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Human milk phospholipids (HMPLs) play an indispensable role in the neurodevelopment and growth of infants. In this study, a total of 37 phospholipid fatty acid (PLFA) species and 139 phospholipid molecular species were detected from human milk and other natural phospholipid sources (including 5 animal-derived species and 2 plant species). Moreover, a similarity evaluation model for HMPLs was established, including phospholipid classes, PLFAs, and phospholipid molecular species, to evaluate their natural substitutes. The closest scores for HMPL substitute in these three dimensions was 0.89, 0.72, and 0.77, which belonged to mare milk, goat milk, and camel milk, respectively. The highest comprehensive similarity score was obtained by camel milk at 0.75, while the lowest score was observed in soybean phospholipid (0.22). Therefore, these results not only monitored the stereochemical structure of HMPLs and their substitutes, but also further provided new insights for the development of infant formulae.
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The study aims to analyze the composition of the gut microbiota in Chinese individuals using metagenomic sequencing technology, with a particular focus on the abundance of Akkermansia muciniphila (Akk). To improve the efficiency of Akk isolation and identification accuracy, modifications were made to the enrichment culture medium and 16S rRNA universal primers. Additionally, potential growth-promoting factors that stimulate Akk growth were explored through in vitro screening. The research results revealed that the abundance of Akk in Chinese fecal samples ranged from 0.004% to 0.4%. During optimization, a type of animal protein peptide significantly enhanced the enrichment efficiency of Akk, resulting in the isolation of three Akk strains from 14 fecal samples. Furthermore, 17 different growth-promoting factors were compared, and four factors, including galactose, sialic acid, lactose, and chitosan, were identified as significantly promoting Akk growth. Through orthogonal experiments, the optimal ratio of these four growth-promoting factors was determined to be 1:1:2:1. After adding 1.25% of this growth-promoting factor combination to the standard culture medium, Akk was cultivated at 37° for 36 h, achieving an OD600nm value of 1.169, thus realizing efficient proliferation and optimized cultivation of Akk. This study provides important clues for a deeper understanding of the gut microbiota composition in Chinese individuals, while also offering effective methods for the isolation and cultivation of Akk, laying the groundwork for its functional and application research in the human body.
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We aimed to obtain the optimal formula for human milk fat substitute (HMFS) through a combination of software and an evaluation model and further verify its practicability through an animal experiment. The results showed that a total of 33 fatty acid (FA) and 63 triglyceride (TAG) molecular species were detected in vegetable oils. Palmitic acid, oleic acid, linoleic acid, 18:1/16:0/18:1, 18:2/16:0/18:2, 18:1/18:1/18:1 and 18:1/18:2/18:1, were the main molecular species among the FAs and TAGs in the vegetable oils. Based on the HMFS evaluation model, the optimal mixed vegetable oil formula was blended with 21.3% palm oil, 2.8% linseed oil, 2.6% soybean oil, 29.9% rapeseed oil and 43.4% maize oil, with the highest score of 83.146. Moreover, there was no difference in the weight, blood routine indices or calcium and magnesium concentrations in the feces of the mice between the homemade mixed vegetable oil (HMVO) group and the commercial mixed vegetable oil (CMVO) group, while nervonic acid (C24:1) and octanoic acid (C8:0) were absorbed easily in the HMVO group. Therefore, these results demonstrate that the mixing of the different vegetable oils was feasible via a combination of computer software and an evaluation model and provided a new way to produce HMFS.
