[Application of discrete element method (DEM) in pharmaceutical process of solid traditional Chinese medicine preparations].

In recent years, the application of numerical simulation in the research and development(R&D) as well as the pharmaceutical processes of new drugs has expanded considerably. The discrete element method(DEM), an important approach among numerical simulation methods, offers an effective tool for the simulation of discontinuous media. Referring to the research progress of DEM and the formulation of solid traditional Chinese medicine(TCM) preparations in recent years, this paper summarizes and analyzes the application of DEM in the pharmaceutical processes of solid TCM preparations, and discusses the challenges of its application in these processes, in order to provide new methods and ideas for promoting the high-quality production of TCM preparations.

Discovery of Biphenyl Derivatives to Target Hsp70-Bim Protein-Protein Interaction in Chronic Myeloid Leukemia by Scaffold Hopping Strategy.

Hsp70-Bim protein-protein interaction (PPI) is the most recently identified specific target in chronic myeloid leukemia (CML) therapy. Herein, we developed a new class of Hsp70-Bim PPI inhibitors via scaffold hopping of S1g-10, the most potent Hsp70-Bim PPI inhibitor thus far. Through structure-activity relationship (SAR) study, we obtained a biphenyl scaffold compound JL-15 with a 5.6-fold improvement in Hsp70-Bim PPI suppression (Kd = 123 vs 688 nM) and a 4-fold improvement in water solubility (29.42 vs 7.19 µg/mL) compared to S1g-10. It maintains comparable apoptosis induction capability with S1g-10 against both TKI-sensitive and TKI-resistant CML cell lines in an Hsp70-Bim-dependent manner. Additionally, through SAR, 1H-15N TRSOY-NMR, and molecular docking, we revealed that Lys319 is a "hot spot" in the Hsp70-Bim PPI interface. Collectively, these results provide a novel chemical scaffold and structural insights for the rational design of Hsp70-Bim PPI inhibitors.

A Case of Life-Threatening Postpartum Necrotizing Pancreatitis Requiring Gastrocystostomy With Serial Pancreatic Necrosectomies.

We present a life-threatening case of postpartum acute necrotizing pancreatitis. The patient is a 37-year-old female with no past medical history who delivered a healthy baby boy via cesarean section. Twenty days later, she presented to the emergency department with acute onset of nausea, non-bloody vomiting, abdominal bloating, and epigastric pain radiating to the back. Less than 24 hours later, she progressed into septic shock despite aggressive resuscitation, requiring vasopressor support in the ICU. Initial CT imaging showed multiple patchy hypodensities throughout the pancreas consistent with severe necrotizing pancreatitis. Her hospitalization was further complicated by difficulty obtaining source control of her infection, Clostridium difficile, and nutritional deficiencies that resulted in gross anasarca. She was discharged from the hospital on day 59 after undergoing multiple percutaneous drain placements, IV antibiotics, and endoscopic gastrocystostomy with four pancreatic necrosectomies. Since discharge, the patient has required readmission twice for complications from her pancreatitis.

Genome Editing VEGFA Prevents Corneal Neovascularization In Vivo.

Corneal neovascularization (CNV) is a common clinical finding seen in a range of eye diseases. Current therapeutic approaches to treat corneal angiogenesis, in which vascular endothelial growth factor (VEGF) A plays a central role, can cause a variety of adverse side effects. The technology of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 can edit VEGFA gene to suppress its expression. CRISPR offers a novel opportunity to treat CNV. This study shows that depletion of VEGFA with a novel CRISPR/Cas9 system inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, subconjunctival injection of this dual AAV-SpCas9/sgRNA-VEGFA system is demonstrated which blocks suture-induced expression of VEGFA, CD31, and α-smooth muscle actin as well as corneal neovascularization in mice. This study has established a strong foundation for the treatment of corneal neovascularization via a gene editing approach for the first time.

[Critical influencing factors on quality of traditional Chinese medicine preparations from perspective of process and equipment].

