Uvaol ameliorates lipid deposition in hyperlipidemic hepatocytes by suppressing protein-tyrosine phosphatase 1B/ER stress signaling.

Uvaol (UV), a pentacyclic triterpene found in olives and virgin olive oil, is known for its anti-inflammatory and antioxidant effects in various disease models. While olive oil is reported to reduce obesity and insulin resistance, the specific impact of UV on liver lipid metabolism and its molecular mechanisms are not fully understood. In this study, hepatic lipid accumulation was measured using oil red O staining, and protein expression levels in liver cells were assessed via Western blot analysis. Apoptosis was evaluated through cell viability and caspase 3 activity assays. UV treatment reduced lipid accumulation, fatty acid uptake, apoptosis, and ER stress in palmitate-treated liver cells. Additionally, UV enhanced fatty acid oxidation. Mechanistically, increased SIRT6 expression and autophagy were observed in UV-treated cells. SIRT6-targeted siRNA or 3-methyladenine blocked the effects of UV in hyperlipidemic cells. In conclusion, UV improves SIRT6/autophagy signaling, reducing lipid deposition and apoptosis in liver cells under high lipid conditions. This in vitro study provides strong evidence for potential therapeutic strategies for hepatic steatosis.

Investigation of film Physical properties under various starch thermal treatments with emphasis on Retrogradation effects.

This study investigated the changes in the physical properties of cornstarch-based films as they were retrogradely aged at different temperatures. Using a casting method, the films were fabricated, and their effects on the mechanical properties, thermal stability, barrier properties, and essential properties were analyzed. With prolonged aging and retrogradation periods, reductions in film thickness, solubility, water content, and water vapor permeability of 5.35%, 9.92%, 29.61%, and 20.94%, respectively, were observed. In addition, the surface roughness decreased by 44.46% for Rq (root-mean-square roughness) and 45.61% for Ra (arithmetic average roughness), while the elongation at break decreased by 72.64%. Conversely, the tensile strength, maximum degradation rate, and maximum degradation temperature increased by 116.98%, 99.5%, and 3.21%, respectively. These results provide a fundamental understanding of the changes that occur in the properties of cornstarch-based films during aging and retrogradation.

Changes in the properties of the corn starch glycerol film in a time-dependent manner during gelatinization.

This study aimed to investigate the fundamental properties, solubility, mechanical properties, barrier performance, and microstructural features of films composed of corn starch and glycerol. Changes in the microstructure were analyzed to understand how they relate to the physical and chemical properties of these films. Specifically, we found that increasing the gelatinization time decreased the film thickness, solubility, water vapor permeability, and maximum degradation temperature and increased the water content. A gradual increase in the water contact angle of the corn starch-glycerol films was observed with increasing gelatinization time. This trend is likely due to the disruptive effect of gelatinization on the crystalline and amorphous structures inherent in corn starch, resulting in reduced film crystallinity, degree of order (DO) and degree of double helix (DD).

Enhancing the perception of saltiness and reducing NaCl levels in snacks through sensory interaction: Immobilizing NaCl nanocrystals in Litsea oleoresin-sunflower seed oleogels.

Excessive dietary salt intake leads to health issues, while reducing NaCl content compromises flavor. Therefore, identifying methods to decrease salt levels without sacrificing flavor is crucial. This study investigated the sensory interaction between the saltiness of NaCl and the pungency of Litsea oleoresin. Glyceryl monostearate (6.6%) and soy lecithin (4.4%) were used as gelling agents to create oleogels, which were then employed to immobilize NaCl nanocrystals, optimizing sensory interactions. NaCl nanocrystals (427.73 ± 61.98 nm) were encapsulated in a Litsea oleoresin-sunflower seed oleogel system with uniform distribution. Sensory evaluation indicated that the NaCl nanocrystal/Litsea oleoresin@oleogel system, with moderate pungency, significantly enhanced perceived saltiness intensity (29.00 ± 1.14, compared to the control, 18.48 ± 1.12) (P < 0.05). When applied to potato chips, this system noticeably increased saltiness perception. This research provides a promising approach for developing low-sodium yet flavorful foods.

Gastroenteropancreatic neuroendocrine neoplasms: current development, challenges, and clinical perspectives.

