辛伐他汀(Simvastatin)是一种临床常用的降血脂药物,其常见的药物不良反应是横纹肌溶解、肌病等肌肉相关症状.SLCO1 B1基因多态性是导致辛伐他汀肌肉不良反应个体化差异的主要原因之一.SLCO1 B1编码的产物参与辛伐他汀的肝摄取,其功能丧失性变异可引起辛伐他汀的血药水平升高,导致更多的药物暴露.对于服用辛伐他汀的患者,检测SLCO1 B1基因型可指导患者的精准用药,但目前尚无研究讨论常规开展SLCO1 B1基因检测的临床意义.对于携带功能降低的等位基因患者,推荐服用小剂量的辛伐他汀或者换用其他他汀类(阿托伐他汀、瑞舒伐他汀等)药物,以降低可能的肌肉不良反应风险.本研究综述了SLCO1 B1基因与辛伐他汀个体化用药的相关研究,为辛伐他汀的合理应用提供参考.

血小板是由骨髓中的巨核细胞产生的一种血细胞,是血液中重要的组成成分.在炎症环境下,血管受损暴露出内皮下基质,随后血小板被释放出的激动药激活并在血管损伤的部位迅速黏附,聚集形成血栓,在促炎细胞因子的激活作用下,血小板止血功能被放大,加速凝血过程.血小板被激活后会进一步释放出血小板衍生颗粒,如α颗粒、溶酶体和致密颗粒,在炎症条件下调控止血和血栓的形成.血小板不仅在止血和血栓形成中发挥作用,也能够和病原体相互作用调节免疫系统.当病原体入侵时,机体被细菌或病毒感染,血小板通过吞噬、作用于细菌或协调免疫细胞进行抗菌,病毒感染后血小板被激活,调节病毒的复制与传播.本文综述了在炎症中血小板发挥的作用,重点关注了血小板的促血栓形成作用,以及血小板与病原体相互作用以调节免疫系统的功能.

人类白细胞抗原B(HLA-B)基因多态性对卡马西平的用药风险有重要影响,携带HLA-B*15:02等位基因的患者在使用卡马西平时,发生严重皮肤不良反应如史蒂文斯-约翰逊综合征和中毒性表皮坏死溶解的风险更高.临床推荐在使用卡马西平之前应当进行基因检测,明确是否携带HLA-B*15:02等位基因,从而进行个体化用药,降低发生严重不良反应的风险.本研究分析了HLA-B基因与卡马西平用药之间的关系,综述了基于HLA-B基因多态性的卡马西平个体化药物治疗现状,整理并总结相关指导意见;同时综述了HLA-B基因在中国使用卡马西平的患者中的相关研究,总结分析卡马西平用于中国患者时的用药风险,为中国患者为精准治疗策略的优化提供参考.

Background The real-world studies on recurrent venous thromboembolism (VTE) and bleeding events of non-vitamin K antagonist oral anticoagulants (NOACs) in VTE patients have reported conflicting findings. Our study aimed to provide the direct comparison evidence of different NOACs for VTE patients in clinical practice settings. Methods Search of the medical literature was conducted using PubMed, Web of Science, EMBASE, Clinical Trials.gov, and the Cochrane Library from inception to March 22, 2021. Among the 19,996 citations retrieved, a total of 63,144 patients from 6 studies were analyzed. Clinical outcomes included recurrent VTE, death, and different bleeding events. Results Adjusted hazard ratio (HR) analysis suggested that apixaban had significant lower bleeding riskthan rivaroxaban (major, minor and any bleeding: HR = 0.61, 0.56, 0.70; p = 0.008, < 0.0001, 0.006, respectively), but no statistics difference found in recurrent VTE events (HR = 1.02, 95% confidence interval (CI) 0.71–1.47, p = 0.93). There was no significant difference of major bleeding between dabigatran and rivaroxaban (odds ratios (OR) = 0.41, 95% CI 0.09–1.90, p = 0.25), apixaban and dabigatran (OR 0.64, 95% CI 0.15–2.72, p = 0.83). No significant difference was found in the comparison of edoxaban and other NOACs in VTE recurrence, major bleeding and composite outcome. Conclusions In the prevention of bleeding events, apixaban was associated with a lower risk than rivaroxaban, but equivalent efficacy for different NOACs in prevention of recurrent VTE. Evidence generated from the meta-analysis based on real-world data can help to guide selection between apixaban and rivaroxaban in routine clinical practice. Trial registration : This systematic review and meta-analysis were conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis and Meta-analysis of Observational Studies in Epidemiology statements and was registered with PROSPERO (CRD42019140553).

