The Mutual Regulatory Role of Ferroptosis and Immunotherapy in Anti-tumor Therapy.

Ferroptosis is a form of cell death that is triggered by the presence of ferrous ions and is characterized by lipid peroxidation induced by these ions. The mechanism exhibits distinct morphological characteristics compared to apoptosis, autophagy, and necrosis. A notable aspect of ferroptosis is its ability to inhibit uncontrolled tumor replication and immortalization, especially in malignant, drug-resistant, and metastatic tumors. Additionally, immunotherapy, a novel therapeutic approach for tumors, has been found to have a reciprocal regulatory relationship with ferroptosis in the context of anti-tumor therapy. A comprehensive analysis of ferroptosis and immunotherapy in tumor therapy is presented in this paper, highlighting the potential for mutual adjuvant effects. Specifically, we discuss the mechanisms underlying ferroptosis and immunotherapy, emphasizing their ability to improve the tumor immune microenvironment and enhance immunotherapeutic effects. Furthermore, we investigate how immunotherapeutic factors may increase the sensitivity of tumor cells to ferroptosis. We aim to provide a prospective view of the promising value of combined ferroptosis and immunotherapy in anticancer therapy by elucidating the mutual regulatory network between each.

The SOX4/EZH2/SLC7A11 signaling axis mediates ferroptosis in calcium oxalate crystal deposition-induced kidney injury.

Epigenetic regulation is reported to play a significant role in the pathogenesis of various kidney diseases, including renal cell carcinoma, acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. However, the role of epigenetic regulation in calcium oxalate (CaOx) crystal deposition-induced kidney injury remains unclear. Our study demonstrated that the upregulation of enhancer of zeste homolog 2 (EZH2)-mediated ferroptosis facilitates CaOx-induced kidney injury. CaOx crystal deposition promoted ferroptosis in vivo and in vitro. Usage of liproxstatin-1 (Lip-1), a ferroptosis inhibitor, mitigated CaOx-induced kidney damage. Single-nucleus RNA-sequencing, RNA-sequencing, immunohistochemical and western blotting analyses revealed that EZH2 was upregulated in kidney stone patients, kidney stone mice, and oxalate-stimulated HK-2 cells. Experiments involving in vivo EZH2 knockout, in vitro EZH2 knockdown, and in vivo GSK-126 (an EZH2 inhibitor) treatment confirmed the protective effects of EZH2 inhibition on kidney injury and ferroptosis. Mechanistically, the results of RNA-sequencing and chromatin immunoprecipitation assays demonstrated that EZH2 regulates ferroptosis by suppressing solute carrier family 7, member 11 (SLC7A11) expression through trimethylation of histone H3 lysine 27 (H3K27me3) modification. Additionally, SOX4 regulated ferroptosis by directly modulating EZH2 expression. Thus, this study demonstrated that SOX4 facilitates ferroptosis in CaOx-induced kidney injury through EZH2/H3K27me3-mediated suppression of SLC7A11.

Coexistence of anti-glomerular basement membrane disease and IgA nephropathy: an illustrative case and comprehensive literature review.

Anti-glomerular basement membrane (GBM) disease is a rare autoimmune condition characterized by the presence of positive anti-GBM autoantibodies, linear deposition of immunoglobulin G (IgG) along the GBM and severe kidney injury. In a limited number of cases, the association of anti-GBM disease with other glomerulonephritis has been reported. Herein, we present the case of a 66-year-old female patient with progressive worsen kidney function and decreased urine output. A renal biopsy revealed crescent glomerulonephritis with lineal IgG deposition along the GBM and mesangial IgA deposition, which supported the diagnosis of concurrent anti-GBM disease and IgA nephropathy (IgAN). In an extensive literature review, we identified a total of thirty-nine patients were reported anti-GBM disease combined with IgAN. The clinical characteristics of these patients demonstrate that the anti-GBM disease combined with IgAN tends to be milder with a more indolent course and a better prognosis than the classic anti-GBM disease, and its potential pathogenesis deserves to be further explored.

A Chinese Multi-Specialty Delphi Consensus to Optimize RAASi Usage and Hyperkalaemia Management in Patients with Chronic Kidney Disease and Heart Failure.

