Escherichia coli BL21(DE3) optimized deletion mutant as the host for whole-cell biotransformation of N­acetyl­D­neuraminic acid.

N­Acetyl­D­neuraminic acid (Neu5Ac) is the crucial compound for the chemical synthesis of antiflu medicine Zanamivir. Chemoenzymatic synthesis of Neu5Ac involves N-acetyl-D-glucosamine 2-epimerase (AGE)-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) to N-acetyl-D-mannosamine (ManNAc), and aldolase-catalyzed condensation between ManNAc and pyruvate. Host optimization plays an important role in the whole-cell biotransformation of value-added compounds. In this study, via single-plasmid biotransformation system, we showed that the AGE gene BT0453, cloned from human gut microorganism Bacteroides thetaiotaomicron VPI-5482, showed the highest biotransformation yield among the AGE genes tested; and there is no clear Neu5Ac yield difference between the BT0453 coupled with one aldolase coding nanA gene and two nanA genes. Next, Escherichia coli chromosomal genes involved in substrate degradation, product exportation and pH change were deleted via recombineering and CRISPR/Cas9. With the final E. coli BL21(DE3) ΔnanA Δnag ΔpoxB as host, a significant 16.5% yield improvement was obtained. Furthermore, precursor (pyruvate) feeding resulted in 3.2% yield improvement, reaching 66.8% molar biotransformation. The result highlights the importance of host optimization, and set the stage for further metabolic engineering of whole-cell biotransformation of Neu5Ac.

The accumulation, histopathology, and intestinal microorganism effects of waterborne cadmium on Carassius auratus gibelio.

Cadmium (Cd) is known to be a potentially toxic heavy metals to the fish health and growth. Carassius auratus gibelio (C. a. gibelio) specimens were exposed to waterborne Cd (0, 0.05, 0.10, 0.15, and 0.20 mg/L CdCl2) for 14 days. Cd accumulation, liver and intestine histopathology, and intestinal microorganism were investigated in the present study. The results indicated that Cd accumulation in the gill, liver, intestine, and muscle gradually decreased as Cd concentration increased. The gill accumulated higher amounts of Cd than other tissues. The histopathology of liver and intestine underwent changes with different Cd concentrations, including hepatocyte hypertrophy, aggregation of blood cells, sinusoids, lipidosis, necrosis of hepatic tissues, the erosion of villi, necrosis in the mucosal layer, the appearance of vacuoles in the lamina propria, hyperplasia, and swelling of goblet cells. Moreover, the core gut microbiota existed in the intestinal microorganism and did not change as Cd concentration increased. However, the diversity of intestinal microorganism was significantly reduced compared with that of the control sample. The present results indicated that C. a. gibelio exposed to Cd suffered toxicity, and Cd could affect the biodiversity of the intestinal microbiota of C. a. gibelio.

How peer relationships affect academic achievement among junior high school students: The chain mediating roles of learning motivation and learning engagement.

BACKGROUND: Despite the recognition of the impact of peer relationships, learning motivation, and learning engagement on academic achievement, there is still a gap in understanding the specific mechanisms through which peer relationships impact academic achievement via learning motivation and learning engagement. METHODS: This study aims to investigate how peer relationships affect junior high school students' academic achievement through the chain mediating roles of learning motivation and learning engagement, employing the self-system model of motivational development as the theoretical framework. In January 2024, 717 participants were selected from two middle schools in eastern China (mean age = 13.49 years, SD = 0.5). The data analysis in this study was performed using the structural equation model (SEM) in AMOS 24.0 and SPSS 24.0. RESULTS: The results showed that peer relationships were directly and significantly related to junior high school students' academic achievement, and that peer relationships were indirectly and positively related to junior high school students' academic achievement via learning motivation and learning engagement respectively. The results also revealed a significant indirect and positive relationship between peer relationships and junior high school students' academic achievement, mediated by the sequential mediating roles of learning motivation and learning engagement. Moreover, the path "peer relationship→learning motivation→academic achievement" has the strongest indirect effect. CONCLUSION: For junior high school students to achieve academic success, the appropriate interventions should be implemented to improve peer relationships, learning motivation, and learning engagement.

