Neoantigen T-Cell Receptor Gene Therapy in Pancreatic Cancer.

A patient with progressive metastatic pancreatic cancer was treated with a single infusion of 16.2×109 autologous T cells that had been genetically engineered to clonally express two allogeneic HLA-C*08:02-restricted T-cell receptors (TCRs) targeting mutant KRAS G12D expressed by the tumors. The patient had regression of visceral metastases (overall partial response of 72% according to the Response Evaluation Criteria in Solid Tumors, version 1.1); the response was ongoing at 6 months. The engineered T cells constituted more than 2% of all the circulating peripheral-blood T cells 6 months after the cell transfer. In this patient, TCR gene therapy targeting the KRAS G12D driver mutation mediated the objective regression of metastatic pancreatic cancer. (Funded by the Providence Portland Medical Foundation.).

Constructing an Artificial Interface as a Bifunctional Promoter for the Li Anode and the NCM Cathode in Lithium Metal Batteries.

The bottleneck of Li metal batteries toward practical applications lies at inferior cyclability as well as Li dendrite issues. As a promising solution, an interface engineering strategy is proposed herein for the Li anode through constructing a hybrid artificial interface. It is assembled onto the Li anode using photocontrolled free radical polymerization (photo-CRP) of polyethylene glycol diacrylate-hexafluorobutyl methacrylate and hexafluorobutyl methacrylate-trifluoroethyl carbonate (PEGDA-HFMBA@HFMBA-FEMC or PH@HF layer). Among such hybrid interfaces, the interior layer of PEGDA-HFMBA exists as a protective shield with flexibility and fracture resistance, while the exterior layer of HFMBA-FEMC plays a role as a LiF reservoir to promote Li mass transfer and its even electrodeposition. In the meantime, some excess HFMBA and FEMC monomers further dissolve into the electrolyte as molecular additives, followed by in situ generation of a thin and robust LiF-rich cathode electrolyte interface (CEI). With the resulting Li anode, Li/NCM811 full cells showcase multifold cyclability amplification in comparison to cells using Bare-Li, covering durable cyclability with a capacity retention of 81.8% after 400 cycles. When the cutoff voltage is elevated to 4.5 V or the working temperature is elevated to 45 °C, the cells still maintain a stable operation for extending 300 cycles.

3D Lithiophilic CuZrAg Metallic Glass Based-Current Collector for High-Performance Lithium Metal Anode.

Lithium metal anodes face several challenges in practical applications, such as dendrite growth, poor cycle efficiency, and volume variation. 3D hosts with lithiophilic surfaces have emerged as a promising design strategy for anodes. In this study, inspiration from the intrinsic isotropy, chemical heterogeneity, and wide tunability of metallic glass (MG) is drew to develop a 3D mesoporous host with a lithiophilic surface. The CuZrAg MG is prepared using the scalable melt-spinning technique and subsequently treated with a simple one-step chemical dealloying method. This resultes in the creation of a host with a homogeneously distributed abundance of lithium affinity sites on the surface. The excellent lithiophilic property and capability for uniform lithium deposition of the 3D CuZrAg electrode have been confirmed through theoretical calculations. Therefore, the 3D CuZrAg electrode displays excellent cyclic stability for over 400 cycles with 96% coulomb efficiency, and ultra-low overpotentials of 5 mV for over 2000 h at 1.0 mA cm-2 and 1.0 mAh cm-2 . Additionally, the full cells partied with either LiFePO4 or LiNi0.8 Co0.1 Mn0.1 O2 cathode deliver exceptional long-term cyclability and rate capability. This work demonstrates the great potential of metallic glass in lithium metal anode application.

Influence of anthropogenic activities and loess dusts on the rainwater hydrochemistry in the Chinese Loess Plateau.

