Neurokinin-1 receptor antagonist aprepitant regulates autophagy and apoptosis via ROS/JNK in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) has a poor prognosis and limited treatment options. Aprepitant, a selective NK-1R antagonist, can inhibit the growth of various tumours in vitro and in vivo. However, it remains unclear whether aprepitant has cytotoxic effects on iCCA. METHODS: We measured the expression of SP/NK-1R in clinical samples of iCCA by immunohistochemistry. Then, we detected the cytotoxic effects of aprepitant on iCCA cells via MTT, EdU and colony formation assay. We constructed a subcutaneous xenograft model of BALB/c nude mice by using HCCC-9810 and RBE cell lines to explore the effects of aprepitant in vivo. To elucidate the potential mechanisms, we explored the pro-apoptotic effect of aprepitant by flow cytometric, western blotting, ROS detection and JC-1 staining. Furthermore, we detected the autophagic level of HCCC-9810 and RBE by western blotting, mRFP-eGFP-LC3 adenovirus transfection and electron microscope. RESULTS: SP/NK-1R is significantly expressed in iCCA. Aprepitant inhibited human iCCA xenograft growth and dose-dependently decreased the viability of RBE and HCCC-9810 cells. Aprepitant-induced mitochondria-dependent apoptosis through ROS/JNK pathway. Additionally, pretreatment with z-VAD-fmk partly reversed the effect of aprepitant on cell viability, while NAC completely attenuated the cytotoxic effects of aprepitant in vitro. Furthermore, we observed the dynamic changes of autophagosome in RBE and HCCC-9810 cells treated with aprepitant. CONCLUSION: SP/NK-1R signalling is significantly activated in iCCA and promotes the proliferation of iCCA cells. By contrast, aprepitant can induce autophagy and apoptosis in iCCA cells via ROS accumulation and subsequent activation of JNK.

How Does Disgust Affect Mpox Prevention? Examining the Underlying Mechanisms of Perceived Severity and Perceived Susceptibility Moderated by Stigma.

The study aims to examine the influencing mechanism of incidental exposure to Disgusting Graphics Information (DGI) about Monkeypox (Mpox) on the intention of prevention behavior. This study first investigates the components of disgust and then examines the mechanism of disgust's influence. The study uses a cross-sectional survey design among respondents who have been incidentally exposed to DGI about Mpox (N = 368). The results showed that disgust toward Mpox is the most effective component among other proposed ones (disgust toward graphics, information sources, and patients). Disgust not only positively influences prevention intention, but also indirectly influences prevention intention through perceived severity rather than perceived susceptibility. Moreover, moderated mediation was found, indicating that stigma toward patients prevents people from adopting preventive behaviors. Both theoretical and practical implications are discussed.

An Intelligent Vascular Disrupting Dendritic Nanodevice Incorporating Copper Sulfide Nanoparticles for Immune Modulation-Mediated Combination Tumor Therapy.

Development of intelligent nanoplatforms that can simultaneously target multiple factors associated with tumor growth and metastasis remains an extreme challenge. Here, an intelligent dendritic nanodevice incorporating both copper sulfide nanoparticles (CuS NPs) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA, a vascular disrupting agent) within the dendrimer internal cavities and surface modified with a targeting agent LyP-1 peptide is reported. The resulting generation 5 (G5) dendrimer-based nanodevice, known as G5-PEG-LyP-1-CuS-DMXAA NPs (GLCD NPs), possess good colloidal stability, pH-sensitive drug release kinetics, and high photothermal conversion efficiency (59.3%). These functional GLCD NPs exert a LyP-1-targeted killing effect on breast tumors by combining CuS-mediated photothermal therapy (PTT) and DMXAA-induced vascular disruption, while also triggering antitumor immune responses through PTT-induced immunogenic cell death and DMXAA-mediated immune regulation via M1 polarization of tumor-associated macrophages and dendritic cell maturation. In addition, with the LyP-1-mediated proapoptotic activity, the GLCD NPs can specifically kill tumor lymphatic endothelial cells. The simultaneous disruption of tumor blood vessels and lymphatic vessels cuts off the two main pathways of tumor metastasis, which plays a two-pronged role in inhibiting lung metastasis of the breast cancer model. Thus, the developed GLCD NPs represent an advanced intelligent nanoformulation for immune modulation-mediated combination tumor therapy with potential for clinical translations.

