A study on molecular mechanism of Xihuang pill in the treatment of glioblastoma based on network pharmacology and validation in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Xihuang pill has been utilized to treat cancer for more than three hundred years in China. The molecular mechanisms of Xihuang pill in treating glioblastoma remains unclear. AIM OF THE STUDY: This study aimed to explore the core molecular mechanisms of Xihuang pill in treating glioblastoma by an integrative pharmacology-based investigation. MATERIALS AND METHODS: The main active compounds of Xihuang pill were identified from TCMSP, BATMAN-TCM, TCMID and CNKI. Glioblastoma-related therapeutic targets were retrieved from GeneCards and UniProt. Subsequently, a protein-protein interaction (PPI) network analysis was constructed using STRING. GO and KEGG enrichment were performed to analyze the intersection targets between the active compounds of Xihuang pill and glioblastoma. Based on the above analysis, we built a CTP network. The in vitro and in vivo experiments were further performed to validate the crucial molecular targets of Xihuang pill for the treatment of glioblastoma. RESULTS: A total of sixty active compounds of Xihuang pill and ten potential targets related to glioblastoma were found. Based on topological analysis, fourteen ingredients were selected as the main active compounds, and MY11 might be the most important metabolite in Xihuang pill. PI3K/Akt signaling pathway and receptor tyrosine kinases were considered as crucial targets for Xihuang pill against glioblastoma through KEGG enrichment and CTP analysis. The present experiments indicated that Xihuang pill suppressed the activation of PI3K/Akt/mTOR signaling pathway in glioblastoma cells and mouse xenografts via modulating the expression of PTEN and Rheb proteins, the interaction between TSC2 and Rheb, and the production of PIP3. Meanwhile, after glioblastoma cells treatment with Xihuang pil, the release of IL-1ß, INF-γ was increased and the production of IL-10, TGF-ß1 was decreased in glioblastoma cells after incubated with Xihuang pill. In addition, the activation of the upstream positive modulators of PI3K/Akt/mTOR pathway including PDGF/PDGFR and FGF/FGFR signaling were down-regulated in glioblastoma cells and mouse xenografts after treatment with Xihuang pill. CONCLUSION: Taken together, Xihuang pill inhibiting glioblastoma cell growth might be partly through down-regulating the activation of PDGF/PDGFR or FGF/FGFR-PI3K/Akt/mTOR signaling axis and improving immuno-suppressive micro-environment of glioblastoma.

Ultra-weak photon emission as a potential tool for evaluating the therapeutic effect of traditional Chinese medicine in patients with type 2 diabetes.

Background: Traditional Chinese medicine (TCM) has been used to prevent and treat type 2 diabetes (T2DM) for thousands of years. The holistic view of TCM and the "multitarget" characteristics of Chinese medicine have unique advantages in the prevention and treatment of T2DM. TCM syndrome differentiation and treatment are effective for T2DM; however, currently, the therapeutic effect of TCM is generally evaluated by asking for patients' subjective feelings, or by checking the changes in relevant indicators. The main problems are that the patient's descriptions are unclear and subjective, and although the self-reported symptoms may have improved significantly, the relevant indicators are sometimes not obvious, which cannot truly reflect the therapeutic effect of TCM. Therefore, it is urgent to develop a novel, sensitive, and noninvasive method to quantitatively evaluate the therapeutic effect of TCM. Methods: In this study, ultra-weak photon emission (UPE) was measured at four sites of hands of T2DM patients with Qi-Yin deficiency before treatment and after 1 and 2 weeks of treatment with TCM. The UPE intensity and spectral distribution were calculated and analyzed using the results measured at these four sites. Spearman's correlation coefficient was used to quantify the correlation between the UPE parameters and the syndrome scores of TCM. Results: The UPE intensity of T2DM patients with Qi-Yin deficiency decreased gradually with the course of the treatment and was significantly lower than that before the treatment. The ratio of photon counts between the wavelength ranges of 495-550 nm and 550-610 nm after the treatment was higher than that before the treatment and negatively correlated with the corresponding syndrome scores so that the degree of symptoms improvement could be characterized by the ratio (495-550 nm/550-610 nm). Conclusions: The therapeutic effect of TCM in T2DM patients with Qi-Yin deficiency can be shown at the level of UPE. UPE is a potential and noninvasive tool for evaluating the therapeutic effect of TCM in patients with T2DM.