The quality of traditional Chinese medicine preparations is directly related to the safety of patients. Among the various factors, the process and corresponding critical equipment are critical factors influencing the quality of the preparations. To improve the quality of traditional Chinese medicine preparations, this article summarizes and analyzes the problems in the process links and corresponding critical equipment in the manufacturing process of traditional Chinese medicine preparations. Furthermore, a critical quality attribute evaluation system is established based on safety and effectiveness combined with the drug properties, preparation process, and preparation characteristics, providing a basis for the process and equipment improvements aimed at quality enhancement.

[Scientific connotation of temperature control in processing of Chinese medicinal materials based on theory of quality evaluation through morphological identification].

Processing of Chinese medicinal materials is an important part in the Chinese medicine heritage, and the temperature control in the processing has a direct impact on the quality and efficacy of traditional Chinese medicines. However, the processing of Chinese medicinal materials has the problems of subjective temperature judgement, determination of the end point based on experience, unclear processing mechanism, unstable quality of products, and inconsistent processing standards. The temperature control in the processing is reflected in the appearance and internal quality of Chinese medicinal materials. The theory of quality evaluation through morphological identification is developed based on the comprehensive evaluation of the shape, color, taste, and components, which is associated with the temperature control in the processing. To solve the problems above, this paper puts forward the following solutions. The first is literature mining. By review of the ancient medical works and pharmaceutical experience, the temperature control in processing and the evolution of processing methods can be revealed. Second, according to the ancient method, the processing principle can be explored, on the basis of which the processing technology can be innovated. Third, the standard operating procedure(SOP) should be established to quantify the fire temperature, providing a theoretical basis for the formulation of Chinese medicinal material processing standards. Moreover, it provides a basis for improving the quality of processed products and increasing the safety and effectiveness of traditional Chinese medicines.

Secondary formation of oxygenated and nitrated polycyclic aromatic compounds under stagnant weather conditions: Drivers and seasonal variation.

Severe air pollution tends to occur under stagnant weather conditions. This study focused on the occurrence and formation of PM2.5-bound polycyclic aromatic compounds (PACs) under stagnant weather conditions, in consideration of their adverse human health effect and ecological toxicity. The concentrations of PACs were higher under stagnant weather conditions than in other situations with averaged values of 46.0 ng/m3 versus 12.3-39.9 ng/m3 for total PACs. Secondary formation contributed to over half of the oxygenated and nitrated polycyclic aromatic compounds (OPAHs and NPAHs). Further analyses revealed different formation mechanisms for secondary OPAHs and NPAHs. Secondary production of OPAHs was sensitive to the variations of both temperature (T) and O3 concentration at T < 22 °C but sustained at a high level despite the fluctuation of temperature and O3 concentration at T > 22 °C. Elevated NO2 concentrations favored the formation of inorganic nitrogen-containing products over NPAHs under lower temperature and higher humidity. Stagnant weather events, accompanied by raised PAC levels occurred in all seasons, but their effects on secondary processes differed among seasons. The elevated temperature, lowered humidity, and increased NO2 level facilitated the secondary formation of OPAHs and/or NPAHs during the stagnant weather events in spring and summer. While under the temperature and humidity conditions in autumn and winter, increased NO2 levels during stagnant weather events promoted the production of secondary inorganic nitrogen-containing compounds over organic products. This study raised concern about the toxic organic pollutants in the atmosphere under stagnant weather conditions and revealed different formation mechanisms between secondary oxygenated and nitrated pollutants as well as among different seasons.

The anti-colorectal cancer effect and metabolites of Agrimonia pilosa Ledeb.

