Molecular mechanisms of tumor resistance to PI3K-mTOR-targeted therapy

Deregulation of the phosphatidylinositide 3-kinase(PI3K) and mammalian target of rapamycin(mTOR) signaling pathway occurs frequently in a wide range of human cancers and is a major driving force in tumorigenesis.Thus,small molecules targeting this pathway are under active development as anticancer therapeutics.Although small-molecule inhibitors of the PI3K-mTOR pathway have shown promising clinical efficacy against human cancers,the emergence of drug resistance may limit their success in the clinic.To date,several resistance mechanisms,including both PI3K-dependent and-independent mechanisms,have been described.Here,we summarize the current understanding of resistance mechanisms to PI3K-mTOR inhibitors and discuss potential strategies for overcoming resistance for potential clinical application.

Incidence,predictors and prognosis of acute kidney injury in acute ST-segment elevation myocardial infarction patients undergoing emergent coronary angiography/primary percutaneous coronary intervention

BACKGROUND Acute kidney injury(AKI) after coronary angiography(CAG) and primary percutaneous coronary intervention(PPCI) is frequently observed, and often interpreted as contrast induced-AKI. This study aimed to investigate the incidence, predictors and outcomes of AKI in acute ST-segment elevation myocardial infarction(STEMI) patients undergoing emergent CAG/PPCI using the control group of STEMI patients who were not exposed to contrast agents within the first 72 h.METHODS We performed a retrospective analysis of 1670 STEMI patients. Of them, 673 patients underwent emergent CAG/PPCI, and 997 patients treated with thrombolysis or no reperfusion therapy who were not exposed to contrast material during the first 72 h. AKI was defined as an increase of serum creatinine ≥ 44.2 mmol/L or ≥ 25% from baseline within 72 h. Patents were then followed up for the occurrence of all-cause mortality for 40 months(interquartile range: 24–55 months).RESULTS After propensity score matching, 505 pairs of patients were matched. Overall, the incidence of AKI was 27.4%, and AKI rates were not significantly different in patients with and without emergent CAG/PPCI procedure(27.5% vs. 27.3%, P = 0.944). Multivariate logistic regression analysis identified that the independent predictors of AKI were female, elevated interleukin-6 level,decreased lymphocyte count, left ventricular ejection fraction < 50% and use of diuretics in patients with emergent CAG/PPCI. Patients with AKI than those without AKI experienced higher incidence of acute heart failure with Killip class Ⅲ(9.4% vs. 3.3%, P =0.005;15.2% vs. 6.8%, P = 0.003, respectively) and mortality(5.8% vs. 1.4%, P = 0.014;12.3% vs. 4.6%, P = 0.002, respectively) in patients with and without emergent CAG/PPCI. Multivariate Cox regression analysis confirmed that AKI was independently associated with long-term mortality rate at 40 months follow-up in patients with and without emergent CAG/PPCI(HR = 1.867, 95% CI:1.086–3.210, P = 0.024;HR = 1.700, 95% CI: 1.219–2.370, P = 0.002, respectively).CONCLUSIONS Approximately 27.0% of STEMI patients experience AKI, which is strongly associated with an increased shortand long-term mortality regardless of emergent CAG/PPCI procedure. The development of AKI is mainly related to female gender, inflammation reaction, heart failure and use of diuretics in STEMI patients undergoing emergent CAG/PPCI.

PPARβ expression in rectus abdominis and abdominal subcutaneous fat of patients with gestational diabetes mellitus and its relationship with glucolipid metabolism