[Simulated processing with vinegar changes chemical structures of main terpenoids in Euphorbiae Ebracteolatae Radix].

This study aims to explore the chemical structure transformation mechanisms of the main terpenoids in the effective fraction of Euphorbiae Ebracteolatae Radix(EER) during the processing with vinegar. The terpenoids including ent-11α-hydroxyabicta-8(14),13(15)-dien-16,12-olide(HAO), jolkinolide B(JNB), fischeria A(FA), and eupractenoid A(EA) were heated at 160 ℃ with 6% acetic acid for 40 min, and then LC-MS/MS was employed to analyze the structural transformation rules of the terpenoids. Further, we analyzed the changes in the relative content of the four compounds and their transformation products in raw and vinegar processed EER to verify the transformation rules during the simulated processing with vinegar. In addition, JNB and FA were processed with single heating, heating with water or heating with acetic acid. We then employed HPLC to compare the content of these two terpenoids and their transformation products before and after processing, so as to investigate the effect of different processing methods on chemical structure transformation. The results showed that the lactone ring of the abietane-type diterpenoids HAO and JNB and the norditerpene lactone FA were opened by heating with acetic acid. When there were hydroxyl groups in the structures, terpenoids were esterized to esters and oxidized to form carbonyl groups. When there was epoxy ring in the structures, ring opening reaction was easy to occur. During the heating with acetic acid, the heterodimeric diterpenoid EA underwent the cleavage of ether bond to produce the rosane-type diterpenoid euphebracteolatin A(EHTA) and another abietane-type diterpenoid. The changes in the relative content of terpenoids and their transformation products in raw and vinegar-processed EER were basically consistent with those of simulated processing of components with vinegar. The HPLC results revealed that the effect of different simulated processing methods on structural transformation varied. Heating with acid can change JNB and FA into new components. Heating with water can also promote the structural transformation, with the efficiency obviously lower than that of heating with acid. Direct heating had no influence on the structure of JNB, while it significantly reduced the relative content of FA. The components treated with direct heating did not produce the products like those of the heating with acid. These results indicated that vinegar plays a key role in the structural transformation of diterpenoids during the processing of EER with vinegar. The structural transformation of diterpenoids in EER during the processing with vinegar may be the material basis for vinegar processed EER to reduce toxicity and preserve effect.

[Effect of different parts of Pinelliae Rhizoma Decoction against airway inflammation and analysis of effective components].

This study investigated the material basis and mechanism of Pinelliae Rhizoma Decoction in the treatment of airway inflammation. The cigarette smoke combined with lipopolysaccharide(LPS) was used to induce an airway inflammation model in mice. The expression levels of IL-6 and IL-8 in the bronchoalveolar lavage fluid(BALF) and the phosphorylation levels of p38 and IκB in the lungs of mice were taken as indexes to screen the effective extracts by system solvent extraction from Pinelliae Rhizoma Decoction(dichloromethane extract, ethyl acetate extract, n-butanol extract, etc.). Meanwhile, the human bronchial epithelial(16-HBE) cell model of cigarette smoke extract(CSE)-induced injury was established, and the mRNA expression levels of IL-6 and IL-8 and the phosphorylation levels of p38 and IκB proteins were also taken as indexes to evaluate the anti-inflammatory effect of different extracts of Pinelliae Rhizoma Decoction. The results showed that Pinelliae Rhizoma Decoction significantly antagonized airway inflammation in mice by down-regulating the expression levels of IL-6 and IL-8 in mice with airway inflammation and 16-HBE cells with CSE-induced injury and inhibiting the phosphorylation levels of p38 and IκB. The dichloromethane and ethyl acetate extracts of Pinelliae Rhizoma Decoction showed significant anti-inflammatory effects, while such effects of other extracts were not prominent. Furthermore, the database of Pinelliae Rhizoma composition was constructed, and the components in effective extracts were analyzed by HPLC-TOF-MS and Nano-LC-MS/MS. As revealed by the results, the compositions of the two effective extracts were similar with 36 common components. They were combined and then divided into Pinelliae Rhizoma alkaloids(PTAs) and Pinelliae Rhizoma non-alkaloids(PTNAs) by 732 cation-exchange resin. Further in vitro investigation confirmed the significant anti-inflammatory effect of PTNAs, while such effect of PTAs was not manifest. The MS analysis showed 172 peptides and 7 organic acids in PTNAs. The peptide content in PTNAs was 63.5% measured by quantitative analysis of BCA assay, and the organic acid content was 9.92% by potentiometric titration method. The findings of this study suggested that Pinelliae Rhizoma Decoction could antagonize airway inflammation in mice by inhibiting phosphorylation of p38 and IκB and blocking the activation of MAPK and NF-κB signaling pathways, and the effective components were related to the peptides and organic acids in PTNAs. The above results lay a foundation for the research on the mechanism and material basis of Pinelliae Rhizoma in antagonizing airway inflammation.

