possibly through activation of PINK1/Parkin-mediated mitophagy.
Animals ; Alzheimer Disease/psychology* ; Mitophagy/drug effects* ; Mitochondria/genetics* ; Caenorhabditis elegans/metabolism* ; Ubiquitin-Protein Ligases/genetics* ; Cognitive Dysfunction/physiopathology* ; Rats ; Protein Kinases/genetics* ; Humans ; Male ; Disease Models, Animal ; Caenorhabditis elegans Proteins/genetics* ; Drugs, Chinese Herbal/administration & dosage*

This study aimed to explore the mechanisms and molecular targets of total flavones of Abelmoschus manihot(TFA) plus empagliflozin(EM) in attenuating diabetic tubulopathy(DT) by targeting mitochondrial homeostasis and pyroptosis-apoptosis-necroptosis(PANoptosis). In the in vivo study, the authors established the DT rat models through a combination of uninephrectomy, administration of streptozotocin via intraperitoneal injections, and exposure to a high-fat diet. Following modeling successfully, the DT rat models received either TFA, EM, TFA+EM, or saline(as a vehicle) by gavage for eight weeks, respectively. In the in vitro study, the authors subjected the NRK52E cells with or without knock-down Z-DNA binding protein 1(ZBP1) to a high-glucose(HG) environment and various treatments including TFA, EM, and TFA+EM. In the in vivo and in vitro studies, The authors investigated the relative characteristics of renal tubular injury and renal tubular epithelial cells damage induced by reactive oxygen species(ROS), analyzed the relative characteristics of renal tubular PANoptosis and ZBP1-mediatted PANoptosis in renal tubular epithelial cells, and compared the relative characteristics of the protein expression levels of marked molecules of mitochondrial fission in the kidneys and mitochondrial homeostasis in renal tubular epithelial cells, respectively. Furthermore, in the network pharmacology study, the authors predicted and screened targets of TFA and EM using HERB and SwissTargetPrediction databases; The screened chemical constituents and targets of TFA and EM were constructed the relative network using Cytoscape 3.7.2 network graphics software; The relative targets of DT were integrated using OMIM and GeneCards databases; The intersecting targets of TFA, EM, and DT were enriched and analyzed signaling pathways by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG) software using DAVID database. In vivo study results showed that TFA+EM could improve renal tubular injury, the protein expression levels and characteristics of key signaling molecules in PANoptosis pathway in the kidneys, and the protein expression levels of marked molecules of mitochondrial fission in the kidneys. And that, the ameliorative effects in vivo of TFA+EM were both superior to TFA or EM. Network pharmacology study results showed that TFA+EM treated DT by regulating the PANoptosis signaling pathway. In vitro study results showed that TFA+EM could improve ROS-induced cell injury, ZBP1-mediatted PANoptosis, and mitochondrial homeostasis in renal tubular epithelial cells under a state of HG, including the protein expression levels of marked molecules of mitochondrial fission, mitochondrial ultrastructure, and membrane potential level. And that, the ameliorative effects in vitro of TFA+EM were both superior to TFA or EM. More importantly, using the NRK52E cells with knock-down ZBP1, the authors found that, indeed, ZBP1 was mediated PANoptosis in renal tubular epithelial cells as an upstream factor. In addition, TFA+EM could regulate the protein expression levels of marked signaling molecules of PANoptosis by targeting ZBP1. In summary, this study clarified that TFA+EM, different from TFA or EM, could attenuate DT with multiple targets by ameliorating mitochondrial homeostasis and inhibiting ZBP1-mediated PANoptosis. These findings provide the clear pharmacological evidence for the clinical treatment of DT with a novel strategy of TFA+EM, which is named "coordinated traditional Chinese and western medicine".
Animals ; Rats ; Mitochondria/metabolism* ; Benzhydryl Compounds/administration & dosage* ; Glucosides/administration & dosage* ; Abelmoschus/chemistry* ; Male ; Homeostasis/drug effects* ; Flavones/administration & dosage* ; Rats, Sprague-Dawley ; Diabetic Nephropathies/physiopathology* ; Drugs, Chinese Herbal/administration & dosage* ; DNA-Binding Proteins/genetics* ; Humans ; Apoptosis/drug effects*

Japanese spotted fever(JSF)is an infectious disease caused by Rickettsia japonica,with nonspecific clinical symptoms and a high risk of misdiagnosis.We reported a case of JSF,in which Rickettsia japonica was detected in blood cells by metagenomic next-generation sequencing.The patient recovered after treatment with doxycycline.This report provides a reference for the clinical diagnosis and treatment of JSF.
Humans ; High-Throughput Nucleotide Sequencing ; Metagenomics ; Rickettsia/isolation & purification* ; Spotted Fever Group Rickettsiosis/microbiology*

Time-of-flight secondary ion mass spectrometer(TOF-SIMS)is a highly sensitive surface analysis instrument with high spatial resolution.Traditional TOF-SIMS instruments for sample targets use high field extraction methods.Although the ion collection efficiency is high,it is prone to issues such as low-energy ion beam defocusing,sample morphology sensitivity,and organic molecule ion dissociation.This study aimed to develope an efficient low-field ion extraction system suitable for TOF-SIMS with a continuous beam source.The SIMION simulation software was used to construct a model of the secondary ion optical extraction system.The key factors affecting the extraction efficiency were studied,and the structural parameters of the extraction cone were optimized.Using an indium target as the sample,an experimental test of the performance of the ion extraction system was carried out on the TOF-SIMS instrument.The influences of the voltages of the ion extraction cone and the single lens on the ion extraction efficiency were consistent with the simulation results.By adopting the technology of deflection and coaxial dynamic compensation,the imaging field of view of the ion extraction system was increased to 500 μm×500 μm.The energy window of the ion extraction system reached 10 eV,and the large imaging depth of field of 400 μm was achieved.In the test of a 5 mg/L cholesterol thin film sample,the signal-to-noise ratio of the characteristic peak[M-OH]+reached 4453.The results showed that this low-field secondary ion extraction system effectively improved the performance of the continuous beam TOF-SIMS instrument.