ETHNOPHARMACOLOGICAL RELEVANCE: Agrimonia pilosa Ledeb. (Rosaceae, A. pilosa) has been used in traditional medicine in China, Japan, Korea, and other Asian countries for treatment of acute and chronic enteritis and diarrhea. Secondary metabolites have been isolated and tested for biological activities. It remains unclear in terms of its potential components of anti-colorectal cancer properties. AIM OF THE STUDY: The study aimed to how extracts from A. pilosa and their components influenced tumor microenvironment and the colorectal tumor growth in vivo on AOM/DSS induced colorectal cancer mice, the metabolites of A. pilosa was also been studied. MATERIALS AND METHODS: Different methods have been used to extract different parts of A. pilosa. And the anti-proliferation effect of these extracts on colon cancer cells have been tested. The components of A. pilosa and its metabolites in vivo were analyzed by UPLC-QTOF-MS/MS. The anti-colorectal cancer (CRC) effects of A. pilosa and its components in vivo were studied on AOM/DSS induced CRC mice. The effects of constituents of A. pilosa on the composition of immune cells in tumor microenvironment (TME) were analyzed by flow cytometry. 16 S rDNA technology was used to analyze the effect of administration on the composition of intestinal microflora. Pathological section staining was used to compare the morphological changes and molecular expression of intestinal tissue in different groups. RESULTS: The constituent exists in root of A. pilosa showed the strongest anti-proliferation ability on colon cancer cells in vitro. The extract from the root of A. pilosa could attenuate the occurrence of colorectal tumors induced by AOM/DSS in a concentration-dependent manner. Administration of the extract from the root of A. pilosa could affect the proportion of γδT cells, tumor associated macrophages and myeloid derived suppressor cells in TME, increasing the proportion of anti-tumor immune cells and decrease the immunosuppressive cells in the TME to promote the anti-tumor immune response. The administration of the extract adjusted the composition of gut microbiota and its components Agrimoniin and Agrimonolide-6-o-glucoside showed the strongest anti-CRC effect in vivo with adjusting the gut microbiota differently. CONCLUSIONS: The extract from root of A. pilosa showed anti-colorectal cancer effects in vivo and in vitro, affecting the composition of gut microbiota and the anti-tumor immune response. Within all components of A. pilosa, Agrimoniin and Agrimonolide-6-o-glucoside showed remarkable anti-CRC efficiency in vivo and in vitro. Besides, the metabolites of extract from root of A. pilosa in gastrointestinal tract mainly composed of two parts: Agrimonolide-related metabolites and Urolithins. The extract from root of A. pilosa could contribute to potential drugs for assisting clinical anti-colon cancer therapy.

An Improved OMP Algorithm for Enhancing the Anti-Interference Performance of Array Antennas.

The demand for precise positioning in noisy environments has propelled the development of research on array antenna radar systems. Although the orthogonal matching pursuit (OMP) algorithm demonstrates superior performance in signal reconstruction, its application efficacy in noisy settings faces challenges. Consequently, this paper introduces an innovative OMP algorithm, DTM_OMP_ICA (a dual-threshold mask OMP algorithm based on independent component analysis), which optimizes the OMP signal reconstruction framework by utilizing two different observation bases in conjunction with independent component analysis (ICA). By implementing a mean mask strategy, it effectively denoises signals received by array antennas in noisy environments. Simulation results reveal that compared to traditional OMP algorithms, the DTM_OMP_ICA algorithm shows significant advantages in noise suppression capability and algorithm stability. Under optimal conditions, this algorithm achieves a noise suppression rate of up to 96.8%, with its stability also reaching as high as 99%. Furthermore, DTM_OMP_ICA surpasses traditional denoising algorithms in practical denoising applications, proving its effectiveness in reconstructing array antenna signals in noisy settings. This presents an efficient method for accurately reconstructing array antenna signals against a noisy backdrop.

Yeast Bxi1/Ybh3 mediates conserved mitophagy and apoptosis in yeast and mammalian cells: convergence in Bcl-2 family.