Aprepitant inhibits the development and metastasis of gallbladder cancer via ROS and MAPK activation.

BACKGROUND: Aprepitant, as a neurokinin-1 receptor (NK-1R) antagonist, originally applied for curing chemotherapy-induced nausea and vomiting, has been reported to have significant antitumor effect on several malignant tumors. However, the effect of aprepitant on gallbladder cancer (GBC) is not clear yet. This study aimed to investigate the anti-tumor activity of aprepitant on GBC and the potential mechanisms. METHODS: The NK-1R expression of gallbladder cancer cells were examined by immunofluorescence. MTT assay, wound healing and transwell migration assay were applied to detect the effect of aprepitant on cell proliferation, migration and invasion. Flow cytometry was used to detect the apoptosis rate. The effects of aprepitant on the expressions of cytokine were examined by real-time quantitative PCR and MAPK activation were detected via immunofluorescence and western blotting. Besides, xenograft model was established to investigate the effect of aprepitant in vivo. RESULTS: Our results indicated that NK-1R was markedly expressed in gallbladder cancer cells and aprepitant effectively inhibited the proliferation, migration and invasion. Furthermore, the apoptosis, ROS and inflammation response were significantly boosted by aprepitant in GBC. Aprepitant induced NF-κB p65 nuclear translocationin and increased the expressions of p-P65, p-Akt, p-JNK, p-ERK and p-P38, as well as the mRNA levels of inflammatory cytokines IL-1ß, IL-6 and TNF-α. Consistently, aprepitant suppressed the growth of GBC in xenograft mice model. CONCLUSION: Our study demonstrated that aprepitant could inhibit the development of gallbladder cancer via inducing ROS and MAPK activation, which suggested that aprepitant may become a promising therapeutic drug against GBC.

A tumor microenvironment-responsive poly(amidoamine) dendrimer nanoplatform for hypoxia-responsive chemo/chemodynamic therapy.

BACKGROUND: Chemodynamic therapy is a promising cancer treatment with specific therapeutic effect at tumor sites, as toxic hydroxyl radical (·OH) could only be generated by Fenton or Fenton-like reaction in the tumor microenvironment (TME) with low pH and high level of endogenous hydrogen peroxide. However, the low concentration of catalytic metal ions, excessive glutathione (GSH) and aggressive hypoxia at tumor site seriously restrict the curative outcomes of conventional chemodynamic therapy. RESULTS: In this study, polyethylene glycol-phenylboronic acid (PEG-PBA)-modified generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers were synthesized as a targeted nanocarrier to chelate Cu(II) and then encapsulate hypoxia-sensitive drug tirapazamine (TPZ) by the formation of hydrophobic Cu(II)/TPZ complex for hypoxia-enhanced chemo/chemodynamic therapy. The formed G5.NHAc-PEG-PBA@Cu(II)/TPZ (GPPCT) nanoplatform has good stability and hemocompatibility, and could release Cu(II) ions and TPZ quickly in weakly acidic tumor sites via pH-sensitive dissociation of Cu(II)/TPZ. In vitro experiments showed that the GPPCT nanoplatforms can efficiently target murine breast cancer cells (4T1) cells overexpressing sialic acid residues, and show a significantly enhanced inhibitory effect on hypoxic cells by the activation of TPZ. The excessive GSH in tumors could be depleted by the reduction of Cu(II) to Cu(I), and abundant of toxic ·OH would be generated in tumor cells by Fenton reaction for chemodynamic therapy. In vivo experiments demonstrated that the GPPCT nanoplatform could specifically accumulate at tumors, effectively inhibit the growth and metastasis of tumors by the combination of CDT and chemotherapy, and be metabolized with no systemic toxicity. CONCLUSIONS: The targeted GPPCT nanoplatform may represent an effective model for the synergistic inhibition of different tumor types by hypoxia-enhanced chemo/chemodynamic therapy.

