Optimization of fermentation conditions and quality evaluation of Chaenomeles sinensis glutinous rice wine.

The present study was conducted to optimize fermentation conditions for preparation of Chaenomeles sinensis Glutinous Rice Wine (CRW). The dynamic changes of main substances in the liquor during fermentation process, aroma components, biologically active substances and antioxidant capacity in the CRW after 6 months of aging were tested. The results showed that under optimized conditions, the yield and alcohol content of wine was 44.97 and 20.00%, respectively. After aging, 64 aroma components were detected in the wine, mainly alcohols and esters. The alcohol content of the CRW was 14.8%. Polyphenols and flavonoids reached 0.23 g/L and 0.037 g/L respectively. Furthermore, the CRW had an excellent free radical scavenging ability. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-024-05934-0.

Oxidation of Toluene over Mesoporous MnO2/CeO2 Nanosphere Catalysts: Effects of the MnO2 Precursor and the Type of Support.

Toluene is the most common volatile organic compound (VOC), and the MnO2-based catalyst is one of the excellent nonprecious metal catalysts for toluene oxidation. In this study, the effects of MnO2 precursors and the support types on the oxidation performance of toluene were systematically explored. The results showed that the 15MnO2/MS-CeO2-N catalyst with Mn(NO3)2·4H2O as the precursor and the mesoporous CeO2 nanosphere (MS-CeO2) as the support exhibited the most excellent performance. To reveal the reason behind this phenomenon, the calcination process of the catalyst precursor and the reaction process of toluene oxidation were investigated by in situ DRIFTS. It was found that the MnO2 precursor and the type of catalyst support could have a large effect on the reaction pathway and the produced intermediates. Therefore, the roles of the MnO2 precursor and the type of support should be key considerations when developing the high-performance MnO2-based toluene oxidation catalyst.

Effects of Modified Dietary Fiber from Fresh Corn Bracts on Obesity and Intestinal Microbiota in High-Fat-Diet Mice.

The effects of insoluble dietary fiber from fresh corn bracts modified by dynamic high-pressure micro-fluidization (DHPM) on the pathological characteristics of obesity, intestinal microflora distribution and production of short-chain fatty acids in high-fat-diet C57BL/6 mice were evaluated. The results show that the DHPM-modified dietary fiber from fresh corn bracts significantly reduces weight gain, insulin resistance and oxidative damage caused by a high-fat diet, and promotes the production of SCFAs, especially acetic acid, propionic acid and butyric acid. These modified dietary fibers also change the proportion of different types of bacteria in the intestinal microflora of mice, reduce the ratio of Firmicutes and Bacteroidota and promote the proliferation of Bifidobacteriales. Therefore, the DHPM-modified dietary fiber from fresh corn bracts can be used as a good intestinal microbiota regulator to promote intestinal health, thereby achieving the role of preventing and treating obesity.

Effects of cereal grain cell wall composition and structure on starch digestion.

BACKGROUND: Wheat is an important food crop, and its characteristics vary depending on the region of cultivation; different environments have varying effects on the composition of the grains. We previously reported the effects of environmental factors on wheat grain cell wall composition and structure. The variations in the structure of aleurone cell walls between different wheat samples were examined to determine the effects of aleurone cell walls on grain starch digestion properties. Ten different varieties of wheat grains with different aleurone cell wall structure and composition constituted a simple research system used to investigate their effect on the starch digestion of bread. RESULTS: The aleurone cell wall thickness ranged from 3.05 µm to 4.67 µm, and the arabinose to xylose ratio of water-extractable arabinoxylan was in the range 0.79-0.97. With the increase in arabinoxylans content or cell wall thickness, the total digestibility of starch within the bread decreased; this phenomenon may be related to the changes in the interaction between polysaccharides and starch granules in this process. CONCLUSION: The results of the present study showed that the wheat cell wall structure has a great impact on starch hydrolysis, indicating that the change in the digestibility of starch in flour and bread may be a result of changes in the cell wall structure leading to different combinations, thus affecting digestibility. The present study showed that the cell wall combines the starch granules during the bread-making process; thus, the diffusion of enzymes through the cell wall was hindered. © 2023 Society of Chemical Industry.

Effects of ginkgolic acid(C15:1)on biofilm formation, pathogenic factor production and quorum sensing of Pseudomonas aeruginosa.