Objective:To study the relationship between peroxisome proliferator-activated receptorβ(PPARβ) expression in rectus abdominis as well as abdominal subcutaneous fat of patients with gestational diabetes mellitus (GDM) and glucolipid metabolism.Methods:The pregnant women who received routine antenatal care and planned to receive selective caesarean section in Obstetrics Department of our hospital between May 2012 and March 2016 were retrospectively analyzed, and 74 healthy pregnant women and 58 pregnant women with GDM were screened and included in the control group and gestational diabetes mellitus group (GDM group) respectively. Rectus abdominis and abdominal subcutaneous fat were collected during Cesarean section to determine the expression of PPARβ was measured;peripheral blood was collected at middle-late pregnancy to determine the content of blood glucose metabolism and lipid metabolism indexes as well as adipocytokines.Results:PARβ mRNA expression and protein expression in rectus abdominis and abdominal subcutaneous fat of GDM group were significantly lower than those of control group (P<0.05);homeostasis model assessment insulin secretion index (HOMA-β), homeostasis model assessment insulin resistance (HOMA-IR) and OGTT glucose curve (AUCG) levels as well as serum low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), cholesterol (TC), Leptin, Resistin and Chemerin content of GDM group were significantly higher than those of control group (P<0.05) while early insulin secretion index (ΔI30/ΔG30) and insulin sensitive index composite (ISIcomp) levels as well as serum high-density lipoprotein cholesterol (HDL-C), Omentin-1 and Omentin-1 and adiponectin (ADPN) content were significantly lower than those of control group (P<0.05);PARβ mRNA expression and protein expression were negatively correlated with HOMA-β, HOMA IR, area under the AUCG, LDL-C, TG, TC, Leptin, Resistin and Chemerin, and positively correlated withΔI30/ΔG30, ISIcomp, HDL-C, and ADPN.Conclusions:PPARβ expression significantly decreases in rectus abdominis and abdominal subcutaneous fat of pregnant womepn with GDM and it is closely related to the abnormal glucolipid metabolism in pregnant women with GDM.

基于深度学习模型的非小细胞肺癌新辅助免疫联合化疗疗效预测

目的:基于深度学习算法,使用临床数据构建人工智能(artificial intelligence,AI)模型,评估其预测非小细胞肺癌(nonsmall cell lung cancer,NSCLC)新辅助免疫联合化疗疗效的可行性。方法:收集2020年1月至2024年1月于首都医科大学附属北京胸科医院确诊且接受新辅助免疫联合化疗的132例NSCLC患者的临床病理数据。对影响新辅助免疫联合化疗疗效的主要因素进行统计学分析。根据统计学分析结果以及查阅相关文献对变量进行筛选,构建变量数据集。基于多层感知机(multi-layer perceptron,MLP)算法采用5折交叉验证建立深度学习模型,应用接收者操作特征(receiver operating characteristic,ROC)曲线评估模型性能。结果:132例患者中单因素分析显示获得主要病理缓解(major pathological response,MPR)组与未获得MPR组的患者在性别(P=0.020)、吸烟史(P=0.004)、癌胚抗原(carcinoembryonic antigen,CEA)(P=0.038)及程序性死亡配体-1(programmed death-ligand 1,PD-L1)是否≥1%(P=0.038)方面差异具有统计学意义;获得完全病理缓解(complete pathological response,pCR)组与未获pCR组患者在肿瘤大小是否≤3 cm(P=0.007)和CEA水平方面(P=0.010)差异具有统计学意义。经5折交叉验证后,MPR预测模型在验证集和测试集的平均受试者工作特征曲线下面积(area under the curve,AUC)值分别为0.72及0.71。结论:该深度学习模型能够有效预测NSCLC患者新辅助免疫联合化疗疗效。

Construction of coal pitch-based HA-K grafted poly condensates and their excellent anti-temperature and viscosity-reducing properties

Humic acids(HAs)are widely used as filtrate and viscosity reducers in drilling fluids.However,their practical utility is limited due to poor stability in salt resistance and high-temperature resistance.Hightemperature coal pitch(CP)is a by-product from coal pyrolysis above 650℃.The substance's molecular structure is characterized by a dense arrangement of aromatic hydrocarbon and alkyl substituents.This unique structure gives it unique chemical properties and excellent drilling performance,surpassing traditional humic acids in drilling operations.Potassium humate is prepared from CP(CP-HA-K)by thermal catalysis.A new type of high-quality humic acid temperature-resistant viscosity-reducer(Graft CP-HA-K polymer)is synthesized with CP-HA-K,hydrolyzed polyacrylonitrile sodium salt(Na-HPAN),urea,formaldehyde,phenol and acrylamide(AAM)as raw materials.The experimental results demonstrate that the most favorable conditions for the catalytic preparation of CP-HA-K are 1 wt%catalyst dosage,30 wt%KOH dosage,a reaction temperature of 250℃,and a reaction time of 2 h,resulting in a maximum yield of CP-HA-K of 39.58%.The temperature resistance of the Graft CP-HA-K polymer is measured to be 177.39℃,which is 55.39℃ higher than that of commercial HA-K.This is due to the abundant presence of amide,hydroxyl,and amine functional groups in the Graft CP-HA-K polymer,which increase the length of the carbon chains,enhance the electrostatic repulsion on the surface of solid particles.After being aged to 120℃ for a specified duration,the Graft CP-HA-K polymer demonstrates significantly higher viscosity reduction(42.12%)compared to commercial HA-K(C-HA-K).Furthermore,the Graft CP-HA-K polymer can tolerate a high salt concentration of 8000 mg.L-1,measured after the addition of optimum amount of 3 wt%Graft CP-HA-K polymer.The action mechanism of Graft CP-HA-K polymer on high-temperature drilling fluid is that the Graft CP-HA-K polymer can increase the repulsive force between solid particles and disrupt bentonite's reticulation structure.Overall,this research provides novelty insights into the synthesis of artificial humic acid materials and the development of temperature-resistant viscosity reducers,offering a new avenue for the utilization of CP resources.