药物基因组学通过研究编码药物代谢酶、药物转运体和作用靶点的基因多态性对药代动力学与药效学的影响,不仅可以指导临床给予患者个体化用药建议,还可以用于新药研发和药政管理等.目前国内外多个重要机构都发布了药物基因组学相关的指南或专家共识.本文旨在通过回顾和梳理国内外药物基因组学相关指南的现状,为我国建立健全适用于中国人群的药物基因组学指南、推动精准医疗实施提供参考和借鉴.

氯吡格雷的抗血小板疗效具有明显的个体差异.影响氯吡格雷药效的因素较多,遗传变异是其中的重要原因.细胞色素P450酶2C19(CYP2C19)基因多态性对氯吡格雷的代谢活化有重要影响.功能缺失等位基因携带者使用氯吡格雷可能增加血栓栓塞事件的风险.临床基因型检测可用于检查是否存在CYP2 C19突变等位基因,预测患者的代谢表型,并可能有助于指导临床医师调整抗血小板治疗方案.本研究从CYP2 C19基因型与临床结局的关系、功能缺失基因携带者的抗血小板治疗方案选择、基因型指导下抗血小板治疗与传统治疗的临床结局比较等方面分析了国内外最新临床研究证据,并整理了临床指南共识、药品说明书及相关指导意见,为抗血小板治疗策略的优化提供参考.

CYP3 A5家族基因在肝含量占CYP3 A总量一半以上,是许多药物代谢的酶.常见的免疫抑制药他克莫司是一种选择性抑制钙调磷酸酶,主要用于器官移植患者的抗排异治疗,经CYP3 A5同工酶进行代谢.CYP3 A5基因突变使酶失活,导致他克莫司不能正常代谢而在体内积聚,产生相关药物不良反应.由于产生充分免疫抑制效果的水平和产生毒性的水平差距很小,他克莫司使用时需要进行治疗药物监测,但是他克莫司的目标浓度因治疗的疾病而异,且操作复杂,初始用药剂量难以准确判定,给临床合理用药带来了困扰.基因型检测可以辅助指导他克莫司个体化治疗,在最大程度保证药物疗效的同时避免药物不良反应的发生.本研究综述了基于CYP3 A5基因多态性的他克莫司个体化药物治疗现状,为抗排异治疗策略的优化提供参考.

Study Objective East Asians have a higher risk of bleeding than Europeans when treated with ticagrelor. This study aimed to explore genetic indicators related to the high bleeding propensity in East Asian patients with acute coronary syndrome (ACS) using ticagrelor. Design A multicenter prospective cohort study. Setting Four sub center hospitals participating the study. Patients Between March 2018 and July 2021, 208 patients with ACS were administered ticagrelor and underwent genetic testing. Invertention Patients were enrolled and followed up for bleeding events for 12 months. Single-nucleotide polymorphisms (SNPs) were detected using whole-exome sequencing. SNPs significantly associated with cumulative bleeding events within 1-, 6-, and 12-month follow-ups were selected (p < 0.01). Among these, SNPs showing a difference of >= 2 fold in their distribution frequency among East Asians and Europeans were selected. Measurements and Main Results Among all patients, 96.60% received ticagrelor plus aspirin or cilostazol, and 42.3% suffered from bleeding events during 12-month follow-up. Furthermore, 22 SNPs of 15 genes were found to have a significant association with cumulative bleeding events within 1-, 6-, and 12-month follow-ups. Among these SNPs, FIG4 rs2295837 (A>T) variant had the strongest association with bleeding events within 1 month (p = 1.28 x 10(-4)), with an increased risk of bleeding in T allele carriers (odds ratio [OR]: 3.07, 95% confidence interval [CI]: 1.68-5.63). PROK2 rs3796224 (C>T) variant was most strongly associated with cumulative bleeding events within 6 months (p = 4.57 x 10(-4)) with an increased risk of bleeding in T allele carriers (OR: 2.16, 95% CI: 1.20-3.89). Moreover, HRNR rs6662450 (C>T) variant showed the strongest relation with cumulative bleeding events within 12 months (p = 4.86 x 10(-4)) with a reduced risk of bleeding in T allele carriers (OR: 0.48, 95% CI: 0.24-0.95). Conclusion Fifteen genes, including PROK2, HRNR, and FIG4, were potential biomarkers of high bleeding propensity in East Asian patients with ACS using ticagrelor.