To complete the investigation of the bacterial inhibitory activity of ginkgolic acid (GA), the quorum sensing activity of GA was investigated to assess the potential of ginkgo biloba exocarp for the production of quorum sensing inhibitor. The effect of GA (C15:1) on the growth and secondary metabolism of p. aeruginosa PAO1 were tested. The results showed that GA (C15:1) had no effect on the growth of p. aeruginosa PAO1, and that GA (C15:1) was able to inhibit the formation of quorum sensing dependent virulence factors, but promoted the formation of biofilms. Finally, the above experimental results were verified by LasR-GFP, RhlR-GFP system and fluorescence quantitative PCR. These results suggested that GA (C15:1) was capable of modulating the quorum sensing system of p. aeruginosa PAO1 and possessed potential as an anti-virulence factor drug, but is not suitable for development as a broad-spectrum biofilm inhibitor.

Study on physicochemical characteristics of lutein nanoemulsions stabilized by chickpea protein isolate-stevioside complex.

BACKGROUND: Chickpea protein isolate (CPI) originating from chickpeas has the advantages of facilitating the stability of food emulsions. Stevioside (STE) exhibits a notable surface activity and can improve the water solubility of numerous hydrophobic nutrients. STE and protein mixtures show great potential as emulsions stabilizers. The present study aimed to prepare a novel nanoemulsion for encapsulating lutein (LUT) by ultrasonic homogenization using chickpea protein isolate-stevioside complex (CPI-STE) as a stabilizer and also to investigate the physicochemical characteristics. RESULTS: The results obtained showed that different preparation conditions demonstrated significant influences on the physicochemical properties of CPI-STE-LUT nanoemulsions. Under the optimal condition, the average particle size of CPI-STE-LUT nanoemulsions was 195.1 nm, and the emulsifying and encapsulation efficiencies of lutein were 91.04% and 87.56%, respectively. CPI-STE-LUT nanoemulsions stabilized by CPI-STE could significantly increase the emulsifying and encapsulation efficiencies of lutein compared to that stabilized by CPI. Fourier transform infrared spectroscopy revealed that hydrogen bond was the main binding force of CPI and lutein, and there was a covalent bond between the two molecules. Furthermore, the stability of CPI-STE-LUT nanoemulsions in gastrointestinal phase was higher than that of CPI-LUT nanoemulsions, which could load lutein more effectively and be more resistant to digestive enzymes. CONCLUSION: The present study reports the physicochemical characterization of CPI-STE-LUT nanoemulsions for the first time. CPI-STE-LUT nanoemulsions were characterized by a small average particle size lower than 200 nm, as well as high emulsifying and encapsulation efficiencies, and good stability. © 2021 Society of Chemical Industry.

Different regulatory mechanisms of plant hormones in the ripening of climacteric and non-climacteric fruits: a review.

KEY MESSAGE: This review contains the regulatory mechanisms of plant hormones in the ripening process of climacteric and non-climacteric fruits, interactions between plant hormones and future research directions. The fruit ripening process involves physiological and biochemical changes such as pigment accumulation, softening, aroma and flavor formation. There is a great difference in the ripening process between climacteric fruits and non-climacteric fruits. The ripening of these two types of fruits is affected by endogenous signals and exogenous environments. Endogenous signaling plant hormones play an important regulatory role in fruit ripening. This paper systematically reviews recent progress in the regulation of plant hormones in fruit ripening, including ethylene, abscisic acid, auxin, jasmonic acid (JA), gibberellin, brassinosteroid (BR), salicylic acid (SA) and melatonin. The role of plant hormones in both climacteric and non-climacteric fruits is discussed, with emphasis on the interaction between ethylene and other adjustment factors. Specifically, the research progress and future research directions of JA, SA and BR in fruit ripening are discussed, and the regulatory network between JA and other signaling molecules remains to be further revealed. This study is meant to expand the understanding of the importance of plant hormones, clarify the hormonal regulation network and provide a basis for targeted manipulation of fruit ripening.

Strain-Specific Effects of Bifidobacterium longum on Hypercholesterolemic Rats and Potential Mechanisms.