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Sustitutos de Grasa , Ácidos Grasos , Leche Humana , Aceites de Plantas , Programas Informáticos , Triglicéridos , Humanos , Animales , Aceites de Plantas/química , Ácidos Grasos/química , Leche Humana/química , Ratones , Triglicéridos/química , Sustitutos de Grasa/química , Aceite de Palma/química , Aceite de Soja/química , Aceite de Linaza/química , Aceite de Brassica napus/química , Aceite de Maíz/química , Caprilatos/química , Ácido Palmítico/química , Ácido Oléico/químicaRESUMEN
Objective: Management of large irregular wounds in children had been confusing plastic and reconstructive surgeons. Herein, this study was aimed to propose a new treatment method based on the principle of adapting different recipient zones to overcome the intractable wounds, simplifying and programing the design process of targeted flaps for covering large irregular soft-tissue defects. Patients and methods: From January 2009 to December 2020, 31 children (9 girls and 22 boys) aged 3-16 years (mean 9.8 years) underwent multiple modular flaps with edge to edge splicing reconstruction of the lower extremities. All the wounds were large with non-adjacent defects and with or without a dead space. Several variants of flaps were harvested according to the needs and reconstruction requirements of patients. Results: A total of 71 flaps were harvested from 31 patients and all flaps donor sites received primary closure. Nine patients underwent split-thickness skin grafting, and three cases of flaps survived from vascular crisis by rebuilding the vessels and the rest accepting LD flap transplants. And five partial necrosis of the distal epidermis flaps recovered using skin grafting and dressing change. No major complication was encountered in other patients and donor sites, except one heel ulcer. During the follow-up (ranging from 16 to 38 months, mean 27.7 months), aesthetic and functional results of reconstructed limbs were satisfactory in all patients. Conclusions: The Individualized design program of multiple flaps for adapting different recipient zones is an alternative for repairing large irregular soft-tissue defects in children, beneficial for plastic and reconstructive surgeons to simplify and program the process of designing and perform multiple flaps to achieve this goal. Level of evidence: III, Retrospective.
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BACKGROUND: A new spray adhesive (KYNA-PF127) was established through the combination of thermosensitive hydrogel (Pluronic F127) and KYNA, aimed to investigate the effect of KYNA-PF127 on multi-territory perforator flaps and its possible molecular mechanism. MATERIALS AND METHODS: 36â¯SD male rats with 250-300â¯g were randomly divided into 3 groups (n = 12): control group, blank glue group and KYNA-PF127 group. KYNA-PF127 hydrogel was prepared and characterized for its morphology and properties using scanning electron microscopy. CCK-8 assay, scratch wound assay, transwell assay, tube formation assay and Ki67 staining were used to study the effect of KYNA-PF127 on the proliferation, migration, and tube formation of HUVECs. VEGF and FGF2 were measured by qPCR to evaluate the angiogenesis capacity of HUVECs in vitro. In vivo, the effect of each group on the survival area of the cross-zone perforator flap was evaluated, and angiogenesis was evaluated by HE and immunofluorescence (CD31 and MMP-9). The effect of inflammation on skin collagen fibers was assessed by Masson. Immunohistochemistry (SOD1, IL-1ß, TNF-α) was used to evaluate the effects of oxidative stress and inflammatory factors on multi-territory flaps. RESULTS: KYNA-PF127 has good sustained release and biocompatibility at 25% concentration. KYNA-PF127 promoted the proliferation, migration, and angiogenesis of HUVECs in vitro. In vivo, the survival area of multi-territory perforator flaps and angiogenic capability have increased after KYNA-PF127 intervention. KYNA-PF127 could effectively reduce the oxidative stress and inflammation of multi-territory perforator flaps. CONCLUSION: KYNA-PF127 promotes angiogenesis through its antioxidant stress and anti-inflammatory effects, and shows potential clinical value in promoting the survival viability and drug delivery of multi-territory perforator flaps.
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Células Endoteliales de la Vena Umbilical Humana , Hidrogeles , Inflamación , Neovascularización Fisiológica , Colgajo Perforante , Ratas Sprague-Dawley , Animales , Masculino , Inflamación/patología , Inflamación/tratamiento farmacológico , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratas , Colgajo Perforante/irrigación sanguínea , Neovascularización Fisiológica/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , AngiogénesisRESUMEN
In this study, Senescence Accelerated Mice (SAMP8) were supplemented with exogenous DHA milk, endogenous DHA milk, normal milk, or 0.9 % saline solution. Enzyme-linked immunosorbent assay (ELISA), gas chromatography (GC), ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI MS/MS), and Morris water maze were used to characterize the effects of diet on oxidative stress and cognition in SAMP8 mice. Supplementation endogenous DHA milk or exogenous DHA milk can enhance the antioxidant capacity of mice organs. Endogenous DHA milk increased the superoxide dismutase (SOD) activity of mice brain and serum than normal milk and 0.9 % saline solution (P ≤ 0.05), as well as increased SOD activity of mice liver and glutathione peroxidase (GSH-Px) activity of mice brain than normal milk (P ≤ 0.05). Exogenous DHA milk increased SOD activity of mice brain than normal milk and 0.9 % saline solution, as well as increased SOD activity of mice serum than 0.9 % saline solution (P ≤ 0.05). Several polar lipid relative content, such as 18:0/18:2 PS, 17:0 Ceramide, and 20:4 LPC in mice brain was affected by dietary supplementation with DHA-containing milk. Lipid oxidation metabolites in mice brain were not affected by DHA-containing milk. Endogenous DHA milk increased the number of platform location crossing times of mice in the Morris water maze test, compared with Exogenous DHA milk, normal milk, and 0.9 % saline solution (P ≤ 0.05).
