Comprehensive regulation of traditional Chinese medicine on proliferation and differentiation of neural stem cells

Since the diccovery of neural stem cells(NSCs)in the embryonic and adult mammalian central nerous system,it provided novel ideas forneurogenesis as the potential of proliferation and differentiation of NSCs.One of the ways to promote the clinical application of neural stem cells(NSCs)is searching effective methods which regulate the proliferation and differentiation.This is also a problem urgently to be solved in medical field.Plenty of earlier studies have shown that traditional chinese medicine can promote the proliferation and differentiation of NSCs by regulating the related signaling pathway in vivo and in vitro.The reports of Chinese and foreign literatures on regulating the proliferation and differentiation of neural stem cells in recent ten years and their target and signaling pathways is analyzed in this review.The traditional chinese medicine regulate proliferation and differentiation of NSCs by the signaling pathways of Notch,PI3K/Akt,Wnt/β-catenin,and GFs.And,those signaling pathways have cross-talk in the regulation progress.Moreover,some traditional Chinese medicine,such as astragalus,has a variety of active ingredients to regulate proliferation and differentiation of NSCs through different signaling pathways.However,to accelerate the clinical application of neural stem cells,the studies aboutthe proliferation and differentiation of NSCs and Chinese medicine should be further deepened,the mechanism of multiple targets and the comprehensive regulation function of traditional Chinese medicine should be clarified.

Studies on the Degranulation of RBL-2H3 Cells Induced by Traditional Chinese Medicine Injections

Aims: To study RBL-2H3 cell degranulation phenomena induced by some TCMIs through cell morphological and ultra-structural observation, released enzyme activity and establish RBL-2H3 cell degranulation test indicated by β- hexosaminidase activity as a method to evaluate TCMIs at nonclinical stage. Methods: RBL-2H3 cells were used to study the degranulation by co-culture with positive control C48/80 and some TCMIs through morphological and ultra-structure observation, β-hexosaminidase activity detection. RBL-2H3 cell degranulation test was established to detect β-hexosaminidase activity caused by 17 kinds of TCMIs and their ingredients. The cytotoxicity effect of some TCMIs on both RBL 2H3 and BRL cells was measured by CCK-8 assay. Results: Toluidine blue staining and ultra-structure of electronic microscope observation of treated RBL-2H3 cells showed degranulation morphologically. Detection of β-hexosaminidase activity in the supernatant of treated cells showed some TCMIs had elevated enzyme release rates. Further analysis of the ingredients and compound in Tanreqing Injection and Shengmai Injection showed Scutellaria baicalensis Georgi in Tanreqing Injection, Red ginseng and Fructus Schisandrae Chinensis in Shengmai Injection were responsible to the degranulation of RBL-2H3 cells. Osmotic pressures and pH influenced RBL-2H3 degranulation. High Osmotic pressure of Tanreqing Injection and low pH of chlorogenic acid at 2.5 and 5.0 mmol/L congcentration might be responsible to high β-hexosaminidase activity. Most of the TCMIs inducing degranulation had cytotoxicity effect for both RBL-2H3 and BRL cells, but some TCMIs inducing degranulation had no cytotoxicity effect. Conclusion: Some TCMIs can induce degranulation of RBL-2H3 cells;RBL-2H3 cell degranulation test can be used in non-clinical stage to detect the risk causing anaphylactoid reactions. Osmotic pressures and pH influenced RBL-2H3 degranulation, and they should be measured before testing. The mechanism of degranulation caused by some TCMIs is cytotoxic, and some are non-cytotoxic and may be through exicytosis.

Nature products of traditional Chinese medicine provide new ideas in γδT cells for tumor immunotherapy

Due to the unique features of innate immune cells, the role of γδT cells in tumor immunity has gradually attracted more and more attention. Previous studies have found that γδT cells play a dual role in tumor immunology: tumor-promoting and tumor-controlling.The anti-tumor therapy of γδT cells has made remarkable success in clinical application. Especially in recent years, researchers have provided some novel effective ways such as γδT cells exosomes and adoptive chimeric antigen receptor-γδT cells immunotherapy. However, some problems remain to be solved, such as low expansion rate, poor targeting, and tumor microenvironment limiting the effectiveness of γδT immunotherapy. Traditional Chinese medicine is expected to play a positive role in the body immune-enhancing function, promoting the proliferation and activation of γδT cells, and inducing the differentiation ofγδT cells. In this review, we summarize the recent research progress and urgent problems of γδT cells in anti-tumor immunotherapy. Moreover, some new strategies of γδT cells for tumor immunotherapy were proposed.

