Background Diffuse large B-cell lymphoma (DLBCL) is a prevalent and aggressive form of non-Hodgkin’s lymphoma with a complex etiology. NOP2/Sun domain 2 (NSUN2) is an RNA methyltransferase that has been linked to the regulation of gene expression in various cancers. However, the function of NSUN2 in DLBCL, specifically its contribution to exosome-driven tumor progression, remains to be thoroughly elucidated. Methods Quantitative real-time polymerase chain reaction was used to analyze the expression of NSUN2 and programmed death ligand 1 variant (PDL1). Western blotting assay was performed to detect the protein levels of NSUN2, PDL1 and Y-box binding protein 1 (YBX1). Cell proliferation was analyzed by cell counting kit-8 and 5-Ethynyl-2’-deoxyuridine assays. Cell apoptosis and CD206-positive cells were quantified by flow cytometry. The levels of tumor necrosis factor-alpha and interferon-γ in cell supernatant were analyzed by enzyme-linked immunosorbent assays. m6A RNA immunoprecipitation and RNA pull-down assays were performed to determine the association between NSUN2 and PDL1. An RNA immunoprecipitation assay was used to analyze the association of YBX1 and PDL1. In vitro findings were validated in a mouse model. Results NSUN2 was overexpressed in DLBCL tissues and cells. DLBCL cell-derived exosomes facilitated the transfer of NSUN2 to DLBCL cells, which in turn promoted tumor cell proliferation, M2 macrophage polarization, and immune escape and inhibited cell apoptosis. In addition, NSUN2 stabilized PDL1 mRNA through an m5C-dependent mechanism and a YBX1-dependent pathway. Moreover, the suppression of PDL1 significantly mitigated the effects induced by NSUN2 within DLBCL cell-derived exosomes on cellular proliferation, apoptosis, M2 macrophage polarization, and immune evasion. Further, DLBCL cell-derived exosomal NSUN2 promoted tumor growth by regulating PDL1. Conclusion NSUN2 in DLBCL cell-derived exosomes stabilized PDL1 in a YBX1-dependent manner and thus promoted tumor immune escape and M2 macrophage polarization. These findings highlight the potential of targeting the NSUN2-PDL1 axis as a novel therapeutic strategy for DLBCL.

Diffuse large B cell lymphoma (DLBCL) expresses abundant programmed death ligand 1 (PD-L1), which shields tumor cells from immune attacks through the PD-L1/PD-1 signaling axis. The mechanism of PD-L1 overexpression includes the deletion of the 3'end of PD-L1, which increases its mRNA stability, and the gain or amplification of PD-L1. Previous studies found two cases of DLBCL carrying an IGH::PD-L1 by whole genome sequencing. We describe two more such cases by a targeted DNA next-generation sequencing (NGS) capable of detecting IGH rearrangements, leading to PD-L1 overexpression. DLBCL with PD-L1 overexpression is often resistant to R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine and prednisolone). Our patients responded to a combination of R-CHOP and a PD-1 inhibitor.

Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Two main subgroups of DLBCL include germinal center B-cell-like (GCB) and activated B-cell-like (ABC). Molecular profiling can further classify DLBCL into four subtypes: MCD (both CD79B and MYD88 L265P), BN2 (NOTCH2 mutation or BCL6 fusion), N1 (NOTCH1 mutation), or EZB (EZH2 mutation or BCL2 fusion). EZH2 inhibitors were recommended for patients with the EZB subtype of DLBCLs; however, little is known about the therapeutic mechanisms. Our results showed that DZNep arrested G1/S phase of GCB-DLBCL cells and inhibited the cell proliferation in vitro through upregulation of p16 by demethylating its promoter. These results suggest that DZNep may have potential as a novel therapeutic agent for DFLBL therapy. This agent may serve as a novel molecular agent to be applied to GCB DLBCL.

