BACKGROUND:Mycoplasma pneumoniae (MP) is a common cause of community-acquired respiratory infections in children, with manifestations ranging from mild upper respiratory tract illnesses to pneumonia. Mycoplasma pneumoniae pneumonia (MPP) represents a more severe form of infection that often necessitates medical intervention. In children diagnosed with MPP, antimicrobial agents active against MP are indicated, and macrolide antibiotics represent the first-line therapy. However, macrolide-resistant MP (MRMP) strains have emerged worldwide, with a notable upward trend in both MP infection incidence and resistance rates, particularly in East Asia. This study aimed to investigate the prevalence of MP infection and the emergence of macrolide resistance in children during a period of elevated incidence, as well as to explore the underlying factors contributing to severe MPP (SMPP) among children in Guangzhou City. METHODS:The study enrolled a total of 10328 children suspected of having MP infection from November 2023 to December 2024. Respiratory tract specimens were collected from the study subjects and then analyzed using real-time polymerase chain reaction (PCR) to detect MP DNA and mutations associated with macrolide resistance at positions A2063G or A2064G. Moreover, demographic data and information on co-infecting common respiratory pathogens were collected during this analysis, and the risk factors for SMPP were analyzed. RESULTS:The positive detection rate of MP was 25.8%, and it decreased from 30.3% in the fourth quarter of 2023 (Q4 2023) to 10.1% in the fourth quarter of 2024 (Q4 2024). The highest positive rate of MP detection was among children aged 5-9 years, followed by those aged 10-14 years and 1-4 years. The detection rate of MRMP in MP-positive cases was 49.4%, and it rose from 37.8% in Q4 2023 to 61.9% in Q4 2024. Among the 1905 pneumonia inpatients who tested positive for MP, 7.4% (141/1905) were found to have co-infection with other common respiratory pathogens. The most prevalent co-infecting pathogens were human rhinovirus (RHV), human adenovirus (HADV), and influenza A virus (IAV). Detection of MRMP (OR = 1.833, 95% CI: 1.189-2.824, p = 0.006), presence of co-infection (OR = 2.209, 95% CI: 1.111-4.391, p = 0.024), and existence of comorbidities (OR = 4.431, 95% CI: 1.833-10.715, p = 0.001) were associated with an increased risk of SMPP. CONCLUSIONS:We reported that during the epidemic period of MP in children, the MRMP positive rate remained persistently high across all age groups in a cohort comprising both outpatients and inpatients. Among hospitalized children with MPP, those with MRMP detection, co-infection with other respiratory pathogens, or underlying comorbidities were more likely to develop severe conditions. These findings suggest that monitoring MRMP in children at potential high risk, particularly those who requiring hospitalization, may be essential for the development of effective prevention and treatment strategies.

Intrahepatic cholangiocarcinoma (ICC) is a lethal hepatic malignancy characterized by a prominent desmoplastic stroma. Here, we demonstrated that ARID1A, a core component of the SWI/SNF protein complex, is upregulated in ICC and plays an oncogenic role. Mechanistically, ARID1A stabilized NICD1 protein by inhibiting AMPK-dependent autophagy and lysosomal degradation, thereby sustaining Notch signaling. TLL1, a metalloproteinase that promotes collagen maturation, was found to be a downstream effector of ARID1A/Notch signaling. Specifically, TLL1 derived from ICC cells activated hepatic stellate cells (HSCs) and thus promoted tumor progression. Accordingly, ablation of TLL1 attenuated cancer-associated fibroblast (CAF) infiltration, collagen accumulation, and tumor progression. Moreover, virtual screening of a bioactive compound library identified acarbose, an effective medicine for glycemic control, as a potent inhibitor for TLL1. Pharmacological inhibition of TLL1 with acarbose suppressed HSC activation, collagen deposition, and tumor development. Collectively, these results establish a tumor-promoting ARID1A/Notch/TLL1 axis in ICC and reveal acarbose as a potential precision therapy for ARID1A-high ICC patients.