Simulated in vitro gastrointestinal digestion of ß-lactoglobulin treated by ultrasound: Detection of peptides profile and the antioxidant activity.

The influence of ultrasonic pretreatment on the release and antioxidant activity of potential antioxidant peptides after in-vitro simulated gastrointestinal digestion of ß-lactoglobulin (BLG) were measured by HPLC-MS/MS, chemical and cellular-based assays. The gastrointestinal digest was fractionated into four fractions by Sephadex G-25 gel filtration column, and fractions showed a considerable ABTS·+ scavenging ability. The fraction with the strongest antioxidant activity was produced by ultrasonicated BLG after gastrointestinal digestion, which relies on ultrasonic-promoted proteolysis to produce many small-molecule antioxidant peptides. The best active fraction has better cellular antioxidant activity and protection of H2O2-induced oxidative HepG2 cell model, which significantly increases the activities of antioxidant enzyme, and is concentration-dependent. HPLC-MS/MS analysis showed that there were more potential antioxidant peptides in the best active fraction. This research will provide a basis for the further application of ultrasonic in dairy products, which can promote the release of more potential antioxidant peptides-derived from gastrointestinal digestion.

Allergenicity assessment of ß-lactoglobulin ferulic acid-glucose conjugates.

We prepared the ß-lactoglobulin (BLG)-ferulic acid (FA)-glucose (Glu) conjugates by alkaline method and Maillard reaction to assess the allergenicity. FA and Glu can form a ternary covalent conjugate with BLG, as evidenced by the shortening of SEC retention time, upward migration of SDS-PAGE protein bands, considerable decrease in free amino and sulfhydryl content, and changes in multistructure. BLG-Glu-FA conjugates weakly bound to immunoglobulin E in allergic sera was weak, reduced interleukin 4 and tumor necrosis factor α levels in RBL-2H3 cells and histamin and interleukin 6 secretion levels in KU812 cells, and inhibited the nuclear factor-κB signaling pathway. In vivo experiments showed that the conjugates regulated T-cell homeostasis in mouse splenic and mesenteric lymphocytes and attenuated splenic and duodenal immune injury. Therefore, the conjugates of BLG with FA combined with Glu altered the epitope structure and exhibited low allergenicity.

Galacto-oligosaccharides modified whey protein isolate ameliorates cyclophosphamide-induced immunosuppression.

The effect of whey protein isolate (WPI)- galacto-oligosaccharides (GOS)/fructo-oligosaccharides (FOS) conjugates on RAW264.7 cells, and further the effect of WPI-GOS conjugates on CTX-induced immunosuppressed mice were investigated. Compared to WPI-FOS conjugates, WPI-GOS conjugates exhibited deeper glycation extent, more pronounced structural unfolding and helix-destabilizing, and obviously improved functional indicators of RAW264.7 macrophages. In addition, WPI-GOS conjugates also repaired immune organ and intestinal barrier and increased IL-1ß and IFN-γ levels in immunosuppressed mice. The alteration of gut microbiota induced by WPI-GOS conjugates changed the serum metabolites, causing the activation of NFκB pathway, which strengthens the immune system. The activation of NFκB pathway maybe associated with the mTOR signal pathway and ABC transporters. However, the precise mechanisms by which NFκB pathway interacts with mTOR signal pathway and ABC transporters to modulate the immune response need for further research.

Gut Microbiome-Serum Metabolism Revealed the Allergenicity of Ferulic Acid Combined with Glucose-Modified ß-Lactoglobulin.

A novel strategy combining ferulic acid and glucose was proposed to reduce ß-lactoglobulin (BLG) allergenicity and investigate whether the reduction in allergenicity was associated with gut microbiome and serum metabolism. As a result, the multistructure of BLG changed, and the modified BLG decreased significantly the contents of IgE, IgG, IgG1, and mMCP-1 in serum, improved the diversity and structural composition of gut microbiota, and increased the content of short-chain fatty acids (SCFAs) in allergic mice. Meanwhile, allergic mice induced by BLG affected arachidonic acid, tryptophan, and other metabolic pathways in serum, the modified BLG inhibited the production of metabolites in arachidonic acid metabolism pathway and significantly increased tryptophan metabolites, and this contribution helps in reducing BLG allergenicity. Overall, reduced allergenicity of BLG after ferulic acid was combined with glucose modification by regulating gut microbiota, the metabolic pathways of arachidonic acid and tryptophan. The results may offer new thoughts alleviating the allergy risk of allergenic proteins.