[Effect of Pinelliae Rhizoma processed with lime and licorice on toxic lectin protein].

The present study was aimed to investigate the effect of lime and licorice processing of Pinelliae Rhizoma on its toxic lectin protein and clarify the scientific detoxification connotation of lime and licorice processing of Pinelliae Rhizoma. Western blot was used to semi-quantitatively analyze the contents of lectin in Pinelliae Rhizoma and Pinelliae Rhizoma Praeparatum. Raw products and lectin were treated by soaking in licorice juice, lime solution or mixture solution of these two to investigate the different processing time on the content of toxic lectin protein. SDS-PAGE gel electrophoresis was used to analyze the changes of lectin protein bands in the solution and precipitates before and after processing. MALDI-TOF technology was used to qualitatively analyze and compare the protein molecular weight before and after processing. The results showed that the contents of lectin in Pinelliae Rhizoma and Pinelliae Rhizoma Praeparatum were 5.01% and 0.04% respectively, indicating that processing could significantly reduce the content of active lectin in raw products. The results also showed that the content of lectin in raw drugs decreased significantly after soaking in lime solution for one day or in licorice juice for three day, and the effect was greatest in mixture solution. Qualitative analysis showed that after being treated by soaking in lime solution, the lectin protein was decomposed into small peptide segments, while after being treated by soaking in licorice juice, the lectin protein was denatured and precipitated. The structure of lectin protein in Pinelliae Rhizoma was broken after being processed with licorice juice and lime solution, which significantly reduced the content of toxic lectinprotein. This is one of the detoxification mechanisms of Pinelliae Rhizoma processing.

[Effect of processing on toxic components lectin from four kinds of Araceae toxic medicines].

The study aimed to investigate the effect of processing on lectin protein in four toxic Chinese medicines tubers of Pinellia ternata,P. pedatisecta,Arisema heterophyllum and Typhonium giganteum. Western blot was used to semi-quantitatively analyze the content of lectin in the four kinds of toxic Chinese medicines and their different processed products. Raw products and lectin were treated by heating or soaking in ginger juice or alum solution. The effects of different excipients and the heating methods on lectin proteins were investigated. The results showed that the content of lectin in raw products of P. pedatisecta,P. ternata,A. heterophyllum,and T. giganteum were 7. 3%,4. 9%,2. 7%,2. 3%,respectively. And the content of lectin in Pinelliae Rhizoma praeparatum cum alumine was 0. 027%. Lectin was not detected in the Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine,Arisaematis Rhizma Praeparatum and Typhonii Rhizoma Praeparatum,which indicated that processing could significantly reduce the content of active lectin in raw products. The results also showed that with the prolongation of soaking and heating time,the content of lectin in raw products decreased gradually,while the content was almost unchanged when soaked in ginger juice alone. The effects of different excipients and heating on lectin were the same as those on raw products. Therefore,the method with alum soaking and heating can reduce the content of active lectin,which is the key to reduce the toxicity of toxic Chinese medicines. In this paper,Western blot was used to study the content of toxic protein in Araceae toxic Chinese medicines as an evaluation method of the processing degree.

Neuroprotective Effect of Dichloromethane Extraction From Piper nigrum L. and Piper longum L. on Permanent Focal Cerebral Ischemia Injury in Rats.

