OBJECTIVE To develop a method for the determination of tacrolimus （TAC） concentration in human whole blood and to apply it in clinical therapeutic drug monitoring. METHODS Whole blood samples were processed by protein precipitation with methanol. The determination was performed using liquid chromatography-tandem mass spectrometry （LC-MS/MS）， with ascomycin serving as the internal standard. Chromatographic separation was carried out on a Kinetex F5 100Å column with a mobile phase consisting of 0.1 mmol/L ammonium acetate containing 0.2 mmol/L formic acid and methanol. Gradient elution was performed at a flow rate of 0.4 mL/min. The injection volume was 5 μL. Detection was conducted using multiple reaction monitoring （ m / z 821.6→768.6 for TAC； m / z 809.4→756.1 for ascomycin） with an electrospray ionization source in positive ion mode. The study focused on 86 whole blood samples collected from 83 pedi atric patients who received TAC therapy at Children’s Hospital of Nanjing Medical University from September 1 to 30， 2025. The aforementioned method was employed to measure the TAC concentration in the whole blood samples. The correlation and agreement between the aforementioned method and the traditional enzyme multiplied immunoassay technique （EMIT） were evaluated through Spearman correlation analysis， Bland-Altman analysis， and Passing-Bablok regression analysis. RESULTS The linear range of TAC was 0.5-100 ng/mL； the evaluation results for accuracy， precision， extraction recovery， matrix effect， and stability tests all met the relevant requirements. Clinical application results showed that the median concentration of TAC in pediatric whole blood measured by LC-MS/MS and EMIT methods were 4.4 and 4.0 ng/mL， respectively. Moreover， the two methods exhibited a strong correlation （correlation coefficient of 0.848 1） and good agreement （average relative deviation of 6.5%）. CONCLUSIONS A reliable LC-MS/MS method for the determination of TAC concentration in human whole blood is successfully established. This method demonstrates strong correlation and good agreement with the EMIT method， making it suitable for clinical therapeutic drug monitoring.

BACKGROUND: Leukocyte telomere length (LTL) shortening, a biomarker of telomere attrition, has been linked to multiple diseases. However, the relationship between LTL and digestive diseases remains uncertain. This study aimed to investigate the association between LTL and the risk of digestive diseases. METHODS: A cohort analysis of over 500,000 participants from the UK Biobank (UKB) between 2006 and 2021 was conducted to estimate the associations of LTL with more than 90 common digestive diseases. LTL was quantified using multiplex quantitative polymerase chain reaction, and cases of each disease were determined according to inpatient and primary care data. Multivariable Cox proportional hazards regression analysis was used to evaluate the associations of LTL with the risk of digestive diseases. Furthermore, such associations were also evaluated after stratification by sex and ethnicity. RESULTS: After a mean follow-up time of 11.8 years, over 20 International Classification of Diseases, 10th Revision ( ICD-10 ) codes were showed to be associated with telomere attrition. LTL shortening is associated with an increased risk of several digestive diseases, including gastroesophageal reflux disease (K21: hazard ratio [HR] = 1.30, 95% confidence interval [95% CI]: 1.19-1.42), esophageal ulcer (K221: HR = 1.81, 95% CI: 1.22-2.71), Barrett's esophagus (K227: HR = 1.58, 95% CI: 1.14-2.17), gastritis (K29: HR = 1.39, 95% CI: 1.26-1.52), duodenal ulcer (K26: HR = 1.55, 95% CI: 1.14-2.12), functional dyspepsia (K30X: HR = 1.36, 95% CI: 1.06-1.69), non-alcoholic fatty liver disease (NAFLD) (K760: HR = 1.39, 95% CI: 1.09-1.78), liver cirrhosis (K74: HR = 4.73, 95% CI: 3.27-6.85), cholangitis (K830: HR = 2.55, 95% CI: 1.30-5.00), and hernia (K43: HR = 1.50, 95% CI: 1.17-1.94; K44: HR = 1.29, 95% CI: 1.17-1.42). The risk of rectal polyps (K621: HR = 0.77, 95% CI: 0.63-0.92) decreased per unit shortening of LTL. CONCLUSIONS This study suggests that LTL shortening is associated with an increased risk of most digestive diseases except for rectal polyps. These findings may provide some clues for understanding the pathogenesis of digestive diseases.