Association of prealbumin with short-term and long-term outcomes in patients with acute ST-segment elevation myocardial infarction

BACKGROUND Prealbumin is considered to be a useful indicator of nutritional status. Furthermore, it has been found to be associated with severities and prognosis of a range of diseases. However, limited data on the association of baseline prealbumin level with outcomes of patients with acute ST-segment elevation myocardial infarction(STEMI) are available.METHODS We analyzed 2313 patients admitted for acute STEMI between October 2013 and December 2020. In-hospital outcomes and mortality during the 49 months(interquartile range: 26–73 months) follow-up period were compared between patients with the low prealbumin level(< 170 mg/L) and those with the high prealbumin level(≥ 170 mg/L).RESULTS A total of 114 patients(4.9%) died during hospitalization. After propensity score matching, patients with the low prealbumin level than those with the high prealbumin level experienced higher incidences of heart failure with Killip class Ⅲ(9.9%vs. 4.4%, P = 0.034), cardiovascular death(8.4% vs. 3.4%, P = 0.035) and the composite of major adverse cardiovascular events(19.2%vs. 10.3%, P = 0.012). Multivariate logistic regression analysis identified that the low prealbumin level(< 170 mg/L) was an independent predictor of in-hospital major adverse cardiovascular events(odds ratio = 1.918, 95% CI: 1.250–2.942, P = 0.003). The cutoff value of prealbumin level for predicting in-hospital death was 170 mg/L(area under the curve = 0.703, 95% CI: 0.651–0.754, P< 0.001;sensitivity = 0.544, specificity = 0.794). However, after multivariate adjustment of possible confounders, baseline prealbumin level(170 mg/L) was no longer independently associated with 49-month cardiovascular death. After propensity score matching, Kaplan-Meier survival curves revealed consistent results.CONCLUSIONS Decreased prealbumin level closely related to unfavorable short-term outcomes. However, after multivariate adjustment and controlling for baseline differences, baseline prealbumin level was not independently associated with an increased risk of long-term cardiovascular mortality in STEMI patients.

微纳米纤维空气净化滤膜的研究进展

微纳米纤维由于比表面积高、长径比高和孔隙率高等优点在制备空气净化滤膜方面极具应用潜力,但始终面临高的过滤效率和低过滤阻力之间的矛盾。文中首先介绍了不同材料制备的微纳米纤维膜的过滤性能,并对不同材料的特性和过滤效果进行了分析;其次总结了典型的微纳米纤维膜结构,包括纤维形貌、纤维膜内结构和层间结构;最后对高效低阻微纳米空气滤膜今后的发展方向进行了展望。

多孔碳包覆铜纳米颗粒的制备及性能表征——推荐一个化学综合实验

以Cu-MOF为前驱体,通过热解法制备氮掺杂多孔碳包覆铜纳米材料,采用X射线粉末衍射仪(XRD)、场发射扫描电子显微镜(SEM)和傅立叶变换红外光谱仪(IR)等仪器对产物的形貌和组成进行表征。考察了不同热解温度对多孔碳材料形貌和组成的影响,探究其在硝基苯还原反应中的催化性能,并优化了反应条件。将材料的制备、表征和性能探究设计成一个综合实验,能够培养学生的科研思维和综合分析能力。