Objective: Variations are present in common clinical practices regarding best practice in managing hyperkalaemia (HK), there is therefore a need to establish a multi-specialty approach to optimal renin-angiotensin-aldosterone system inhibitors (RAASi) usage and HK management in patients with chronic kidney disease (CKD) & heart failure (HF).This study aimed to establish a multi-speciality approach to the optimal use of RAASi and how to manage HK in patients with CKD and HF.Methods: A steering expert group of cardiology and nephrology experts from across China convened to discuss challenges to HK management through a nominal group technique (NGT). The group then created a list of 41 statements for a consensus questionnaire, which was distributed for a further survey of in extended panel group of cardiologists and nephrologists across China. Consensus was assessed using a modified Delphi technique, with agreement defined as "strong" (≥75% and <90%) and "very strong" (≥90%). The steering group, data collection, and analysis were aided by an independent facilitator. Results: A total of 150 responses from 21 provinces across China were recruited in the survey. Respondents were comprised of an even split (n=75, 50%) between cardiologists and nephrologists. All 41 statements achieved the 75% consensus agreement threshold, of which 27 statements attained very strong consensus (≥90% agreement) and 14 attained strong consensus (agreement between 75% and 90%). Conclusions: Based on the agreement levels from respondents, the steering group agreed a set of recommendations intended to improve patient outcomes in the use of RAASi therapy and HK management in China.

Susceptibility of renal fibrosis in diabetes: Role of hypoxia inducible factor-1.

Diabetes may prevent kidney repair and sensitize the kidney to fibrosis or scar formation. To test this possibility, we examined renal fibrosis induced by unilateral ureteral obstruction (UUO) in diabetic mouse models. Indeed, UUO induced significantly more renal fibrosis in both Akita and STZ-induced diabetic mice than in nondiabetic mice. The diabetic mice also had more apoptosis and interstitial macrophage infiltration during UUO. In vitro, hypoxia induced higher expression of the fibrosis marker protein fibronectin in high glucose-conditioned renal tubular cells than in normal glucose cells. Mechanistically, hypoxia induced significantly more hypoxia-inducible factor-1 α (HIF-1 α) in high glucose cells than in normal glucose cells. Inhibition of HIF-1 attenuated the expression of fibronectin induced by hypoxia in high-glucose cells. Consistently, UUO induced significantly higher HIF-1α expression along with fibrosis in diabetic mice kidneys than in nondiabetic kidneys. The increased expression of fibrosis induced by UUO in diabetic mice was diminished in proximal tubule-HIF-1α-knockout mice. Together, these results indicate that diabetes sensitizes kidney tissues and cells to fibrogenesis probably by enhancing HIF-1 activation.

Identification of distinct clinical phenotypes of cardiogenic shock using machine learning consensus clustering approach.

BACKGROUND: Cardiogenic shock (CS) is a complex state with many underlying causes and associated outcomes. It is still difficult to differentiate between various CS phenotypes. We investigated if the CS phenotypes with distinctive clinical profiles and prognoses might be found using the machine learning (ML) consensus clustering approach. METHODS: The current study included patients who were diagnosed with CS at the time of admission from the electronic ICU (eICU) Collaborative Research Database. Among 21,925 patients with CS, an unsupervised ML consensus clustering analysis was conducted. The optimal number of clusters was identified by means of the consensus matrix (CM) heat map, cumulative distribution function (CDF), cluster-consensus plots, and the proportion of ambiguously clustered pairs (PAC) analysis. We calculated the standardized mean difference (SMD) of each variable and used the cutoff of ± 0.3 to identify each cluster's key features. We examined the relationship between the phenotypes and several clinical endpoints utilizing logistic regression (LR) analysis. RESULTS: The consensus cluster analysis identified two clusters (Cluster 1: n = 9,848; Cluster 2: n = 12,077). The key features of patients in Cluster 1, compared with Cluster 2, included: lower blood pressure, lower eGFR (estimated glomerular filtration rate), higher BUN (blood urea nitrogen), higher creatinine, lower albumin, higher potassium, lower bicarbonate, lower red blood cell (RBC), higher red blood cell distribution width (RDW), higher SOFA score, higher APS III score, and higher APACHE IV score on admission. The results of LR analysis showed that the Cluster 2 was associated with lower in-hospital mortality (odds ratio [OR]: 0.374; 95% confidence interval [CI]: 0.347-0.402; P < 0.001), ICU mortality (OR: 0.349; 95% CI: 0.318-0.382; P < 0.001), and the incidence of acute kidney injury (AKI) after admission (OR: 0.478; 95% CI: 0.452-0.505; P < 0.001). CONCLUSIONS: ML consensus clustering analysis synthesized the pattern of clinical and laboratory data to reveal distinct CS phenotypes with different clinical outcomes.