Growing evidence shows that cancer progression links with both heterogeneity of the tumor microenvironment and dysregulated activity of immune cells. Cancer-secreted exosomes are being recognized as indispensable mediators of the exchange cargo between cancer and immune cells. The M2-phenotype tumor-associated macrophages have the function of promoting tumor progression and drug resistance. Diffuse large B-cell lymphoma(DLBCL) is a highly heterogeneous and very common malignant non-Hodgkin's lymphoma. Here, we demonstrate that different subtype DLBCL cell-derived exosomes are internalized by macrophages, which can affect macrophages polarization. The mechanism of DLBCL-derived exosomes on macrophage polarization remains unclear currently. This study showed that DLBCL-secreted exosomes could induce the transformation of macrophages to a protumor M2-like phenotype, and block the drug-induced apoptosis of DLBCL cells in an indirect co-culture system. Different DLBCL-derived exosomes could change the phenotype of macrophages through the STAT3 signaling, which upregulated the expression of oncogenic genes and classical markers of M2-like phenotype macrophages, such as IL-10, CD206, and CD163. The addition of DLBCL-derived exosomes resulted in the activation of the STAT3 signaling pathway of M0/M2 macrophages in an indirect co-culture system. GP130 was highly enriched in DLBCL-derived exosomes, which triggered the activation of STAT3 of macrophages and subsequently induced the downstream targets such as BCL2, SURVIVIN, and BAX. The parallel changes of STAT3 and GP130 in macrophages confirmed that GP130 of DLBCL-derived exosomes promoted macrophage polarization by activating STAT3 signaling. Furthermore, all of these effects could be reversed by the GP130 inhibitor SC144. The data indicated that DLBCL-derived exosomes could trigger macrophages polarization into a pro-survival M2-like phenotype, which was at least partially through the GP130/STAT3 signaling pathway. Collectively, this study showed that DLBCL-derived exosomes could promote macrophages transformation to protumor M2-like phenotype in the tumor microenvironment.

Background Mesenchymal stem cells (MSCs) represent a subset of non-hematopoietic adult stem cells, which can also fuse with other cells spontaneously in bone marrow and capable of adopting the phenotype of other cells. The fusion of somatic cells with stem cells can reprogram somatic cells to a pluripotent state. Our research on the fusion of bone marrow mesenchymal stem cells(BM-MSCs) and MM cells demonstrate that the fused cells can exhibit stemness and cancer cell-like characteristics. Results We successfully produced a hybrid cells that acquired larger size and multinucleation, in which partial chromatin condensation, a visible nucleolus, and one or more round or oval nucleus. Experiments results showed that the stemness markers highly expressed in these fused cells and there were much more chromosomes in fused cells than those in parental cells as well as exhibited increased resistance to drug treatment. Conclusions Our results suggest that cell fusion between BM-MSCs and MM cells could contribute it genomic heterogeneity and play a role on disease progression. Methods We fused human BM-MSCs with MM cells lines RPMI 8226 or XG1 in vitro by polyethylene glycol (PEG), and the hybrid cells were sorted by sedimentation assays. The growth, migration, cell cycle, chromosome and drug sensitive of hybrids were assessed by cell counting, cell colony formation, transwell assays, cytogenetic assay and flow cytometry (FCM). The proteins and genes related to stemness and cytokines were tested by western blot and/or real-time quantitative RT-PCR.

BACKGROUND:Rheumatoid arthritis (RA) is an auto-immune disease characterized by chronic inflammation of multiple joints. Hypoxia is a constant feature of synovial microenvironment in RA. Fibroblast-like synoviocytes (FLSs), which are potent effector cells in RA. It has been reported that large numbers of monocytes are recruited to the synovium and play an important role in synovial inflammation and tissue destruction in RA. However, the mechanism is still unclear. The aim of this study is to explore the role of hypoxia microenvironment on the recruitment of monocytes and then promote the development of RA.METHODS:Rheumatoid arthritis model was constructed. Monocytes and FLSs were isolated from rheumatoid arthritis mice. RT-PCR, western blot and ELISA were used to detect the expression of SDF-1 in FLSs. CXCR4 expression in monocytes was examined by cell immunofluorescence and flow cytometry analysis. Transwell assay was performed to evaluate the potential of cell migration.RESULTS:We demonstrated that hypoxia microenvironment enhanced SDF-1 production of FLSs, which attracted the recruitment of CXCR4-expressing monocytes to the synovium and induced monocytes differentiation into tissue macrophages. Moreover, these macrophages secreted inflammatory factors including IL-6, TNF-α, IL-1β and MMP-3, which contributed to the synovial inflammation and tissue destruction in RA.CONCLUSION:The results of this study suggested that hypoxia microenvironment played an important role in enhancing SDF-1 production of FLSs. SDF-1/CXCR4 axis was involved in the recruitment of monocytes in RA synovium and it might be a possible way of inhibiting inflammation and bone erosion in RA.