硫唑嘌呤属于硫嘌呤类药物,是一种免疫抑制药.硫嘌呤甲基转移酶(thiopurine S-methyltransferase,TPMT)是参与硫唑嘌呤代谢的重要酶,TPMT的基因多态性是导致个体间用药差异的重要因素.TPMT基因遗传多态性导致酶活性存在个体差异,从而使接受硫唑嘌呤治疗的人群表现出不同的疗效和药物不良反应.TPMT的遗传多态性能够影响到TPMT酶的活性,而不同TPMT活性的患者在服用硫唑嘌呤后的药效学不同.越来越多的研究证实,在服用硫唑嘌呤之前进行基因型检测,针对不同TPMT活性的患者进行硫唑嘌呤个体化用药指导,可以有效地提高疗效,降低药物不良反应.本研究综述了基于TPMT基因多态性的硫唑嘌呤个体化用药治疗现状,通过讨论TPMT基因型指导下对不同患者服用硫唑嘌呤后的临床结局的影响,以及精准用药的建议,尤其关注了中国人群中TPMT用药的研究现状,以期为不同人群进行硫唑嘌呤治疗提供用药建议.

(1) Background: The purpose of this study was to evaluate the effect of gene polymorphisms on prothrombin time (PT) and activated partial thromboplastin time (APTT) in a healthy Chinese population. (2) Methods: A total of 403 healthy volunteers from a series of novel oral anticoagulants (NOACs) bioequivalence trials in China were included. Coagulation tests for PT and APTT were performed in the central lab at Peking University First Hospital. Whole-exome sequencing (WES) and genome-wide association analysis were performed. (3) Results: In the correlation analysis of PT, 105 SNPs from 84 genes reached the genome-wide significance threshold (p < 1 × 10−5). Zinc Finger Protein 594 (ZNF594) rs184838268 (p = 4.50 × 10−19) was most significantly related to PT, and Actinin Alpha 1 (ACTN1) was found to interact most with other candidate genes. Significant associations with previously reported candidate genes Aurora Kinase B (AURKB), Complement C5(C5), Clock Circadian Regulator (CLOCK), and Histone Deacetylase 9(HDAC9) were detected in our dataset (p < 1 × 10−5). PiggyBac Transposable Element Derived 2(PGBD2) rs75935520 (p = 4.49 × 10−6), Bromodomain Adjacent To Zinc Finger Domain 2A(BAZ2A) rs199970765 (p = 5.69 × 10−6) and Protogenin (PRTG) rs80064850 (p = 8.69 × 10−6) were significantly correlated with APTT (p < 1 × 10−5). The heritability values of PT and APTT were 0.83 and 0.64, respectively; (4) Conclusion: The PT and APTT of healthy populations are affected by genetic polymorphisms. ZNF594 and ACTN1 variants could be novel genetic markers of PT, while PRTG polymorphisms might be associated with APTT levels. The findings could be attributed to ethnic differences, and need further investigation.

华法林是目前临床上使用广泛、效果确切的抗凝药,但其治疗时间窗较窄、个体差异较大,故提高华法林个体化用药的合理性具有重要的临床意义.CYP2 C9和VKORC1基因多态性对华法林的代谢及敏感性有重要影响.CYP2 C9和VKORC1等位基因突变影响华法林抗凝治疗的有效性和安全性.临床基因型检测可用于检查是否存在CYP2 C9和VKORC1突变等位基因,预测患者的代谢表型及用药敏感性,并可能有助于指导临床医师调整抗凝治疗方案.经过临床基因检测,可使得达到国际标准化比值的时间明显缩短且出血频率明显降低.本研究综述了基于CYP2 C9和VKORC1基因多态性的华法林个体化药物治疗现状,整理了相关指导意见,为抗凝治疗策略的优化提供参考.