Objective To investigate the diagnostic value of 4 novel inflammatory markers related to routine blood tests,namely neutrophil-to-lymphocyte ratio(NLR),red blood cell distribution width(RDW),hemoglobin-to-RDW ratio(HRR)and systemic immune-inflammation index(SII),in elderly patients with chronic cardiovascular disease(CVD)complicated with frailty.Methods Retrospectively analyze 110 patients with chronic stable CVD who were hospitalized in the cadre ward of cardiovascular medicine at the Air Force Characteristic Medical Center from January 2022 to June 2023.According to the assessment results of the Fried scale,they were divided into three groups:non-frailty group(Fried score=0,n=30),the pre-frailty group(Fried score 1 or 2,n=40)and frailty group(Fried score≥3,n=40).The differences in general information,the impairment rate of daily living activities,miniature nutritional assessment-short form(MNA-SF)scores,mini-mental state examination(MMSE)scores,and the indicators such as NLR,RDW,HRR,and SII among the three groups were compared.Spearman rank correlation was used to analyze the correlation between NLR,RDW,HRR,SII and frailty scores as well as each frailty indicator.Multivariate logistic regression analysis was performed to identify the independent risk factors for frailty in elderly patients with chronic CVD,and the receiver operating characteristic(ROC)curve was used to assess the clinical diagnostic value of NLR and HRR in elderly patients with chronic CVD complicated with frailty.Results Compared with non-frailty group and pre-frailty group,patients in frailty group were older,with higher impaired rates of daily living activities,NLR,RDW,and SII,and lower MNA-SF scores,MMSE scores,and HRR,and differences were statistically significant(P<0.05).Spearman rank correlation analysis showed that the frailty score was positively correlated with NLR(rs=0.354,P<0.001),and RDW(rs=0.448,P<0.001),negatively correlated with HRR(rs=-0.232,P=0.024),and had no significant correlation with SII(rs=0.144,P=0.167).Further analysis of the correlation between the above novel inflammatory markers and the 5 components of frailty showed that NLR was positively correlated with fatigue(rs=0.228,P=0.017),slowed walking speed(rs=0.299,P<0.001),and low physical function(rs=0.319,P<0.001);RDW was positively correlated with decreased grip strength(rs=0.321,P<0.001),slowed walking speed(rs=0.422,P<0.001),and low physical function(rs=0.246,P=0.001);and HRR was negatively correlated with slowed walking speed(rs=-0.230,P=0.025),and low physical function(rs=-0.299,P=0.003).Multivariate logistic regression analysis showed that MNA-SF score(OR=0.577,95%CI 0.342-0.973)was an independent protective factor for pre-frailty in elderly patients with chronic CVD(P<0.05);NLR(OR=7.866,95%CI 1.101-56.185)was an independent risk factor for frailty,while HRR(OR=0.344,95%CI 0.120-0.983)and MNA-SF score(OR=0.292,95%CI 0.146-0.580)were independent protective factors for frailty in elderly CVD patients(P<0.05).The area under the ROC curve of NLR and HRR for diagnosing frailty in elderly patients with chronic CVD were 0.778 and 0.749,respectively.Conclusion NLR and HRR have high clinical diagnostic value for frailty in elderly patients with chronic CVD,and are expected to become effective inflammatory markers for screening elderly patients with chronic CVD complicated with frailty.

Objective To observe the difference of bone micro-structure in different regions of proximal femur,micro-CT scanning was performed on 30 proximal femur specimens to explain the mechanism of proximal femur fracture and to provide anatomical basis for prosthesis design.Methods Totally 30 intact proximal femur specimens were obtained from 60-80 year-old cadavers.Micro-CT scanning was used to measure the trabecular thickness(Tb.Th),trabecular number(Tb.N),trabecular space(Tb.Sp),connectivity(Conn)and bone mineral density(BMD)and other parameters in 7 regions of proximal femur,including proximal pressure trabecular(PPT),distal pressure trabecular(DPT),femoral head-neck junction(FHNJ),head and neck of femoral neck(HNFN),the base of femoral neck(BPFN),intertrochanteric line(IL)and greater trochanter(GT).Results The bone mineral density of IL and GT were higher than those of BPFN,FHNJ,DPT and PPT.The trabecular thickness of GT was the largest,followed by IL,BPFN and HNFN,and the smallest was FHNJ,DPT and PPT.The trabecular space of IL was larger than that of GT,and the data of both were larger than those of other parts,among which DPT and PPT were the smallest.The trabecular number of IL and GT were the smallest,BPFN,HNFN and FHNJ were larger,and DPT was the largest.The volume fraction of IL was the smallest,BPFN and HNFN were larger,DPT and PPT were the largest.Conclusion The bone density,trabecular thickness,bone volume,and total volume of GT and IL in the proximal femur of elderly patients are all relatively large,so the reason for the high incidence of fractures is not due to weak internal bone microstructure;The bone density,trabecular thickness,and trabecular gap at the proximal and distal ends of the vertical trabecular bone are relatively small.If it is necessary to perform core decompression for prosthesis filling at this location,the design should be conducive to the mechanical conduction of the prosthesis and the regeneration of surrounding bone tissue.