Development of the reading literacy questionnaire for EFL learners at primary schools.

Previous studies have indicated that there are a variety of factors influencing reading literacy assessment, including linguistic, cognitive, and affective factors, but little has been done on how to integrate these influential factors reasonably in a reading literacy instrument. As such, the purpose of this study is to develop and validate an English Reading Literacy Questionnaire (ERLQ) for English as foreign language (EFL) learners at the elementary level. The ERLQ was designed and revised through three rounds of validation with a sample of 784 pupils (Grades 3-6) in six primary schools from six provinces in China. Validity and reliability tests of the questionnaire were conducted with item analysis, Exploratory Factor Analysis (EFA), Confirmatory Factor Analysis (CFA), reliability test, and the analysis of criterion validity in SPSS 26.0 and AMOS 23.0. Results indicated that the revised ERLQ had high internal consistency, ranging from 0.729 to 0.823. The criterion validity of the ERLQ was supported by significant correlations to the Chinese Students' English Rating Scale verified by the authoritative department, with a correlation coefficient of 0.871. The study shows that the revised questionnaire, with 3 dimensions of 14 items, has high reliability and validity, which can be used as an assessment instrument for the intended audience. It also suggests that modifications may be made for further use in other regions and countries, depending on the background information of the learners.

The Clinical Application and Accuracy Evaluation of Noninvasive Prenatal Testing for Common Trisomy and Sex Chromosome Aneuploidy.

BACKGROUND: Noninvasive prenatal testing (NIPT) has been widely adopted in prenatal examination for fetal chromosomal aneuploidy. The present study aimed to evaluate the clinical features of NIPT for both common trisomy and sex chromosome aneuploidy (SCA). METHODS: A total of 24,164 pregnant women with NIPT testing from July 2020 to June 2022 were recruited at the Linping Maternity and Child Health Care Hospital. RESULTS: Ninety cases showed high risk of trisomy 21/18/13 with karyotype results available, and the sensitivity, specificity, and positive predictive value (PPV) were 98.41%, 99.88% and 68.89%, respectively. The three most important reasons for screening were advanced maternal age (AMA, 28.06%), intermediate risk of prenatal screening (20.34%) and Multiple of medium (MoM) abnormality of prenatal screening (17.38%). High risk of NIPT results with Z-score ≥15 have a higher PPV when compared to those with 3 ≤ Z-score < 10, and 10 ≤ Z-score < 15. Meanwhile, 97 pregnant women received positive results for fetal sex chromosome aneuploidy (SCA) in NIPT. In addition, the rate for further diagnostics of SCA was 64.95% and the PPV of SCA was 50.79%. CONCLUSIONS: Our data show that NIPT has a promising future in prenatal screening for genetic abnormalities of the fetus, and that the accuracy of NIPT is closely related to Z-score.

Molecular structure, IgE binding capacity and gut microbiota of ovalbumin conjugated to fructose and galactose:A comparative study.

Ovalbumin (OVA) was modified by fructose (Fru) and galactose (Gal) to study the structure, IgG/IgE binding capacity and effects on human intestinal microbiota of the conjugated products. Compared with OVA-Fru, OVA-Gal has a lower IgG/IgE binding capacity. The reduction of OVA is not only associated with the glycation of R84, K92, K206, K263, K322 and R381 in the linear epitopes, but also with conformational epitope changes, manifested as secondary and tertiary structural changes caused by Gal glycation. In addition, OVA-Gal could alter the structure and abundance of gut microbiota at phylum, family, and genus levels and restore the abundance of bacteria associated with allergenicity, such as Barnesiella, Christensenellaceae_R-7_group, and Collinsela, thereby reducing allergic reactions. These results indicate that OVA-Gal glycation can reduce the IgE binding capacity of OVA and change the structure of human intestinal microbiota. Therefore, Gal glycation may be a potential method to reduce protein allergenicity.

