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.

Aging is a natural process in which tissue and organ function declines as organisms age. Aging is the inevitable outcome of the organism and is irreversible. The aging process is accompanied by degenerative changes in the morphological structure of multiple organs of the body, leading to an increase in the incidence of chronic diseases such as hypertension, cardiovascular and cerebrovascular diseases, hyperlipidemia, and diabetes year by year. In recent years, natural phytochemicals have attracted widespread attention from the public due to their advantages such as non-toxicity or low toxicity, low cost, and various biological activities. Epigallocatechin gallate (EGCG) is a dietary polyphenol extracted from green tea, which has a variety of biological functions including anti-oxidation, anti-inflammation, hypoglycemic, hypolipidemic and anti-aging activities. The EGCG-mediated anti-aging mechanism has been investigated in many studies with different aging models. This article reviews the research progress on rodents, nematodes, fruit flies, and cell aging models, focusing on summarizing the lifespan extension and physiological changes of rodents, nematodes and fruit flies after EGCG intervention from multiple angles, and exploring potential mechanism by which EGCG delays aging and extends lifespan. This review provides a theoretical reference for the study of the anti-aging mechanism of phytochemicals.

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*

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 investigate the epidemiology of elderly respiratory tract infection (RTI) cases in a hospital in Xuzhou region from 2020 to 2022. Methods The cases of RTI patients in a hospital were screened from May 2020 to December 2022, and 548 cases that met the criteria were included in the study. Patient case data were analyzed for symptoms, pathogen distribution, and differences in patient distribution under different screening conditions (age, disease, and season). Results More than 90.00% of the included RTI patients presented with symptoms of cough, sputum, wet rales and pleural effusion was less common. The top three comorbidities were cardiovascular disease (153 patients, 27.92%), cerebrovascular disease (133 patients, 24.27%), and gastrointestinal disease (105 patients, 19.16%).All 548 elderly patients tested positive for respiratory pathogens (100.00%). There were 540 cases of single pathogen infection (98.54%) and 8 cases of mixed infection (1.46%). The top five single pathogen infections were Pseudomonas aeruginosa (92 cases, 16.76%), Escherichia coli (78 cases, 14.21%), drug-resistant Staphylococcus aureus (69 cases, 12.57%), Klebsiella pneumoniae (65 cases, 11.84%), and Mycoplasma pneumoniae (46 cases, 8.38%). The highest detection rate of respiratory pathogens was found in patients >90 years old, whose main pathogens were Pseudomonas aeruginosa, Klebsiella pneumoniae and drug-resistant Staphylococcus aureus. The next highest rates of pathogen detection were found in patients aged 86-91 and 81-85 years, unlike patients >90 years, who had a higher rate of Escherichia coli detection. Unlike other age groups, patients <75 years old had a higher percentage of influenza B virus detection. The highest incidence of pneumonia was found in 45.62% (250 cases). Escherichia coli had the highest detection rate in acute bronchitis/episodes and pneumonia, respiratory syncytial virus had the highest detection rate in wheezing bronchitis, Klebsiella pneumoniae had the highest detection rate in bronchopneumonia, and Pseudomonas aeruginosa had the highest detection rate in fever. The highest detection rate of pathogens was found in fall (36.50%), followed by spring (27.01%). The distribution of pathogen infections in all seasons was matched with the results of pathogenicity testing. Streptococcus oxysporus had the highest number of infections in the fall (χ²=20.33, P<0.001). Conclusion Elderly respiratory tract infections in this region are most common in patients over 90 years old, with the highest incidence of pneumonia and high incidence in fall, and the pathogens are mainly Pseudomonas aeruginosa, Escherichia coli and drug-resistant Staphylococcus aureus. Attention to distinguish the above characteristics can provide some support for early diagnosis and treatment of respiratory infections in the elderly in this region.