BACKGROUND: Piper nigrum L. and Piper longum L. consist a classic formula in traditional Chinese Hui medicine and are widely used in treatment of stroke. To examine the therapeutic effect of neuron injury after apoplexy, we used a permanent middle cerebral artery occlusion model in rats to investigate the effects of dichloromethane fraction (DF) of Piper nigrum L. and Piper longum L. MATERIALS AND METHODS: After subjecting the rats to permanent middle cerebral artery occlusion, DF (100 and 200 mg/kg) were administered for 14 days. Neurological deficits and the degree of cerebral tissue injury was detected by 2,3,5-Triphenyltetrazolium Chloride Staining Hematoxylin and eosin staining and Nissl staining. Postsynaptic density protein 95 (PSD-95), synapsin-I (syn-I), and α-synuclein (α-syn) were stained by immunohistochemistry. PSD-95, Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMK II), phosphorylated CaMK II (p-CaMK II), CaM, N-methyl D-aspartate receptor subtype 2B (NR2B) expression were detected by Western blot. Meanwhile, phytochemical profile of DF was determined through ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). RESULTS: DF alleviated neurological deficits and markedly prevented ischemia-induced cellular damage. Immunohistochemical micrographs revealed that PSD-95 and syn-I proteins increased, and α-syn presented reduced expression in brain samples from the sham group. Western blot analyses revealed that the model group exhibited a noticeable reduction in PSD-95, p-CaMK II, CaM, and NR2B. The DF-treated model group exhibited increased PSD-95, p-CaMK II, CaM, and NR2B. UPLC-Q-TOF/MS analysis revealed eight main components of DF, of which piperine accounted for the largest proportion.

[Effects of toxic fractions of Euphorbiae Ebracteolatae Radix on toxicity of mice intestinal tract and colon aquaporins expression level before and after vinegar processing].

To investigate the toxicity changes of Euphorbiae Ebracteolatae Radix (EER) before and after vinegar processing, toxic diterpenoids were concentrated with chloroform as extraction solvent from EER. Then the residue was extracted for non-chloroform extract with 95% ethanol and water after extraction with chloroform. The chloroform extraction of vinegar processed EER was prepared with the same method. The mice received the drug by oral administration. Moisture content in mice feces, duodenum and colon tissue, aquaporin AQP1, AQP3, AQP4 protein expression levels were assayed as the indexes to investigate the toxicity variation of chloroform fraction, non-chloroform fraction, as well as intestinal tract toxicity before and after vinegar processing of EER. The results showed that the chloroform fraction extracted from EER could significantly increase the moisture content in mice feces, duodenum and colon, and decrease AQP1 protein expression level, increase AQP3 and AQP4 protein expression levels in the colon. The intestinal toxicity of the chloroform extract was significantly higher than that of non-chloroform extract. The moisture content in mice feces, duodenum and colon was significantly decreased, and the AQPs protein expression tended to be normal in the colon after vinegar processing. The results showed that the chloroform fraction extracted from EER could lead to diarrhea, intestinal edema, and the intestinal toxicity action was associated with interfering AQPs protein expression and promoting intestinal fluid transport disorder in mice. Vinegar-processing could reduce intestinal toxicity of EER, so vinegar processing was considered to be the scientific processing method of EER.

Report: Regional variation in the chemical composition and antioxidant activity of Rosmarinus officinalis L. from China and the Mediterranean region.

Rosemary is an aromatic evergreen shrubby herb that is native to the Mediterranean region. This herb is now widely cultivated in many regions of the world. Rosemary is widely used in traditional Chinese medicines, foods, nutraceuticals and cosmetics. Hydro distilled essential oils, obtained from rosemary in China and the Mediterranean region, were analysed by gas chromatography-mass spectrometry (GC-MS). Thirty-seven compounds accounting for 94.97%-99.72% of the oils were identified. The majority of the compounds in the essential oils exhibited no significant differences (table 1 and fig. 1). The extracts were prepared with three solvents of different polarity (dichloromethane, ethyl acetate and aqueous). The ethyl acetate fractions exhibited the highest phenol content and were found to be significantly more active than the dichloromethane and aqueous fractions (fig. 2). Antioxidant activity (by DPPH radical scavenging, ferric ion reduction (FRAP) and thiobarbituric acid reactive substance (TBARS)) was also assessed. The ethyl acetate extracts of Yunnan had the highest amount of antioxidant capacity from China by DPPH and TBARS, with the lowest IC50 values being 0.0011 mg/ml, and 1.6611 mg/ml, respectively. In conclusion, the antioxidant activities of the essential oils and ethyl acetate extracts from rosemary obtained by three different testing methods revealed higher antioxidant activity from rosemary grown in China than in the Mediterranean region. These results suggested that Chinese rosemary should be widely used in food, traditional medicine, cosmetics and perfume products, as well as other chemical industries.