Dendritic Cells as a Pharmacological Target of Traditional Chinese Medicine

Dendritic cells （DCs） represent a heterogeneous population of professional antigen-presenting cells （APCs） that play a central role in the initiation and regulation of immune responses. There is considerable evidence that DCs can be used as therapeutic targets for pharmacological modulation of immune responses. Traditional Chinese medicine （TCM） has a long-standing history of using herbal medicine in the treatment of variety of human diseases. Many of the clinical effects of TCM have reportedly been attributed to the up- or down-regulation of immune responses. Accumulating evidence indicates that TCM and its components can interfere with immune responses at the earliest stage by targeting key functions of DCs. Here, we review those published studies of TCM with respect to their effects on immunobiological functions of DCs. Investigations based on both chemical entities derived from TCM as well as TCM herbal mixtures are presented. These studies suggest that various TCM herbal medicines have the capacity to inhibit or promote major functions of DCs, such as differentiation, maturation, cytokine production, survival, antigen uptake and presentation as well as trafficking. These studies have revealed novel biological effects of TCM and documented the utility of this approach to discover novel biological modifier of DC functions derived from natural sources.

Research progress of protective effect of traditional Chinese medicine on brain cells

Objective:To review the protective effect of traditional Chinese medicine(TCM)on brain cells.Methods:The domestic journals and literature are searched and reviewed,classifies them according to the mechanisms of TCM,summaries their protective effect on brain cells.Results:Some single medicine and prescriptions possess a variety of biological activity,and has a protective effect on brain cells.Conclusion:Some TCM possess a variety of biological activity and have a protective effect on brain cells,so there are broad prospects to develop and apply these TCM.

Experimental Study of the Anti-human Papilloma Virus Mechanisms by a Traditional Chinese Medicine “Keyouling”

To investigate the anti-human papilloma virus (HPV) effect of the external liniment of traditional Chinese medicine “Keyouling”, the efficacy of this drug acting upon the proliferation and growth of the normal epidermis cells of rabbits′ penis prepuce in different concentrations was observed. An experimental model of pathology of histocytes infected with HPV in vitro was established by using the human HPV infected prepuce epidermis cells as virus carrier to infect the sub-cultured epidermis cells of rabbits′ penis prepuce. The direct killing effect and the blocking effect of infections of epidermis cells in rabbits′ penis prepuce induced by HPV with different concentrations of “Keyouling” were determined by 3 kinds of culture methods: (1) the infected epidermis cells of rabbits′ penis prepuce were challenged by co-culture with HPV suspension and with culture medium containing drug; (2) the co-culture with epidermis cells of rabbits′ penis prepuce and HPV suspension was challenged with drug; (3) co-culture with culture medium containing drug and epidermis cells of rabbits′ penis prepuce was challenged with HPV suspension. It was found that “Keyouling” had little effect on the proliferation and growth of the normal epidermis cells of rabbits′ penis prepuce, but it had direct killing effect on HPV, and blocked effectively the infection of the epidermis cells of rabbits′ penis prepuce by HPV. It concludes that “Keyouling” has significant killing effect to HPV, but not normal histocytes in vitro , indicating that it is safe for clinical use. Its blocking effect on the HPV infection of rabbits′ penis prepuce epidermis cells suggested “Keyouling” may be clinically used to prevent the development of tumors or to inhibit the further progression or metastasis of tumors.