Xper computed tomography-guided translumbar inferior vena cava catheterization for long-term hemodialysis: A case report and literature review.

Hemodialysis is the most widely used renal replacement therapy for end-stage renal disease patients. Exhausted vascular access due to repeated indwelling central venous catheters is becoming a challenging clinical problem, which also contributes to reduced survival of the hemodialysis patients. Lack of conventional peripheral and central venous access mandates the use of alternative strategies. We present a case of translumbar dialysis catheter (TLDC) for long-term hemodialysis in a patient with central venous occlusion refractory to conventional endovascular techniques. After a careful literature review, totally 10 cohort studies including 216 cases through TLDC were reported. The incidence of procedure-related complications was very low. The catheter-related infection rate of TLDC was comparable with overall tunneled cuffed catheters (TCCs) reported by clinical practice guidelines for vascular access. Although the patency might be relatively low due to the catheter-related complications, TLDC could be rescued by multiple systemic and topical medications and interventional therapies. Percutaneous translumbar placement of a cuffed tunneled hemodialysis catheter directly into the inferior vena cava (IVC) can provide a relatively safe salvage when traditional central venous sites such as the internal jugular, femoral, subclavian veins are unavailable. Xper computed tomography together with real-time fluoroscopic guidance can reduce the intraoperative risks and complications.

Comparison of Outcomes between Percutaneous and Surgical Placement of Peritoneal Dialysis Catheters in Uremic Patients: A Meta-Analysis.

BACKGROUND: The optimal technique for inserting peritoneal dialysis catheters in uremic patients remains debated. This meta-analysis aimed to summarize the current evidence evaluating the efficacy and safety of percutaneous insertion methods compared to surgical methods. METHOD: A literature search was performed in the PubMed, EMBASE, Cochrane, and Web of Science databases. The primary outcome was defined as catheter survival. The secondary outcomes were mechanical and infectious complications related to catheter insertion. RESULTS: Twenty studies were finally identified, including 2 randomized controlled trials. The pooled results of catheter survival, overall mechanical complications, and infectious complications were not significant (odds ratio [OR] = 1.10, 95% confidence interval (CI) = 0.76-1.57, p = 0.62; OR = 0.73, 95% CI = 0.48-1.11, p = 0.14; and OR = 0.64, 95% CI = 0.37-1.09, p = 0.14, respectively). Comparison stratified by the blind percutaneous method versus open surgery indicated a lower overall number of mechanical complications (OR = 0.54, 95% CI = 0.31-0.93, I2 = 72%) and malposition rate (OR = 0.56, 95% CI = 0.34-0.90, I2 = 0%). The leakage rate was higher in the blind percutaneous group than in the open surgery group (OR = 2.55, 95% CI = 1.72-3.79, I2 = 0%); the guided percutaneous method achieved a similar leakage risk to the surgical methods. CONCLUSIONS: The blind percutaneous method performed better with fewer overall mechanical complications and less malposition than open surgery. The leakage risk was higher in the blind percutaneous group, while the guided percutaneous placement group showed similar outcomes to the surgical method groups. Percutaneous methods also had a lower infection risk, which needs further evidence to be confirmed.