Doxorubicin Encapsulated in TPGS-Modified 2D-Nanodisks Overcomes Multidrug Resistance.

Multidrug resistance (MDR) is regarded as a main obstacle for effective chemotherapy, and P-glycoprotein (P-gp)-mediated drug efflux has been demonstrated to be the key factor responsible for MDR. In this study, a novel pH-responsive hybrid drug delivery system was developed by conjugating d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), a kind of P-gp inhibitor, on the surface of laponite nanodisks to overcome MDR. The prepared LM-TPGS display excellent colloidal stability, a high encapsulation efficiency of doxorubicin (DOX), and a pH-responsive drug release profile. In vitro experiments verified that LM-TPGS/DOX could exhibit significantly enhanced therapeutic efficacy in treating DOX-resistant breast cancer cells (MCF-7/ADR) through inhibiting the activity of P-gp-mediated drug efflux and effectively accumulating DOX within cancer cells. In vivo results revealed that LM-TPGS/DOX outstandingly suppressed MCF-7/ADR tumors with low side effects. Therefore, the high drug payload, enhanced inhibition efficacy to drug-resistant cells, and low side effects make the LM-TPGS/DOX a promising nanoplatform to reverse MDR for effective chemotherapy.

Poly(amidoamine) Dendrimer-Coordinated Copper(II) Complexes as a Theranostic Nanoplatform for the Radiotherapy-Enhanced Magnetic Resonance Imaging and Chemotherapy of Tumors and Tumor Metastasis.

The development of a powerful nanoplatform to realize the simultaneous therapy and diagnosis of cancer using a similar element for theranostics remains a critical challenge. Herein, we report such a theranostic nanoplatform based on pyridine (Pyr)-functionalized generation 5 (G5) poly(amidoamine) dendrimers complexed with copper(II) (Cu(II)) for radiotherapy-enhanced T1-weighted magnetic resonance (MR) imaging and the synergistic radio-chemotherapy of both tumors and tumor metastasis. In this study, amine-terminated G5 dendrimers were covalently linked with 2-pyridinecarboxylic acid, acetylated to neutralize their remaining terminal amines, and complexed with Cu(II) through both the internal tertiary amines and the surface Pyr groups to form the G5.NHAc-Pyr/Cu(II) complexes. We show that the complexes are able to inhibit the proliferation of different cancer cell lines with half-maximal inhibitory concentrations ranging from 4 to 10 µM and induce significant cancer cell apoptosis. Due to the presence of Cu(II), the G5.NHAc-Pyr/Cu(II) complexes display an r1 relaxivity of 0.7024 mM-1 s-1, enabling effective in vivo MR imaging of tumor xenografts and lung metastatic nodules. Further, under radiotherapy (RT) conditions, the tumor MR imaging sensitivity can be significantly enhanced, and the G5.NHAc-Pyr/Cu(II) complexes enable the enhanced chemotherapy of both a xenografted tumor model and a blood-vessel metastasis model. With the demonstrated theranostic potential of the dendrimer-Cu(II) nanocomplexes without additional agents or elements for RT-enhanced MR imaging and chemotherapy of tumor and tumor metastasis, this novel Cu(II)-based nanohybrids may hold great promise for the theranostics of different cancer types and metastases.

Quantitative analysis of amino acids in human and bovine colostrum milk samples through iTRAQ labeling.