Influenza-associated encephalopathy and acute necrotizing encephalopathy (ANE) are rare but devastating complications of pediatric influenza, and early differentiation from severe pneumonia without neurological involvement is challenging at admission. We aimed to develop and internally validate an early prediction model to distinguish severe influenza A pneumonia from influenza-associated central nervous system (CNS) involvement using routine admission laboratory tests. The intended clinical use is early triage: to prioritize neurological evaluation (e.g., EEG/neuroimaging when indicated), closer monitoring, and escalation of care for children predicted to be at higher risk of CNS involvement. In this retrospective cohort at a tertiary pediatric center, consecutive children hospitalized with RT-PCR–confirmed influenza A from 1 January 2023 to 1 June 2025 were classified as mild pneumonia (Group 1), severe pneumonia without neurological involvement (Group 2), or CNS involvement (Group 3) using prespecified clinical, neurophysiological, and neuroimaging criteria. The primary prediction contrasted Group 2 versus Group 3 using routine laboratory tests obtained within 6 h of admission. Several machine-learning algorithms were trained and tuned using internal resampling and a held-out validation set. Bootstrap stability selection identified robust markers, and a parsimonious elastic-net logistic regression model was derived. We assessed discrimination, calibration, and clinical net benefit at prespecified risk thresholds (0.15, 0.25 and 0.35). Among 227 eligible children (median age 4.2 years), 95 (41.9

Wound healing is a dynamic process involving multiple cell types and signaling pathways. Dermal sheath cells (DSCs), residing surrounding hair follicles, play a critical role in tissue repair, yet their regulatory mechanisms remain unclear. This study used single-cell proteomics with the AcanCreER;R26LSL-tdTomato-DTR mouse model to explore DSC function across different healing stages. All animal procedures were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines. Gene set enrichment analysis (GSEA) and temporal clustering (Mfuzz) were employed to reveal dynamic functional shifts. GSEA identified enriched gene sets related to interferon-gamma response, inflammatory response, ultraviolet response, myogenesis, and xenobiotic metabolism. Temporal clustering revealed eight distinct clusters: clusters associated with the early contracting and proliferative phases were linked to metabolic activation and oxidative stress, while clusters from the later remodeling phase emphasized extracellular matrix remodeling and structural reorganization. The dynamic expression of epithelial-mesenchymal transition-related genes and keratins supported DSCs' dual epithelial and mesenchymal traits. Additionally, keratins, collagens, integrins, and actin proteins emerged as promising markers or signature molecules for DSCs. This study reveals DSCs' dual traits during wound repair, providing a basis for therapies to enhance healing.

The conserved histone variant H3.3 plays pivotal roles in heterochromatin formation and retrotransposon silencing.However,the molecular mechanism underlying H3.3-primed heterochromatin regulation remains elusive.Here,we demonstrate that H3.3-specific Ser31 phosphorylation and Lys27 trimethyla-tion synergistically promote H3K9me3-heterochromatin formation.Mechanistically,polycomb protein chromobox homolog 7(CBX7)preferentially binds Ser31-phosphorylated H3.3K27me3 nucleosomes and then recruits KRAB-associated protein 1(KAP1),which may further engage the histone lysine 9 methyltransferase to establish H3K9me3-associated heterochromatin.Remarkably,H3K9me3 is signifi-cantly impaired when the H3.3-CBX7 interaction is disrupted,accompanied by the activation of retro-transposons.Moreover,during X-chromosome inactivation(XCI),H3K9me2/3 fails to accumulate at the inactive X(Xi)when blocking the H3.3-CBX7-KAP1 axis.Taken together,our results reveal a novel molecular mechanism by which H3.3 Ser31 phosphorylation(H3.3Ser31p)facilitates H3K9me3-heterochromatin formation during retrotransposon silencing and XCI via the H3.3K27me3-CBX7-KAP1 axis.

Infectious diseases have been spreading rapidly across the globe, posing serious impacts on human health and the global economy. At present, the long-term use of antiviral drugs is accompanied by issues such as drug resistance and adverse reactions. Therefore, developing novel antiviral agents is particularly important. Selenium is an essential trace element for humans and animals. Individuals or animals with low selenium levels are more susceptible to viruses, which can lead to more severe illnesses. In the prevention and treatment of viral infections, selenium exerts antioxidant activity, regulates the immune system, protects the stability of endothelial cells, and participates in the regulation of signaling pathways. The correlation between selenium and infectious diseases has attracted considerable attention. This article reviews the antiviral mechanisms of selenium, new antiviral targets of selenium, and the current methods of selenium supplementation, aiming to provide new ideas and medication approaches for the prevention and treatment of viral diseases.