别嘌醇被广泛用于痛风和高尿酸血症的一线治疗,在使用过程中疗效显著、价格低廉,但随其在临床的广泛应用,有关其不良反应的报道也逐渐增加.HLA-B基因多态性对别嘌醇的使用有重要影响.HLA-B*58:01等位基因携带者应用别嘌醇可能增加严重皮肤不良反应的风险.HLA-B*58:01基因是检测别嘌醇诱导的皮肤药物不良反应的高度特异性和有效的遗传标记,基因检测可用于检查是否携带HLA-B*58:01等位基因,有助于指导临床医生调整痛风及高尿酸血症患者的治疗方案.筛查HLA-B*58:01基因可能有助于患者防止患者别嘌醇诱导的严重皮肤不良反应的发生.本研究综述了基于HLA-B基因多态性的别嘌醇个体化药物治疗现状,整理了相关指导意见,为治疗策略的优化提供参考.

WHAT IS KNOWN AND OBJECTIVE:Limited data are available for the comparison between different non-vitamin K antagonist oral anticoagulants (NOACs) on clinical outcomes. We aimed to provide evidence of different NOACs for patients with non-valvular atrial fibrillation (NVAF).METHODS:Electronic databases were searched from inception through 22 March 2020 to identify eligible studies in which clinical outcomes (stroke, systemic embolism [SE], bleeding or death events) were directly compared between different NOACs.RESULTS:29 real-world studies enrolled more than 700,000 patients were included. Compared with dabigatran, apixaban had higher risk of death (OR 1.07), major bleeding (1.43), GI bleeding (1.64), ischaemic stroke and stroke/SE events (1.10); rivaroxaban had higher risk of death (1.28), major bleeding (1.24), GI bleeding (1.14) and ischaemic stroke (1.08). Compared with rivaroxaban, apixaban had lower risk of death (0.8), major bleeding (0.56) and ischaemic stroke events (0.71). Compared with edoxaban, rivaroxaban had higher risk of major bleeding (2.83), GI bleeding (5.18) and ischaemic stroke (2.28).WHAT IS NEW AND CONCLUSION:In view of the global burden of disease and the routine use of NOACs worldwide, the findings have immediate and important implications. Our data suggested that apixaban might be the priority choice in prevention of bleeding and stroke and dabigatran could be the priority choice in prevention of death events.TRIAL REGISTRATION:This systematic review and meta-analysis were conducted and reported according to the Preferred Reporting Items for Systematic Reviews (PRISMA), Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines and was registered with PROSPERO (CRD42019140553).

Low-dose rivaroxaban is often given to patients with atrial fibrillation (AF) around the world, but the rationale for its use remains unclear. We aimed to compare the efficacy and safety of standard- or low-dose rivaroxaban in patients with AF through systematic review of literature with meta-analysis. We searched PubMed, Web of Science, EMBASE, Clinical Trials.gov, the Cochrane Library, and Bayer trial website from inception of each database until June 2020. Randomized controlled trials (RCTs) and cohort studies were included in the meta-analysis. A random-effects model was employed to calculate the pooled effect estimates. Two RCTs and 17 cohort studies were included in the qualitative analysis. Indirect comparison of RCTs showed no significant difference between the two rivaroxaban dosages in risk of efficacy or safety outcomes (p > 0.05). Indirect comparison of cohort studies showed a lower risk of MACE among Caucasians in standard-dose group (HR 0.779; 95% CI 0.687–0.884; p < 0.001). Bleeding outcomes did not differ significantly between the two dosage regimens in Asian or Caucasian populations, except that the standard dose was associated with higher risk of major bleeding among elderly Caucasian patients (HR 1.329; 95% CI 1.141–1.547; p < 0.001). The quality of evidence was rated ranging from very low to low for all the efficacy and safety outcomes. In Caucasians with AF, standard-dose rivaroxaban may prevent MACE significantly better than low-dose treatment. Further studies in Asians are needed to verify the advantages of the standard dose.