Thrombotic thrombocytopenic purpura developed after pegylated interferon treatment for hepatitis B infection.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare and life-threatening thrombotic microangiopathy characterized by microangiopathic hemolytic anemia, severe thrombocytopenia, and organ ischemia. It is related to severe deficiency in ADAMTS13, which is usually acquired via ADAMTS13 autoantibodies or inherited via mutations of the ADAMTS13 gene. The etiology of acquired TTP including HIV infection, pregnancy, autoimmune disease, organ transplantation, drugs, malignancy and so on. Here, we firstly reported a patient diagnosed as acquired TTP after pegylated interferon therapy for hepatitis B and COVID-19 vaccination. CASE PRESENTATION: A 36-year-old male attended to our unit with a five-day history of intermittent hematuria and progressive fatigue on January 5th, 2022. He had a 13 years history of hepatitis B infection and undergone pegylated interferon treatment (which was paused for two months because of COVID-19 vaccination) for nearly 3 years. Laboratory evaluation revealed a haemoglobin level of 61 g/L, platelet count of 11 × 109/L, lactate dehydrogenase 2133 U/L. The direct and indirect Coombs test were both negative. On a peripheral blood smear, there were about 18.8% schistocytes. Meanwhile, the results of ADAMTS 13 activity and antibody were < 5% and 181.34 ng/ml (131.25-646.5), respectively CONCLUSION: This case firstly reported the rare complication of TTP after pegylated interferon treatment for hepatitis B and COVID-19 vaccine injection. This unique sign warrants more attention as an early cue of diagnosis of TTP and be aware of the rarity adverse effect of interferon therapy and COVID-19 vaccination.

p-Cresyl sulfate predicts clinical outcomes in sustained peritoneal dialysis: a 5-year follow-up cohort study and meta-analysis.

BACKGROUND: The impact of p-cresyl sulfate (PCS) and indoxyl sulfate (IS) on the prognosis of patients with uremia remains controversial. We performed a prospective study on peritoneal dialysis (PD) to investigate the relationship between PCS or IS levels with clinical outcomes. METHODS: This prospective cohort study investigated the association of serum PCS and IS with clinical outcomes in patients undertaking PD. We performed a correlations analysis to explore the influencing factors of PCS an IS. Meta-analysis was conducted to objectively evaluate the prognostic effects of PCS and IS on different stages of CKD patients. RESULTS: A total of 127 patients were enrolled consecutively and followed with an average period of 51.3 months. Multivariate Cox regression showed that serum total PCS not only contributed to the occurrence of PD failure event (HR: 1.05, 95% CI = 1.02 to 1.07, p < 0.001), but also increased the risk of cardiovascular event (HR: 1.08, 95% CI = 1.04 to 1.13, p < 0.001) and PD-associated peritonitis (HR: 1.04, 95% CI = 1.02 to 1.08, p = 0.001). Dividing the total PCS level by 18.99 mg/L, which was calculated from the best cutoff value of the ROC curve, patients with total PCS higher than 18.99 mg/L had worse prognosis. Meta-analysis confirmed its value in cardiovascular event in PD. CONCLUSION: The serum total PCS concentration was a detrimental factor for higher PD failure event, cardiovascular event, and PD-associated peritonitis. It could be used as an innovative marker in predicting poor clinical outcome in PD.

NS398 as a potential drug for autosomal-dominant polycystic kidney disease: Analysis using bioinformatics, and zebrafish and mouse models.

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by uncontrolled renal cyst formation, and few treatment options are available. There are many parallels between ADPKD and clear-cell renal cell carcinoma (ccRCC); however, few studies have addressed the mechanisms linking them. In this study, we aimed to investigate their convergences and divergences based on bioinformatics and explore the potential of compounds commonly used in cancer research to be repurposed for ADPKD. We analysed gene expression datasets of ADPKD and ccRCC to identify the common and disease-specific differentially expressed genes (DEGs). We then mapped them to the Connectivity Map database to identify small molecular compounds with therapeutic potential. A total of 117 significant DEGs were identified, and enrichment analyses results revealed that they are mainly enriched in arachidonic acid metabolism, p53 signalling pathway and metabolic pathways. In addition, 127 ccRCC-specific up-regulated genes were identified as related to the survival of patients with cancer. We focused on the compound NS398 as it targeted DEGs and found that it inhibited the proliferation of Pkd1-/- and 786-0 cells. Furthermore, its administration curbed cystogenesis in Pkd2 zebrafish and early-onset Pkd1-deficient mouse models. In conclusion, NS398 is a potential therapeutic agent for ADPKD.

PKD2 gene variants in Chinese patients with autosomal dominant polycystic kidney disease.