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Antioxidantes , Cognición , Ácidos Docosahexaenoicos , Leche , Estrés Oxidativo , Superóxido Dismutasa , Animales , Estrés Oxidativo/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Cognición/efectos de los fármacos , Leche/química , Ratones , Superóxido Dismutasa/metabolismo , Masculino , Antioxidantes/metabolismo , Antioxidantes/farmacología , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Suplementos Dietéticos , Aprendizaje por Laberinto/efectos de los fármacos , Hígado/metabolismo , Hígado/efectos de los fármacosRESUMEN
ZW10 interacting kinetochore protein (ZWINT), an essential part of the kinetochore complex, plays a crucial role in maintaining genome stability by correcting improper attachments between the kinetochore and microtubules. An initial analysis of The Cancer Genome Atlas and Gene Expression Omnibus databases revealed that ZWINT is significantly expressed across a diverse range of tumor types. We subsequently investigated the influence of ZWINT on clinical outcomes and potential signaling pathways. A multidimensional analysis of ZWINT revealed significant statistical associations between ZWINT expression and clinical outcomes, as well as the E2F1 oncogenic signature. Experimental validation confirmed the increased expression of ZWINT in both pancreatic cancer cell lines and pancreatic adenocarcinoma tissues. Furthermore, our findings indicate that ZWINT promotes the proliferation of PANC-1 cells through cell cycle regulation. This comprehensive analysis of ZWINT suggests a strong correlation between its expression and various types of tumors, especially pancreatic adenocarcinoma (PAAD), indicating its potential oncogenic role. These findings enhance our understanding of the function of ZWINT in carcinogenesis.
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Milk samples were collected from 3625 Chinese Holstein cows to assess the effects of κ-casein (κ-CN) and ß-lactoglobulin (ß-LG) genetic variants on its milk coagulation properties. The results show that Chinese Holstein cows have a higher frequency of the κ-CN AA and AB variants, and ß-LG of the AB and AA variants. Of these, κ-CN B variants, the ß-LG AA and BB variants were more frequent in milk showing good coagulation. The effects of the genetic variants on milk composition, milk proteome, and protein phosphorylation sites were studied. The results showed that higher concentrations of protein and dry matter were found in κ-CN BE variant. Moreover, large variations in milk proteome among different κ-CN and ß-LG variants were observed. Highly phosphorylated for κ-CN, especially Ser97, was observed in cows with the κ-CN BE variant, but no effect of ß-LG variants on phosphorylation site was found. Of the various factors examined, variation of κ-CN phosphorylation sites Ser97 may be the most important in affecting casein structure and milk coagulation ability. Some milk protein contents were found to be negative factors for milk coagulation. In summary, this study showed that κ-CN genetic variants contained different milk compositions and phosphorylation site Ser97 influenced milk coagulation.
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Leche , Proteoma , Animales , Femenino , Bovinos , Proteoma/metabolismo , Fosforilación , Leche/química , Proteínas de la Leche/química , Caseínas/química , Lactoglobulinas/genética , Lactoglobulinas/metabolismo , GenotipoRESUMEN
BACKGROUND: Polydatin has shown considerable pharmacological activities in ischemia-reperfusion injuries of various organs. However, its effects and mechanisms in spinal cord ischemia-reperfusion injury have not been fully established. In this study, the mechanisms of polydatin against spinal cord ischemia-reperfusion injury were investigated via network pharmacology, molecular docking and molecular dynamics simulation. METHODS: Spinal cord ischemia-reperfusion injury-related targets were obtained from the GeneCards database, while polydatin-related action targets were obtained from the CTD and SwissTarget databases. A protein-protein interaction network of potential targets was constructed using the String platform. After selecting the potential key targets, GO functional enrichment and KEGG pathway enrichment analyses were performed via the Metascape database, and a network map of "drug-target-pathway-disease" constructed. The relationships between polydatin and various key targets were assessed via molecular docking. Molecular dynamics simulation was conducted for optimal core protein-compound complexes obtained by molecular docking. RESULTS: Topological analysis of the PPI network revealed 14 core targets. GO functional enrichment analysis revealed that 435 biological processes, 12 cell components and 29 molecular functions were enriched while KEGG pathway enrichment analysis revealed 91 enriched signaling pathways. Molecular docking showed that polydatin had the highest binding affinity for MAPK3, suggesting that MAPK3 is a key target of polydatin against spinal cord ischemia-reperfusion injury. Molecular dynamics simulations revealed good binding abilities between polydatin and MAPK3. CONCLUSIONS: Polydatin exerts its effects on spinal cord ischemia-reperfusion injury through multiple targets and pathways. MAPK3 may be a key target of polydatin in spinal cord ischemia-reperfusion injury.