Lung Cancer Treatment in Traditional Chinese Medicine: History, Current Status, and Development

Objective:This article discusses the following aspects,including the history of traditional Chinese medicine(TCM)in the treatment of lung cancer,the breakthrough in the theory of TCM in treatment of lung cancer,clinical study of TCM in treating lung cancer,microscientific interpretation of TCM treatment for lung cancer and the prospect of TCM in treating lung cancer.Materials and Methods:In this paper,through a systematic search,combing traditional Chinese medicine prevention and treatment of lung cancer ancient books and modern literature.Results:A series of large sample and multi-centered clinical studies have proved that TCM comprehensive treatment significantly improves the clinical efficacy for lung cancer.“TCM treatment system for non-small cell lung cancer”based on staged and standardized integration of TCM and Western medicine has been well developed and popularized in practice.Furthermore,in virtue of the international cooperation platform established by the National Cancer Institute of the United States,the scientific connotation of TCM in the prevention and treatment of lung cancer is systematically and deeply studied,thereby promoting the research and development of new Chinese drugs for lung cancer.Conclusions:Nowadays,TCM has realized a landmark breakthrough in treating lung cancer,bringing benefits to all lung cancer patients.

Comparison of intestinal absorption characteristics between rhubarb traditional Chinese medicine preparation and activity ingredients using in situ and in vitro model

Objective: The intestinal absorption characteristics of active ingredients are very important for oral administration of traditional Chinese medicine(TCM) to achieve the desired therapeutic effect.However, a deeper understanding about active ingredients absorption characteristics is still lack. The aim of this study was to investigate the absorption properties and mechanism of rhubarb active ingredients in TCM preparation and pure form.Methods: The intestinal absorption behavior of active ingredients in Shenkang extract(SKE) and rhubarb anthraquinone ingredients(RAI) were investigated by in situ single-pass intestinal perfusion model. And the bidirectional transport characteristics of these active ingredients were assessed by in vitro Caco-2 cell monolayer model.Results: In situ experiment on Sprague-Dawley rats, the effective permeability coefficient values of aloeemodin, emodin and chrysophanol in RAI were higher than those in SKE, and the value of rhein in RAI was lower than that in SKE. But the easily absorbed segments of intestine were consistent for all ingredients,whether in SKE or in RAI. In vitro experiment, the apparent permeability coefficient values of rhein, emodin and chrysophanol in RAI were higher than those in SKE, and this value of aloe-emodin in RAI was lower than that in SKE. But their efflux ratio(ER) values in SKE and RAI were all similar.Conclusion: Four rhubarb anthraquinone ingredients in SKE and RAI have similar absorption mechanism and different absorption behavior, and the microenvironment of the study models influenced their absorption behavior. The results may provide an aid for understanding of the absorption characteristics of the TCM active ingredients in complex environments and the complementarities of different research models.

Advances in mesenchymal stem cells combined with traditional Chinese medicine therapy for liver fibrosis

Liver fibrosis is a primary cause of liver cirrhosis, and even hepatocarcinoma. Recently, the usage of mesenchyrnal stem cells （MSCs） has been investigated to improve liver fibrosis. It has been reported that the differentiation, proliferation and migration of MSCs can be regulated by traditional Chinese medicine treatment; however, the mechanisms are still unclear. In this article, the authors review the characteristics of MSCs such as multidirectional differentiation and homing, and its application in animal experiments and clinical trials. The authors also list areas that need further investigation, and look at the future prospects of clinical application of MSCs.

Accurate construction of cell membrane biomimetic graphene nanodecoys via purposeful surface engineering to improve screening efficiency of active components of traditional Chinese medicine

Biomimetic nanoengineering presents great potential in biomedical research by integrating cell membrane(CM) with functional nanoparticles. However, preparation of CM biomimetic nanomaterials for custom applications that can avoid the aggregation of nanocarriers while maintaining the biological activity of CM remains a challenge. Herein, a high-performance CM biomimetic graphene nanodecoy was fabricated via purposeful surface engineering, where polyethylene glycol(PEG) was used to modifying magnetic graphene oxide(MGO) to improve its stability in physiological solution, so as to improve the screening efficiency to active components of traditional Chinese medicine(TCM). With this strategy, the constructed PEGylated MGO(PMGO) could keep stable at least 10 days, thus improving the CM coating efficiency. Meanwhile, by taking advantage of the inherent ability of He La cell membrane(HM) to interact with specific ligands, HM-camouflaged PMGO showed satisfied adsorption capacity(116.2 mg/g) and selectivity. Finally, three potential active components, byakangelicol, imperatorin,and isoimperatorin, were screened from Angelica dahurica, whose potential antiproliferative activity were further validated by pharmacological studies. These results demonstrated that the purposeful surfaceengineering is a promising strategy for the design of efficient CM biomimetic nanomaterials, which will promote the development of active components screening in TCM.