Hypercholesterolemia is an independent risk factor of cardiovascular disease, which is among the major causes of death worldwide. The aim of this study was to explore whether Bifidobacterium longum strains exerted intra-species differences in cholesterol-lowering effects in hypercholesterolemic rats and to investigate the potential mechanisms. SD rats underwent gavage with each B. longum strain (CCFM 1077, I3, J3 and B3) daily for 28 days. B. longum CCFM 1077 exerted the most potent cholesterol-lowering effect, followed by B. longum I3 and B3, whereas B. longum B3 had no effect in alleviating hypercholesterolemia. Divergent alleviation of different B. longum strains on hypercholesterolemia can be attributed to the differences in bile salt deconjugation ability and cholesterol assimilation ability in vitro. By 16S rRNA metagenomics analysis, the relative abundance of beneficial genus increased in the B. longum CCFM 1077 treatment group. The expression of key genes involved in cholesterol metabolism were also altered after the B. longum CCFM 1077 treatment. In conclusion, B. longum exhibits strain-specific effects in the alleviation of hypercholesterolemia, mainly due to differences in bacterial characteristics, bile salt deconjugation ability, cholesterol assimilation ability, expressions of key genes involved in cholesterol metabolism and alterations of gut microbiota.

Preparation of a functional beverage with α-glucosidase inhibitory peptides obtained from ginkgo seeds.

To obtain α-glucosidase inhibitory peptides from ginkgo seeds and use it to develop beverages, papain hydrolysis was used to hydrolyze and extract ginkgo seed peptides. Through ultrafiltration and semi-preparative high performance liquid chromatography, peptide fragments which were molecular weight of < 10 KDa with high α-glucosidase inhibition rate were separated and purified to prepare beverages. At the same time, the A1, A2, B1, and B2 peptide fragments purified by semi-preparative high performance liquid chromatography were analyzed for amino acid composition. All four peptide fragments have glutamate. Studies have shown that amino acids such as glutamate can promote postprandial insulin secretion and reduce glucose levels. The result indicates that the amino acid composition may be related to the inhibition rate of α-glucosidase. After orthogonal experiment design, analysis of variance and principal component analysis, when 5% xylitol and 0.3% citric acid were added, and the glycine content was 1.2%, the ginkgo polypeptides beverage had the best flavor.

Systematic characterization of the branch point binding protein, splicing factor 1, gene family in plant development and stress responses.

BACKGROUND: Among eukaryotic organisms, alternative splicing is an important process that can generate multiple transcripts from one same precursor messenger RNA, which greatly increase transcriptome and proteome diversity. This process is carried out by a super-protein complex defined as the spliceosome. Specifically, splicing factor 1/branchpoint binding protein (SF1/BBP) is a single protein that can bind to the intronic branchpoint sequence (BPS), connecting the 5' and 3' splice site binding complexes during early spliceosome assembly. The molecular function of this protein has been extensively investigated in yeast, metazoa and mammals. However, its counterpart in plants has been seldomly reported. RESULTS: To this end, we conducted a systematic characterization of the SF1 gene family across plant lineages. In this work, a total of 92 sequences from 59 plant species were identified. Phylogenetic relationships of these sequences were constructed, and subsequent bioinformatic analysis suggested that this family likely originated from an ancient gene transposition duplication event. Most plant species were shown to maintain a single copy of this gene. Furthermore, an additional RNA binding motif (RRM) existed in most members of this gene family in comparison to their animal and yeast counterparts, indicating that their potential role was preserved in the plant lineage. CONCLUSION: Our analysis presents general features of the gene and protein structure of this splicing factor family and will provide fundamental information for further functional studies in plants.

Methyl jasmonate induces the resistance of postharvest blueberry to gray mold caused by Botrytis cinerea.