The process of degrading unwanted or damaged mitochondria by autophagy, called mitophagy, is essential for mitochondrial quality control together with mitochondrial apoptosis. In mammalian cells, pan-Bcl-2 family members including conical Bcl-2 members and non-conical ones are involved in and govern the two processes. We have illustrated recently the BH3 receptor Hsp70 interacts with Bim to mediate both apoptosis and mitophagy. However, whether similar pathways exist in lower eukaryotes where conical Bcl-2 members are absent remained unclear. Here, a specific inhibitor of the Hsp70-Bim PPI, S1g-10 and its analogs were used as chemical tools to explore the role of yeast Bxi1/Ybh3 in regulating mitophagy and apoptosis. Using Om45-GFP processing assay, we illustrated that yeast Ybh3 mediates a ubiquitin-related mitophagy pathway in both yeast and mammalian cells through association with Hsp70, which is in the same manner with Bim. Moreover, by using Bax/Bak double knockout MEF cells, Ybh3 was identified to induce apoptosis through forming oligomerization to trigger mitochondrial outer membrane permeabilization (MOMP) like Bax. We not only illustrated a conserved ubiquitin-related mitophagy pathway in yeast but also revealed the multi-function of Ybh3 which combines the function of BH3-only protein and multi-domain Bax protein as one.

Giant Modulation of the Second Harmonic Generation by Magnetoelectricity in Two-Dimensional Multiferroic CuCrP2S6.

Multiferroic materials have attracted considerable attention owing to their unique magnetoelectric or magnetooptical properties. The recent discovery of few-layer van der Waals multiferroic crystals provides a new research direction for controlling the multiferroic properties in the atomic layer limit. However, research on few-layer multiferroic crystals is limited and the effect of thickness-dependent symmetries on those properties is less explored. In this study, the symmetries and magnetoelectric responses of van der Waals multiferroic CuCrP2S6 are investigated by optical second harmonic generation (SHG). Structural and magnetic phase transitions are successfully probed by the temperature-dependent SHG signals, revealing significant changes by applying the magnetic field reflecting the magnetoelectric effect. Moreover, it is found that symmetries and resultant magnetoelectric responses can be modulated by the number of layers. These results offer a new principle of controlling the multiferroicity and indicate that 2D van der Waals multiferroic material is a promising building block for functional nanodevices.

[Current situation and development trend of digital traditional Chinese medicine pharmacy].

The 21st century is a highly information-driven era, and traditional Chinese medicine(TCM) pharmacy is also moving towards digitization and informatization. New technologies such as artificial intelligence and big data with information technology as the core are being integrated into various aspects of drug research, manufacturing, evaluation, and application, promoting interaction between these stages and improving the quality and efficiency of TCM preparations. This, in turn, provides better healthcare services to the general population. The deep integration of emerging technologies such as artificial intelligence, big data, and cloud computing with the TCM pharmaceutical industry will innovate TCM pharmaceutical technology, accelerate the research and industrialization process of TCM pharmacy, provide cutting-edge technological support to the global scientific community, boost the efficiency of the TCM industry, and promote economic and social development. Drawing from recent developments in TCM pharmacy in China, this paper discussed the current research status and future trends in digital TCM pharmacy, aiming to provide a reference for future research in this field.

Epitaxial Growth of Two-Dimensional Nonlayered AuCrS2 Materials via Au-Assisted Chemical Vapor Deposition.

Two-dimensional (2D) nonlayered transition metal dichalcogenide (TMD) materials are emergent platforms for various applications from catalysis to quantum devices. However, their limited availability and nonstraightforward synthesis methods hinder our understanding of these materials. Here, we present a novel technique for synthesizing 2D nonlayered AuCrS2 via Au-assisted chemical vapor deposition (CVD). Our detailed structural analysis reveals the layer-by-layer growth of [AuCrS2] units atop an initial CrS2 monolayer, with Au binding to the adjacent monolayer of CrS2, which is in stark contrast with the well-known metal intercalation mechanism in the synthesis of many other 2D nonlayered materials. Theoretical calculations further back the crucial role of Cr in increasing the mobility of Au species and strengthening the adsorption energy of Au on CrS2, thereby aiding the growth throughout the CVD process. Additionally, the resulting free-standing nanoporous AuCrS2 (NP-AuCrS2) exhibits exceptional electrocatalytic properties for the hydrogen evolution reaction.

Small molecule inhibitor targeting the Hsp70-Bim protein-protein interaction in estrogen receptor-positive breast cancer overcomes tamoxifen resistance.