Neuroendocrine neoplasms (NENs) are highly heterogeneous and potentially malignant tumors arising from secretory cells of the neuroendocrine system. Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are the most common subtype of NENs. Historically, GEP-NENs have been regarded as infrequent and slow-growing malignancies; however, recent data have demonstrated that the worldwide prevalence and incidence of GEP-NENs have increased exponentially over the last three decades. In addition, an increasing number of studies have proven that GEP-NENs result in a limited life expectancy. These findings suggested that the natural biology of GEP-NENs is more aggressive than commonly assumed. Therefore, there is an urgent need for advanced researches focusing on the diagnosis and management of patients with GEP-NENs. In this review, we have summarized the limitations and recent advancements in our comprehension of the epidemiology, clinical presentations, pathology, molecular biology, diagnosis, and treatment of GEP-NETs to identify factors contributing to delays in diagnosis and timely treatment of these patients.

A novel sustainable immunoassay for sensitive detection of atrazine based on the anti-idiotypic nanobody and recombinant full-length antibody.

Immunoassays have been widely used to determine small-molecule compounds in food and the environment, meeting the challenge of obtaining false positive or negative results because of the variance in the batches of antibodies and antigens. To resolve this problem, atrazine (ATR) was used as a target, and anti-idiotypic nanobodies for ATR (AI-Nbs) and a recombinant full-length antibody against ATR (ATR-rAb) were prepared for the development of a sustainable enzyme-linked immunosorbent assay (ELISA). AI-Nb-7, AI-Nb-58, and AI-Nb-66 were selected from an immune phage display library. ATR-rAb was produced in mammalian HEK293 (F) cells. Among the four detection methods explored, the assay using AI-Nb-66 as a coating antigen and ATR-rAb as a detection reagent yielded a half maximal inhibitory concentration (IC50) of 1.66 ng mL-1 for ATR and a linear range of 0.35-8.73 ng mL-1. The cross-reactivity of the assay to ametryn was 64.24%, whereas that to terbutylazine was 38.20%. Surface plasmon resonance (SPR) analysis illustrated that these cross-reactive triazine compounds can bind to ATR-rAb to varying degrees at high concentrations; however, the binding/dissociation kinetic curves and the response values at the same concentration are different, which results in differences in cross-reactivity. Homology modeling and molecular docking revealed that the triazine ring is vital in recognizing triazine compounds. The proposed immunoassay exhibited acceptable recoveries of 84.40-105.36% for detecting fruit, vegetables, and black tea. In conclusion, this study highlights a new strategy for developing sustainable immunoassays for detecting trace pesticide contaminants.

Steady-State Delivery and Chemical Modification of Food Nutrients to Improve Cancer Intervention Ability.

Cancer is a crucial global health problem, and prevention is an important strategy to reduce the burden of the disease. Daily diet is the key modifiable risk factor for cancer, and an increasing body of evidence suggests that specific nutrients in foods may have a preventive effect against cancer. This review summarizes the current evidence on the role of nutrients from foods in cancer intervention. It discusses the potential mechanisms of action of various dietary components, including phytochemicals, vitamins, minerals, and fiber. The findings of epidemiological and clinical studies on their association with cancer risk are highlighted. The foods are rich in bioactive compounds such as carotenoids, flavonoids, and ω-3 fatty acids, which have been proven to have anticancer properties. The effects of steady-state delivery and chemical modification of these food's bioactive components on anticancer and intervention are summarized. Future research should focus on identifying the specific bioactive compounds in foods responsible for their intervention effects and exploring the potential synergistic effects of combining different nutrients in foods. Dietary interventions that incorporate multiple nutrients and whole foods may hold promise for reducing the risk of cancer and improving overall health.

Innovative orthogonal two-dimensional reversed-phase liquid chromatography × supercritical fluid chromatography with a phenyl/tetrazole stationary phase for the preparative isolation of diarylheptanoids.

In this investigation, we successfully isolated and purified natural diarylheptanoids using an orthogonal offline two-dimensional RPLC × SFC approach, employing only the phenyl/tetrazole stationary phase. First, a styrene-divinylbenzene matrix medium pretreatment liquid chromatography system effectively processed chlorophyll-containing plant extract solution with a recovery rate of 33.8 %, obviating the need for concentration steps. Subsequently, an offline two-dimensional RPLC × SFC employing only the phenyl/tetrazole stationary phase achieved a remarkable 96.38 % orthogonality and was established and utilized in the preparative separation and purification of natural products. Finally, the constructed single stationary phase highly orthogonal RPLC × SFC system was successfully applied in the preparative separation and purification of natural diarylheptanoids from the Saxifraga tangutica target fraction and yielded four diarylheptanoids with purities exceeding 95 %.