目的 分析地西他滨联合小剂量阿糖胞苷治疗老年急性髓系白血病(AML)的效果.方法 选取我院2013年6月～2017年1月收治的老年急性髓系白血病患者72例作为观察对象,按照单双号法分为对照组和治疗组各36例,对照组采用单纯阿糖胞苷治疗,治疗组采用地西他滨联合小剂量阿糖胞苷治疗,观察并比较两组疗效和不良反应情况.结果 治疗组的治疗总有效率与对照组进行统计比较,对照组低于治疗组,差异有统计学意义(P<0.05);两组不良反应发生率比较,治疗组与对照组之间比较,差异无统计学意义(P>0.05).结论 地西他滨联合小剂量阿糖胞苷治疗老年急性髓系白血病具有效果确切,不良反应发生率低等优点,具有积极的临床使用和推广意义.

INTRODUCTION:Gap junctions (GJs) represent the best known intercellular communication (IC) system and are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules to pass from cell to cell. In this study, we constructed an amino terminus of human Cx43 (Cx43NT-GFP), verified the overexpression of Cx43-NT in HUVEC cells and explored the impact of gap junctions (GJs) on multiple myeloma (MM).MATERIAL AND METHODS:The levels of phosphorylated Cx43(s368) and the change of MAPK pathway associated molecules (ERK1/2, JNK, p38, NFκB) were also investigated in our cell models. Cx43 mRNA and proteins were detected in both MM cell lines and mesenchymal stem cells (MSCs). Dye transfer assays demonstrated that gap junction intercellular communication (GJIC) occurring via Cx43 situated between MM and MSCs or MM and HUVECCx43NT is functional.RESULTS:Our results present evidence for a channel-dependent modulator action of connexin 43 on the migratory activity of MM cells toward MSCs or HUVECCx43-N was higher than those of spontaneous migration (p < 0.05) and protection them from apoptosis in the presence of dexamethasone via cytokines secretion. In the meantime, the migration of MM cells involves an augmented response of p38 and JNK signaling pathway of carboxyl tail of the protein.CONCLUSIONS:Our data suggest that GJIC between MM and MSCs is one of the essential factors in tumor cell proliferation and drug sensitivity, and is implicated in MM pathogenesis.

Objective:To explore the clinical characteristics and laboratory data of aggressive non-Hodgkin's lymphoma(NHL)compli-cated by autoimmune hemolytic anemia(AIHA).Methods:Data of six patients with aggressive NHL complicated by AIHA treated at the Second Affiliated Hospital of Suzhou University between September 2013 and July 2016 were reviewed retrospectively. The onset symptoms,disease progression,therapy,and prognostic factors were analyzed.Results:From September 2013 to July 2016,155 pa-tients with aggressive NHL were treated in our hospital.Six of them were complicated by AIHA(3.9%),with three males and three fe-males,aged from 62 to 74.The median age was 67 years.The first clinical symptoms included the following:five presented with lymph-adenectasis,three had fever,and one presented with multiple bone destruction and bone pain,all complicated by progressive hemo-globin decrease.Histological examination of the six patients revealed three cases with diffuse large B-cell lymphoma(DLBCL),including one case with positive Bcl-2,Bcl-6,and C-myc,and one case with positive CD5;one case was peripheral T-cell lymphoma-not other-wise specified(PTCL-NOS);and two cases with angioimmunoblastic T-cell lymphoma(AITCL).Epstein-Barr virus(EBV)-mRNA(EBER) was detected by chromogenic in situ hybridization(CISH).Up to 100%(5/5)cases were EBER-positive.Eastern Cooperative Oncology Group scores of the six patients were three to four.All six cases were in the Ann-Arbor stagesⅢ-Ⅳand International Prognostic Index score 4-5.All cases belonged to the high risk group.At the time when lymphoma was confirmed by pathology,the median level of he-moglobin was 56(34-79)g/L.The median ratio of reticulocytes was 6.7(0.2-21.0)%.The positive rate of Coombs test was 100%.All cas-es showed autoantibodies against C3(1:64-1:2 048).Four cases showed antoantibodies against G antigen(±~1:16).The plasma con-centration of EBV DNA of three patients was detected and all increased.Except that 1 case gave up treatment,five patients received the chemotherapy with CHOP or R-CHOP.Four patients received prednisone between chemotherapy intermittent period.Two cases showed sustained complete response(CR).The overall survival(OS)was 20 and 14 months.Another patient with DLBCL and one pa-tient with PTCL-NOS died from secondary severe pulmonary infection and heart failure during the myelosuppression.The OS times were 1.5 and 2 months,respectively.One patient with AITCL died in the disease progression after four cycles of chemotherapy.The OS was 4.5 months.Conclusion:Aggressive NHL complicated by AIHA is common in older patients with poor prognosis.The incidence rate of EBV infection was high,and hemolysis is rapid and serious.The patients'tolerance to chemotherapy was poor.Early diagnosis and effective chemotherapy may improve patients'prognosis.