BACKGROUND: The effects of postharvest methyl jasmonate (MeJA) treatment (50 µmol L-1 ) on the control of gray mold caused by Botrytis cinerea in blueberry fruit were evaluated by analyzing (i) the levels of disease resistance signals; (ii) the activity of enzymes involved in antioxidant system, disease resistance and phenylpropanoid pathway, and (iii) the secondary metabolite content. RESULTS: The results indicated that MeJA treatment significantly restrained the development of gray mold decay in blueberries. The treatment induced a nitric oxide (NO) burst and increased the endogenous hydrogen peroxide (H2 O2 ) content in the earlier period of storage. The enhanced NO and H2 O2 generation by MeJA treatment might serve as a signal to induce resistance against B. cinerea infection. Furthermore, in inoculated fruit, MeJA treatment significantly promoted antioxidant enzymes and defense-related enzyme activity, which included superoxide dismutase, catalase, ascorbate peroxidase, chitinase, and ß-1,3-glucanase, and the degree of membrane lipid peroxidation was reduced. The MeJA treatment enhanced the phenylpropanoid pathway by provoking phenylalanine ammonialyase, cinnamate 4-hydroxylase, and 4-coumarate CoA ligase activity, which was accompanied by elevated levels of phenolics and flavonoids in blueberry fruit. CONCLUSION: These results suggested that MeJA could induce the disease resistance of blueberries against B. cinerea by regulating the antioxidant enzymes, defense-related enzymes, and the phenylpropanoid pathway through the activation of signaling molecules.

Right ventricle may be involved in regional diastolic dysfunction earliest in primary hypertension patients.

BACKGROUND: Impaired ventricular diastolic function is common in hypertensive patients and is one of the major causes of heart failure. Both left and right ventricle diastolic dysfunction have been reported, but the order of involvement is not clear. METHOD: A total of 161 primary hypertensive patients and 40 healthy volunteers were enrolled. Pulsed wave tissue Doppler was used to measure regional diastolic dysfunction (defined as early peak diastolic [Em] and late diastolic [Am] velocity ratios (Em/Am) < 1) at right ventricular tricuspid valve annulus lateral side (RAVP1), right ventricular tricuspid valve annulus septum side (RAVP2), left ventricular mitral valve annulus septum side (LAVP1) and left ventricular mitral annulus lateral side (LAVP2). RESULTS: The prevalence of regional diastolic dysfunction at RAVP1, RAVP2, LAVP1, and LAVP2 was all higher in the hypertensive group (P < .001 for all). In both the hypertensive group and the control group, more cases were presented with RAVP1 diastolic dysfunction, while the least number of cases had LAVP2 diastolic dysfunction. In patients with stage 1 hypertension, most cases had RAVP1, or RAVP1 and RAVP2/LAVP1 diastolic dysfunction, while in patients with more advanced hypertension stages, significantly more cases had both RAVP1 and LAVP2, or all four locations diastolic dysfunction (P < .001). A similar trend was observed in patients with longer hypertension duration (duration of 6-9.9 years and 10-18 years compared with 2-5.9 years of duration, P < .001). CONCLUSIONS: With a more advanced stage and longer duration of hypertension, the range of regional diastolic dysfunction increased, showing a trend from the right ventricular wall, to the septum and left the ventricular wall. In primary hypertension, regional diastolic dysfunction in the right ventricle might happen earlier than that in the septum and the left ventricle.

Carbon Dioxide Captured by Metal Organic Frameworks and Its Subsequent Resource Utilization Strategy: A Review and Prospect.

The carbon dioxide (CO2) is notorious as the greenhouse gas, which could cause the global warming and climate change. Therefore, the reduction of the atmospheric CO2 emissions from power plants and other industrial facilities has become as an increasingly urgent concern. In the recent years, CO2 capture and storage technologies have received a worldwide attention. Adsorption is considered as one of the efficient options for CO2 capture because of its cost advantage, low energy requirement and extensive applicability over a relatively wide range of temperature and pressure. The metal organic frameworks (MOFs) show widely potential application prospects in CO2 capture and storage owing to their outstanding textural properties, such as the extraordinarily high specific surface area, tunable pore size, ultrahigh porosity (up to 90%), high crystallinity, adjustable internal surface properties, and controllable structure. Herein, the most important research progress of MOFs materials on the CO2 capture and storage in recent years has been comprehensively reviewed. The extraordinary characteristics and CO2 capture performance of Zeolitic Imidazolate Frameworks (ZIFs), Bio-metal organic frameworks (bio-MOFs), IL@MOFs and MOF-composite materials were highlighted. The promising strategies for improving the CO2 adsorption properties of MOFs materials, especially the low-pressure adsorption performance under actual flue gas conditions, are also carefully summarized. Besides, CO2 is considered as an abundant, nontoxic, nonflammable, and renewable C1 resource for the synthesis of useful chemicals and fuels. The potential routes for resource utilization of the captured CO2 are briefly proposed.