Humans ; Male ; Female ; Middle Aged ; Cohort Studies ; Leukocytes/metabolism* ; Telomere/genetics* ; Proportional Hazards Models ; Adult ; Digestive System Diseases/genetics* ; Aged ; Risk Factors ; Telomere Shortening

ObjectiveTo investigate the efficacy and underlying mechanisms of Gynostemma pentaphyllum alcohol extract in improving glucose and lipid metabolism disorders in db/db mice through transcriptomics and gut microbiota analysis. MethodsEighteen db/db mice were randomly assigned to the model（DM） group， metformin（MET） group， and G. pentaphyllum alcohol extract（GP） group， with six mice in each group， based on stratification of fasting blood glucose and body weight. An additional six db/m mice were selected as the normal control（NC） group. Mice in the NC and DM groups were administered deionized water （10 mL·kg^-1） daily. The MET group received metformin （0.195 g·kg^-1） by gavage. The GP group was treated with G. pentaphyllum alcohol extract （3.9 g·kg^-1） by gavage for six weeks. Fasting blood glucose was measured every two weeks. After six weeks of intervention， serum levels of total cholesterol （TC）， triglycerides （TG）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C）， aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， creatinine （CREA）， and blood urea nitrogen （BUN） were assessed. Enzyme-linked immunosorbent assay （ELISA） was used to measure insulin （FINS）， adiponectin （ADP）， and tumor necrosis factor-α （TNF-α）. Hematoxylin-eosin （HE） staining was used to observe liver histomorphology， periodic acid-Schiff （PAS） staining was employed to assess hepatic glycogen synthesis， and Oil Red O staining was used to detect hepatic lipid deposition. Liver transcriptomic data were used to identify differentially expressed genes in the liver and conduct enrichment analysis. Real-time PCR was employed to verify the expression levels of adiponectin gene （Adipoq）， peroxisome proliferator-activated receptor gamma coactivator-1α （PGC-1α）， AMP-activated protein kinase （AMPK）， peroxisome proliferator-activated receptor α （PPARα）， glucokinase （GCK）， forkhead box （Fox）O1， FoxO3， phosphoenolpyruvate carboxykinase （PEPCK）， and glucose-6-phosphatase （G6PC）. Metagenomic sequencing was conducted to analyze changes in gut microbiota composition. ResultsCompared with the NC group， the DM group exhibited significantly elevated fasting blood glucose （P<0.01）， serum AST， ALT， TC， TG， LDL-C， and HDL-C （P<0.01）. FINS， homeostatic model assessment for insulin resistance （HOMA-IR）， and the inflammatory cytokine TNF-α were significantly increased （P<0.01）， while ADP was significantly decreased （P<0.05）. Histological analysis confirmed severe hepatic steatosis and excessive lipid accumulation in the DM group， along with markedly reduced glycogen synthesis. Compared with the DM group， the GP group showed significantly decreased fasting blood glucose （P<0.01）， reduced serum TC， LDL-C， and HDL-C levels （P<0.05）， significantly decreased serum TG and AST levels （P<0.01）， significantly reduced FINS， HOMA-IR， and TNF-α levels （P<0.01）， and significantly increased ADP （P<0.01）. Hepatic steatosis and lipid deposition were significantly alleviated， while glycogen synthesis was markedly enhanced. Transcriptomic differential and enrichment analyses suggested that the mechanisms by which G. pentaphyllum alcohol extract improved hepatic glucose and lipid metabolism in db/db mice may involve regulation of the AMPK and FoxO signaling pathways. Real-time PCR results confirmed that expression of PGC-1α， PEPCK， G6PC， FoxO1， and FoxO3 was significantly downregulated following treatment with G. pentaphyllum alcohol extract （P<0.05， P<0.01）， whereas mRNA expression of Adipoq， PPARα， GCK， and AMPK was significantly upregulated （P<0.05， P<0.01）. Metagenomic analysis showed that the relative abundance of Lactobacillus， Alistipes， and Akkermansia species was higher in the GP group than in the DM group. ConclusionG. pentaphyllum alcohol extract may improve glucose and lipid metabolism disorders in db/db mice by regulating the hepatic AMPK/PPARα pathway to suppress lipid deposition and alleviate hepatic steatosis， by inhibiting gluconeogenesis through the AMPK/PGC-1α and FoxO pathways to lower fasting blood glucose， and by increasing the abundance of beneficial gut bacteria such as Lactobacillus， Alistipes， and Akkermansia to restore gut microbiota balance.