BACKGROUND: The types and quantity of proteins vary widely between bovine and human milk, with corresponding differences in free and hydrolytic amino acids. In this study, the free and hydrolytic amino acids of bovine and human colostrum were for the first time qualitatively and quantitatively determined using isobaric tags for relative and absolute quantification technology combined with liquid chromatography tandem mass spectrometry detection. RESULTS: Total free amino acid content was 0.32 g L-1 and 0.63 g L-1 in bovine and human colostrum respectively, with free amino acid content in human colostrum twice that of bovine colostrum. However, total hydrolytic amino acid content was 4.2 g L-1 and 2.2 g L-1 in bovine and human colostrum respectively. We found that the hydrolytic amino acid content in bovine colostrum was higher than that in human colostrum; however, the amount of free amino acids and the overall amino acid content in human colostrum were respectively substantially higher and more varied than in bovine colostrum. CONCLUSION: Our findings revealed differences between bovine and human colostrum, with these data providing the basis for further research into amino acid metabolomics and infant formula. © 2018 Society of Chemical Industry.

Depth-Profiling the Nuclease Stability and the Gene Silencing Efficacy of Brush-Architectured Poly(ethylene glycol)-DNA Conjugates.

PEGylation of an oligonucleotide using a brush polymer can improve its biopharmaceutical characteristics, including enzymatic stability and biodistribution. Herein, we quantitatively explore the nuclease accessibility of the nucleic acid as a function of "depth" toward the backbone of the brush polymer. It is found that protein accessibility decreases as the nucleotide is located closer to the backbone. Thus, by moving the conjugation point from the terminus of the nucleic acid strand to an internal position, much smaller brushes can be used to achieve the same level of steric shielding. This finding also makes it possible to assess antisense gene regulation efficiency of these brush-DNA conjugates as a function of their nuclease stability.

Modulating the Cellular Immune Response of Oligonucleotides by Brush Polymer-Assisted Compaction.

Unwanted stimulation of the innate immune system by foreign nucleic acids has been one of the major barriers preventing bioactive sequences from reaching market. Foreign nucleic acids can be recognized by multiple pattern recognition receptors (PRRs), which trigger a signaling cascade to activate host defense systems, leading to a range of side effects. This study demonstrates that polyethylene glycol (PEG)-modified DNA strands can greatly reduce the activation of the innate immune system, and the extent of reduction is dependent upon polymer architecture. Highly branched brushes with long PEG side chains achieve the best suppression by blocking PRR interactions via a local steric effect. Interestingly, the brush polymer creates little barrier toward DNA-DNA interaction. Quantification of inflammatory cytokines in both mRNA and protein levels as well as the extent of cellular uptake shows a direct correlation between steric congestion and reduction of cellular immune response. These results suggest that the brush architecture offers unique advantages for PEGylating oligonucleotides in the context of minimizing unwanted immune system activation.

Enhanced simultaneous overlap extension-PCR by gold nanoparticles.

Methods to fuse multiple DNA fragments are extremely useful in synthetic biology and protein engineering. Here, we report a gold nanoparticle-mediated simultaneous overlap extension-PCR (AuNP-mediated SOE-PCR) method that enables the fusion of multiple DNA fragments simultaneously with their amplification in a single reaction using typical PCR conditions. Using greater concentrations of rTaq DNA polymerase and AuNPs significantly improves the performance of SOE-PCR especially for the fusion of more than three DNA fragments. We show that up to six lambda DNA fragments can be simultaneously fused by AuNP-mediated SOE-PCR.

Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

Effect of PEG Architecture on the Hybridization Thermodynamics and Protein Accessibility of PEGylated Oligonucleotides.

PEGylation is an attractive approach to modifying oligonucleotides intended for therapeutic purposes. PEG conjugation reduces protein interactions with the oligonucleotide, and helps to overcome their intrinsic biopharmaceutical shortcomings, such as poor enzymatic stability, rapid body clearance, and unwanted immunostimulation. However, the effect of PEG architecture and the manner in which the PEG component interferes with the hybridization of the oligonucleotide remain poorly understood. In this study, we systematically compare the hybridization thermodynamics and protein accessibility of several DNA conjugates involving linear, Y-shaped, and brush-type PEG. It is found that PEGylated DNA experiences two opposing effects: local excluded volume effect and chemical interactions, the strengths of which are architecture-dependent. Notably, the brush architecture is able to offer significantly greater protein shielding capacity than its linear or Y-shaped counterparts, while maintaining nearly identical free energy for DNA hybridization compared with free DNA.