Chemotherapy in conjunction with traditional Chinese medicine for survival of elderly patients with advanced non-small-cell lung cancer:protocol for a randomized double-blind controlled trial

BACKGROUND： Traditional Chinese medicine （TCM） is considered an important complementary therapy with beneficial effects for cancer patients. Elderly patients with non-small-cell lung cancer （NSCLC） are a complex patient group with increasing co-morbidity and shrinking physiological reserve, and may derive substantial benefit from the supportive aspects of TCM Researchers from Shanghai Longhua Hospital found that qi and yin deficiency is a common syndrome in patients with stage III or IV lung cancer. This project was designed to study the combination of single-agent chemotherapy with TCM methods of benefiting qi and yin in elderly patients with advanced NSCLC. METHODS AND DESIGN： This is a double-blind controlled, multi-center, and prospective study with randomly selected participants from elderly NSCLC patients in China. Seventy-six patients who meet the inclusion criteria will be allocated into two groups, which will receive treatments of 3-week single-agent chemotherapy with TCM or placebo for four cycles Progression-free survival （PFS） is the primary end point, and the secondary end points are overall survival, objective response rate, time-to-progression, and quality of life （EORTC QLQ-LC43, and TCM syndrome score） Meanwhile, other end points such as toxicity, side effects and safety of the treatments will be assessed. DISCUSSION： Results from this study may provide evidence on the effectiveness, and parameters for the usage of single-agent chemotherapy combined with or without TCM on PFS of elderly patients with NSCLC.

Progress in diagnosis and treatment of small cell lung cancer with integrated traditional Chinese and Western medicine

Lung cancer is one of the most common major diseases that seriously threaten human health,lung cancer includes small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).Although patients with SCLC account for about 20%of the total number of patients with lung cancer,the mortality rate is much higher than that of patients with NSCLC.Integrated traditional Chinese and Western medicine has obvious advantages in the treatment of patients with SCLC.According to the relevant literature reports on the treatment of SCLC in recent years,this article will summarize the research progress of integrated traditional Chinese and western medicine in the treatmentof SCLC from the aspects of traditional Chinese medicine(TCM)combined with surgery,chemotherapy,radiotherapy,and molecular targeted therapy.

Progress in research of single-flavor Chinese medicine extracts in treatment of non-small cell lung cancer

Lung cancer is one of the diseases with high morbidity and mortality,and non-small cell lung cancer accounts for a large proportion of it.Traditional Chinese medicine has a long history in treating tumors.More and more researchers have paid more attention to it.At present,many studies have proved that traditional Chinese medicine extracts such as astragalus,turmeric,ginseng,coix seed,and gallbladder can inhibit tumor cell proliferation,and have synergistic effects when used in combination with radiotherapy and chemotherapy Its mechanism of action is mostly related to regulating the cell cycle,inhibiting the expression of cancer-related genes,and inducing apoptosis.It is believed that with the development of purification technology,Chinese medicine extracts will play a greater role.

Organic anion transporter 1 and 3 contribute to traditional Chinese medicine-induced nephrotoxicity

With the internationally growing popularity of traditional Chinese medicine(TCM), TCM-induced nephropathy has attracted public attention. Minimizing this toxicity is an important issue for future research. Typical nephrotoxic TCM drugs such as Aristolochic acid, Tripterygium wilfordii Hook. f, Rheum officinale Baill, and cinnabar mainly damage renal proximal tubules or cause interstitial nephritis. Transporters in renal proximal tubule are believed to be critical in the disposition of xenobiotics. In this review, we provide information on the alteration of renal transporters by nephrotoxic TCMs, which may be helpful for understanding the nephrotoxic mechanism of TCMs and reducing adverse effects. Studies have proven that when administering nephrotoxic TCMs, the expression or function of renal transporters is altered, especially organic anion transporter 1 and 3. The alteration of these transporters may enhance the accumulation of toxic drugs or the dysfunction of endogenous toxins and subsequently sensitize the kidney to injury.Transporters-related drug combination and clinical biomarkers supervision to avoid the risk of future toxicity are proposed.