Effect of Ginkgo biloba seed exopleura extract and chitosan coating on the postharvest quality of ginkgo seed.

BACKGROUND: The effects of Ginkgo biloba seed exopleura extract (GSEE) and chitosan (CH) coating on the preservation of ginkgo seeds were investigated. RESULTS: Results showed that CH could alleviate the decay rate, and that CH combined with GSEE (CH-GSEE) treatment further inhibited the development of seed decay due to the additional antifungal activity of GSEE. The nutrient contents, including water, starch and soluble protein, were maintained by both CH-GSEE and CH treatments. CH-GSEE treatment led to better restriction on oxidative stress by decreasing superoxide anion production rate, membrane permeability, malondialdehyde content, respiration rate and ethylene production rate. The antioxidant enzyme activities of peroxidase, superoxide dismutase and catalase in ginkgo seeds were maintained by treatment with CH-GSEE at a higher level. These results were consistent with the enhanced visual appearance, qualities and storability of the CH-GSEE-treated seeds. Principal component analysis provided a global view of the internal relations of the ginkgo seeds with different treatments. CONCLUSIONS: The postharvest qualities of CH-GSEE-treated seeds were better than those of other treatment groups. Therefore, CH-GSEE is an effective and alternative way for inhibiting decay, maintaining quality and extending the postharvest life of ginkgo seeds. © 2018 Society of Chemical Industry.

Physical adsorption of patulin by Saccharomyces cerevisiae during fermentation.

Patulin (PAT), a mycotoxin mainly produced by various species of fungi, is frequently detected in moldy fruit- and vegetable-based products, which pose a health risk to the consumer. Over the past decades, a few studies reported that PAT content could be significantly decreased by microbial fermentation process. However, the physical adsorption mechanism between PAT and yeast during fermentation is still unclear. In this paper, we focused on the physical adsorption of PAT by Saccharomyces cerevisiae CCTCC 93161 during fermentation in aqueous solutions. Firstly, morphology of differently treated yeast cells were analyzed by scanning electron microscope, then the interactions between PAT and yeast cells were investigated by infrared absorption spectra of differently treated S. Cerevisiae cells before and after the adsorption of PAT. The results showed that the efficiency of PAT removal raised significantly with the increase of fermentation temperature and time, whereas it decreased significantly with the increase of initial PAT concentration in the fermentation system. The proteins and polysaccharides in the cell walls of yeast interacted with PAT and accounted for the physical adsorption. The current work would possibly provide some new insights on PAT control for fermented foods.

Novel C15 Triene Triazole, D-A Derivatives Anti-HepG2, and as HDAC2 Inhibitors: A Synergy Study.

A series of novel C15 urushiol derivatives were designed by introducing a pechmann structure and F-, Cl-, and Br-nitro substituents with different electronic properties into its alkyl side chain, as well as a triazolyl functional group in its aromatic oxide. Their chemical structures were determined based on the analysis of the NMR (nuclear magnetic resonance) spectroscopic and mass spectrometric data. The results showed that compound 4 exhibited a strong inhibition of the HepG2 cell proliferation (half maximal inhibitory concentration (IC50): 2.833 µM to human hepatocellular carcinoma (HepG2), and 80.905 µM to human normal hepatocytes (LO2)). Furthermore, it had an excellent synergistic effect with levopimaric acid. The nitrogen atom of the triazole ring formed a hydrogen-bonding interaction with Gly103, Gly154, and Tyr308, which made compound 4 bind to histone deacetylase (HDAC)2 more tightly. One triazole ring and His33 formed a π⁻π stacking effect; the other, whose branches were deep into the pocket, further enhanced the interaction with HDAC2. Meanwhile, compound 4 involved a hydrophobic interaction with the residues Phe210 and Leu276. The hydrophobic interaction and π⁻π stacking provided powerful van der Waals forces for the compounds.

The antibacterial activity and mechanism of ginkgolic acid C15:1.