INTRODUCTION: Estrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance. METHODS: A specific inhibitor of Hsp70-Bim protein-protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 µM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days. RESULTS: It was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume. CONCLUSIONS: The in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.

Exploring the binding mechanism of a small molecular Hsp70-Bim PPI inhibitor through molecular dynamic simulation.

CONTEXT: The interface of Hsp70-Bim protein-protein interaction (PPI) has been identified as a specific target for Chronic Myeloid Leukemia (CML) therapy and the specific inhibitors were developed to exhibit in vivo anti-leukemia activities. Herein, we explored the binding mechanism of a Hsp70-Bim inhibitor, 6-(cyclohexylthio)-3-((2-morpholinoethyl) amino)-1-oxo-1H-phenalene-2-carbonitrile (S1g-6), to Hsp70 at the atomic level by MD simulation. TYR-149, THR-222, ALA-223, and GLY-224 on Hsp70 were identified as four key residues that contribute to Hsp70/S1g-6 complex. Moreover, the site mutation validation demonstrated the TYR-149 of Hsp70 is a "hot-spot" in the Hsp70-Bim PPI interface. These results could benefit the design of further inhibitors to occupy the Bim binding site on the Hsp70 surface. METHODS: The binding mechanism of S1g-6 and Hsp70 was predicted through the molecular dynamics (MD) method by Gromacs-2021.3. The MD simulation was performed with 100-ps NVT and 100-ps NPT ensemble, and the force field was chosen as the Charmm36 force field. The temperature was set as 300 K, the time step was 2 fs and the total MD simulation time was 500 ns.

Analyzing urban influence on extreme winter precipitation through observations and numerical simulation of two South China case studies.

This study investigates the impact of urbanization on extreme winter rainfall in the South China Greater Bay Area (GBA) through the analysis of hourly station observations and simulations using the Weather Research and Forecasting Model with the Single Layer Urban Canopy Model (WRF-SLUCM). Data from 2008 to 2017 reveal that urban areas in the GBA experience lower 99th percentile hourly winter rainfall intensity compared to surrounding rural regions. However, urban locations exhibit higher annual maximum hourly rainfall (Rmax) and very extreme rainfall events (99.99th percentile) in winter, suggesting a positive influence of urbanization on extreme winter precipitation. A case study further underscores the role of the Urban Heat Island (UHI) effect in enhancing extreme rainfall intensity and probability in the GBA urban areas. Additionally, two extreme cases were dynamically downscaled using WRF-SLUCM, involving four parallel experiments: replacing urban land use with cropland (Nourban), using historical urban land use data from 1999 (99LS), projecting near-future urban land use for 2030 (30LS), and considering 2030 urban land use without anthropogenic heat (AH) (30LS-AH0). Synoptic analysis demonstrates that cold air intrusion suppresses the GBA UHI in Case 2013 but not in Case 2015. Reduced evaporation and humidity induced by urban surfaces significantly decrease urban precipitation in Case 2013. In contrast, the persistent UHI in Case 2015 enhances local convection and land-ocean circulation, leading to increased moisture flux convergence and amplified urban precipitation intensity and probability in 30LS compared to Nourban. This amplification is primarily attributed to AH, while the change in 99LS remains insignificant. These findings suggest that urban influences on extreme precipitation in the GBA persist during winter, particularly when the UHI effect is maintained.

Effect of empathy nursing combined with SBAR communication system on the negative emotions and nursing quality of children with tracheotomy.

BACKGROUND: Situation, Background, Assessment, and Recommendation (SBAR) is a structured method for communicating critical information that requires immediate attention and action. OBJECTIVE: To study the effects of empathy nursing combined with the SBAR communication system on the negative emotions and nursing quality of children undergoing tracheotomy. METHODS: This is a clinical observational study. A total of 100 tracheotomy patients who were cared for in the pediatric intensive care unit (subsequent treatment in the tracheotomy clinic or otolaryngology ward) of our hospital from September 2021 to June 2022 were recruited and assigned at a ratio of 1:1 either into a control group (empathic care) or an observation group (empathic care combined with SBAR) using a randomized method. Further, the postoperative anxiety self-rating scale scores, negative emotions, hope index, and nursing quality were compared between the two groups. RESULTS: After nursing, the psychological resilience scale score of the observation group was higher than that of the control group, whereas the anxiety self-rating scale score was significantly lower than that of the control group (all P< 0.05). Basic and special nursing, knowledge awareness, and safety management of the two groups of patients improved significantly, with higher results in the observation group than in the control group (P< 0.05). CONCLUSION: Empathy nursing combined with the SBAR communication system considerably improves postoperative negative emotions and enhances the quality of nursing care for patients undergoing tracheotomy.