Heat stress and excessive maturity of fruiting bodies suppress GABA accumulation by modulating GABA metabolism in Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm.

GABA is a health-promoting bioactive substance. Here, the GABA biosynthetic pathways were investigated, and then the dynamic quantitative changes in GABA and the expression levels of genes related to GABA metabolism under heat stress or at different developmental stages of fruiting bodies in Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm were determined. We found that the polyamine degradation pathway was the main route of GABA production under growth normal condition. The accumulation of GABA and the expression of most genes related to GABA biosynthesis, including genes encoding glutamate decarboxylase (PoGAD-2), polyamine oxidase (PoPAO-1), diamine oxidase (PoDAO) and aminoaldehyde dehydrogenase (PoAMADH-1 and PoAMADH-2), were significantly suppressed by heat stress and the excessive maturity of fruiting bodies. Finally, the effects of GABA on the mycelial growth, heat tolerance and the morphogenesis and development of fruiting bodies were studied, the results showed that the deficiency of endogenous GABA inhibited the mycelial growth and primordial formation and aggravated heat damage, whereas exogenous application of GABA could improve thermotolerance and promote the development of fruiting bodies.

A comparative study on the allergenic potential of ß-lactoglobulin conjugated to glucose, caffeic acid and caffeoyl glucopyranose.

This work intends to perform a comparative study on the allergenic potential of ß-lactoglobulin (BLG)-glucose, BLG-caffeic acid and BLG-caffeoyl glucopyranose conjugates. The modifications changed the molecular weight and multi-structure of BLG and destroyed the allergenic epitope, which resulted in a decrease in the IgE binding level and the release ability of histamine and IL-6 in KU812 cells. Compared with BLG, the conjugates reduced the serum levels of IgG, IgE, ß-Hex and IL-4 in vivo, while increasing the level of interferon-γ, which caused an imbalance of Th1/Th2 immune response. Meanwhile, these conjugates not only increased the relative abundance of allergy-related gut flora, such as Lachnospiraceae, norank_o_Clostridia_UCG-014, Erysipelotrichaceae, Turicibacter and Lachnospiraceae_NK4A136_group, but also improved the level of short-chain fatty acids (SCFAs). Caffeoyl glucopyranose with a large molecular weight and long carbon chains exerted a great influence on the allergy-related gut flora and SCFAs. Therefore, the changes in the Th1/Th2 balance and SCFA level produced by the allergy-related gut flora were responsible for reducing the potential allergy of BLG.

The association between peer relationship and learning engagement among adolescents: The chain mediating roles of self-efficacy and academic resilience.

Previous studies have shown that peer relationship affects learning engagement. And learning engagement plays a vital role in promoting knowledge acquisition and production, enhancing adolescents' academic success. However, few studies have focused on the mechanism between peer relationship and learning engagement. As such, based on Social Cognitive Theory, this study attempts to explore how peer relationship of adolescents is linked to learning engagement through the chain mediating roles of self-efficacy and academic resilience. The participants were 250 students who were selected via random sampling in a public middle school, in Eastern China, in June 2021. All the participants filled in the structured self-report questionnaires on peer relationship, self-efficacy, academic resilience, and learning engagement. The data were analyzed with structural equation modeling (SEM) in SPSS 24.0 and AMOS 24.0. Results indicated that peer relationship was directly and positively associated with learning engagement. Results also indicated that peer relationship was indirectly and positively associated with learning engagement via self-efficacy and academic resilience, respectively, and sequentially. More importantly, it was found that the direct effect was much lower than the indirect effects of which self-efficacy was the greatest. It is suggested that appropriate interventions and support should be provided to facilitate adolescents' peer relationship, self-efficacy, and academic resilience, thus promoting their learning engagement and academic success.

Ultrasound Improved the Non-Covalent Interaction of ß-Lactoglobulin with Luteolin: Regulating Human Intestinal Microbiota and Conformational Epitopes Reduced Allergy Risks.