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Simulación de Dinámica Molecular , Daño por Reperfusión , Médula Espinal , Humanos , Simulación del Acoplamiento Molecular , Farmacología en Red , Daño por Reperfusión/tratamiento farmacológicoRESUMEN
Background: Macrophages play an important role in regulating the course of spinal tuberculosis within the bone microenvironment. This study aimed to investigate the differential expression of miRNA in macrophage-derived exosomes within the tuberculosis-infected bone microenvironment, to identify miRNAs that hold potential as diagnostic markers and therapeutic targets. Methods: We established study cohorts for spinal tuberculosis, collected bone marrow blood samples, isolated macrophage exosomes, and performed exosome miRNA sequencing. A miRNA-mRNA co-expression network was constructed using WGCNA analysis. Gene GO analysis and KEGG pathway enrichment analysis were performed using KOBAS software. Target miRNAs were selected based on fold change, P-value, and false discovery rate, and their validation was carried out using qRT-PCR and ROC curve studies. Subsequently, we constructed a target gene network for these miRNAs and performed KEGG pathway enrichment analysis to explore the potential signaling mechanisms involved in regulating the disease course of spinal tuberculosis. Results: Our findings revealed that macrophages from the tuberculosis-infected bone microenvironment exhibited an M1 phenotype. The successful extraction of exosomes from macrophage supernatants was confirmed through electron microscopy, particle size analysis, and protein blot analysis. Exosome miRNA-seq demonstrated that 28 miRNAs were up-regulated, while 34 miRNAs were down-regulated in individuals with spinal tuberculosis. GO analysis and KEGG pathway enrichment analysis indicated that the differentially expressed miRNAs were involved in various biological processes, cell components, molecular functions, and signaling pathways, which collectively contribute to the regulation of the disease course of spinal tuberculosis. Notably, miRNA-125b-5p was successfully selected based on fold change, p-value, and false discovery rate. qRT-PCR validation further confirmed the significant up-regulation of miRNA-125b-5p in spinal tuberculosis. The ROC curve revealed that miR-125b-5p is a potential diagnostic biomarker for spinal tuberculosis. Moreover, construction of the miRNA-125b-5p target gene network and subsequent KEGG enrichment analysis highlighted the importance of MAPK, TNF, Ras, Rap1, and the PI3K-Akt signaling pathways in the regulation of the disease course of spinal tuberculosis. Conclusion: Our study demonstrates differential expression of miRNAs in macrophage-derived exosomes in the tuberculosis-infected bone microenvironment. Specifically, MiRNA-125b-5p is significantly up-regulated in spinal tuberculosis and shows potential as a diagnostic biomarker for spinal tuberculosis.
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Thermal-denatured whey protein-milk fat emulsion gels with different degrees of pre-emulsification were prepared by pre-emulsifying milk fat with thermal-denatured whey protein and used in the preparation of reduced-sodium processed cheeses. The effect of the thermal-denatured whey protein pre-emulsification process on the texture and microstructure of reduced-sodium processed cheeses was evaluated by studying the composition, color, texture, functional properties, microstructure and sensory analysis of the processed cheeses. The results showed that compared with cheese without pre-emulsified fat (1.5% ES control), the moisture content of cheese with pre-emulsified 100% fat (1.5% ES100) increased by 5.81%, the L* values increased by 7.61%, the hardness increased by 43.24%, and the free oil release decreased by 38%. The microstructure showed that the particle size of fat was significantly reduced, and the distribution was more uniform. In addition, compared with the cheese added with 3% emulsifying salt (3% ES control), the amount of emulsifying salt in the 1.5% ES100 decreased by 50%, but the fat distribution of the two kinds of cheese tended to be consistent, and there was no obvious change in texture characteristics and meltability. Sensory scores increased with the increase in pre-emulsification degree. Overall, the pre-emulsification of milk fat with thermal-denatured whey protein can reduce the sodium content of processed cheese and improve its quality.