Hsp70-Bim incoherent feedforward loop contributes to cell-fate heterogeneity and fractional killing.

BACKGROUND AND PURPOSE: Although chemotherapeutics or molecular targeted drugs often elicit profound initial responses, fractional killing capable of driving acquired resistance can persist. Identifying stress-induced negative feedback or an incoherent feedforward loop (IFFL), which may contribute to fractional killing, is urgently needed. EXPERIMENTAL APPROACH: Mathematical modelling was used to identify how and to what extent a recently reported Hsp70-Bim protein-protein interaction (PPI) contributes to the adaptation of the Bcl-2 network. Experimental validation was made by using a specific inhibitor of Hsp70-Bim PPI, S1g-2, as chemical tool. Bifurcation analysis and stochastic simulation were used for the theoretical study of the impact of Hsp70-Bim PPI on cell-fate heterogeneity and factional killing. KEY RESULTS: The Hsp70-Bim-AKT circuit forms an IFFL that greatly contributes to the adaptation of the Bcl-2-regulated apoptosis network, thus leading to fractional killing. This adaptive programme enhances noise-induced cell-fate heterogeneity by shifting from a saddle-node to a saddle-collision transition scenario. CONCLUSION AND IMPLICATIONS: Hsp70-Bim IFFL serves as a molecular pathway induced by DNA damaging drugs or tyrosine kinase inhibitors that enabled fractional killing, whereby acquired resistance emerges. A synergistic strategy is unveiled for overcoming fractional killing by suppressing Hsp70-Bim PPI.

Ectopic BH3-Only Protein Bim Associates with Hsp70 to Regulate Yeast Mitophagy.

Mitophagy, a form of selective autophagy, plays an essential role to maintain a population of healthy and functional mitochondria for normal cellular metabolism. Acting mainly as one of the B-cell lymphoma 2 (Bcl-2) family pro-apoptotic members, Bim (also known as BCL2L11) was identified to be a co-chaperone of Hsp70 to promote mitophagy in mammalian cells. Herein, with the help of a specific Hsp70/Bim disruptor and Om45-GFP processing assay, we illustrated that ectopic BimEL is able to promote mitophagy through Hsp70/Bim interaction in yeast, where Bax/Bak is absent. The Hsp70/Bim-mediated mitophagy is conserved in eukaryotes, from yeast to humans.

Emergent zero-field anomalous Hall effect in a reconstructed rutile antiferromagnetic metal.

The anomalous Hall effect (AHE) that emerges in antiferromagnetic metals shows intriguing physics and offers numerous potential applications. Magnets with a rutile crystal structure have recently received attention as a possible platform for a collinear-antiferromagnetism-induced AHE. RuO2 is a prototypical candidate material, however the AHE is prohibited at zero field by symmetry because of the high-symmetry [001] direction of the Néel vector at the ground state. Here, we show AHE at zero field in Cr-doped rutile, Ru0.8Cr0.2O2. The magnetization, transport and density functional theory calculations indicate that appropriate doping of Cr at Ru sites reconstructs the collinear antiferromagnetism in RuO2, resulting in a rotation of the Néel vector from [001] to [110] while maintaining a collinear antiferromagnetic state. The AHE with vanishing net moment in the Ru0.8Cr0.2O2 exhibits an orientation dependence consistent with the [110]-oriented Hall vector. These results demonstrate that material engineering by doping is a useful approach to manipulate AHE in antiferromagnetic metals.