ObjectiveTo investigate the efficacy and underlying mechanisms of Gynostemma pentaphyllum alcohol extract in improving glucose and lipid metabolism disorders in db/db mice through transcriptomics and gut microbiota analysis. MethodsEighteen db/db mice were randomly assigned to the model（DM） group， metformin（MET） group， and G. pentaphyllum alcohol extract（GP） group， with six mice in each group， based on stratification of fasting blood glucose and body weight. An additional six db/m mice were selected as the normal control（NC） group. Mice in the NC and DM groups were administered deionized water （10 mL·kg^-1） daily. The MET group received metformin （0.195 g·kg^-1） by gavage. The GP group was treated with G. pentaphyllum alcohol extract （3.9 g·kg^-1） by gavage for six weeks. Fasting blood glucose was measured every two weeks. After six weeks of intervention， serum levels of total cholesterol （TC）， triglycerides （TG）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C）， aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， creatinine （CREA）， and blood urea nitrogen （BUN） were assessed. Enzyme-linked immunosorbent assay （ELISA） was used to measure insulin （FINS）， adiponectin （ADP）， and tumor necrosis factor-α （TNF-α）. Hematoxylin-eosin （HE） staining was used to observe liver histomorphology， periodic acid-Schiff （PAS） staining was employed to assess hepatic glycogen synthesis， and Oil Red O staining was used to detect hepatic lipid deposition. Liver transcriptomic data were used to identify differentially expressed genes in the liver and conduct enrichment analysis. Real-time PCR was employed to verify the expression levels of adiponectin gene （Adipoq）， peroxisome proliferator-activated receptor gamma coactivator-1α （PGC-1α）， AMP-activated protein kinase （AMPK）， peroxisome proliferator-activated receptor α （PPARα）， glucokinase （GCK）， forkhead box （Fox）O1， FoxO3， phosphoenolpyruvate carboxykinase （PEPCK）， and glucose-6-phosphatase （G6PC）. Metagenomic sequencing was conducted to analyze changes in gut microbiota composition. ResultsCompared with the NC group， the DM group exhibited significantly elevated fasting blood glucose （P<0.01）， serum AST， ALT， TC， TG， LDL-C， and HDL-C （P<0.01）. FINS， homeostatic model assessment for insulin resistance （HOMA-IR）， and the inflammatory cytokine TNF-α were significantly increased （P<0.01）， while ADP was significantly decreased （P<0.05）. Histological analysis confirmed severe hepatic steatosis and excessive lipid accumulation in the DM group， along with markedly reduced glycogen synthesis. Compared with the DM group， the GP group showed significantly decreased fasting blood glucose （P<0.01）， reduced serum TC， LDL-C， and HDL-C levels （P<0.05）， significantly decreased serum TG and AST levels （P<0.01）， significantly reduced FINS， HOMA-IR， and TNF-α levels （P<0.01）， and significantly increased ADP （P<0.01）. Hepatic steatosis and lipid deposition were significantly alleviated， while glycogen synthesis was markedly enhanced. Transcriptomic differential and enrichment analyses suggested that the mechanisms by which G. pentaphyllum alcohol extract improved hepatic glucose and lipid metabolism in db/db mice may involve regulation of the AMPK and FoxO signaling pathways. Real-time PCR results confirmed that expression of PGC-1α， PEPCK， G6PC， FoxO1， and FoxO3 was significantly downregulated following treatment with G. pentaphyllum alcohol extract （P<0.05， P<0.01）， whereas mRNA expression of Adipoq， PPARα， GCK， and AMPK was significantly upregulated （P<0.05， P<0.01）. Metagenomic analysis showed that the relative abundance of Lactobacillus， Alistipes， and Akkermansia species was higher in the GP group than in the DM group. ConclusionG. pentaphyllum alcohol extract may improve glucose and lipid metabolism disorders in db/db mice by regulating the hepatic AMPK/PPARα pathway to suppress lipid deposition and alleviate hepatic steatosis， by inhibiting gluconeogenesis through the AMPK/PGC-1α and FoxO pathways to lower fasting blood glucose， and by increasing the abundance of beneficial gut bacteria such as Lactobacillus， Alistipes， and Akkermansia to restore gut microbiota balance.