AIM: To investigate the role of pyroptosis in the development of diabetic retinopathy(DR)and to explore the regulatory mechanism by which circular RNA(circRNA)and its targeted microRNA(miRNA)mediate pyroptosis in DR, providing new therapeutic targets and strategies for the prevention and treatment of DR.METHODS: A streptozotocin(STZ)-induced model of type 1 diabetes in SD rats was established. The expression of circRNA_0076631, miR-214, and pyroptosis-related factors were measured in retinal tissues. CCK-8 and tube formation assays were used to detect the effect of different concentration of glucose on cell proliferation and angiogenic abilities of human retinal microvascular endothelial cells(HRMECs). The expression levels of circRNA_0076631, miR-214, and pyroptosis-related markers were evaluated through qRT-PCR and Western blot analysis, with additional experiments conducted following circRNA_0076631 knockdown to assess its effect on pyroptosis markers. Previous bioinformatics analysis and luciferase reporter assays identified a shared binding site among circRNA_0076631, miR-214, and caspase-1. To clarify the interaction between these molecules, co-transfection experiments using circRNA_0076631 inhibitors(ASO-circRNA_0076631), miR-214 overexpression transfection reagent, and miR-214 inhibitors(AMO-miR-214)were conducted to elucidate the regulatory pathway involved in DR.RESULTS: Both the diabetic rat model and D-glucose-treated HRMECs showed significantly elevated expression of circRNA_0076631 and pyroptosis-related factors(NLRP3, caspase-1, and IL-1β), while miR-214 expression was reduced(all P<0.05). The mRNA expression of pyroptosis-related factors caspase-1 was reduced after the overexpression of miR-214, and it was upregulated after the inhibition of miR-214(all P<0.05). Knockdown of circRNA_0076631 reduced the mRNA expression of pyroptosis markers caspase-1(P<0.05). Co-transfection experiments revealed that the inhibition circRNA_0076631 suppressed pyroptosis(all P<0.05), but this suppression was reversed upon co-transfection with miR-214 inhibitors, leading to increased mRNA expression of the pyroptosis marker caspase-1(all P<0.05).CONCLUSION: The circRNA_0076631 and pyroptosis play critical roles in the pathogenesis of DR, and circRNA_0076631 may regulate pyroptosis by modulating miR-214, which in turn influences the expression of caspase-1 in the pyroptosis signaling pathway, thereby contributing to DR progression. The circRNA_0076631 may serve as a novel therapeutic target, providing new insights for the prevention and treatment of DR.

AIM: To investigate the role of pyroptosis in the development of diabetic retinopathy(DR)and to explore the regulatory mechanism by which circular RNA(circRNA)and its targeted microRNA(miRNA)mediate pyroptosis in DR, providing new therapeutic targets and strategies for the prevention and treatment of DR.METHODS: A streptozotocin(STZ)-induced model of type 1 diabetes in SD rats was established. The expression of circRNA_0076631, miR-214, and pyroptosis-related factors were measured in retinal tissues. CCK-8 and tube formation assays were used to detect the effect of different concentration of glucose on cell proliferation and angiogenic abilities of human retinal microvascular endothelial cells(HRMECs). The expression levels of circRNA_0076631, miR-214, and pyroptosis-related markers were evaluated through qRT-PCR and Western blot analysis, with additional experiments conducted following circRNA_0076631 knockdown to assess its effect on pyroptosis markers. Previous bioinformatics analysis and luciferase reporter assays identified a shared binding site among circRNA_0076631, miR-214, and caspase-1. To clarify the interaction between these molecules, co-transfection experiments using circRNA_0076631 inhibitors(ASO-circRNA_0076631), miR-214 overexpression transfection reagent, and miR-214 inhibitors(AMO-miR-214)were conducted to elucidate the regulatory pathway involved in DR.RESULTS: Both the diabetic rat model and D-glucose-treated HRMECs showed significantly elevated expression of circRNA_0076631 and pyroptosis-related factors(NLRP3, caspase-1, and IL-1β), while miR-214 expression was reduced(all P<0.05). The mRNA expression of pyroptosis-related factors caspase-1 was reduced after the overexpression of miR-214, and it was upregulated after the inhibition of miR-214(all P<0.05). Knockdown of circRNA_0076631 reduced the mRNA expression of pyroptosis markers caspase-1(P<0.05). Co-transfection experiments revealed that the inhibition circRNA_0076631 suppressed pyroptosis(all P<0.05), but this suppression was reversed upon co-transfection with miR-214 inhibitors, leading to increased mRNA expression of the pyroptosis marker caspase-1(all P<0.05).CONCLUSION: The circRNA_0076631 and pyroptosis play critical roles in the pathogenesis of DR, and circRNA_0076631 may regulate pyroptosis by modulating miR-214, which in turn influences the expression of caspase-1 in the pyroptosis signaling pathway, thereby contributing to DR progression. The circRNA_0076631 may serve as a novel therapeutic target, providing new insights for the prevention and treatment of DR.