Interleukin 38 improves insulin resistance in hyperlipidemic skeletal muscle cells via PPARδ/SIRT1-mediated suppression of STAT3 signaling and oxidative stress.

The cytokine interleukin-38 (IL-38), a recently discovered member of the IL-1 family, has been shown to regulate inflammation and improve hepatic endoplasmic reticulum stress and lipid metabolism in individuals with obesity. However, its impact on insulin signaling in skeletal muscle cells and the underlying mechanisms remain unclear. In vitro obesity models were established using palmitate treatment, and Western blot analysis was performed to assess target proteins. Commercial kits were used to measure glucose uptake in cultured myocytes. Our study showed that IL-38 treatment alleviated the impairment of insulin signaling, including IRS-1 and Akt phosphorylation, and increased glucose uptake in palmitate-treated C2C12 myocytes. Increased levels of STAT3-mediated signaling and oxidative stress were observed in these cells following palmitate treatment, and these effects were reversed by IL-38 treatment. In addition, IL-38 treatment upregulated the expression of PPARδ, SIRT1 and antioxidants. Knockdown of PPARδ or SIRT1 using appropriate siRNAs abrogated the effects of IL-38 on insulin signaling, oxidative stress, and the STAT3-dependent pathway. These results suggest that IL-38 alleviates insulin resistance by inhibiting STAT3-mediated signaling and oxidative stress in skeletal muscle cells through PPARδ/SIRT1. This study provides fundamental evidence to support the potential use of IL-38 as a safe therapeutic agent for the treatment of insulin resistance and type 2 diabetes.

Formation, characterization, and application of natural bioactive phytosterol-based oleogels: A review.

Oleogels are innovative structured fat systems that can replace detrimental lipids and saturated fats. Among the various gelators used to construct oleogels, phytosterols are regarded as potential oleogelators due to ability to lower blood cholesterol levels and protect patients from cardiovascular illnesses, although little research has been conducted on phytosterols. This article examines the formation, characterization, and application of phytosterol-based oleogels in detail. The oleogelation behaviors of phytosterol-based oleogels are affected by their formulation, which includes phytosterol type, combined oleogelator, proportion, concentration and oil type. These oleogels exhibit potential applications as solid fat substitutes without affecting the texture or sensory properties of food products or as effective delivery vehicles. To encourage the research and implementation of phytosterol-based oleogels, we will ultimately not only highlight problems related to their use in food processing, but also provide a few viewpoints, with the goal of providing fresh insights for advancing trends.

Aster glehni Extract, Including Caffeoylquinic Acids as the Main Constituents, Induces PPAR ß/δ-Dependent Muscle-Type Change and Myogenesis in Apolipoprotein E Knockout Mice.

To probe the functions of Aster glehni (AG) extract containing various caffeoylquinic acids on dyslipidemia, obesity, and skeletal muscle-related diseases focused on the roles of skeletal muscle, we measured the levels of biomarkers involved in oxidative phosphorylation and type change of skeletal muscle in C2C12 cells and skeletal muscle tissues from apolipoprotein E knockout (ApoE KO) mice. After AG extract treatment in cell and animal experiments, western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used to estimate the levels of proteins that participated in skeletal muscle type change and oxidative phosphorylation. AG extract elevated protein expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), phosphorylated 5'-AMP-activated protein kinase (p-AMPK), peroxisome proliferator-activated receptor beta/delta (PPARß/δ), myoblast determination protein 1 (MyoD), and myoglobin in skeletal muscle tissues. Furthermore, it elevated the ATP concentration. However, protein expression of myostatin was decreased by AG treatment. In C2C12 cells, increments of MyoD, myoglobin, myosin, ATP-producing pathway, and differentiation degree by AG were dependent on PPARß/δ and caffeoylquinic acids. AG extract can contribute to the amelioration of skeletal muscle inactivity and sarcopenia through myogenesis in skeletal muscle tissues from ApoE KO mice, and function of AG extract may be dependent on PPARß/δ, and the main functional constituents of AG are trans-5-O-caffeoylquinic acid and 3,5-O-dicaffeoylquinic acid. In addition, in skeletal muscle, AG has potent efficacies against dyslipidemia and obesity through the increase of the type 1 muscle fiber content to produce more ATP by oxidative phosphorylation in skeletal muscle tissues from ApoE KO mice.

Fabrication of polysaccharide-coated oleanolic acid-curcumin-coassembled nanoparticles (OA/Cur NPs): Enhancement of colloidal stability and water solubility.