Background Community-acquired pneumonia (CAP) in children is a major public health issue due to its morbidity and economic impact, primarily caused by viral pathogens, highlighting the need to understand their prevalence and patterns for effective management and strategies. Methods This study analyzed 14,442 pediatric CAP patients in Guangzhou from 2023 to 2024 to evaluate viral prevalence, demographics, and seasonal trends, and their association with disease severity to elucidate their impact on children's health outcomes. Results Our analysis found 5,610 positive viral cases, with a 38.85% positivity rate; RSV was the most common at 26.58%, followed by ADV (18.42%) and RHV (15.67%). Infection rates for ADV and RHV rose in 2024. Children aged 0-3 made up 68.2% of cases, with severe CAP mainly affecting males (62.4%) and infants (57.3%). RSV peaked in winter, while ADV and RHV peaked in spring. Conclusions The study highlights the dynamic viral epidemiology in pediatric CAP, emphasizing RSV's threat and emerging risks from RHV and ADV, advocating for better diagnostics and targeted vaccinations for vulnerable groups to improve clinical management and reduce the misuse of antibiotics.

Introduction:COVID-19-related non-pharmaceutical interventions and their subsequent relaxation may alter the epidemiology of pediatric respiratory pathogens. However, hospital-based data from subtropical southern China using a consistent multiplex PCR platform remain limited. This study described changes in pediatric respiratory pathogen detection patterns in Guangzhou, with emphasis on a season-matched comparison before and after the lifting of restrictions. Methodology:We retrospectively analyzed 2,213 children 0 to <16 years who presented with fever or respiratory symptoms at Guangzhou Women and Children's Medical Center. Nasopharyngeal swabs collected during the pre-lifting period (November 2021-October 2022) and post-lifting period (February-May 2023) were tested using the same specimen type, nucleic-acid extraction system, PCR instrument, and 12-pathogen multiplex PCR panel. To reduce seasonal bias, the primary pathogen comparison was restricted to February-May 2022 vs. February-May 2023. Categorical variables were compared using the Chi-square test or Fisher's exact test, as appropriate. Results:Overall, 843 of 2,213 children (38.1%) tested positive for at least one pathogen. Compared with February-May 2022, February-May 2023 showed higher detection rates of Influenza A (0.64% vs. 16.60%), RSV (5.72% vs. 14.67%), and HRV (1.06% vs. 5.79%), and lower detection rates of Influenza B (7.84% vs. 0.39%) and HMPV (10.59% vs. 0.39%). The post-lifting cohort included a higher proportion of infants (<1 year: 14.29% vs. 8.50%) and higher proportions of fever, wheezing, and lower respiratory tract diagnoses; these clinical comparisons should be interpreted cautiously because health-care-seeking behavior and admission thresholds may have changed after lifting restrictions. Conclusions:After the lifting of COVID-19 restrictions, pediatric respiratory pathogen detection in Guangzhou shifted toward increased Influenza A, RSV, and HRV activity in the season-matched comparison, with a younger patient composition and more lower-respiratory clinical diagnoses among tested children. These findings support continued multiplex surveillance and age-targeted prevention strategies, but causal attribution to the lifting of restrictions should be made cautiously because of residual confounding by seasonality, SARS-CoV-2 circulation, and health-care-seeking behavior.