WWOX suppresses KLF5 expression and breast cancer cell growth

The WW domain-containing oxidoreductase（WWOX） is a tumor suppressor in a variety of cancers, including breast cancer. Reduced WWOX expression is associated with the basal-like subtype and a relatively poor disease-free survival rate among breast cancer patients. Though several WWOX partners have been identified, the functional mechanisms of WWOX＇s role in cancers have not been fully addressed to date. In the current study, we found WWOX suppresses expression of KLF5—an oncogenic transcription factor—at protein level, and suppresses cancer cell proliferation in both bladder and breast cancer cell lines. Furthermore, we demonstrated that WWOX physically interacts with KLF5 via the former＇s WW domains and the latter＇s PY motifs. Interestingly, we found the expression of WWOX negatively correlates with KLF5 expression in a panel of breast cancer cell lines. Taken together, we conjecture that WWOX may suppress cancer cell proliferation partially by reducing the expression of KLF5.

Anacardic acid(6-pentadecylsalicylic acid) induces apoptosis of prostate cancer cells through inhibition of androgen receptor and activation of p53 signaling

Anacardic acid （AA） is a mixture of 2-hydroxy-6-alkylbenzoic acid homologs. It is widely regarded as a non-specific histone acetyltransferase inhibitor of p300. The effects and the mechanisms of AA in LNCaP cells （prostate cancer cells） remain unknown. To investigate the effect of AA on LNCaP cells, we had carried out several experiments and found that AA inhibits LNCaP cell proliferation, induces G1/S cell cycle arrest and apoptosis of LNCaP cell. The mechanisms via which AA acts on LNCaP cells may be due to the following aspects. First, AA can regulate p300 transcription and protein level except for its mechanisms regulating function of p300 through post-translational modification in LNCaP cells. Second, AA can activate p53 through increasing the phosphorylation of p53 on Serl 5 in LNCaP cells. AA can selectively activate p21 （target genes of p53）. Third, AA can down-regulates androgen receptor （All） through supressing p300. Our study suggests that AA has multiple anti-tumor activities in LNCaP cells and warrants further investigation.

Microstructure refinement and second phase particle regulation of Mo-Y_(2)O_(3) alloys by minor TiC additive

The oxide dispersion strengthened Mo alloys(ODS-Mo)prepared by traditional ball milling and subsequent sintering technique generally possess comparatively coarse Mo grains and large oxide particles at Mo grain boundaries(GBs),which obviously suppress the corresponding strengthening effect of oxide addition.In this work,the Y_(2)O_(3) and TiC particles were simultaneously doped into Mo alloys using ball-milling and subsequent low temperature sintering.Accompanied by TiC addition,the Mo-Y_(2)O_(3) grains are sharply refined from 3.12 to 1.36μm.In particular,Y_(2)O_(3) and TiC can form smaller Y-Ti-O-C quaternary phase particles(~230 nm)at Mo GBs compared to single Y_(2)O_(3) particles(~420 nm),so as to these new formed Y-Ti-O-C particles can more effectively pin and hinder GBs movement.In addition to Y-Ti-O-C particles at GBs,Y_(2)O_(3),TiOx,and TiCx nanoparticles(<100 nm)also exist within Mo grains,which is significantly different from traditional ODS-Mo.The appearance of TiOx phase indicates that some active Ti within TiC can adsorb oxygen impurities of Mo matrix to form a new strengthening phase,thus strengthening and purifying Mo matrix.Furthermore,the pure Mo,Mo-Y_(2)O_(3),and Mo-Y_(2)O_(3)-TiC alloys have similar relative densities(97.4%-98.0%).More importantly,the Mo-Y_(2)O_(3)-TiC alloys exhibit higher hardness(HV0.2(425±25))compared to Mo-Y_(2)O_(3) alloys(HV0.2(370±25)).This work could provide a relevant strategy for the preparation of ultrafine Mo alloys by facile ball-milling.