Natural terpenoid carriers, such as oleanolic acid (OA), can enhance the water solubility and stability of hydrophobic compounds such as curcumin (Cur). However, improving the colloidal stability of nanoparticle emulsions and resolving the redispersion problem of freeze-dried nanoparticle powders remain significant challenges. In this study, we fabricated coassembled oleanolic acid-curcumin nanoparticles (OA/Cur NPs) and applied a polysaccharide surface coating method to improve their colloidal stability and water solubility. The results showed that the optimal ratio of Cur/OA for preparing OA/Cur NPs was 4:10, resulting in a high encapsulation efficiency (EE) of Cur (75.2%). Additionally, TEM, contact angle tests, Turbiscan TOWER optical stability analysis of the polysaccharide-coated OA/Cur NP emulsions and redispersion tests of their lyophilized powders verified the advantages of carboxymethyl chitosan/ß-cyclodextrin (CMC/ß-CD) coating over other polysaccharides. This study indicated that polysaccharide coating is an effective method for enhancing the colloidal stability, water solubility, and redispersibility of OA/Cur NPs.

An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.

Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 101-2 × 109 CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.

Enhancing gut barrier integrity: Upregulation of tight junction proteins by chitosan oligosaccharide through the ERK1/2 signaling pathway.

OBJECTIVES: This study aimed to explore the protective mechanism of chitosan oligosaccharide (COS) against lipopolysaccharide (LPS)-induced inflammatory responses in IEC-6 cells and dextran sodium sulfate (DSS)-induced colitis in mice. METHODS: The cell inflammation model was constructed by LPS in vitro and enteritis model by DSS in vivo. RESULTS: Following LPS exposure, IEC-6 cell proliferation significantly decreased, epithelial cell integrity was compromised, and TNF-α and IL-1ß levels were increased. However, COS pretreatment reversed these changes. In vivo, DSS-treated mice exhibited evident pathological alterations, including heightened inflammatory levels and significantly decreased expression of tight junction proteins and critical proteins in the Mitogen activated proteins kinase signaling pathway. Nevertheless, COS administration notably reduced inflammatory levels and increased the expression of tight junction proteins and key proteins in the Mitogen activated proteins kinase signaling pathway. CONCLUSIONS: Our findings suggest that COS safeguards gut barrier integrity by upregulating tight junction proteins through the ERK1/2 signaling pathway. Therefore, COS has emerged as a promising candidate for novel drug interventions against inflammatory bowel disease.

Design and preparation of NMN nanoparticles based on protein-marine polysaccharide with increased NAD+ level in D-galactose induced aging mice model.

Nicotinamide mononucleotide (NMN) is being investigated for its ability to address the decline in NAD+ level during aging. This study aimed to construct a delivery system based on ovalbumin and fucoidan nanoparticles to ameliorate the bioaccessibility of NMN by increasing NAD+ level in aging mouse model. The NMN-loaded ovalbumin and fucoidan nanoparticles (OFNPs) were about 177 nm formed by the interplay of hydrogen bonds between ovalbumin and fucoidan. Compared with free NMN, NMN-loaded OFNPs intervention could obviously improve the antioxidant enzyme activity of senescent cell induced by D-galactose. The NMN-loaded OFNPs treatment could ameliorate the loss of weight and organ index induced by senescence, and maintain the water content for the aging mice. The Morris maze test indicated that hitting blind side frequency and escape time of NMN-loaded OFNPs group decreased by 13% and 35% compared with that of free NMN group. Furthermore, the NMN-loaded OFNPs significantly alleviated the age-related oxidative stress and increased the generation of NAD+ 1.34 times by improving the bioaccessibility of NMN. Our data in this study supplied a strategy to enhance the bioavailability of NMN in senescence treatment.

AMPK/autophagy-mediated alleviation of tendinopathy by IL-38: A novel strategy for the treatment of obesity-related tendinopathy.