The transition of cancer cells from epithelial state to mesenchymal state awarded hepatocellular carcinoma (HCC) stem cell properties and induced tumorigenicity, drug resistance, and high recurrence rate. Reversing the mesenchymal state to epithelial state by inducing mesenchymal-epithelial remodeling could inhibit the progression of HCC. Using high-throughput screening, chrysin was selected from natural products to reverse epithelial-mesenchymal transition (EMT) by selectively increasing CDH1 expression. The target identification suggested chrysin exerted its anti-HCC effect through covalently and specifically binding threonine 205 (Thr205) of alpha-enolase (ENO1). For the first time, we revealed that ENO1 bound β-catenin mRNA, and recruited YTHDF2 to identify the m6A modified β-catenin in the 3'-UTR region to degrade β-catenin mRNA. Eventually, the CDH1 gene expression was improved through the regulation of β-catenin mRNA. ENO1/β-catenin mRNA interaction might be a promising target for cellular plasticity reprogramming. Moreover, chrysin could mediate mesenchymal‒epithelial remodeling through increasing degradation of β-catenin mRNA by promoting the binding of ENO1 and β-catenin mRNA. To the best of our knowledge, chrysin is the first reported small molecule inducing β-catenin mRNA degradation through binding to ENO1. The water-soluble derivative of chrysin may be a natural product-derived lead compound for circumventing metastasis, recurrence, and drug resistance of HCC by mediating mesenchymal‒epithelial remodeling.

Screening carbonyl reductases with the ability to catalyze the reduction of complex carbonyl compounds is of great significance for the biosynthesis of R-tolvaptan(R-TVP). In this study, the target carbonyl reductase in the crude enzyme extract of rabbit liver was separated, purified, and identified by ammonium sulfate precipitation, gel-filtration chromatography, ion exchange chromatography, affinity chromatography, and protein mass spectrometry. With the rabbit liver genome as the template, the gene encoding the carbonyl reductase rlsr5 was amplified by PCR and the recombinant strain was successfully constructed. After RLSR5 was purified by affinity chromatography, its enzymatic properties were characterized. The results indicated that the gene sequence of rlsr5 was 972 bp, encoding a protein with a molecular weight of 40 kDa. RLSR5 was a dimeric protein, and each monomer was composed of a (α/β)₈-barrel structure. RLSR5 could asymmetrically reduce 7-chloro-1-[2-methyl-4-[(2- methylbenzoyl)amino]benzoyl]-5-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine (prochiral ketone, PK) to synthesize R-TVP. The specific activity of the enzyme was 36.64 U/mg, and the optical purity of the product was 99%. This enzyme showcased the optimal performance at pH 6.0 and 30 °C. It was independent of metal ions, with the activity enhanced by Mn²⁺. This study lays a foundation for the biosynthesis of tolvaptan of optical grade.