Apoptosis of bladder transitional cell carcinoma T24 cells induced by adenovirus-mediated inducible nitric oxide synthase gene transfection

Objectives：To investigate the effects of adenovirus-mediated inducible nitric oxide synthase gene transfection on bladder transitional cell carcinoma T24 cells,and to provide novel insights and approaches to clinical therapies against bladder transitional cell carcinoma.Methods：Firstly,construct recombinant adenovirus vector pAd-iNOS of iNOS,followed by transfection of pAd-iNOS into HECK293 packaging cells.Thirdly,harvest recombinant adenovirus rAd-iNOS after amplification and purification procedures.Finally,transfect the recombinant adenovirus rAd-iNOS into human bladder carcinoma T24 cells and examine the effect of rAd-iNOS transfection on apoptosis of T24 and possible mechanism.Results：As shown by this study,the recombinant adenovirus rAd-iNOS was constructed successfully.The virus titer was 5.8×108 PFU/mL and recombinant was verified by PCR analysis.Transfection of adenovirus rAd-iNOS into T24 cells could induce secretion of high NO concentration,P53 protein expression upregulation,as well as promotion ofT24 cell apoptosis.Conclusions：The transfection of human bladder carcinoma T24 cells from recombinant adenovirus rAdiNOS was confirmed to induce intracellular iNOS over-expression,high production of NO,up-regulation of intracellular P53 expression and promotion of cell apoptosis.

Fe−C micro-alloying effect on properties of Zr_(53)Al_(11.6)Ni_(11.7)Cu_(23.7)bulk metallic glass

(Zr_(53)Al_(11.6)Ni_(11.7)Cu_(23.7))_(1−x)(Fe_(77.1)C_(22.9))_(x)(x=0−2.2,at.%)bulk metallic glasses(BMGs)were prepared by copper mold suction casting method.Their glass forming ability and physical and chemical properties were systematically investigated.The glass forming ability is firstly improved with increasing x,and then decreased when x exceeds 0.44 at.%.Both glass transition temperature and crystallization temperature are increased,while the supercooled liquid region is narrowed,with Fe−C micro-alloying.The hardness,yielding and fracture strength,and plasticity firstly increase and then decrease when x reaches up to 1.32 at.%.The plasticity of the BMG(x=1.32 at.%)is six times that of the Fe-free and C-free BMG.In addition,by the Fe−C micro-alloying,the corrosion potential is slightly decreased,while the corrosion current density increases.The pitting corrosion becomes increasingly serious with the increase of Fe and C content.

Process intensification in gas/liquid/solid reaction in trickle bed reactors: A review

As an important form of reactors for gas/liquid/solid catalytic reaction,trickle bed reactors (TBRs) are widely applied in petroleum industry,biochemical,fine chemical and pharmaceutical industries because of their flexibility,simplicity of operation and high throughput.However,TBRs also show inefficient production and hot pots caused by non-uniform fluid distribution and incomplete wetting of the catalyst,which limit their further application in chemical industry.Also,process intensification in TBRs is necessary as the decrease in quality of processed crude oil,caused by increased exploitation depths,and more restrictive environmental regulations and emission standards for industry,caused by increased environment protection consciousness.In recent years,lots of strategies for process intensification in TBRs have been proposed to improve reaction performance to meet the current and future demands of chemical industry from the environmental and economic perspective.This article summarizes the recent progress in techniques for intensifying gas/liquid/solid reaction in TBRs and application of intensified TBRs in petroleum industry.

浅析基层公共图书馆针对特殊人群的阅读推广服务——以广州市基层公共图书馆为例

阅读推广是图书馆的生命,广州市基层公共图书馆明确特殊人群阅读推广的服务定位,针对特殊群体,一是提供精细化服务;二是创新手段与方法;三是形成社会化合作机制。但在总体向好的情势下,更应注意馆藏资源建设,开展经典阅读,重视阅读推广服务队伍素质培养。

The relation between serum phosphorus levels and long-term mortality in Chinese patients with ST-segment elevation myocardial infarction