Rainwater hydrochemistry is an important indicator for tracing anthropogenic input on air quality. As the fastest economically developing city in the northwestern China and the Chinese Loess Plateau, rainwater chemistry, sources of dissolved solutes, and the influence of loess dust on rainwater chemistry in Xi'an city is unclear. Inorganic ions, δD and δ18O of two years' rainwater samples were measured to decipher the above issues. Rainwater samples were weakly alkaline (pH = 7.2) with the mean total dissolved solids (TDS) values of 43 mg/L. NH4+ and Ca2+ dominated in the cations and SO42- and NO3- dominated in the anions. The wet deposition of sulfur (S) and nitrogen (N) was 70.9 ± 67 mg·(m2·month)-1 and 244.8 ± 270.9 mg·(m2·month)-1, respectively. The meteoric water line in Xi'an was δD = 7.29δ18O+3.72 (R2 = 0.99). δD, δ18O, and d-excess analysis indicated the influence of evaporation on the dissolved solutes in rainwater, especially in the dry season. Rainwater acidity in the Xi'an city was mainly neutralized by Ca2+ and NH4+, and the neutralization ability in Xi'an city is higher than the southern China cities. Correlation analysis (CA), positive matrix factorization (PMF), and the backward air masses trajectory model identified high NH4+ and Ca2+ in rainwater were mainly originated from local agricultural activities and loess dust, while NO3- and SO42- were associated with local coal combustion and vehicle exhaust sources. High inputs of dusts and coal combustion in spring and winter resulted in elevated values of pH and major ions in Xi'an. Due to the air pollution control policy, air quality in Xi'an is getting better in recent years. Our study highlights the influence of anthropogenic activities and loess dusts on the rainwater hydrochemistry in Xi'an and provides important dataset for air pollution control for other cities in semi-arid and arid regions.

Electrochemical aptasensor based on Mo2C/Mo2N and gold nanoparticles for determination of chlorpyrifos.

Two-dimensional Mo2C/Mo2N composites were synthesized by high temperature ball milling and used as support materials for fabricating a chlorpyrifos (CPF) aptasensor. Gold nanoparticles (Au NPs) were electrodeposited on the surface of a Mo2C/Mo2N-modified electrode to connect with the ferrocene (Fc) probe via Au-S bonds. The Fc probe can hybridize with the aptamer probe to form a double-stranded structure. The addition of CPF made the double strands melt and the Fc probe approached the surface of the electrode, thereby resulting in amplification of the electrochemical response. The current response of the aptasensor for detecting CPF in solutions linearly varied from 0 to 400 ng mL-1 (with a maximum at 0.98 V vs. Ag/AgCl). The Au NPs/Mo2C/Mo2N composites exhibited satisfactory electrochemical behavior due to their excellent electrical conductivity and large surface area. This ultrasensitive aptasensor showed a low limit of detection of 0.036 ng mL-1. It was applied to determine CPF in real samples with acceptable recoveries from 94.7 to 116.7%, and the relative standard deviation was from 2.57 to 7.08%.Graphical abstract Schematic diagram of the manufacturing process of the aptasensor. Electrochemical aptasensor based on Mo2C/Mo2N/Au NP composites show excellent performance in detecting CPF.

A boron nitride electrode modified with a nanocomposite prepared from an ionic liquid and tungsten disulfide for voltammetric sensing of 4-aminophenol.

Boron nitride (BN) was used as a support and covered with an ionic liquid (IL) and tungsten disulfide (WS2) nanoparticles to obtain an electrode for the determination of 4-aminophenol (4-AP). BN was prepared using a "solvent cutting" method, and the BN-IL-WS2 nanocomposite was obtained by an ultrasonic method. BN and its hybrids were characterized by scanning electron microscopy, transmission electron microscopy and X-ray diffraction. When the BN-IL-WS2 composites were coated on the surface of the electrode, the response to 4-AP was strongly amplified due to the strong synergetic effect between the three materials. The voltammetric response of the modified sensor (with a maximum at 0.29 V vs. Ag/AgCl) in solutions with a pH of 6 is linear in the 0.01-50 µΜ 4-AP concentration range, and the limit of detection is 3 nM. A modified glassy carbon electrode was applied for the determination of 4-AP in seawater and dispersions containing paracetamol tablets. The results were consistent with those obtained by HPLC. Graphical abstract Schematic representation of the voltammetric determination process of 4-aminophenol (4-AP). The electrochemical sensor based on the glassy carbon electrode modified with boron nitride (BN), ionic liquid (IL) and tungsten disulfide (WS2) nanomaterials. They, exhibit an excellent performance compared with other electrodes.

Electrochemical determination of 2,4-dichlorophenol by using a glassy carbon electrode modified with molybdenum disulfide, ionic liquid and gold/silver nanorods.