The present study compared performances of the three major methods used for assessing platelet reactivity (PR)-VerifyNow, light transmission aggregometry (LTA) and thromboelastography (TEG)-to predict ischaemic events in patients receiving clopidogrel. PubMed, EMBASE and the Cochrane Library were searched from their inception to April 2019 for prospective studies that examined PR using VerifyNow, LTA or TEG and the incidence of ischaemic events. The investigated diagnostic indices include sensitivity, specificity, positive (PLR) and negative likelihood ratio (NLR), diagnostic odds ratio and area under the receiver operating characteristic curves (AUC) of VerifyNow, LTA and TEG, respectively. A total of 26 prospective studies involving 22 185 patients were included in the analysis. The pooled AUC was 0.71 (95% CI: 0.67-0.75) for VerifyNow, 0.60 (95% CI: 0.55-0.64) for LTA and 0.81 (95% CI: 0.77-0.84) for TEG. Results of indirect comparisons indicated the AUC of VerifyNow was higher than that of LTA (1.18, 95% CI: 1.08-1.30) and lower than that of TEG (0.88, 95% CI: 0.82-0.94). TEG outperformed the other two methods for assessing PR in all predictive measures, including sensitivity, specificity, PLR and NLR. Despite a lack of studies that directly compared the three methods, our findings suggest that TEG should be recommended.

Background Preventing thrombosis is an important part of atrial fibrillation (AF) treatment. However, it may increase the risk of bleeding, and bleeding risk assessment tools' predictive value remains unclear. This network meta-analysis investigated the sensitivity and specificity of HAS-BLED, and other bleeding risk assessment tools, to predict major bleeding events in AF patients. Methods The PubMed, EMBASE, and Cochrane Central Register of Controlled Trials databases were searched using keywords, including "AF," "bleeding," and "HAS-BLED," for results published through 30 November 2018. The predictive sensitivity and specificity of each bleeding risk assessment tool was analyzed by network meta-analysis. Results Our analysis included 18 studies, recruiting a total of 321 888 people. The bleeding risk assessment tools analyzed in this study included the ABC-bleeding score, ATRIA, European score, GARFIELD-AF, HAS-BLED, HEMORR2HAGES, ORBIT, Shireman, and mOBRI. A comprehensive analysis of sensitivity and specificity, based on an inconsistency model, showed that European score, ABC, and mOBRI have relatively high-sensitivity but low-specificity tools, whereas HAS-BLED and HEMORR2HAGES have balanced sensitivity and specificity. ORBIT, ATRIA, Shireman, and GARFIELD-AF had relatively high specificity but low sensitivity. A consistency model analysis showed similar results. Conclusions HAS-BLED is a balanced bleeding risk assessment tool in terms of sensitivity and specificity, whereas the European score, ABC, and mOBRI are high-sensitivity tools and ORBIT, ATRIA, Shireman, and GARFIELD-AF are high-specificity tools.

PURPOSE:A meta-analysis was performed to evaluate the correlation between single-nucleotide polymorphisms (SNPs) and risk of statin-induced myopathy (SIM).METHODS:We retrieved the studies published on SIM until April 2019 from the PubMed, Embase, and Cochrane Library databases. We collected data from 32 studies that analyzed 10 SNPs in five genes and included 21,692 individuals and nine statins.RESULTS:The analysis of the heterozygous (p = 0.017), homozygous (p = 0.002), dominant (p = 0.005), and recessive models (p = 0.009) of SLCO1B1 rs4149056 showed that this SNP increases the risk of SIM. Conversely, heterozygous (p = 0.048) and dominant models (p = 0.030) of SLCO1B1 rs4363657 demonstrated that this SNP is associated with a reduced risk of SIM. Moreover, an increased risk of SIM was predicted for carriers of the rs4149056 C allele among simvastatin-treated patients, whereas carriers of the GATM rs9806699 A allele among rosuvastatin-treated patients had a lower risk of SIM.CONCLUSION:The meta-analysis revealed that the rs4149056 and rs4363657 SNPs in SLCO1B1 and the rs9806699 SNP in GATM are correlated with the risk of SIM.