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In this study, we built classification models using machine learning techniques to predict the bioactivity of non-covalent inhibitors of Bruton's tyrosine kinase (BTK) and to provide interpretable and transparent explanations for these predictions. To achieve this, we gathered data on BTK inhibitors from the Reaxys and ChEMBL databases, removing compounds with covalent bonds and duplicates to obtain a dataset of 3895 inhibitors of non-covalent. These inhibitors were characterized using MACCS fingerprints and Morgan fingerprints, and four traditional machine learning algorithms (decision trees (DT), random forests (RF), support vector machines (SVM), and extreme gradient boosting (XGBoost)) were used to build 16 classification models. In addition, four deep learning models were developed using deep neural networks (DNN). The best model, Model D_4, which was built using XGBoost and MACCS fingerprints, achieved an accuracy of 94.1% and a Matthews correlation coefficient (MCC) of 0.75 on the test set. To provide interpretable explanations, we employed the SHAP method to decompose the predicted values into the contributions of each feature. We also used K-means dimensionality reduction and hierarchical clustering to visualize the clustering effects of molecular structures of the inhibitors. The results of this study were validated using crystal structures, and we found that the interaction between the BTK amino acid residue and the important features of clustered scaffold was consistent with the known properties of the complex crystal structures. Overall, our models demonstrated high predictive ability and a qualitative model can be converted to a quantitative model to some extent by SHAP, making them valuable for guiding the design of new BTK inhibitors with desired activity.
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Introduction: Glycosylation is one of the essential post-translational modifications that influences the function of milk proteins. Methods: In the present study, 998 proteins and 764 glycosylated sites from 402 glycoproteins were identified in human milk by TMT labeling proteomics. Compared to human milk proteins, the glycoproteins were mainly enriched in cell adhesion, proteolysis, and defense/immune process. Results: The abundance of 353 glycosylated sites and their 179 parent proteins was quantified. After normalization to their parent protein's abundance, 78 glycosylated sites in 56 glycoproteins and 10 glycosylated sites in 10 glycoproteins were significantly higher in colostrum and mature milk, respectively. These changed glycoproteins were mainly related to host defense. Intriguingly, one glycosylated site (Asp144) in IgA and two glycosylated sites (Asp38 and Asp1079) in tenascin are significantly upregulated even though their protein abundance was downregulated during lactation. Discussion: This study helps us figure out the critical glycosylated sites in proteins that might influence their biological function in an unbiased way.
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Milk samples were collected from 10 cows, in the colostrum (3-4 days) and mature (90 days) lactation stage, to assess the differential expression of all whey proteins and N-glycoproteins. In total, 240 whey proteins and 315 N-glycosylation sites on 214 glycoproteins were quantified. GO annotations, KEGG pathway analysis, and protein classification were performed to understand the similarities and differences of the biological functions of whey proteins and N-glycoproteins among different lactation stages in bovine milk. Furthermore, differential expression of whey proteins and whey N-glycosylated proteins was found between different lactation stages. The related changes of biological functions in differentially expressed proteins were discussed. For example, the increased frequency of glycosylation on lactoferrin and folate receptor alpha occurring in bovine colostrum may provide protection and stimulate development of the newborn calf. Our study thereby improves understanding of variations of glycosylation sites on milk glycoproteins among lactation stages.