Molybdenum disulfide (MoS2) was used as an electrically conductive skeleton and functionalized with an ionic liquid and gold/silver nanorods. The resulting composite was characterized by scanning electron microscopy, transmission electron microscopy and UV-vis spectroscopy. The composites were used to modify a glassy carbon electrode (GCE) to obtain a sensor for 2,4-dichlorophenol (2,4-DCP). The results show that the oxidation power and electrocatalytic activity of the modified GCE towards 2,4-DCP are enhanced compared to a bare GCE and other modified GCEs. Response is linear in the 0.01 to 50 µM 2,4-DCP concentration range, with a 2.6 nM detection limit. The sensor is highly sensitive and long-term stable. It was successfully applied to the determination of 2,4-DCP in spiked water samples and gave satisfactory recoveries. Graphical abstract Schematic of an electrochemical sensor for the differential pulse voltammetric (DPV) determination of 2,4-dichlorophenol. It is based on the use of an MoS2-ionic liquid-Au/Ag nanorod composite.

[Estimation of soil moisture and organic matter content in saline alkali farmland by using CARS algorithm combined with covariates]. / 利用CARS算法联合协变量估算盐碱农田土壤水分和有机质含量.

Rapid acquisition of the data of soil moisture content (SMC) and soil organic matter (SOM) content is crucial for the improvement and utilization of saline alkali farmland soil. Based on field measurements of hyperspectral reflectance and soil properties of farmland soil in the Hetao Plain, we used a competitive adaptive reweighted sampling algorithm (CARS) to screen sensitive bands after transforming the original spectral reflectance (Ref) into a standard normal variable (SNV). Strategies Ⅰ, Ⅱ, and Ⅲ were used to model the input variables of Ref, Ref SNV, Ref-SNV+ soil covariate (SC), and digital elevation model (DEM). We constructed SMC and SOM estimation models based on random forest (RF) and light gradient boosting machine (LightGBM), and then verified and compared the accuracy of the models. The results showed that after CARS screening, the sensitive bands of SMC and SOM were compressed to below 3.3% of the entire band, which effectively optimized band selection and reduced redundant spectral information. Compared with the LightGBM model, the RF model had higher accuracy in SMC and SOM estimation, and the input variable strategy Ⅲ was better than Ⅱ and Ⅰ. The introduction of auxiliary variables effectively improved the estimation ability of the model. Based on comprehensive analysis, the coefficient of determination (Rp2), root mean square error (RMSE), and relative analysis error (RPD) of the SMC estimation model validation based on strategy Ⅲ-RF were 0.63, 3.16, and 2.01, respectively. The SOM estimation models based on strategy Ⅲ-RF had Rp2, RMSE, and RPD of 0.93, 1.15, and 3.52, respectively. The strategy Ⅲ-RF model was an effective method for estimating SMC and SOM. Our results could provide a new method for the rapid estimation of soil moisture and organic matter content in saline alkali farmland.

IL-17A and TNF-α-induced Dectin-1 expression may promote keratinocyte proliferation in psoriatic lesions.

Psoriasis is a common skin disease with a high recurrence rate. Aberrant keratinocyte proliferation is a significant pathogenic characteristic of psoriatic lesions, and studies have revealed that the development of psoriasis is significantly influenced by pro-inflammatory cytokines, such as IL-17A and TNF-α. Biologics targeting these cytokines have been widely used in psoriasis treatment and achieve remarkable effects, however, the underlying mechanism of how IL-17A and TNF-α specifically regulate keratinocyte proliferation has not been fully elucidated. Dectin-1 is an essential membrane protein that is directly related to the immune microenvironment and the proliferation of multiple cell types. To elucidate how IL-17A and TNF-α may promote keratinocyte proliferation in psoriatic lesions and whether Dectin-1 is involved. The expression of Dectin-1 in keratinocytes from psoriatic lesions was detected by real-time PCR, western blot and immunofluorescence. Correlation analysis and cytological experiments were then performed to determine the relationship between Dectin-1 and IL-17A/TNF-α in psoriatic lesions. Finally, we investigated the signalling pathway through which Dectin-1 may promote keratinocyte proliferation. Dectin-1 was significantly increased in keratinocytes from psoriatic lesions. Moreover, IL-17A and TNF-α effectively induced the expression of Dectin-1 in HaCaT cells, which was shown to activate the Syk/NF-κB signalling pathway and promote the proliferation of keratinocytes. IL-17A and TNF-α may promote the proliferation of keratinocytes in psoriatic lesions through induction of Dectin-1, indicating that Dectin-1 could be a potential therapeutic target for the treatment of psoriasis.