INTRODUCTION:Different coagulation indices for direct oral anticoagulants (DOACs) exist in clinical practice, but limited data are available for the diagnostic power of these indices. This review and meta-analysis aims to explore the diagnostic value of coagulation indices for DOACs. MATERIALS AND METHODS:PubMed, Web of Science, EMBASE, Clinical Trials.gov, and the Cochrane Library were searched from inception of each database to 15 February 2020. Studies reporting a relationship between coagulation indices and the gold standard (liquid chromatography/tandem mass spectrometry) were included in the analysis. RESULTS:Sixteen articles from 9169 citations evaluating the performance of coagulation indices were included in this review. A total of 236, 273, 273 rivaroxaban samples were included to assess the diagnostic power of anti-Xa activity (AXA), prothrombin time (PT), combined PT and activated partial thromboplastin time, respectively. A total of 268 dabigatran samples were included to assess the diagnostic performance of diluted thromboplastin time (dTT). AXA calibrated by rivaroxaban showed a sensitivity of 0.98 (95% confidence interval (CI): 0.91-0.99) and a specificity of 0.98 (95% CI: 0.94-0.99) at the threshold of 30 ng/mL. For dabigatran, the combined sensitivity of dTT was 0.76 (95% CI: 0.66-0.84) and combined specificity was 0.97 (95% CI: 0.92-0.99). CONCLUSIONS:DOAC-specific calibrated AXA was a good index to indicate concentration for rivaroxaban and apixaban. More studies on edoxaban and betrixaban are in need. Diluted TT, thrombin inhibitor assay, and ecarin-based assays were potential to measure dabigatran concentration. Due to the limited data, results should be validated in the future.

Objective. The drug efficacy may differ among different statins, and evidence from head-to-head comparisons is sparse and inconsistent. The study is aimed at comparing the lipid-lowering/increasing effects of 7 different statins in patients with dyslipidemia, cardiovascular diseases, or diabetes mellitus by conducting systematic review and network meta-analyses (NMA) of the lipid changes after certain statins’ use. Methods. In this study, we searched four electronic databases for randomized controlled trials (RCTs) published through February 25, 2020, comparing the lipid-lowering efficacy of no less than two of the included statins (or statin vs. placebo). Three reviewers independently extracted data in duplicate. Firstly, mixed treatment overall comparison analyses, in the form of frequentist NMAs, were conducted using STATA 15.0 software. Then, subgroup analyses were conducted according to different baseline diseases. At last, sensitivity analyses were conducted according to age and follow-up duration. The trial was registered with PROSPERO (number CRD42018108799). Results. As a result, seven statin monotherapy treatments in 50 studies (51956 participants) were used for the analyses. The statins included simvastatin (SIM), fluvastatin (FLU), atorvastatin (ATO), rosuvastatin (ROS), lovastatin (LOV), pravastatin (PRA), and pitavastatin (PIT). In terms of LDL-C lowering, rosuvastatin ranked 1st with a surface under cumulated ranking (SUCRA) value of 93.1%. The comparative treatment efficacy for LDL-C lowering was ROS>ATO>PIT>SIM>PRA>FLU>LOV>PLA. All of the other ranking and NMA results were reported in SUCRA plots and league tables. Conclusions. According to the NMAs, it can be concluded that rosuvastatin ranked 1st in LDL-C, ApoB-lowering efficacy and ApoA1-increasing efficacy. Lovastatin ranked 1st in TC- and TG-lowering efficacy, and fluvastatin ranked 1st in HDL-C-increasing efficacy. The results should be interpreted with caution due to some limitations in our review. However, they can provide references and evidence-based foundation for drug selection in both statin monotherapies and statin combination therapies.

Aim: We aimed to identify genetic variants associated with ACE inhibitor (ACEI)-induced cough. Materials & methods: A nested case-control study was performed among hypertensive Chinese patients receiving enalapril-only therapy. Whole-exome sequencing and genome-wide association analysis were performed. Results: We identified that PNPT1 rs13015243 (odds ratio [OR]: 0.47; 95% CI: 0.34-0.66; p = 7.45 × 10-6), PNPT1 rs13009649 (OR: 0.48; 95% CI: 0.35-0.67; p = 9.96 × 10-6) and PCGF3 rs1044147 (OR: 2.67; 95% CI: 1.71-4.17; p = 9.91 × 10-6) were significantly associated with ACEI-induced cough. Nearly genome-wide significant associations in previously reported candidate risk genes CLASP1, ACE, CES1, CPN1, XPNPEP1, PDE11A or SLC38A were detected in our dataset. Conclusion: Our results suggest that ACEI-induced cough is associated with noncoding SNPs of PNPT1 and PCGF3, all of which are independent of the bradykinin pathway. Study registration: NCT03259399.