Objective: This study aims to investigate the correlation between blood leukocyte, CRP, LDH, and cytokine levels and the severity of illness in children with adenovirus pneumonia. Methods: A total of 100 children with adenovirus pneumonia (55 mild cases and 45 severe cases) who were treated at Guangzhou Women and Children’s Medical Center from January 2022 to January 2024, and 40 healthy children as a control group, were selected. Clinical data, some laboratory test data, and serum cytokine levels detected by flow cytometry were collected, and statistical methods were used to analyze the correlation between relevant indicators and the severity of the illness. Results: The research showed that among general clinical manifestations, the proportions of children with fever, dyspnea, pleural effusion, and moist rales in the severe group were all higher than those in the mild group (p < 0.05). Among the collected laboratory test data, indicators such as WBC, neutrophils, and LDH were significantly higher than in the mild group and the control group (p < 0.05) and were positively correlated with the severity of the disease. Regarding the tested cytokines, most children with adenovirus pneumonia showed elevated levels, and cytokines such as IL-6, IL-2, and IL-8 were significantly positively correlated with the disease. In the ROC curve analysis, NEU 6.03 × 109/L (sensitivity 82.2%, specificity 72.7%, AUC 0.830) and IL-6 41.823 pg/mL (sensitivity 75.6%, specificity 81.8%, AUC 0.833) demonstrated certain value in the early identification of children with severe disease. Conclusion: In this study, laboratory indicators (C-reactive protein, lactate dehydrogenase, neutrophils, etc.) and changes in the levels of specific cytokines (TNF-β, IL-2, IL-6, IL-8, etc.) in children with adenovirus pneumonia were closely related to the severity of the disease. Notably, neutrophil count and interleukin-6 were significantly positively correlated with disease severity and had high AUC values, suggesting they may be important parameters for early prediction of the progression of mild adenovirus infection to severe disease.

Human adenovirus (HAdV)-114 (P7H3F3) is a newly identified recombinant genotype, while recent studies suggest that it likely represents a previously circulating HAdV-3 strain. A total of 413 strains previously identified as HAdV-3 from 20 Chinese provinces during 2000-2024, along with 125 GenBank-derived HAdV-3 strains (1988-2023), underwent genotype reconfirmation and genetic evolutionary analysis using the penton base, hexon, and fiber genes. Furthermore, 97 representative strains were selected for whole-genome analysis to determine their genetic recombination patterns. Of the 538 analyzed strains, 537 were identified as HAdV-114 using three genes, with only one 2010 U.S strain classified as HAdV-3. Phylogenetic analysis revealed three HAdV-114 clades (Clades 1-3): Clade 1 (1988-2019) displayed sporadic circulation, Clade 2 (2023-2024) appeared geographically restricted to three Chinese provinces, and Clade 3 (2000-2024) likely represented the globally dominant strain. Genetic evolutionary analyses indicated that, although HAdV-114 originated from recombination between HAdV-7 and HAdV-3, its three Clades diverged from the parental trajectories, with Clades 1 and 3 sharing a common evolutionary origin and Clade 2 forming an independent terminal cluster. Genomic recombination analysis identified two recombination patterns across the three clades. All clades shared a 3' terminal genomic region (nt 18,794-end) derived from HAdV-3, whereas their 5' ends comprised gene fragments recombined from unknown sources and epidemic HAdV-7 strains, with varied breakpoints. HAdV-114, historically identified as HAdV-3, is the predominant genotype associated with HAdV-related respiratory infections. Therefore, enhanced surveillance of recombinant HAdV strains is imperative for effective disease control.

The mammalian early embryo development requires translation of maternal mRNA inherited from the oocyte. While poly(A) tail length influences mRNA translation efficiency during the oocyte-to-embryo transition (OET), molecular mechanisms regulating maternal RNA poly(A) tail length are not fully understood. In this study, we identified MARTRE, a previously uncharacterized protein family (MARTRE1-MARTRE6), as regulators expressed during mouse OET that modulate poly(A) tail length. MARTRE inhibits deadenylation through the direct interaction with the deadenylase CCR4-NOT, and ectopic expression of Martre stabilized mRNA by attenuating poly(A) tail shortening. Deletion of the Martre gene locus results in shortened poly(A) tails and decreased translation efficiency of actively translated mRNAs in mouse zygotes, but does not affect maternal mRNA decay. MARTRE proteins thus fine-tune maternal mRNA translation by negatively regulating the deadenylating activity of CCR4-NOT. Moreover, Martre knockout embryos show delayed 2-cell stage progression and compromised preimplantation development. Together, our findings highlight protection of long poly(A) tails from active deadenylation as an important mechanism to coordinate translation of maternal mRNA.