PKD2 gene variants account for 4.5% to 20% of patients with autosomal dominant polycystic kidney disease (ADPKD). Little is known about the clinical characteristics of PKD2 variants in Chinese patients with ADPKD. Herein, we performed a comprehensive search for variants of PKD2 gene in 44 Chinese ADPKD pedigrees and a total of 37 variants were identified. Of these 37 variants, 18 were nonsense variants, 10 frameshift variants, 4 missense variants, and 5 splice site variants. 11/37 variants were detected for the first time. The median age at diagnosis was 30.5 years, and positive family history was detected in 77.27% patients, liver cysts in 68.18%, hypertension in 45.45%, nephrolithiasis in 31.82%, macro-hematuria in 22.73%, and proteinuria in 13.63%. The level of estimated glomerular filtration rate in 8/39 patients were blow 60 ml/min/1.73m2 . 11/17 patients were classified as rapid progression by Mayo Clinic classification. The end stage renal disease (ESRD) events were reported in 9/22 pedigrees, and the presence of nephrolithiasis and macro-hematuria were significantly associated with ESRD in the pedigrees with PKD2 variants. The identified variants and clinical features will facilitate the early diagnosis and prognosis prediction in Chinese ADPKD patients with PKD2 variants.

New-onset glucose disorders in peritoneal dialysis patients: a meta-analysis and systematic review.

BACKGROUND: Peritoneal dialysis (PD) patients are at high risk of developing glucose metabolism disturbance (GMD). The incidence and prevalence of new-onset GMD, including diabetes mellitus (DM), impaired glucose tolerance (IGT) and impaired fast glucose (IFG), after initiation of PD, as well as their correlated influence factors, varies among studies in different areas and of different sample sizes. Also, the difference compared with hemodialysis (HD) remained unclear. Thus we designed this meta-analysis and systematic review to provide a full landscape of the occurrence of glucose disorders in PD patients. METHODS: We searched the MEDLINE, Embase, Web of Science and Cochrane Library databases for relevant studies through September 2018. Meta-analysis was performed on outcomes using random effects models with subgroup analysis and sensitivity analysis. RESULTS: We identified 1124 records and included 9 studies involving 13 879 PD patients. The pooled incidence of new-onset DM (NODM) was 8% [95% confidence interval (CI) 4-12; I2 = 98%] adjusted by sample sizes in PD patients. Pooled incidence rates of new-onset IGT and IFG were 15% (95% CI 3-31; I2 = 97%) and 32% (95% CI 27-37), respectively. There was no significant difference in NODM risk between PD and HD [risk ratio 0.99 (95% CI 0.69-1.40); P = 0.94; I2 = 92%]. PD patients with NODM were associated with an increased risk of mortality [hazard ratio 1.06 (95% CI 1.01-1.44); P < 0.001; I2 = 92.5%] compared with non-DM PD patients. CONCLUSIONS: Around half of PD patients may develop a glucose disorder, which can affect the prognosis by significantly increasing mortality. The incidence did not differ among different ethnicities or between PD and HD. The risk factor analysis did not draw a definitive conclusion. The glucose tolerance test should be routinely performed in PD patients.

Coronavirus-associated kidney outcomes in COVID-19, SARS, and MERS: a meta-analysis and systematic review.

OBJECTIVES: A meta-analysis and systematic review was conducted on kidney-related outcomes of three recent pandemics: SARS, MERS, and COVID-19, which were associated with potentially fatal acute respiratory distress syndrome (ARDS). METHODS: A search of all published studies until 16 June 2020 was performed. The incidence/prevalence and mortality risk of acute and chronic renal events were evaluated, virus prevalence, and mortality in preexisting hemodialysis patients was investigated. RESULTS: A total of 58 eligible studies involving 13452 hospitalized patients with three types of coronavirus infection were included. The reported incidence of new-onset acute kidney injury (AKI) was 12.5% (95% CI: 7.6%-18.3%). AKI significantly increased the mortality risk (OR = 5.75, 95% CI 3.75-8.77, p < 0.00001) in patients with coronavirus infection. The overall rate of urgent-start kidney replacement therapy (urgent-start KRT) use was 8.9% (95% CI: 5.0%-13.8%) and those who received urgent-start KRT had a higher risk of mortality (OR = 3.43, 95% CI 2.02-5.85, p < 0.00001). Patients with known chronic kidney disease (CKD) had a higher mortality than those without CKD (OR = 1.97, 95% CI 1.56-2.49, p < 0.00001). The incidence of coronavirus infection was 7.7% (95% CI: 4.9%-11.1%) in prevalent hemodialysis patients with an overall mortality rate of 26.2% (95% CI: 20.6%-32.6%). CONCLUSIONS: Primary kidney involvement is common with coronavirus infection and is associated with significantly increased mortality. The recognition of AKI, CKD, and urgent-start KRT as major risk factors for mortality in coronavirus-infected patients are important steps in reducing future mortality and long-term morbidity in hospitalized patients with coronavirus infection.