The present study aims to investigate the effects of ultrasound on the non-covalent interaction of ß-lactoglobulin (ß-LG) and luteolin (LUT) and to investigate the relationship between allergenicity and human intestinal microbiota. After treatment, the conformational structures of ß-LG were changed, which reflected by the decrease in α-helix content, intrinsic fluorescence intensity and surface hydrophobicity, whereas the ß-sheet content increased. Molecular docking studies revealed the non-covalent interaction of ß-LG and LUT by hydrogen bond, van der Walls bond and hydrophobic bond. ß-LG-LUT complex treated by ultrasound has a lower IgG/IgE binding ability and inhibits the allergic reaction of KU812 cells, depending on the changes in the conformational epitopes of ß-LG. Meanwhile, the ß-LG-LUT complex affected the composition of human intestinal microbiota, such as the relative abundance of Bifidobacterium and Prevotella. Therefore, ultrasound improved the non-covalent interaction of ß-LG with LUT, and the reduction in allergenicity of ß-LG depends on conformational epitopes and human intestinal microbiota changes.

Simulated in vitro digestion of α-lactalbumin modified by phosphorylation: Detection of digestive products and allergenicity.

The effects of phosphorylation on the allergenicity of bovine α-lactalbumin (BLA) and digestive products were studied in vitro digestion. Two components with different molecular weight and conformation were obtained from natural and phosphorylated BLA. In vivo and in vitro assessment of allergenicity showed that phosphorylation prior to digestion significantly decreased the IgE/IgG binding capacity and allergic response in KU812 cells, and reduced the levels of IgG, IgE, IL-4 and histamine, with an increase in IFN-γ levels in mouse serum, depending on the changes in BLA structures, producing numerous small peptides. There were four phosphorylated sites (S22, T29, S47 and S70) in the high molecular weight components of phosphorylated BLA after digestion. These phosphorylated sites could mask the linear epitopes of digestive products, resulting in reduced allergic activity. Phosphorylation prior to digestion of dairy products can reduce the risk of anaphylaxis in patients with milk allergy to some extent.

Investigation into predominant peptide and potential allergenicity of ultrasonicated ß-lactoglobulin digestion products.

The effect of ultrasonicated ß-lactoglobulin on the allergenic potential of predominant peptide was studied in vitro digestion. Gastrointestinal (GI) digestion of ultrasonicated ß-lg was fractionated into four fractions, which have different molecular weight and allergenic potentials. The lowest allergenicity of fraction was produced by ultrasonicated ß-Lg after GI digestion, depending on the changes in the structure of ß-Lg by ultrasonic and the promotion of its proteolysis, resulting in the production of numerous small peptides with significantly reduced IgE activity and basophil histamine release. Mass spectrometry analysis showed that ultrasonic can promote the further hydrolysis of large intermediate peptides, Y42, L54, L57/L58, L95, L104/F105, L122 were target residues that became more available to protease by the pretreatment of ultrasonic, thus have a smaller molecular weight with reduced allergenic potential. Ultrasonic processing of milk products alone could reduce the risk of an allergenic reaction in milk allergy patients to some extent.

Mechanism of Reduction in Allergenicity and Altered Human Intestinal Microbiota of Digested ß-Lactoglobulin Modified by Ultrasonic Pretreatment Combined with Glycation.

The effects of ultrasound combined with glycation (UCG) on the allergenicity and human microbial community of ß-Lg during in vitro digestion were studied by ELISA, cell experiments, and 16S rRNA high-throughput sequencing. UCG modification and subsequent digestion significantly reduced allergenicity. The decrease in the allergenicity of ß-Lg depended not only on the low digestibility of glycated ß-Lg, which led to the decrease of some peptides with complete immunogenicity, but also the masking effect of glycation on allergen epitopes of ß-Lg. Meanwhile, UCG modification and subsequent digestion could alter the structures of intestinal microbiota and the community abundance at phylum, family, and genus levels, such as Bacteroidota, Fusobacteriota, Enterobacteriaceae, Bacteroidaceae, Ruminococcaceae, Bacteroides, and Faecalibacterium. These results show that simulated in vitro digestion of modified ß-Lg reduces allergenicity and alters human intestinal microbiota, which could provide a theoretical basis for studying the relationship between intestinal dysbiosis and cow's milk allergy.