Effective Antisense Gene Regulation via Noncationic, Polyethylene Glycol Brushes.

Negatively charged nucleic acids are often complexed with polycationic transfection agents before delivery. Herein, we demonstrate that a noncationic, biocompatible polymer, polyethylene glycol, can be used as a transfection vector by forming a brush polymer-DNA conjugate. The brush architecture provides embedded DNA strands with enhanced nuclease stability and improved cell uptake. Because of the biologically benign nature of the polymer component, no cytotoxicity was observed. This approach has the potential to address several long-lasting challenges in oligonucleotide therapeutics.

Poly(amidoamine) Dendrimers Modified with 1,2-Epoxyhexane or 1,2-Epoxydodecane for Enhanced Gene Delivery Applications.

We report a new non-viral gene delivery system based on hydrophobically modified poly(amidoamine) (PAMAM) dendrimers. In this study, the periphery of amine-terminated generation 5 (G5) PAMAM dendrimers was partially reacted with 1,2-epoxyhexane and 1,2-epoxydodecane, respectively. The formed hydrophobically modified G5 dendrimers (denoted as G5.NH2-C6 or G5.NH2-C12) were used to complex two different plasmid DNAs (pDNAs) encoding luciferase (Luc) and enhanced green fluorescent protein (EGFP), respectively for gene transfection studies. The polyplexes formed between vectors and pDNA were characterized by gel retardation assay, dynamic light scattering, and zeta potential measurements. We show that the G5.NH2-C6 and G5.NH2-C12 vectors are able to effectively compact the pDNA, allowing for highly efficient gene transfection into a model cell line (HeLa cells) as demonstrated by both Luc assay and confocal microscopic imaging of the EGFP expression. Under the studied N/P ratios (the molar ratio of primary amines of the dendrimers to phosphates in the pDNA backbone) at 2.5 or 5, the transfection efficiency of the dendrimer-based vectors followed the order of G5.NH2-C12 > G5.NH2-C6 > G5.NH2. This enhanced gene transfection capacity is believed to be associated with the enhanced hydrophobic interaction between the vector/pDNA complexes and the relatively hydrophobic cell membranes. The developed hydrophobically modified dendrimers may be used as a promising non-viral vector for enhanced gene delivery applications.

Underwater versus conventional endoscopic mucosal resection for ≥10 mm sessile or flat colorectal polyps: A systematic review and meta-analysis.

BACKGROUND AND AIM: Underwater endoscopic mucosal resection (UEMR) has been an emerging substitute for conventional EMR (CEMR). This systematic review and meta-analysis aimed at comparing the efficiency and safety of the two techniques for removing ≥10 mm sessile or flat colorectal polyps. METHODS: PubMed, Cochrane Library and Embase databases were searched up to February 2023 to identify eligible studies that compared the outcomes of UEMR and CEMR. This meta-analysis was conducted on the en bloc resection rate, R0 resection rate, complete resection rate, procedure time, adverse events rate and recurrence rate. RESULTS: Nine studies involving 1,727 colorectal polyps were included: 881 were removed by UEMR, and 846 were removed by CEMR. UEMR was associated with a significant increase in en bloc resection rate [Odds ratio(OR) 1.69, 95% confidence interval(CI) 1.36-2.10, p<0.00001, I2 = 33%], R0 resection rate(OR 1.52, 95%CI 1.14-2.03, p = 0.004, I2 = 31%) and complete resection rate(OR 1.67, 95%CI 1.06-2.62, p = 0.03, I2 = 0%) as well as a significant reduction in procedure time(MD ‒4.27, 95%CI ‒7.41 to ‒1.13, p = 0.008, I2 = 90%) and recurrence rate(OR 0.52, 95%CI 0.33-0.83, p = 0.006, I2 = 6%). Both techniques were comparable in adverse events rate. CONCLUSION: UEMR can be a safe and efficient substitute for CEMR in removing ≥10 mm sessile or flat colorectal polyps. More studies verifying the advantages of UEMR over CEMR are needed to promote its application.