[Effect of ligustilide on oxygen and glucose deprivation/reperfusion-induced mitochondria fission in PC12 cells].

This study aimed to investigate the effect and mechanism of ligustilide, the main active ingredient in Ligusticum wallichii, on mitochondria fission after PC12 cell injury induced by oxygen and glucose deprivation/reperfusion(OGD/R). In the experiment, an OGD/R model was established in vitro, and PC12 cells were pre-treated with ligustilide for 3 h, and then the cell viability was detected by CCK-8 method. The effect of different concentrations of ligustilide on the morphology of PC12 cells after OGD/R injury was observed under an inverted microscope. Transmission electron microscopy was used to observe the mitochondrial fission of PC12 cells after OGD/R injury. DCFH-DA immunofluorescence staining method was used to detect intracellular reactive oxygen species(ROS) changes. Changes in mitochondria membrane potential(MMP) were detected by flow cytometry. Hochest 33258 was used to observe the apoptosis of PC12 cells. Western blot was used to detect changes in cytochrome C(Cyt C) content in mitochondria and cytoplasm, and mitochondrial fission-related proteins Drp 1 and Fis 1. All results showed that compared with the model group, ligustilide significantly increased the survival rate of PC12 cells and the number of cells. Further experiments showed that ligustilide inhibited the release of ROS and decline of mitochondrial membrane potential in PC12 cells after OGD/R injury. Moreover, ligustilide reduced the release of Cyt C and promoted the expressions of Drp1 and Fis1 in mitochondrial fission proteins. Verification experiments showed that mitochondrial fission inhibitor mdivi-1 decreased cell survival rate and inhibited fission. The results indicated that ligustilide exerted neuro-protective effects by promoting mitochondrial fission and reducing cell damage. It preliminary proves that the mechanism of ligustilide on ischemic brain injury may be related to the promotion of mitochondrial fission and the maintenance of cell homeostasis.

Clinical Characteristics and Outcomes of Community-Acquired versus Hospital-Acquired Acute Kidney Injury: A Meta-Analysis.

BACKGROUND: The different clinical characteristics of community-acquired acute kidney injury (CA-AKI) versus hospital-acquired AKI (HA-AKI) have remained inconclusive, and thus, a meta-analysis was conducted to summarize and quantify the clinical significance distinguishing the 2 types of AKI. METHODS: We identified observational studies reporting the clinical characteristics and prognosis of HA-AKI and CA-AKI. ORs and mean differences (MDs) were extracted for each outcome and the results aggregated. The primary outcome was defined as the mortality rate; renal recovery, oliguria incidence, dialysis, intensive care unit (ICU) requirement, and length of hospital stay were secondary outcomes. RESULTS: Fifteen eligible studies involving 46,157 patients (22,791 CA-AKI patients and 23,366 HA-AKI patients) were included. Mortality was significantly lower in CA-AKI than in HA-AKI patients, with an OR of 0.43 (95% CI 0.35-0.53). The incidence of oliguria and need for ICU were also lower in CA-AKI patients (OR 0.58, 95% CI 0.38-0.88; OR 0.24, 95% CI 0.14-0.40, respectively). CA-AKI patients had a shorter hospital stay (MD -9.42, 95% CI -13.73 to -5.12). The renal recovery rate and dialysis need between CA- and HA-AKI were similar (OR 1.27, 95% CI 0.53-3.02; OR 1.05, 95% CI 0.82-1.34, respectively). CONCLUSIONS: CA-AKI showed better clinical manifestations with a lower incidence of oliguria, reduced risk of ICU treatment, and shorter hospital stay. Mortality associated with CA-AKI was lower compared with HA-AKI, indicating a better prognosis. The rate of renal recovery and need for dialysis showed no significant difference between the 2 groups.