BACKGROUND: The present study investigated the antibacterial activity and underlying mechanisms of ginkgolic acid (GA) C15:1 monomer using green fluorescent protein (GFP)-labeled bacteria strains. RESULTS: GA presented significant antibacterial activity against Gram-positive bacteria but generally did not affect the growth of Gram-negative bacteria. The studies of the antibacterial mechanism indicated that large amounts of GA (C15:1) could penetrate GFP-labeled Bacillus amyloliquefaciens in a short period of time, and as a result, led to the quenching of GFP in bacteria. In vitro results demonstrated that GA (C15:1) could inhibit the activity of multiple proteins including DNA polymerase. In vivo results showed that GA (C15:1) could significantly inhibit the biosynthesis of DNA, RNA and B. amyloliquefaciens proteins. CONCLUSION: We speculated that GA (C15:1) achieved its antibacterial effect through inhibiting the protein activity of B. amyloliquefaciens. GA (C15:1) could not penetrate Gram-negative bacteria in large amounts, and the lipid soluble components in the bacterial cell wall could intercept GA (C15:1), which was one of the primary reasons that GA (C15:1) did not have a significant antibacterial effect on Gram-negative bacteria.

Solubility-permeability interplay of a supersaturated lutein delivery system constructed by glycosylated stevioside and hydroxypropyl-methylcellulose.

This study investigated the solubilizing capacity of glycosylated stevioside/hydroxypropyl-methylcellulose (stevia-G-HPMC) complexes with varying mass ratios on lutein. The impact on the steady-state flux and permeability coefficient of intracellular lutein was also explored through the construction of a Caco-2 cellular transport model. The results indicated that the equilibrium solubility of lutein linearly increased with an increase in stevia-G amount. The stability constants of the ternary system surpassed those of the binary system. Molecular dynamics simulation revealed a tight and stable structure in lutein supersaturated complexes. Meanwhile, lutein-stevia-G-HPMC complexes demonstrated superior cumulative penetrations, with the peak Papp (AP â†’ BL) value being (3.24 ± 0.89) × 10-5 cm·s-1. There was a slight decrease in Papp (BL â†’ AP), which improved the forward transport of lutein. Highly soluble lutein in aqueous environments saturated the extracellular transport proteins on the AP side of cell membranes, thereby maintaining the high permeability transport. Notably, the permeability trend of lutein in Caco-2 cells negatively correlated with the equilibrium solubility and matched the single exponential growth model. When the mass ratio of lutein, stevia-G and HPMC was 1:21:5, the solubility-permeability trade-off of lutein was effectively maintained.

Preparation and characterization of fine and stable short amylose nanocarriers for curcumin using a highly efficient and convenient method.

To prepare fine and stable nanocarriers for curcumin using a highly efficient and convenient method, nanoprecipitation combined with ultrasonication and a high-speed dispersion (US+HSS) method were used to prepare short amylose nanoparticles with pre-formed helical structures. Their morphology, structural characteristics, and embedding effects for curcumin were investigated. The results showed that the optimal ratio of ethanol to short amylose solution and ultrasonic time was 4:1 and 4 min, respectively. The nanoparticles showed a small size (82.43 nm), relatively high loading capacity (11.57 %), and a peak gelatinization temperature of 97.74 °C. Compared to the nanoprecipitation method, the short amylose nanoparticles prepared using the US+HSS method possessed a higher V-type crystalline structure ratio. In addition, the US+HSS method was easier to use to prepare nanoparticles with high stability against NaCl, and the stable nanoparticles showed the best in vitro sustained release effect for curcumin. The Peppas-Sahlin model was the optimal model that matched curcumin release from nanoparticles during digestion.

Effects of ultraviolet C on the quality and aroma volatile in peach fruit during postharvest storage.

The study investigated the impact of UV-C irradiation on peach fruit quality during postharvest storage, with a focus on aroma changes and the mechanisms involving lipoxygenase metabolism. Results showed that UV-C irradiation at a dosage of 1.5 kJ/m2 was found to preserve the quality attributes of peach fruit during ambient storage, as evidenced by high flesh firmness, inhibition of weight loss and respiration rate, as well as high values of L* and ascorbic acid. Meanwhile, UV-C irradiation led to an increase in the contents of aroma-related volatiles, particularly esters and lactones, compared to non-irradiated fruit. Our results suggested that the enhanced emission of aroma-related volatiles in UV-C irradiated peach fruit was linked to elevated levels of unsaturated fatty acids. Besides, UV-C induced the expressions and activities of enzymes in the lipoxygenase pathway, thus promoting the synthesis of esters and lactones, which contribute to the enhanced aroma in peach fruit.