Targeting immunometabolism by active ingredients derived from traditional Chinese medicines for treatment of rheumatoid arthritis

Rheumatoid arthritis(RA),the most common inflammatory arthropathy word wild,is a systemic autoimmune disease that mainly affects the synovium of joints with a high disability rate.Metabolic misregulation has emerged as a fundamental pathogenesis of RA linked to immune cell dysfunction,while targeting immunometabolism provides a new and effective approach to regulate the immune responses and thus alleviate the symptom of RA.Recently,natural active compounds from traditional Chinese medicines(TCMs)have potential therapeutic effects on RA and regulating immunometabolism.In this review,in addition to updating the connection between cellular metabolism and cell function in immune cells of RA,we summarized that the anti-inflammatory mechanisms of the potential natural compounds from TCM by targeting metabolic reprogramming of immune cells,and discusses them as a rich resource for providing the new potential paradigm for the treatment of RA.

Effect of Yang-activating and stasis-eliminating decoction from Traditional Chinese Medicine on intestinal mucosal permeability in rats with ulcerative colitis induced by dextran sulfate sodium

OBJECTIVE: To evaluate the effect of decoction prepared with Yang-activating and stasis-eliminating medicinals from Traditional Chinese Medicine(TCM) on the intestinal mucosal permeability in rats with ulcerative colitis induced by dextran sulfate sodium(DSS).METHODS: Totally 55 male Wistar rats(body weight of 170-190 g) were randomly divided into the blank group(n = 10) and the model duplication group(n = 45). The blank group was not intervened, while the other was modeled with 5% dextran sulfate sodium by gavaging in a dosage of4 m L per day to induce ulcerative colitis, a total of7 days. Then, the model rats were divided into model blank group, mesalazine group and TCM group,and each group was consisted of 15 rats. They were given retention enema 10 min with normal saline,mesalazine enema(0.036 g/m L), and Yang-activating and stasis-eliminating decoction [0.54 g/m L of a decoction boiled by Puhuang(Pollen Typhae), Xiebai(Bulbus Allii Macrostemonis) and Wulingzhi(Faeces Trogopteri)] for 10 days respectively. Afterwards,all of the rats were evaluated by disease activity index(DAI), histological changes of distal colon, expression of occludin protein and ultrastructure of intestinal epithelial cells. Furthermore, ratio of lactulose to mannitol(L/M) discharged in urine was evaluated.RESULTS: Comparing the results between TCM and model control groups, scores of DAI and histological lesions decreased significantly(P = 0.000 <0.01), ultrastructures of intestinal epithelial cells and tight junctions were more complete. The expression of occludin protein(P = 0.001 < 0.01) increased while the L/M value decreased significantly(P = 0.000 < 0.01) in TCM group. There was no statistical difference between the TCM and mesalazine groups in results of each item(P > 0.05).CONCLUSION: The decoction prepared with Yang-activating and stasis-eliminating TCM medicianls can restore intestinal mucosal epithelial cells and tight junctions the model rats with ulcerative colitis; it can reduce histological lesions and protect the permeability of intestinal mucosa barrier in the rats as well.

Celastrol activates caspase-3/GSDME-dependent pyroptosis in tumor cells by inducing endoplasmic reticulum stress Author links open overlay panel

Objective:To investigate the pyroptosis-inducing effects of celastrol on tumor cells and to explore the potential mechanisms involved,specifically focusing on the role of the caspase-3/gasdermin E(GSDME)signaling pathway and the impact of endoplasmic reticulum(ER)stress and autophagy.Methods: Necrostatin-1(Nec-1),lactate dehydrogenase release(LDH)assay,and Hoechst/propidium iodide(PI)double staining were employed to validate the mode of cell death.Western blot was used to detect the cleavage of GSDME and the expression of light chain 3(LC3)and BIP.Results: Celastrol induced cell swelling with large bubbles,which is consistent with the pyroptotic phenotype.Moreover,treatment with celastrol induced GSDME cleavage,indicating the activation of GSDME-mediated pyroptosis.GSDME knockout via CRISPR/Cas9 blocked the pyroptotic morphology of celastrol in HeLa cells.In addition,cleavage of GSDME was attenuated by a specific caspase-3 inhibitor in celastrol-treated cells,suggesting that GSDME activation was induced by caspase-3.Mechanistically,celastrol induced endoplasmic reticulum(ER)stress and autophagy in HeLa cells,and other ER stress inducers produced effects consistent with those of celastrol.Conclusion: These findings suggest that celastrol triggers caspase-3/GSDME-dependent pyroptosis via activation of ER stress,which may shed light on the potential antitumor clinical applications of celastrol.