BACKGROUND: Effective treatment for severed acute radiation sickness (over 8 Gy) has not been obtained at present. Mesenchymal stem cells, which are shown to secrete hematopoietic cytokines and support hematopoietic progenitors, play an important role in cute radiation sickness. OBJECTIVE: To investigate the therapeutic potential of non-adherent bone marrow-derived stem cells in the treatment of acute radiation injury induced by 8.5 Gy X-ray irradiation, as wel as the mechanisms involved. METHODS: Non-adherent marrow-derived stem cells from the long bone of fetal limbs were col ected for analyzing surface antigens, cel cycle, osteogenic and adipogenic differentiation potential, and expressions of vascular endothelial growth factors and Annexin A2. After being exposed to 8.5 Gy total body irradiation, BALB/C mice were randomly assigned into transplantation group and control group. Mice in the transplantation group were given 3×106 CFDA-SE labeled human non-adherent bone marrow-derived stem cells, and those in the control group were given 0.3 mL normal saline. Then, the survival rate, peripheral white blood cells at different time, pathologic change and angiogenesis of the bone marrow were observed. RESULTS AND CONCLUSION: After X-ray irradiation, transplanted non-adherent mesenchymal stem cells appeared to have a homing to the site of injury. The survival rate of mice in the transplantation group was much higher than that in the control group. Compared with the control group, the white blood cells in the transplantation group decreased more slowly while recovered more rapid: the nadir appeared at day 14 after transplantation while it recovered within 30 days. The bone marrow of mice in the transplantation group regenerated more actively and had more hematopoietic islands than those in the control group on day 21. In addition, bone marrow angiogenesis of the transplantation group was more obvious than that of the control group. In conclusion, human fetal non-adherent bone marrow-derived stem cells could promote bone marrow angiogenesis in a mouse model of acute radiation injury, through which they could play an important role in tissue regeneration of acute radiation injury.

BACKGROUND: There are non-adhesion stem cells in bone marrow stem cells, and the morphology and phenotype characteristics of the cells are similar to adhesion bone marrow mesenchymal stem cells. OBJECTIVE: To explore the characteristics of fetal non-adherent bone marrow stromal cells (NA-BMSCs). METHODS: The fetal NA-BMSCs were cultivated by the method of adherent separation repeatly ("pull-offs"), the suspended cells were incubated in another culture bottle every 24 hours for 4 times. The growth curve of the passage 1, 3, 5 NA-BMSCs was detected by MTT assay, and the phenotype of the NA-BMSCs was determined with flow cytometry. Passage 1 NA-BMSCs were preformed with hematopoietic colony assay and passage 3 NA-BMSCs were preformed with osteogenic and adipogenic induction. RESULTS AND CONCLUSION: After four times’ "pull-off", there were still asherent cells and colony formation; the resulting cells were continuing to culture for 7 days and those cells were the primary NA-MSCs (P0). The morphology of the cells was uneven; they highly expressed the integrin and the surface markers of endothelial cells with a middle level of CD106. Interestingly, they also expressed the surface markers of hematopoietic cells CD34. Those cells had multi-differentiation potency in vitro and they could differentiate into osteoblasts and hematopoietic cells under different inducing medium, but the capability of lipid differentiation was weak.