Characterization and Comparison Analysis of Milk Fat Globule Membrane Proteins between Human and Porcine Milk.

This study aimed to explore the differences in milk fat globule membrane (MFGM) proteins between human milk (HM) and porcine milk (PM) using a label-free quantitative proteomic approach. A total of 3920 and 4001 MFGM proteins were identified between PM and HM, respectively. Among them, 3520 common MFGM proteins were detected, including 956 significant differentially expressed MFGM proteins (DEPs). Gene ontology (GO) enrichment analysis showed that the DEPs were highly enriched in the lipid metabolic process and intrinsic component of membrane. Kyoto Encyclopedia of Genes and Genomes pathways suggested that protein processing in the endoplasmic reticulum was the most highly enriched pathway, followed by peroxisome, complement, and coagulation cascades. This study reflects the difference in the composition of MFGM proteins between HM and PM and provides a scientific and systematic reference for the development of MFGM protein nutrition.

Comparative Site-Specific O-Glycosylation Analysis of the Milk Fat Globule Membrane Proteome in Donkey Colostrum and Mature Milk.

Donkey milk fat globule membrane (MFGM) proteins are a class of membrane-bound secreted proteins with broad-spectrum biofunctional activities; however, their site-specific O-glycosylation landscapes have not been systematically mapped. In this study, an in-depth MFGM O-glycoproteome profile of donkey milk during lactation was constructed based on an intact glycopeptide-centered, label-free glycoproteomics pipeline, with 2137 site-specific O-glycans from 1121 MFGM glycoproteins and 619 site-specific O-glycans from 217 MFGM glycoproteins identified in donkey colostrum and donkey mature milk, respectively. As lactation progressed, the number of site-specific O-glycans from three glycoproteins significantly increased, whereas that of 11 site-specific O-glycans from five glycoproteins significantly decreased. Furthermore, donkey MFGM O-glycoproteins with core-1 and core-2 core structures and Lewis and sialylated branch structures may be involved in regulating apoptosis. The findings of this study reveal the differences in the composition of donkey MFGM O-glycoproteins and their site-specific O-glycosylation modification dynamic change rules during lactation, providing a molecular basis for understanding the complexity and biological functions of donkey MFGM protein O-glycosylation.

Proteomic and phosphoproteomic reveal immune-related function of milk fat globule membrane in bovine milk of different lactation periods.

Information regarding protein expression and phosphorylation modifications in the bovine milk fat globule membrane is scarce, particularly throughout various lactation periods. This study employed a complete proteome and phosphoproteome between bovine colostrum and mature milk. A total of 11 proteins were seen in both protein expression and phosphorylation levels. There were 400 proteins identified in only protein expression, and 104 phosphoproteins identified in only phosphorylation levels. A total of 232 significant protein characteristics were identified within the proteome and significant phosphorylation sites within 86 phosphoproteins of the phosphoproteome. Biological activities and pathways primarily exhibited associations with the immune system. Simultaneously, a comprehensive analysis of proteins and phosphorylation sites using a multi-omics approach. Hence, the data we have obtained has the potential to expand our understanding of how the bovine milk fat globule membrane might be utilized as a beneficial component in dairy products.

Label-free-based proteomic analysis reveals differential whey proteins of porcine milk during lactation.

In this study, label-free proteomic technology was applied to analyze and compare the whey proteomes of porcine colostrum and mature milk. In total, 2993 and 2906 whey proteins were detected in porcine colostrum and mature milk, respectively. A total of 2745 common proteins were identified in the two milk samples, and 280 proteins were found to be significantly differentially expressed whey proteins in porcine milk. Gene Ontology analysis demonstrated that the differentially expressed whey proteins were primarily enriched in lipid homeostasis, oxidoreductase activity, and the collagen trimer. Kyoto Encyclopedia of Genes and Genomes analysis suggested that the phagosome and endocytosis were the crucial pathways. This study provides systematic and in-depth insight into the compositions and functional properties of whey proteins in porcine milk during different periods of lactation, which may be beneficial for the development of porcine whey proteins in the future.