The effect of interleukin-38 (IL-38), a recently identified member of the IL-1 family with potential applications in various inflammation-related conditions, on ER stress has not been explored. Furthermore, its role in obesity-associated tendinopathy has not been investigated. In this study, human primary tenocytes were treated with palmitate (200 or 400 µM) and palmitate plus IL-38 (0-50 ng/mL) for 24 h. Western blotting was used to assess ER stress and tendinopathogenic markers in tenocytes. Monodansylcadaverine (MDC) staining was used to evaluate autophagosomes. Apoptosis was determined by cell viability assays, caspase 3 activity assays and TUNEL assays. Cell migration was evaluated by a cell scratch assay. Small interfering (si) RNA transfection was used for target gene silencing. Treatment of tenocytes with IL-38 attenuated apoptosis, restored the balance between MMPs and TIMP-1, and alleviated ER stress under palmitate conditions. IL-38 treatment enhanced AMPK phosphorylation and promoted the expression of autophagy markers related to LC3 conversion, p62 degradation, and autophagosome formation in cultured tenocytes. The effects of IL-38 on ER stress, apoptosis, and MMP-9, MMP-13, and TIMP-1 expression in palmitate-treated tenocytes were abrogated by AMPK siRNA or 3-methyladenine (3MA). These results suggest that IL-38 alleviates ER stress through the AMPK/autophagy pathway, thereby reducing apoptosis and preventing extracellular matrix (ECM) degradation in tenocytes under hyperlipidemic conditions. This study provides a promising therapeutic avenue for treating obesity-related tendinopathy using an endogenous compound such as IL-38.

Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water.

High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 µg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment.

Interleukin-27 as a novel player in alleviating hepatic steatosis: Mechanistic insights from an in vitro analysis.

Interleukin-27 (IL-27) is a recently discovered cytokine that has been implicated in inflammatory and metabolic conditions, such as atherosclerosis and insulin resistance. However, the mechanisms by which IL-27 attenuates hepatic lipid accumulation in hyperlipidemic conditions and counteracts endoplasmic reticulum (ER) stress, a known risk factor for impaired hepatic lipid metabolism, have not been elucidated. This in vitro study was designed to examine the effect of IL-27 on hepatic lipid metabolism. The study included the evaluation of lipogenesis-associated proteins and ER stress markers by Western blotting, the determination of hepatic lipid accumulation by Oil Red O staining, and the examination of autophagosome formation by MDC staining. The results showed that IL-27 treatment reduced lipogenic lipid deposition and the expression of ER stress markers in cultured hepatocytes exposed to palmitate. Moreover, treatment with IL-27 suppressed CD36 expression and enhanced fatty acid oxidation in palmitate-treated hepatocytes. The effects of IL-27 on hyperlipidemic hepatocytes were attenuated when adenosine monophosphate-activated protein kinase (AMPK) or 3-methyladenine (3 MA) were inhibited by small interfering RNA (siRNA). These results suggest that IL-27 attenuates hepatic ER stress and fatty acid uptake and stimulates fatty acid oxidation via AMPK/autophagy signaling, thereby alleviating hepatic steatosis. In conclusion, this study identified IL-27 as a promising therapeutic target for nonalcoholic fatty liver disease (NAFLD).

Protection against myocardial ischemia/reperfusion injury in mice by 3-caffeoylquinic acid isomers isolated from Saxifraga tangutica.

The development of ischemic heart disease (IHD) involves a variety of pathophysiological responses, such as mitochondrial dysfunction. Many compounds with antioxidant activity isolated from natural products have been shown to have significant effects on the prevention and treatment of cardiovascular diseases. However, little is known about the palliative effects of 3-caffeoylquinic acid isomers isolated from Saxifraga tangutica (S. tangutica) on myocardial ischemia/reperfusion injury (MIRI). Three isomers of 3-caffeoylquinic acid were isolated from S. tangutica and identified as neochlorogenic acid (Fr2-4-1-1, 18.5 mg), chlorogenic acid (Fr2-5-1-1, 81.7 mg) and cryptochlorogenic acid (Fr2-5-2-1, 15.0 mg) using medium-pressure liquid chromatography-high-pressure two-dimensional liquid chromatography. An in vitro DPPH assay showed that cryptochlorogenic acid (CCGA), neochlorogenic acid (NCGA) and chlorogenic acid (CGA) (in order of activity from strongest to weakest) possessed superior antioxidant activity. Langendorff's in vitro model was utilized to explore the protective effects of 3 caffeoylquinic acid isomers against MIRI. The ex vivo MIRI assay demonstrated that CCGA significantly improved hemodynamic function (P < 0.05), hemodynamic function-related indices (LVDP, RPP, +dP/dt and -dP/dt), and cell morphology in I/R myocardium tissues. In addition, the results of western blot analysis showed that mitochondrial biogenesis was significantly increased in I/R myocardial tissues after treatment with CCGA. In contrast, the activities of CGA and NCGA were lower. This is the first demonstration of efficient preparative isolation of 3-caffeoylquinic acid isomers (CGA, NCGA and CCGA) from S. tangutica. CCGA may be a promising approach for the treatment of cardiac I/R injury, especially for the regulation of mitochondrial biogenesis after MIRI.