<正>原发肺非霍奇金淋巴瘤(PPNHL)是起源于肺内淋巴组织的恶性淋巴瘤,本病较罕见,仅占结外淋巴瘤的3.6%,其中69%~78%起源于支气管黏膜相关的淋巴组织,即原发肺黏膜相关淋巴组织淋巴瘤(pri mary pul monary mucosa-associated

背景：建立相关动物模型是研究多发性骨髓瘤病理生理机制及药物治疗的重要方法。目的：为研究多发性骨髓瘤细胞在人胎骨微环境内的生长特性,建立新的Balb/c-nu-hu人鼠嵌合的浆细胞瘤动物模型。方法：取孕16周人女性胎儿1cm左右胎骨植入Balb/c-nu裸鼠皮下,建立新的Balb/c-nu-hu鼠-人嵌合体;胎骨植入3周后将白细胞介素6依赖的人多发性骨髓瘤细胞株XG-7悬液注入胎骨内建立人浆细胞瘤Balb/c-nu-hu模型;观察裸鼠及肿瘤的生长情况。40d后,取胎骨、肿瘤及裸鼠重要组织行苏木精-伊红染色及抗人CD34、CD59、CD138和VEGF免疫组织化学染色;X射线片前后对比检查模型裸鼠体内胎骨骨密度的变化。结果与结论：胎骨表面及胎骨内见新生血管生成,且具有高度活性;XG-7细胞株能在植入人胎骨的裸鼠体内生长、浸润,形成髓外肿瘤,具有多种与浆细胞瘤相似的病理学特征,其中免疫组织化学结果显示CD138、CD59阳性说明其具有浆细胞表面标记,血管CD34阳性表明其为人源性;模型裸鼠出现恶病质,终末人骨髓瘤细胞浸润播散至鼠外周血及淋巴结,X射线片显示胎骨出现骨破坏。说明Balb/c-nu-hu鼠-人嵌合体模型可作为一种建立肿瘤模型可靠的基础动物载体,利用Balb/c-nu-hu鼠-人嵌合体可以成功建立人浆细胞瘤模型。

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMSCs) are a potentially useful source for cell replacement therapy following spinal cord injury. However, the homing characteristics of BMSCs in vivo remain unclear. Low-dose radiation has been shown to promote homing of BMSCs to exposed sites.OBJECTIVE: To investigate the effects of low-dose local radiation to non-injured areas on the ability of human BMSCs to home to the injured mouse spinal cord, as well as recovery of spinal cord injury.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Central Laboratory, Second Affiliated Hospital of Soochow University between October 2007 and October 2008.MATERIALS: BMSCs were isolated from four adult, human donors.METHODS: Fifty adult, female, Balb/c mice were subjected to adjusted weight-drop impact resulting in complete paraplegia. Three days later, mice were randomly assigned to a radiation + transplantation group (n = 23) and a transplantation group (n = 20). In total, 2 x 106 carboxyfluorescein diacetate succinimidyl ester-labeled BMSCs were injected into each mouse via the caudal vein. Mice in the radiation + transplantation group received 2.5 Gy local X-ray irradiation 2 hours before BMSCs injection.MAIN OUTCOME MEASURES: The homing of BMSCs to injured cord and irradiated skin after transplantation was observed by fluorescence microscope; the structure recovery of injured cord was assessed by magnetic resonance imaging.RESULTS: Compared with the transplantation group, at 24 hours after transplantation, the number of BMSCs was significantly increased in the injured area and the exposed site (P < 0.05), and inflammation and edema were significantly alleviated in the injured cord in the radiation + transplantation group.CONCLUSION: Local low-dose radiation has the potential to promote homing of BMSCs and recovery of spinal cord injury, although the radiated region was not injured area.