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Calostro , Leche , Animales , Bovinos , Femenino , Embarazo , Calostro/química , Glicoproteínas/metabolismo , Lactancia , Leche/química , Proteínas de la Leche/metabolismo , Proteoma/metabolismo , Suero Lácteo/química , Proteína de Suero de Leche/metabolismoRESUMEN
Background: Surgical intervention is necessary for resolving the symptoms of the spinal cord and nerve compression caused by symptomatic metastatic epidural spinal cord compression. However, surgeons are constantly seeking ways to improve surgical efficiency and safety. This study aims to evaluate the efficacy of 3D simulation/printing-assisted surgery for symptomatic metastatic epidural spinal cord compression of the posterior column. Methods: We retrospectively analyzed the clinical data of patients who underwent surgical treatment for symptomatic metastatic epidural spinal cord compression of the posterior column in our hospital from January 2015 to January 2020. The simulated group underwent a 3D digital simulation of the lesion area using imaging data before surgery. Twelve patients in the simulated group also received 3D printing, while the direct surgery group did not receive any 3D simulation or printing. All patients were followed up for at least 2 years. We collected clinical data, including operation time, intraoperative blood loss, pedicle screw adjustment rate, intraoperative fluoroscopy times, the incidence of dural injury and cerebrospinal fluid leakage, VAS score, postoperative neurological function improvement, and tumor recurrence. Statistical analysis was performed using SPSS23.0, and P < 0.05 was considered statistically significant. Results: A total of 46 patients were included in this study, with 20 in the simulated group and 26 in the non-simulated group. The simulated group had better operation time, intraoperative blood loss, screw adjustment rate, fluoroscopy times, and incidence of dural injury/cerebrospinal fluid leakage compared to the non-simulated group. The VAS scores of the two groups improved significantly after the operation and at the last follow-up compared to before the operation. However, there was no statistically significant difference between the two groups. There was also no statistically significant difference in neurological function improvement between the two groups. In the simulated group, 25% of patients relapsed, while in the non-simulated group, 34.61% of patients relapsed. However, there was no statistical difference between the two groups. Conclusion: Preoperative 3D simulation/printing-assisted surgery is a practical and feasible approach for treating symptomatic metastatic epidural spinal cord compression of the posterior column.
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Equol is the most potent soy isoflavone metabolite and is produced by specific intestinal microorganisms of mammals. It has promising application possibilities for preventing chronic diseases such as cardiovascular disease, breast cancer, and prostate cancer due to its high antioxidant activity and hormone-like activity. Thus, it is of great significance to systematically study the efficient preparation method of equol and its functional activity. This paper elaborates on the metabolic mechanism of equol in humans; focuses on the biological characteristics, synthesis methods, and the currently isolated equol-producing bacteria; and looks forward to its future development and application direction, aiming to provide guidance for the application and promotion of equol in the field of food and health products.
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Heat shock protein family A member 5 (HSPA5) plays a pivotal role in the endoplasmic reticulum (ER) stress response and unfolded protein response (UPR), both of which are crucial for protein folding, assembly, and quality control within cells. In response to ER stress, HSPA5 becomes overexpressed to preserve cellular homeostasis. A previous study revealed a robust association between HSPA5 expression and various cancers. However, the prognostic function of HSPA5 and its involvement in tumor formation remain largely unknown. In this study, we integrated HSPA5 expression data from databases such as the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and The Cancer Genome Atlas (TCGA) to conduct a comprehensive pan-cancer analysis of HSPA5. Our findings revealed that HSPA5 is overexpressed in various tumor types and is significantly associated with poor prognosis. Additionally, HSPA5 expression is significantly correlated with immune checkpoints, stromal infiltration, and consequent alterations in the immune landscape. Verification was conducted on samples from patients with various tumor types, including breast and liver cancers. Additionally, we also performed verification in vitro. In conclusion, HSPA5 may offer a potential target for cancer treatment.
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This study aimed to determine chemical composition and assess exposure in flavored milk among Chinese residents, based on risk assessment methodologies of acceptable daily intake (ADI) and toxicological concern threshold (TTC). Esters (32.17%), alcohols (11.19%), olefins (9.09%), aldehydes (8.39%), and ketones (7.34%) comprised the majority of the flavoring samples. Methyl palmitate (90.91%), ethyl butyrate (81.82%), and dipentene (81.82%) had the highest detection rates in flavor samples. This study screened fifteen flavor components of concern and discovered that 2,3,5-trimethylpyrazine, furfural, benzaldehyde, and benzenemethanol were detected in 100% of flavored milk samples. Benzenemethanol was found in the highest concentration (14,995.44 µg kg-1). The risk assessment results revealed that there was no risk for Chinese residents in consuming flavored milk, and the maximum per capita daily consumption of 2,3,5-trimethylpyrazine, furfural, and benzenemethanol were 226.208 g, 140.610 g, and 120.036 g, respectively. This study could provide guidelines for amounts of flavor additive ingredients in milk.