Background Despite a series of attempts during the last decades, the prognosis for esophageal squamous cell carcinoma (ESCC) remains poor. Although clinical immunotherapy trials have shown encouraging results, their benefits are limited. This study aims to identify novel targets for immunotherapy in ESCC.Experimental design ESCC cell lines and mouse models were used to identify the tumor-promoting function of pancreatic progenitor cell differentiation and proliferation factor (PPDPF) and evaluate the effect of blockade of CD24. RNA sequencing was performed to profile transcriptomic changes upon PPDPF deficiency. Fluorescence microscopy-based phagocytosis assay and flow cytometry were employed to analyze macrophage phagocytosis. Immunoblotting, glutathione S-transferase-pulldown assay and co-immunoprecipitation assay were conducted to investigate the mechanism underlying the tumor-promoting role of PPDPF in ESCC. Clinical samples were analyzed to further validate the findings from preclinical models.Results The expression of PPDPF was significantly upregulated in ESCC. Deficiency of PPDPF inhibited the development of ESCC in mice. Mechanistically, PPDPF interfered with the c-Myc-GSK3β interaction and enhanced the protein stability of c-Myc, which increased the expression of CD24 and therefore promoted immune escape from macrophage phagocytosis. Positive correlations between PPDPF, c-Myc, and CD24 were observed in clinical samples. Anti-CD24 monotherapy effectively inhibited the ESCC tumor growth in mice.Conclusions PPDPF acts as an oncoprotein in ESCC by positively regulating the c-Myc/CD24 axis. These findings provide a potential effective target for immunotherapy in ESCC.

Epigenetic mechanisms are essential for gene expression regulation. Recent advances have revealed how cells not only stabilize transcriptional states but also actively prepare for future gene expression. This review explores four processes in epigenetic preparation for future gene induction: priming, reining, transcriptional memory, and transcriptional tolerance. Priming establishes chromatin configurations that facilitate future gene activation without immediate transcription. Conversely, reining balances responsiveness with transcriptional stability to prevent premature gene activation or overexpression. Transcriptional memory facilitates faster and stronger responses to recurrent stimuli by reflecting past activation events, whereas transcriptional tolerance imposes restraint on subsequent activation. We examine how these mechanisms, involving DNA methylation, histone modification, and chromatin remodeling, integrate with signaling pathways and transcription factors to orchestrate future gene induction. Leveraging recent insights from mammalian systems, this review highlights the emerging role of epigenetic preparation in adaptive cellular responses, with implications for development, disease, and cellular memory in mammals.

Decitabine (DAC), a well-recognized DNA hypomethylating agent, has been applied to treat acute myeloid leukemia. However, clinic investigations revealed that DNA methylation reduction does not correlate with a clinical response, and relapse is prevalent. To gain a better understanding of its anti-tumor mechanism, we perform a temporally resolved CRISPR-Cas9 screen to identify factors governing the DAC response. We show that DNA damage generated by DNMT-DNA adducts and 5-aza-dUTP misincorporation through the dCMP deaminase DCTD act as drivers of DAC-induced acute cytotoxicity. The DNA damage that arises during the next S phase is dependent on DNA replication, unveiling a trans-cell cycle effect of DAC on genome stability. By exploring candidates for synthetic lethality, we unexpectedly uncover that KDM1A promotes survival after DAC treatment through interactions with ZMYM3 and CoREST, independent of its demethylase activity or regulation of viral mimicry. These findings emphasize the importance of DNA repair pathways in DAC response and provide potential biomarkers.

To better understand the epidemiological characteristics of human adenovirus (HAdV) infections in China during and after the coronavirus disease 2019 (COVID-19) pandemic, respiratory specimens were collected from 17,562 enrolled patients with acute respiratory infections (ARIs) in 14 sentinel surveillance provinces during 2020-2023. Eight common respiratory viruses were detected using commercially available nucleic acid detection kits. HAdV-positive cases were statistically analyzed for detection rates, geographic distribution, seasonal patterns, demographic characteristics, and co-infection status. The results of this study showed that the overall HAdV detection rate was 5.09 % (894/17,562) during 2020-2023, with a gradual decrease in the annual detection rate from 6.66 % in 2020 to 3.89 % in 2022 and a rebound in 2023 (5.19 %). The overall HAdV detection rate was significantly higher in the southern region (6.15 %) than in the northern region (4.76 %) (P < 0.001). The median age of patients with HAdV infection was 3 years, with infants aged 0-2 years accounting for the majority (41.39 %). HAdV-positive cases were detected throughout the year, with no clear seasonal pattern, and the HAdV epidemic in China during 2020-2023 may have been driven primarily by the virus infection in the southern region. Co-infections were frequent in HAdV-positive cases (overall rate: 36.01 %), primarily consisting of dual infections (79.28 %), with human rhinovirus and human respiratory syncytial virus being the most common coinfecting pathogens. In conclusion, this study suggested the significant regional and temporal variation in HAdV detection rate in China during 2020-2023, and thus ongoing surveillance should be conducted to elucidate the epidemiological dynamics of HAdV infections.

