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

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.

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.

OBJECTIVE To explore the correlation between childhood obstructive sleep apnea hypopnea syndrome and otitis media with effusion,to provide evidence-based basis for clinical diagnosis and treatment.METHODS A total of 985 children were diagnosed in the Department of Otolaryngology,Heping Hospital Affiliated to Changzhi Medical College from September 2019 to March 2025.Combined or no OME patients for multivariate logistics regression analysis and construct related risk prediction model,obtain data of OSAHS(exposure factors)and OME(outcome)from the public database of genome-wide association studies(GWAS),two-sample Mendelian randomization(MR)analysis.RESULTS The risk prediction model for OME in children with OSAHS,Hosmer-Lemeshow goodness of fit test(χ2=12.541,P=0.157),C index of 0.749,Calibration calibration curve fitting,and correlation between children OSHAS and OME.Furthermore,the MR analysis using inverse variance weighting observed a positive causal relationship between OSAHS and OME in children(OR=1.05,95%CI=0.999-1.051,P=0.005).MR Egger test results(Q=42.358,P=0.716)and IVW(Q=43.271,P=0.759)showed no heterogeneity in the analysis,MR Egger intercept(intercept=-0.004,P=0.921)showed no horizontal pleiotropy,and the MR analysis results were stable.CONCLUSION There is a correlation between pediatric OSAHS and OME,and it increases the risk of OME.

Pancreatic cancer(PC)is a highly malignant digestive system tumor with extremely high mortality and recurrence rates.While traditional surgical resection and chemotherapy remain the main treatment options,challenges such as high postoperative recurrence and poor prognosis persist.Therefore,exploring more effective comprehensive treatment strategies is crucial for improving patient survival and prognosis.This review systematically summarizes recent advances in systemic therapy for PC,with a focus on the application and efficacy of chemotherapy,targeted therapy,and immunotherapy.Additionally,it discusses the potential of neoadjuvant therapy,integrated traditional Chinese and Western medicine approaches,and conversion therapy in enhancing the effects of conventional chemotherapy.Studies have shown that targeted therapy can enhance antigen presentation and reduce side effects,while immune checkpoint inhibitors,cancer vaccines,and adoptive cell immunotherapy help mitigate tumor immune evasion and improve the tumor microenvironment.Despite continuous innovation in treatment approaches,clinical management of PC,particularly for advanced-stage patients,still faces significant challenges.Future research should focus on early diagnosis,precision medicine,and personalized treatment strategies to further improve cure rates and patient survival quality,providing more effective therapeutic options for clinical practice.

Objective:To study the influence of methyltransferases like 3(METTL3)on the radiosensitivity of oral squamous cell carcinoma cells(OSCCs).Methods:The apoptosis level of OSCCs CAL27,SCC9 and SCC15 treated with X-ray radiation doses of 2,4 and 8 Gy respectively was compared by flow cytometry,the expression of methylated gene RNA and protein in the cells were examined by qRT-PCR and Western blot.m6A in the cells was quantified by LC/LC-MS method.qRT-PCR and Western blot were used to investigate the expression of methylated gene RNA and protein in the cells.Flow cytometry was used to examine the cell apoptosis level of CAL27 and SCC15 cells treated with METTL3 overexpression and knockdown respectively.The clone forma-tion of CAL27 and SCC15 cells after knockdown and overexpression of target genes followed by radiation treatment was observed by clonogenic assays.Results:The apoptosis rate of all the cell lines increased with the increase dose of radiation at each dose,CAL27 cells showed the highest and SCC15 showed the lowerst apoptosis rate.The RNA and protein expression levels of METTL3 in CAL27 were significantly lower than those of SCC15.m6A quantification showed that the methylation modification in CAL27 cells was lower than that in SCC15.The expression of METTL3 was increased in CAL27 and SCC15 cells after radiation treatment.Knockdown of METTL3 increaced the apoptosis rate and decreased the clonogenesity,overession of METTL3 the decreaced the ap optosis rate and increased the clonogenecity of the cells.Conclusion:Regulation of METTL3 can affect the radiotherapy sensitivity of OSCCs,METTL3 may become a new target for radiosensitization of OSCCs.

Cholangiocarcinoma(CCA)is a malignant tumor originating from the epithelial cells of the bile ducts,with its incidence and mortality rates rising in recent years,particularly in Asia.Because the early symptoms of CCA are often insidious and nonspecific,most patients are diagnosed at an advanced stage and lose the opportunity for radical surgical treatment.Although chemotherapy remains the main first-line therapy,its efficacy is limited.With the rapid development of next-generation sequencing technologies and the growing emphasis on individualized precision medicine,our understanding of the molecular biology of CCA has deepened substantially.Research on molecular targeted therapy,immunotherapy,and combination strategies integrating both approaches has made remarkable progress.This article provides an overview of the molecular targets,immune microenvironment,immune checkpoint inhibitors,and therapeutic strategies related to CCA.