Moral Threshold Model is Universal? Initial Evidence from China's Collectivist Culture.

PURPOSE: People choose actions that maintain their moral self-regard. This paper explains how one's moral actions influence moral self-regard. The moral threshold model (MTM) has been proposed by scholars and tested using a limited sample. However, whether the MTM is universally applicable among people is still unknown. PARTICIPANTS AND METHODS: Our sample comprised 1761 individuals living in China, a collectivist culture, who were recruited to test the two main hypotheses of the MTM. Participants engaged in a hypothetical investment task. RESULTS: 1) When the beneficiary was a prosocial cause, participants showed stronger preferences for smaller guaranteed positive payouts over larger uncertain ones; 2) as compared to making decisions for charities, when a participant made decisions exclusively for themselves, the maximum potential benefit was more likely to influence participant behavior. CONCLUSION: The current study provides initial evidence for the validity of using the worst outcome avoidance (WOA) hypothesis of MTM among members of China's collectivist culture, indicating that the WOA hypothesis of MTM may have universal application.

Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway.

Objective: CA125/MUC16 is an O-glycosylated protein that is expressed on the surfaces of ovarian epithelial cells. This molecule is a widely used tumor-associated marker for diagnosis of ovarian cancer. Recently, CA125 was shown to be involved in ovarian cancer metastasis. The purpose of this study was to investigate the mechanism of CA125 during ovarian cancer metastasis. Methods: We analyzed the Oncomine and CSIOVDB databases to determine the expression levels of DKK1 in ovarian cancer. DKK1 expression levels were upregulated or downregulated and applied with CA125 to Transwell and Western blot assays to ascertain the underlying mechanism by which CA125 stimulates cell migration via the SGK3/FOXO3 pathway. Anti-mesothelin antibodies (anti-MSLN) were used to block CA125 stimulation. Then the expression levels of DKK1were tested by enzyme-linked immunosorbent assay (ELISA) to eliminate the blocking effect of anti-MSLN to CA125 stimulation. Xenograft mouse models were used to detect the effects of CA125 and anti-MSLN on ovarian cancer metastasis in vivo. Results: DKK1 levels were downregulated in ovarian tumor tissues according to the analyses of two databases and significantly correlated with FIGO stage, grade and disease-free survival in ovarian cancer patients. DKK1 levels were downregulated by CA125 stimulation in vitro. Overexpression of DKK1 reversed the ability of exogenous CA125 to mediate cell migration by activating the SGK3/FOXO3 signaling pathway. Anti-MSLN abrogated the DKK1 reduction and increased the apoptosis of ovarian cancer cells. The use of anti-MSLN in xenograft mouse models significantly reduced tumor growth and metastasis accelerated by CA125. Conclusions: These experiments revealed that the SGK3/FOXO3 pathway was activated, wherein decreased expression of DKK1 was caused by CA125, which fuels ovarian cancer cell migration. Mesothelin is a potential therapeutic target for the treatment of ovarian cancer metastasis.

Influence of ultrasonic pretreatment on the structure, antioxidant and IgG/IgE binding activity of ß-lactoglobulin during digestion in vitro.

The influence of ultrasonic pretreatment on the structure, antioxidant activity, and IgG/IgE binding activity of ß-lactoglobulin (ß-Lg) during digestion in vitro were studied by spectroscopy, chromatography and ELISA. After ß-Lg was treated by ultrasonic prior to digestion in vitro, the treated ß-Lg showed high intrinsic fluorescence emission, more of the hydrolytic products and high antioxidant activity, which depend on the proteolysis promoted by ultrasonic, and produced many small-molecule antioxidant peptides. Native ß-Lg was resistant to gastric digestion and retained its allergenicity. However, the allergenicity of ultrasonicated ß-Lg after gastric digestion was increased due to ultrasonic promotes the production of peptides with intact structure and immunogenicity. Subsequent gastrointestinal digestion, ultrasonicated ß-Lg has a diametrically opposite results because the increase of small peptides with the decreasing of immunogenicity. Therefore, the structural changes of ß-Lg by ultrasonic and gastrointestinal digestion were responsible for improving the antioxidant activity and reducing the IgG/IgE binding activity.