AIM: To evaluate the safety and efficacy of allogeneic intrastromal lenticule implantation combined with corneal collagen cross-linking(CXL)in patients with moderate to advanced keratoconus.METHODS: A retrospective case series analysis was conducted. A total of 19 patients(20 eyes)with moderate to advanced keratoconus who underwent combined allogeneic intrastromal lenticule implantation and CXL at the Jinan Mingshui Eye Hospital from June 2021 to December 2023 were included. The uncorrected distance visual acuity(UCVA), thinnest corneal thickness, central corneal epithelial thickness, anterior corneal flat keratometry(Kf), steep keratometry(Ks), and mean keratometry(Km), as well as the first applanation time(A1T), the first applanation length(A1L), the velocity during the first applanation moment(VIN), the second applanation time(A2T), the second applanation length(A2L), the velocity during the second applanation moment(VOUT), highest concavity time(HCT), highest concavity radius(HCR), peak distance(PD), deformation amplitude(DA), stiffness parameter at first applanation(SP-A1), integrated radius(IR), central corneal thickness(CCT), intraocular pressure(IOP), corneal thickness-corrected IOP, biomechanically intraocular pressure IOP(bIOP), and corneal thickness variation rate(ARTH)were compared between the two groups before surgery and at 1 wk, 1, 3 and 6 mo after surgery.RESULTS: All patients successfully completed the surgery without any intraoperative complications. No significant differences were observed between pre-operative and post-operative measurements for UCVA or the corneal biomechanical parameters, including A1L, A2L, PD, A1T, A2T, VIN, VOUT, DA, IOP, and bIOP(all P>0.05). Significant differences were found between pre-operative and post-operative values for corneal thinnest point thickness, central corneal epithelial thickness, Kf, Ks, Km, and the corneal biomechanical parameters, including HCT, HCR, SP-A1, ARTH, IR, and CCT(all P<0.05). The anterior corneal curvature demonstrated an initial increase followed by a decrease post-operatively. Furthermore, significant differences were observed between pre-operative and post-operative values for HCT, HCR, SP-A1, ARTH, IR, and CCT(all P<0.005).CONCLUSION: Allogenic intrastromal lenticule implantation combined with corneal collagen cross-linking demonstrates favorable safety and stability in treating moderate-to-advanced keratoconus. This combined procedure effectively increases corneal thickness and rigidity, resulting in corneas that are more resistant to deformation postoperatively.

Objective:To investigate the distribution of CGG repeat numbers in the FMR1 gene among reproductive-age women in China, providing data reference for carrier screening and genetic counseling of Fragile X syndrome. Methods:This cross-sectional study recruited 16 610 reproductive-age women from 12 medical institutions between July 2022 and October 2023. Peripheral venous blood samples (3 mL) were collected, and genomic DNA was extracted. The number of CGG repeats in the FMR1 gene was determined using the triplet-primed polymerase chain reaction (TP-PCR) combined with capillary electrophoresis technology. Statistical analyses were performed to assess the prevalence and distribution of CGG repeat expansions. Results:Among 16 610 women of childbearing age, 5 684 (34.220%) women had the same number of CGG repeats in the two alleles of FMR1 gene, and 10 926 (65.780%) women had different numbers of repeats in the two alleles. Among the 33 220 FMR1 alleles in 16 610 women of reproductive age, the most common CGG repeat numbers were 29 [48.645% (16 160/33 220)] and 30 [26.276% (8 729/33 220)], while the most frequent CGG genotype was CGG 29/29 [24.726% (4 107/16 610)]. The CGG repeat numbers of FMR1 gene were normal in 16 498 women (99.326%). Among the 112 women (0.674%) with CGG repeat abnormities, 96 (0.578%) women were classified as intermediate carriers, 15 (0.090%) as premutation carriers, and 1 (0.006%) as a full mutation carrier, whose CGG genotype was (36, >200). Conclusion:In the general reproductive-age female population in China, the normal CGG repeat numbers of the FMR1 gene account for 99.326%, while the intermediate carrier rate is 0.578%, and the combined carrier rate of the premutation and full mutation types is 0.096%.