Tri-anion solvation structure electrolyte improves the electrochemical performance of Li||LiNi0.8Co0.1Mn0.1O2 batteries.

Li||LiNi0.8Co0.1Mn0.1O2 batteries,which consist of lithium  metal anode (LMA) matched with NCM811 cathode, have an energy density more than twice that of lithium ion battery (LIB). However, the unstable electrode/electrolyte interface still hinders its practical application.Ether electrolytes show promise in improving the stability of LMA and NCM811 cathodes.However, a robust and stable electrode/electrolyte interface in Li||NCM811 batteries cannot be easily and efficiently achieved with most of the ether electrolytes reported in present studies. Herein, we present a straightforward and efficient tri-anion synergistic strategy to overcome this bottleneck. The addition of ClO4- and NO3- anions to LiFSI-based ether electrolytes forms a unique solvation structure with tri-anion (FSI-/ClO4-/NO3-) participation (LB511).This structure not only enhances the electrochemical window of the ether electrolytes but also achieves a stable Li||NCM811 batteries interface.The interaction between electrode and electrolyte is suppressed and an inorganic-rich (LiF/Li3N/LiCl) SEI/CEI layer is formed. Meanwhile, the coordination structure in the LB511 electrolyte increases the overpotential for Li deposition, resulting in a uniform and dense layer of deposition.Therefore, the Li||Cu cells using the LB511 electrolyte have an average CE of 99.6%.The Li||NCM811 batteries was cycled stably for 250 cycles with a capacity retention of 81% in the LB511 electrolyte (N/P = 2.5, 0.5 C).

A Versatile Strategy for the Controlled Synthesis of Atomically Precise Palladium Nanoclusters.

The study of the structures, applications, and structure-property relationships of atomically precise metal nanoclusters relies heavily on their controlled synthesis. Although great progress has been made in the controlled synthesis of Group 11 (Cu, Ag, Au) metal nanoclusters, the preparation of Pd nanoclusters remains a grand challenge. Herein, a new, simple, and versatile synthetic strategy for the controlled synthesis of Pd nanoclusters is reported with tailorable structures and functions. The synthesis strategy involves the controllable transformations of Pd4(CO)4(CH3COO)4 in air, allowing the discovery of a family of Pd nanoclusters with well-defined structure and high yield. For example, by treating the Pd4(CO)4(CH3COO)4 with 2,2-dipyridine ligands, two clusters of Pd4 and Pd10 whose metal framework describes the growth of vertex-sharing tetrahedra have been selectively isolated. Interestingly, chiral Pd4 nanoclusters can be gained by virtue of customized chiral pyridine-imine ligands, thus representing a pioneering example to shed light on the hierarchical chiral nanostructures of Pd. This synthetic methodology also tolerates a wide variety of ligands and affords phosphine-ligated Pd nanoclusters in a simple way. It is believed that the successful exploration of the synthetic strategy would simulate the research enthusiasm on both the synthesis and application of atomically precise Pd nanoclusters.

WTAP-mediated m6A modification of IFNE is required for antiviral defense in condyloma acuminata.

BACKGROUND: IFN-ε is essential in combating viral infections, particularly in epithelial cells and protected mucosal tissues. Its protective effects have been demonstrated against HSV2, Zika virus, HIV and SARS-COV2. However, the specific expression and role of IFN-ε in skin keratinocytes and HPV infection are still not fully understood and require further investigation. OBJECTIVE: In this study, we aimed to investigate the functions and expression mechanism of IFN-ε in keratinocytes during HPV infection and the progression of condyloma acuminata. METHODS: Keratinocytes isolated from biopsied CA warts and normal skins samples were analyzed by MeRIP-seq analysis. IFN-ε and WTAP in CA warts and normal skins were analyzed by immunostaining and qPCR. RESULTS: In this study, we identified IFN-ɛ was markedly upregulated in CA warts and HPV-infected keratinocytes. IFN-ɛ expression also showed negatively correlation with the size of CA warts (R=-0.4646, P = 0.009). IFN-ɛ suppressed the susceptibility of HPV infection directly. m6A analysis reveals WTAP is a key m6A writer promoting the m6A modification of IFNE mRNA. CONCLUSION: Our research suggests that IFN-ɛ is an important Type I IFN cytokine involved in the development of genital warts. Furthermore, we found that HPV infection affects the m6A modifications of IFNE through a mechanism dependent on WTAP. This study provides insights into the innate immune response of the host to HPV infection and may contribute to the development of future strategies for regulating innate immunity to treat genital warts.