Animals ; Rabbits ; Alcohol Oxidoreductases/biosynthesis* ; Recombinant Proteins/metabolism* ; Escherichia coli/metabolism* ; Liver/enzymology*

Beta-amylases (BAMs), key enzymes in starch hydrolysis, play an important role in plant growth, development, and resistance to abiotic stress. To mine the saline-alkali tolerance-related BAM genes in alfalfa (Medicago sativa L.), we identified MsBAM genes in the whole genome. The physicochemical properties, phylogeny, gene structures, conserved motifs, secondary structures, promoter cis-acting elements, chromosome localization, and gene replication relationships of BAM gene family members were analyzed. RNA-seq and quantitative real-time PCR (qRT-PCR) were employed to analyze the expression patterns of BAM family members under saline-alkali stress. The results showed that 54 BAM genes were identified in the genome, which were classified into 8 subgroups according to the phylogenetic tree. The members of the same subgroup had similar gene structures except that those of subgroups 1 and 7 had large differences. Conserved motif analysis showed that all MsBAM proteins had a typical glycohydrolysis domain. The chromosome localization analysis showed that MsBAM gene family members were unevenly distributed on 27 chromosomes. The duplication of gene segments led to the increase in BAM gene number in alfalfa. The promoters of BAM genes contained a large number of elements in response to plant hormones and stress. Transcriptome data and qRT-PCR results showed that the expression levels of most MsBAM genes were up-regulated in response to saline-alkali stress. Under the saline-alkali stress, the expression levels of 28 genes, including MsBAM6, were up-regulated on days 1 and 7, and those of 5 genes, including MsBAM9, were up-regulated by over 2 folds. In addition, under salt-alkali stress, BAM activity and soluble sugar content were significantly increased. These results indicate that BAM genes play a key role in alfalfa in response to saline-alkali stress, laying a foundation for further research in this field.
Medicago sativa/physiology* ; beta-Amylase/metabolism* ; Phylogeny ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics* ; Multigene Family ; Alkalies ; Plant Proteins/genetics*

Objective:To detect the biological markers related to Alzheimer's disease(AD)in the peripheral blood of AD patients,and to explore the activities and levels of the antioxidant function indexes and the expressions of related genes and proteins in the blood of AD patients and the changes after intervention of selenium-rich food.Methods:The Mini-Mental State Examination(MMSE)combined with electroencephalogram or brain CT and clinician diagnosis were used for screening AD.Fifty-six elderly patients with AD aged 75-90 years old were selected.Among them,28 cases were selected as normal diet group for AD(AD group),and 28 cases were selected as dietary selenium intervention group(Se-AD group).The patients in Se-AD group were given daily dietary selenium supplementation(increaseing dietary selenium by 15-20 μg per day)for 3 months.Meanwhile,30 people with the same age were selected as healthy control group.The activities of serum superoxide dismutase(SOD),cholinesterase(CHE),and glutathione peroxidase(GSH-Px)and the levels of serum malondialdehyde(MDA),homocysteine(Hcy),and nitric oxide(NO)as well as reagent kit the levels of serum β-amyloid protein(Aβ),and microtubule-associated protein(Tau)and phosphorylated microtubule-associated protein(p-Tau)of the subjects in various groups were detected by and enzyme-linked immunosorbent assay(ELISA)method;the expression levels of apolipoprotein E4(ApoE4),presenilin 1(PS1),presenilin 2(PS2),cysteinyl aspartate specific proteinase 3(Caspase3),sorting associated protein receptor 1(SORL1),β-site amyloid precursor protein cleaving enzyme 1(BACE1),hypoxia-inducible factor 1(HIF1),nuclear factor-kappa B(NF-κB),β-amyloid precursor protein(APP),protein kinase C(PKC),and Aβ mRNA in peripheral blood of the subjects various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method.Results:Compared with healthy control group,the serum SOD activities of the patients in Se-AD group and AD group were significantly decreased(P＜0.05),while serum CHE activity and the levels of MDA and Hcy were significantly increased(P＜0.05);the serum GSH-Px activity of the patients in AD group was significantly decreased(P＜0.05),and the level of NO was significantly increased(P＜0.05).Compared with Se-AD group,serum CHE activity and the level of Hcy of the patients in AD group were significantly increased(P＜0.05).The expression levels of ApoE4,PS1,Caspase3,BACE1,NF-κB and APP mRNA of the patients in Se-AD group and AD group were significantly increased(P＜0.05),and the expression levels of PKC mRNA were significantly decreased(P＜0.05);the expression level of PS2 mRNA of the patients in AD group was significantly increased(P＜0.05),and the expression levels of Aβ mRNA of the patients in Se-AD group and AD group were significantly increased(P＜0.05).Conclusion:The activities of serum SOD,GSH-Px and CHE and the levels of MDA,Hcy and NO,the levels of Aβ,Tau and p-Tau proteins,and the expression levels of ApoE4,PS1,Caspase3,BACE1,NF-κB,PKC,PS2,Aβ and APP mRNA in peripheral blood of the AD patients may vary and can be used for clinical diagnosis of the AD patients.Selenium-rich food can improve AD to some extent,and its mechanism is related to reducing the oxidative damage of brain tissue and decreasing the expression of AD related genes PS2 and Aβ.