[Intestinal toxicity of different processed products of Crotonis Fructus and effect of processing on fatty oil and total protein].

To study the effect of different processes of Crotonis Fructus on fatty oil, total protein and intestinal toxicity, three kinds of processed products (heat Crotonis Semen Pulveratum, non-heat Crotonis Semen Pulveratum and diluted Crotonis Semen Pulveratum) were prepared. Mice were orally given Crotonis Fructus. The content of DAO and D-lactic acid in the serum were measured by ELISA to investigate the change of intestinal permeability in mice. Western blot was used to determine the expressions of tight junction proteins (occludin, claudin-1) in different intestinal tract, so as to observe the effect of Crotonis Fructus and its processed products on intestinal epithelial barrier. These results showed that Crotonis Fructus could significantly increase the intestinal permeability and reduce the expression of tight junction proteins in duodenum and jejunum, but with little impact on the ileum and colon. The intestinal permeability and the expression of tight junction proteins became normal after processing. However, the order of the toxicity of Crotonis Semen Pulveratum from high to low was non-heat Crotonis Semen Pulveratum > diluted Crotonis Semen Pulveratum≈4heat Crotonis Semen Pulveratum. According to the results of composition, the composition of fatty oil did not change during the processing, but the content and composition of total protein in Crotonis Semen Pulveratum changed significantly. The order of total protein content from high to low was that non-heat Crotonis Semen Pulveratum > heat Crotonis Semen Pulveratum > diluted Crotonis Semen Pulveratum. The molecular weight distribution of the total protein bands of non-heat Crotonis Semen Pulveratum and diluted Crotonis Semen Pulveratum was consistent, but the composition of total protein of heat Crotonis Semen Pulveratum significantly changed as evidenced by decreased and thin some stripes. This indicated that heating and dilution could reduce the content of total protein, and heating could cause partial protein denaturation and inactivation. In conclusion, both dilution and heating can reduce the toxicity of Crotonis Fructus, but the heating shows a more significant attenuation effect, indicating that heating is the key step in Crotonis Semen Pulveratum preparation.

Anti-inflammatory activity and chemical composition of dichloromethane extract from Piper nigrum and P. longum on permanent focal cerebral ischemia injury in rats

Abstract White pepper (Piper nigrum L.) and long pepper (Piper longum L.) belong to family Piperaceae and are commonly used as household spices and traditional medicine worldwide, specifically in China and Southeast Asia. In Traditional Chinese Hui Medicine, these herbs are widely used for treatment of stroke. Our present study investigated effects of these herbs on inflammation in rat model with cerebral ischemia. After subjecting the rats to permanent middle cerebral artery occlusion (pMCAO) for 6 h, at doses of 100 and 200 mg/kg, dichloromethane fraction from white pepper and long pepper, respectively, was intragastrically administered once a day for seven consecutive days. Cerebral cortical and hippocampal tissues were collected after seven days. Superoxide dismutase, malonaldehyde, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 were measured by spectrophotometer. Phytochemical profile of dichloromethane fraction was determined through HPLC. Dichloromethane fraction exhibited anti-inflammatory activity by suppressing expression or production of IL-1β, IL-6, and TNF-α. By contrast, dichloromethane fraction showed activity against pMCAO injury by reducing oxygen-free radicals through increased superoxide dismutase activity and decreased malonaldehyde level. HPLC analysis revealed piperine as major component of dichloromethane fraction. These results show that dichloromethane fraction provides protection against cerebral ischemia. The possible mechanism is related to anti-inflammatory activity and reduction in oxygen-free radicals.

Alisma orientale: Ethnopharmacology, Phytochemistry and Pharmacology of an Important Traditional Chinese Medicine.