Spatial distribution and potential sources of arsenic and water-soluble ions in the snow at Ili River Valley, China.

Trace elements and water-soluble ions in snow can be used as indicators to reveal natural and anthropogenic emissions. To understand the chemical composition, characteristics of snow and their potential sources in the Ili River Valley (IRV), snow samples were collected from 17 sites in the IRV from December 2018 to March 2019. Inverse distance weighting, enrichment factor (EF) analysis, and backward trajectory modelling were applied to evaluate the spatial distributions and sources of water-soluble ions and dissolved arsenic (As) in snow. The results indicate that Ca2+ and SO42- were the dominant ions, and the concentrations of As ranged from 0.09 to 0.503 µg L-1. High concentrations of As were distributed in the northwest and middle of the IRV, and the concentrations of the major ions were high in the west of the IRV. The strong correlation of As with F-, SO42-, and NO2- demonstrates that As mainly originated from coal-burning and agricultural activities. Principal component analysis showed that the ions originated from a combination of anthropogenic and crustal sources. The EFs showed that K+, SO42-, and Mg2+ were mainly influenced by human activities. Backward trajectory cluster analysis suggested that the chemical composition of snow was affected by soil dust transport from the western air mass, the unique terrain, and local anthropogenic activities. These results provide important scientific insights for atmospheric environmental management and agricultural production within the IRV.

Transcriptomic profiles of neoantigen-reactive T cells in human gastrointestinal cancers.

Tumor-infiltrating neoantigen-reactive T cells can mediate regression of metastatic gastrointestinal cancers yet remain poorly characterized. We performed immunological screening against personalized neoantigens in combination with single-cell RNA sequencing on tumor-infiltrating lymphocytes from bile duct and pancreatic cancer patients to characterize the transcriptomic landscape of neoantigen-reactive T cells. We found that most neoantigen-reactive CD8+ T cells displayed an exhausted state with significant CXCL13 and GZMA co-expression compared with non-neoantigen-reactive bystander cells. Most neoantigen-reactive CD4+ T cells from a patient with bile duct cancer also exhibited an exhausted phenotype but with overexpression of HOPX or ADGRG1 while lacking IL7R expression. Thus, neoantigen-reactive T cells infiltrating gastrointestinal cancers harbor distinct transcriptomic signatures, which may provide new opportunities for harnessing these cells for therapy.

Electrochemical Biosensor Based on Well-Dispersed Boron Nitride Colloidal Nanoparticles and DNA Aptamers for Ultrasensitive Detection of Carbendazim.

A selective electrochemical biosensor was developed for detecting carbendazim (CBZ) based on well-dispersed colloidal boron nitride (BN) nanocrystals and gold nanoparticles (Au NPs). BN was synthesized by "solvent cutting" to modify a glassy carbon electrode (GCE), and Au NPs were then electrodeposited. A single-stranded oligonucleotide with methylene blue (MB) was modified to the electrode surface through gold-sulfur bonds. A double-stranded DNA was formed in the presence of an aptamer. The aptamer chain can specifically bind to the target CBZ. When the aptamer binds to CBZ, the electroactive substance MB labeled at one end of the complementary chain can effectively contact the electrode surface. Detection of CBZ is realized by simultaneously monitoring the MB signal enhancement. The CBZ concentration was determined in a wide linearity range from 0.1 ng mL-1 to 100 µg mL-1, with a low detection limit of 0.019 ng mL-1. This biosensor exhibited excellent selectivity and acceptable repeatability and was applied in cucumber, kiwifruit, and water samples with good recoveries, demonstrating that the strategy has remarkable potential and offers a good platform for CBZ detection.