Background Elevated serum phosphorus levels may be associated with adverse outcomes in cardiovascular disease. This study aimed to investigate the relation between serum phosphorus levels and risk of all-cause mortality in Chinese patients with ST-segment elevation myocardial infarction (STEMI) who had preserved renal function at baseline. Methods We enrolled patients with STEMI who had preserved renal function at baseline in Xuanwu Hospital from January 2011 to December 2016. Those patients were divided into four groups based on serum phosphorus levels. All-cause mortality rates were compared between groups. Mean duration of follow up was 54.6 months. We used Cox proportional-hazards models to examine the relation between serum phosphorus levels and all-cause mortality after adjustment for potential confounders. Results 1989 patients were involved and 211 patients (10.6%) died during follow-up. Based on serum phosphorus levels, patients were categorized into the following groups:< 2.50 mg/dL (n = 89), 2.51–3.50 mg/dL (n = 1066), 3.51–4.50 mg/dL (n = 672) and > 4.50 mg/dL (n = 162), respectively. The lowest mortality occurred in patients with serum phosphorus levels between 2.51–3.50 mg/dL, with a multivariable-adjusted hazard ratio of 1.19 (95% CI: 0.64–1.54), 1.37 (95% CI: 1.22–1.74), and 1.46 (95% CI: 1.35–1.83) in patients with serum phosphorus levels of < 2.50 mg/dL, 3.51–4.50 mg/dL and > 4.50 mg/dL, respectively. Conclusions Elevated serum phosphorus levels were associated with all-cause mortality in Chinese patients with STEMI who had preserved renal function at baseline.

A new look at cardiac pathophysiology difference in type 2 diabetic mice by comparative analysis

Objective To clarify the differences in cardiac structure,cardiac function,and myocardial metabolism in type 2 diabetes mellitus mice with obesity or non-obesity and to elucidate the key molecular mechanisms leading to this difference.Methods Db/db mice and low-dose STZ injection combined with HFD-induced diabetes mellitus mice were used in this study as the model of type 2 diabetes mellitus with obesity or non-obesity.

An Improved Algorithm for Imbalanced Data and Small Sample Size Classification

Traditional classification algorithms perform not very well on imbalanced data sets and small sample size. To deal with the problem, a novel method is proposed to change the class distribution through adding virtual samples, which are generated by the windowed regression over-sampling (WRO) method. The proposed method WRO not only reflects the additive effects but also reflects the multiplicative effect between samples. A comparative study between the proposed method and other over-sampling methods such as synthetic minority over-sampling technique (SMOTE) and borderline over-sampling (BOS) on UCI datasets and Fourier transform infrared spectroscopy (FTIR) data set is provided. Experimental results show that the WRO method can achieve better performance than other methods.

Quality Evaluation of Threshing and Redrying Process Based on Analytic Hierarchy Process

Improving the stability of the homogenization process to achieve the homogeneity of tobacco products is one of important targets for the redrying industry.According to the specification for threshing and redrying process,a total of 14 indicators in three categories that affect the quality of the threshing and redrying process were selected.Using analytic hierarchy process,combined with expert experiences,a judgment matrix was constructed to conduct consistency test.The weights of indices in production were obtained.This will help in evaluating the actual production quality,finding the weak links of process and adjusting the parameters of the corresponding links in a targeted manner,thereby improving the quality of production process.

Solid-state NMR spectroscopy at ultrahigh resolution for structural and dynamical studies of MOFs

To characterize the structure and dynamics of metal--organic frameworks(MOFs)indepth at the molecular level,it is necessary to pursue high-resolution solid-state magic angle spinning(MAS)nuclear magnetic resonance(NMR)spectroscopy.Spectral resolution is usually affected by the quality of materials and various experimental conditions,of which magic angle(MA)accuracy is a crucial determinant.The current industrial criteria for MA calibration based on the common standard of KBr were found insufficient in guaranteeing optimal resolution MAS NMR for highly ordered MOFs.To drive towards higher-resolution MAS NMR spectroscopy,we propose_a calibration protocol for more accurate MA with a higher-precision criterion based on 79Br MAS NMR of KBr,where the linewidth ratio of the fifth-order spinning sideband to the central band of KBr should be less than 1.00.As a result,ultrahigh-resolution 13C cross-polarization(CP)MAS NMR of MOF-5 is achieved with minimal linewidths as low as 4 Hz,and therefore MOF-5 can be used as a new standard convenient for verifying MA accuracy and also optimizing 13c CP conditions.Maintaining high-precision MA under variable temperature(VT)was found challenging on certain commercial MAS NMR probes,as was systematically investigated by VT NMR using KBr and MOF-5.Nevertheless,ultrahigh-resolution MAS NMR spectroscopy with stable MA under VT is employed to reveal fine structures and linker dynamics of a series of Zn-based MOFs with highly regulated structures.The ultrahigh-resolution NMR methodcan be generally applied to study a broad range of MOFs and other materials.