Anti-tumor and Phenotypic Regulation Effect of Matrine on Dendritic Cells through Regulating TLRs Pathway

Objective To investigate the effects of matrine on antigen presentation of dendritic cells(DCs),and to explore the pharmacological mechanism of matrine on anti-tumor effect.Methods Different concentrations(0,1,2,4,8 and 16µg/mL)of matrine were co-cultured with DCs,the harvested DCs were co-cultured with antigens of Lewis lung cancer(LLC)cells,and then DCs and T cells were co-cultured to produce DCs-activated killer(DAK)cells,which have significant tumor-killing activity.The expression of cytokines,mRNA and protein of toll-like receptors(TLRs)in DCs were detected by enzyme linked immunosobent assay,polymerase chain reaction and Western blot,respectively.And the killing effect of DAK were measured by MTT assay.Results Matrine significantly increased the mRNA expression of TLR7,TLR8,myeloid differentiation factor 88(MyD88),tumor necrosis factor receptor-associated factor 6(TRAF-6)and IκB kinase(IKK),as well as the protein expression of TLR7 and TLR8,and up-regulated the levels of interleukin-12(IL-12),IL-6 and tumor necrosis factor-α(TNF-α),meanwhile,it also increased the expressions of MHC-II,CD54,CD80 and CD86 in DCs.DCs-activated effector T cells had significant tumor-killing activity.When the concentration of matrine was more than 4µg/mL,all indices had significant difference(P<0.01 or P<0.05).Conclusion Matrine plays an anti-tumor role by regulating TLRs signal transduction pathway,promoting the secretion of inflammatory cytokines and enhancing immune function.

Therapeutic potential of natural compounds from Chinese medicine in acute and subacute phases of ischemic stroke

Stroke is one of the leading causes of death and disability in adults worldwide,resulting in huge social and financial burdens.Extracts from herbs,especially those used in Chinese medicine,have emerged as new pharmaceuticals for stroke treatment.Here we review the evidence from preclinical studies investigating neuroprotective properties of Chinese medicinal compounds through their application in acute and subacute phases of ischemic stroke,and highlight potential mechanisms underlying their therapeutic effects.It is noteworthy that many herbal compounds have been shown to target multiple mechanisms and in combinations may exert synergistic effects on signaling pathways,thereby attenuating multiple aspects of ischemic pathology.We conclude the paper with a general discussion of the prospects for novel natural compound-based regimens against stroke.

How do Chinese medicines that tonify the kidney inhibit dopaminergic neuron apoptosis?

Wistar rats were intragastrical y perfused with Chinese medicines used for tonifying the kidney. These included 0.180 g/mL of Herba Epimedi （Epimedium）, Semen Cuscutae （Dodder Seed）, or Herba Cistanches （Desertliving Cistanche）, 0.04 mg/mL monoamine oxidase-B inhibitor selegiline, or distil ed water for 14 consecutive days to prepare drug-containing serum or blank serum. MES23.5 cells in the logarithmic phase were cultured in media supplemented with 15%drug-containing serum for 24 hours, fol owed by incubation in culture solution containing 100μmol/L H2O2 for 3 hours. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） and flow tometry results showed that al drug-containing serums improved the survival rate of H 2 O 2-injured MES23.5 cells, inhibited pro-apoptotic FasL and caspase-3 expression, promoted anti-apoptotic Bcl-2 expression. However, drug-containing serums had little influence on Fas expression in H 2 O 2-injured MES23.5 cells. Enzyme-linked immunosorbent assay results showed that serum containing Herba Cistanches or Herba Epimedi increased the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cellline-derived neurotrophic factor in injured MES23.5 cells;serum containing Semen Cuscutae only increased brain-derived neurotrophic factor expres-sion; while expression of the above neurotrophic factors remained the same in cells treated with serum containing selegiline. These findings indicate that Chinese medicines used to tonify the kid-ney can protect nerve cells by regulating the expression of apoptosis-related factors and neuro-trophic factors in MES23.5 cells.