Preparation, Characterization, and Application of Magnetic Fe-SBA-15 Mesoporous Silica Molecular Sieves.

Magnetic Fe-SBA-15 mesoporous silica molecular sieves were prepared, characterized, and used for magnetic separation. Wet impregnation, drying, and calcination steps led to iron inclusion within the mesopores. Iron oxide was reduced to the metal form with hydrogen, and the magnetic Fe-SBA-15 was obtained. Fourier-transform infrared spectroscopy confirmed the preparation process from the oxide to metal forms. The structure of magnetic materials was confirmed by Mössbauer spectra. Powder X-ray diffraction data indicated that the structure of Fe-SBA-15 retained the host SBA-15 structure. Brunauer-Emmett-Teller analysis revealed a decrease in surface area and pore size, indicating Fe-SBA-15 coating on the inner surfaces. Scanning electron micrographs confirmed the decrease in size for modified SBA-15 particles. From scanning electron micrographs, it was found that the size of the modified SBA-15 particles decreased. Transmission electron micrographs also confirmed that modified SBA-15 retained the structure of the parent SBA-15 silica. Fe-SBA-15 exhibited strong magnetic properties, with a magnetization value of 8.8 emu g(-1). The iron content in Fe-SBA-15 was determined by atom adsorption spectroscopy. Fe-SBA-15 was successfully used for the magnetic separation of three aromatic compounds in water. Our results suggest wide applicability of Fe-SBA-15 magnetic materials for the rapid and efficient separation of various compounds.

The diagnostic value of magnetic resonance urography for detecting ureteric obstruction: a systematic review and meta-analysis.

OBJECTIVE: To evaluate the diagnostic accuracy of magnetic resonance urography (MRU) and determine its value for detecting ureteric obstruction. METHODS: The electronic databases, including PubMed, Embase and the Cochrane library, were systematically searched for studies published throughout September 2018. The summary of sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and receiver operating characteristic (ROC) curves was assessed to evaluate the diagnostic accuracy of MRU. Subgroup analyses were conducted based on the mean age of the included patients (adults or children). RESULTS: Eight studies with a total of 594 patients were included. The summary of the sensitivity and specificity of MRU for diagnosing ureteric obstruction was 0.94 and 0.87, respectively. Furthermore, the pooled PLR and NLR were 7.33 and 0.07, respectively. The DOR of MRU for detecting ureteric obstruction was 95.12. In addition, the summary of the area under the ROC of MRU was 0.96. Finally, the specificity, PLR and area under the ROC of MRU for diagnosing ureteric obstruction in adults were higher than children, while the sensitivity of MRU in adults was lower than children. CONCLUSIONS: These findings suggested a relatively high diagnostic value of MRU for detecting ureteric obstruction. Moreover, the diagnostic accuracy of MRU in adults was higher than in children. KEY MESSAGE Magnetic resonance urography (MRU) in detecting ureteric obstruction has relatively better sensitivity, specificity, PLR, NLR, DOR and AUC. The diagnostic value, including specificity, PLR and AUC of MRU in adults, was higher than in children, while the sensitivity of MRU in adults was lower than in children.

One-Step Solution-Immersion Process of Hydrophobic Octyl Graphene Oxide-Modified Nickel Foam for Highly Efficient Oil-Water Separation.

Hydrophobic foam was prepared by immersing nickel foam in a dispersion of octyl group-grafted graphene oxide (GO) and used to clean up oil-water mixtures. Octyl graphene oxide (C8-GO) was synthesized using GO and triethoxyoctylsilane by a solvothermal method. The resulting coverage of the foam was characterized, and the surface morphology of the foam was also investigated. The static water contact angle (SWCA) was measured to evaluate the change in wettability of the developed foam. The pristine smooth microstructure of the nickel foam became rough after being covered with the C8-GO nanosheets. The SWCA of C8-GO nickel foam (C8-GO NF) surface was approximately 147°. The C8-GO NF can float on the surface of the water in contrast to the easy-sinking unmodified foam, demonstrating good hydrophobicity. Furthermore, the C8-GO NF showed outstanding performance in organic compound adsorption and excellent efficiency in oil-water separation. The reusability and durability of the obtained foam are evaluated to highlight its usability in more complicated scenarios. The use of C8-GO NF was proved to be a promising strategy for oil-water separation under harsh circumstance.