Increasing evidence indicates that RNA-binding proteins and the reprogramming of lipid metabolism play crucial roles in tumorigenesis. However, the extent to which members of these families contribute, and whether targeting metabolic genes to affect overall protein production in cancer cells, remains largely unknown. This study analyzes CRC tissue samples and databases to reveal the high expression of hnRNP A1 in CRC and its critical role in tumorigenesis, proliferation, migration, and prognosis. Through combined sequencing analyses, we identified a novel mechanism by which PPARα regulates lipid metabolism. Our data indicate that hnRNP A1 is central to lipid metabolism reprogramming in CRC, promoting lipid accumulation by regulating PPARα mRNA stability, thereby influencing cell proliferation and apoptosis. Overall, hnRNP A1 could serve as a novel target for CRC therapy. Its involvement in cancer development offers new biological insights and potential therapeutic strategies. As a potential biomarker and therapeutic target, it presents novel approaches for the clinical management of CRC.

After the waning impact of the new coronaviruses, influenza viruses have become prevalent again in the last two years. H3N2 is a subtype of the influenza A virus and has become the dominant and most watched strain of seasonal influenza in the past decade. Ebselen, an organic selenium compound, plays a role in cardiovascular health and cancer prevention due to its antimicrobial and antioxidant effects. In our latest study, ebselen was found to effectively inhibit H3N2-mediated apoptosis in MDCK cells in vitro, as well as restore body weight and ameliorate lung inflammation and apoptosis in H3N2-infected mice in vivo. Further investigation revealed that ebselen inhibits mitochondria-mediated apoptosis, thereby preventing H3N2-mediated apoptosis. In summary, this study suggests that ebselen has potential as anti-H3N2 influenza virus drug.

The initiation of major zygotic genome activation (ZGA) is crucial for human early embryogenesis. However, the transcription factors (TFs) regulating major ZGA in humans remain largely unknown. Here, we performed a CRISPR-based activation screen of 1,603 human TFs in human extended pluripotent stem cells (hEPSCs), which identified 132 candidates as potential regulators of major ZGA. Further evaluation of these candidates revealed that the KRAB-containing TFs ZIM3 and ZNF394 activated totipotent features in hEPSCs upon overexpression. Importantly, simultaneous knockdown of these two TFs arrested human embryo development prior to the eight-cell embryo stage. Mechanistically, the KRAB domains contributed to ZIM3- and ZNF394-mediated totipotency induction in vitro, accompanied by the suppression of a set of four-cell embryo enriched genes. Our study provided valuable resources for totipotency and major ZGA regulation, suggesting an un-reported role of KRAB-containing TFs in major ZGA in humans.

Abstract Selenium (Se) is a trace element that fulfills vital functions in the organism. Over the past few years, numerous studies have revealed the profound connection between Selenium and its therapeutic potential in addressing various viral infectious diseases. Selenium can boost the activity of immune cells and counteract oxidative damage caused by free radicals to prevent the replication of viruses. Studies have shown that Selenium protects against a wide range of viruses such as DNA viruses, hepatitis B virus, RNA viruses, human immunodeficiency virus and coxsackie virus. Selenium is probably one of the research directions for antiviral drugs in the future. This review summarizes the use of Selenium in antiviral therapy and its research progress.

Long interspersed nuclear element-1 (LINE-1 or L1), the only autonomously active retrotransposon in humans today, constitutes a large proportion of the genome and continues to evolve the genome and impact fundamental biological processes. L1 retrotransposition critically depends on its endonuclease and reverse transcriptase subunit open reading frame 2 protein (ORF2p), which targets genomic loci and nicks DNA using an evolutionarily distinct yet not fully understood mechanism. Our structural and biochemical analyses revealed that ORF2p is a structure-dependent endonuclease. It binds a double-stranded DNA region upstream of the nicking site and recognizes a downstream forked or flap structure for efficient DNA nicking. This discovery suggests that L1 mobilization piggybacks on chromosomal processes with noncanonical DNA structure intermediates.