Plantar fasciitis(PF)is a primary cause of heel pain(HP).Platelet-rich plasma(PRP),as an emerging biological treatment method,possesses unique biological characteristics,showing significant dual therapeutic advantages in suppressing inflammation and promoting tissue repair.This article reviews the pathogenesis of PF,the biological characteristics of PRP and its application in pain management and functional prognosis assessment of PF,particularly focusing on PRP in improving plantar fascia thickness(PFT).

Objective:To analyze the iodine nutrition status of children and adolescents and influencing factors in Zhejiang Province, providing scientific basis for optimizing iodine deficiency disorders (IDD) prevention and control strategies.Methods:In June 2022, a multistage stratified sampling method was used to divide 16 counties (cities, districts, abbreviated as counties) in Zhejiang Province into three categories based on their geographical locations (average distance from the coastline): coastal areas (including Dinghai District, Jiaojiang District, Sanmen County, Cixi City and Lucheng District), sub-coastal areas (including Wuxing District, Haining City, Linping District, Fuyang District and Fenghua District), and inland areas(including Suichang County, Changshan County, Shengzhou City, Jindong District, Dongyang City and Yongjia County). One county was selected from each category, and one township (street) was selected from each county. Two administrative villages (neighborhood committees) were selected from each township (street). Ten households including all children and adolescents aged 6-17 in each household were selected from each administrative village (neighborhood committee). Demographic information and personal dietary characteristics were collected via questionnaires, while household salt and a random urine sample were tested for iodine level. Trend analysis was conducted using a χ 2trend test, and a multivariate logistic stepwise regression model was used to analyze the influencing factors of urinary iodine levels. Results:A total of 755 children and adolescents aged 6-17 were selected, including 387 males (51.26%) and 368 females (48.74%), with an age of (11.24 ± 3.32) years. There were 269 children and adolescents in coastal areas (35.63%) and 409 children and adolescents in urban areas (54.17%). A total of 755 household salt samples were collected, with a median salt iodine concentration of 21.80 mg/kg. These included 263 non-iodized salt samples, 38 unqualified iodized salt samples, and 454 qualified iodized salt samples. The coverage rate of iodized salt was 65.17% (492/755), and the consumption rate of qualified iodized salt was 60.13% (454/755). The distribution of salt iodine quality among children and adolescents in different geographical locations showed statistically significant differences (χ 2 = 111.95, P < 0.001), with the proportion of non-iodized salt gradually decreasing from coastal areas to inland areas (χ 2trend = 90.17, P < 0.001). A total of 755 urine samples were collected, with a median urinary iodine concentration of 186.60 μg/L. The proportions of urinary iodine < 100, 100-199, 200-299, and ≥300 μg/L were 16.95% (128/755), 37.62% (284/755), 24.37% (184/755), and 21.06% (159/755), respectively. The χ 2trend test revealed a nonlinear positive correlation between salt iodine level and urinary iodine level (χ 2regression = 21.98, P < 0.001; χ 2partial = 6.96, P < 0.001). The frequency distribution of urinary iodine in children and adolescents from different geographical locations and between urban and rural areas showed statistically significant differences (χ 2 = 29.63, 16.56, P < 0.001). Among them, the proportion of children and adolescents with urinary iodine < 100 μg/L gradually decreasing from coastal areas to inland areas (χ 2trend = 6.15, P = 0.013). The results of multivariate logistic regression analysis revealed that sub-coastal regions, inland regions, and urban-rural regions ( OR = 1.57, 1.53, 1.64, 95% CI: 1.11-2.24, 1.03-2.27, 1.17-2.32, P < 0.05) were significantly associated with urinary iodine levels in children and adolescents aged 6-17. Conclusions:In 2022, the iodine nutrition of children and adolescents in Zhejiang Province is generally suitable, but there is a risk of iodine deficiency among coastal children and adolescents. Geographic location and urban/rural areas are influencing factors on iodine nutrition status of children and adolescents in Zhejiang Province.