Introduction Myeloma bone disease (MBD) is the result of the increased activity of osteoclasts (OCs), which is not accompanied by a comparable increase of osteoblast (OB) function, thus leading to enhanced bone resorption. Osteoblasts can also regulate osteoclast activity through expression of cytokines, such as receptor activator of nuclear factor-κB ligand (RANKL), which activates osteoclast differentiation, and osteoprotegerin (OPG), which inhibits RANKL by acting as a decoy receptor. Material and methods Based on a series of 21 patients with multiple myeloma (MM) and human osteoblast cell line HFOB1.19, we provide evidence that the bone marrow-derived mesenchymal stem cells (BMMSCs) of patients with MM exhibit normal phenotype, but showed reduced efficiency to differentiate into OBs as compared with normal controls. Results In vitro assays showed that MM cells inhibited the potential of osteogenic differentiation of BMMSCs from healthy controls and rendered the OBs sensitive to TRAIL-induced apoptosis. There was no evidence of the formation of tartrate-resistant acid phosphatase positive OCs. The osteogenic differentiation of HFOB1.19 was also inhibited in the presence of RPMI 8266 or XG7 MM cells, as confirmed by von Kossa and ALP staining. Osteoblast s induced from BMMSCs supported survival and proliferation of MM cells, especially when the MM cells were cultured in medium containing rhTRAIL and dexamethasone. Multiple myeloma cells proliferated and grew well in the presence of residual OBs. Conclusions Besides OCs, our results demonstrated that OBs and MM cells were dependent upon each other and made a microenvironment suitable for MM cells.

BACKGROUND: Establishment of a plasmacytoma model plays an important role in study of the physiological mechanism and medication for multiple myeloma. OBJECTIVE: To establish a novel plasmacytoma model of Balb/c-nu-hu for further investigation of the growth of multiple myeloma (MM) cell line in human fetal bone. METHODS: Subcutaneously transplantation of 1-cm long segment of 16 weeks old human fetal thigh or tibia bone into Balb/c-nu mice to develop a novel chimeric model based on the Balb/c-nu-hu, 3 weeks later inoculated the interleukin-6 (IL-6)-dependent human MM cell line XG-7 into the human fetal bone. Human fetal bone implanted as well as extramedullary macro-tumors and important organs of the mouse model respectively stained by hematoxylin-eosin staining and monoclonal mouse anti-human CD34, CD59, CD138 and VEGF followed by morphological examination. X-ray film was used to examine the changes of bone density of fetal bone in vivo. RESULTS AND CONCLUSION: New vessels could be observed on the surface and in fetal bones, with highly activity. The MM cell line XG-7 could grow, infiltrate and migrate in nude mice and formed extramedullary tumor, which exhibited similar pathological features to plasmacytoma. The positive expression of CD138 and CD59 demonstrated that the tumor had surface marker of plasma cell, and the positive CD34 showed it was derived from human. X-ray film showed the resorption of the human bones. The model was characterized by cachexia, terminal scatter and migration of myeloma cells. The Balb/c-nu-hu chimeric model is a novel vector for researching hominine hematopoiesis and bone based on animal. The MM cell can growth successfully based on the Balb/c-nu-hu chimera.

目的:探讨原发结外非霍奇金淋巴瘤(PE-NHL)的发病情况、临床特点、诊断及治疗.方法:回顾性分析苏州大学附属第二医院2001年1月～2008年5月收治的PE-NHL 110例.结果:110例PE-NHL占同期收治恶性淋巴瘤患者的60.11%(110/183);原发部位广泛,常见部位依次为胃肠道21.82%(24/110)、Waldeye环10.91%(12/110)、鼻腔和软组织各9.10%(10/110)、纵隔7.27%(8/110)、其他少见部位41.82%(46/110);首发表现以原发器官肿大或肿块为主(77.27%),无特异性;90例患者国际预后指教(IPI)评分:低、中、高危组分别占41.11%、44.44%、14.44%.93例进行免疫分型:B细胞型69.90%,T细胞型30.10%;95例接受治疗,CR 61.05%,PR 16.84%,总有效率77.89%,中位生存时间30个月(2～86个月),5年总生存率27%.24例原发胃肠道非霍奇金淋巴瘤(PGIL),其临床特征与非原发胃肠道组比较无统计学差异,治疗23例,CR 65.21%,PR 17.39%,总有效率82.60%,中位生存时间24个月(2～78个月),5年总生存率30%.结论:本组资料中PE-NHL比例高达60.11%,超过结内淋巴瘤;其首发部位广泛,临床表现多样但无特异性;全面认识本病、遵循合理的诊治模式是提高疗效的关键.