Alisma orientale (Sam.) Juzep. (Alismataceae) is a traditional and famous Chinese medicinal herb. Its rhizomes, which possess versatile bioactivities, are commonly used to treat oliguria, edema, gonorrhea with turbid urine, leukorrhea, diarrhea and dizziness. Approximately 120 compounds have been isolated from A. orientale. Terpenoids have been identified as A. orientale's characteristic constituents, which include protostane triterpenoids and guaiane sesquiterpenoids. The traditional medical uses of A. orientale in TCM have been evaluated in modern pharmacological studies, which have shown that A. orientale and its active constituents exhibit a wide range of bioactivities, such as diuretic, anti-urolithiatic, antinephritic, anti-atherosclerotic, immunomodulatory, and hepatoprotective activities. The medicinal potential of A. orientale makes it an ideal candidate for new drug development. Further studies are still required to identify its bioactive constituents, and elucidate the structure-activity relationship and detailed mechanisms of action. Additionally, the use of the other medicinal parts of A. orientale may reduce resource waste and afford novel secondary metabolites.

Anti-inflammatory and antinociceptive activities of non-alkaloids fractions from Aconitum flavum in vivo

Aconitum flavum Hand.-Mazz., Ranunculaceae, has been used for the treatment of rheumatism, traumatic injury in folk and clinical medicine, but the alkaloids has high toxicity. This study was designed to investigate the acute toxicity, anti-inflammatory and antinociceptive activities of non-alkaloids fractions from A. flavum in rodents. The anti-inflammatory activity was evaluated by inflammatory models of dimethylbenzene-induced ear vasodilatation and acetic acid-induced capillary permeability enhancement test in mice and carrageenan-induced paw edema in rats whereas the antinociceptive activity was evaluated using acetic acid-induced writhes, hot plate test and formalin test in mice. The result showed that the LD50 value of BtOH and EtOAc fractions could not be determined as no lethality was observed up to 40 g/kg (p.o.) in mice. BtOH fraction significantly decreased the dimethylbenzene-induced ear vasodilatation, carrageenan-induced paw edema and acetic acid-induced capillary permeability. EtOAc fraction only significantly attenuated paw edema and capillary permeability at the dose of 500 mg/kg. In antinociceptive test, BtOH and EtOAc fractions significantly reduced the writhing number evoked by acetic acid injection and the licking time in both phases of the formalin test. Meanwhile BtOH and EtOAc fractions had significant effect on hot plate test after 90 min. Our data indicate that the BtOH and EtOAc fractions of NAF are no toxicity. BtOH and EtOAc fractions not only inhibit inflammatory and peripheral inflammatory pain but also have central antinociceptive effect.

Anti-inflammatory, antinociceptive activity of an essential oil recipe consisting of the supercritical fluid CO2 extract of white pepper, long pepper, cinnamon, saffron and myrrh in vivo.

This study was designed to investigate the anti-inflammatory and antinociceptive activities of essential oil recipe (OR) in rodents. The anti-inflammatory activity was evaluated by inflammatory models of dimethylbenzene (DMB)-induced ear vasodilatation and acetic acid-induced capillary permeability enhancement in mice whereas the antinociceptive activity was evaluated using acetic acid-induced writhes and hot plate test methods in mice. Additionally, the chemical composition of OR has been also analyzed by gas chromatography and mass spectrometry (GC/MS). 37 compounds, representing 74.42% of the total oil content, were identified. ß-Selinene (7.38%), aromadendrene (5.30%), ß-elemene (5.22%), cis-piperitol (5.21%), cis-ß-guaiene (4.67%), ylangene (3.70%), 3-heptadecene (3.55%), δ-cadinene (3%) and ß-cadinene (2.87%) were found to be the major constituents of the oil. Oral pretreatment with OR (62.5-1000 mg/kg) not only decreased the DMB-induced ear vasodilatation but also attenuated capillary permeability under acetic acid challenge in mice. OR significantly reduced the writhing number evoked by acetic acid injection. All test samples showed no significant analgesic activity on the hot plate pain threshold in mice. These data demonstrated that the OR inhibits inflammatory and peripheral inflammatory pain. These results may support the fact that the essential oil of traditional Hui prescription played a role in the inflammation of stroke.