ObjectiveTo prepare a recombinant PETase with a dual-catalytic-triad and to evaluate its efficiency in the biodegradation of polyethylene terephthalate （PET）. MethodsBased on the crystal structure of wild-type PETase， point mutations （T88H/L117D） were introduced via site-directed mutagenesis. The recombinant protein was prepared using prokaryotic expression and chromatography purification techniques. The enzymatic hydrolysis of the mutant PETase was assessed by relatively quantifying the products mono （2-hydroxyethyl） terephthalate （MHET） and terephthalic acid （TPA）. ResultsBoth wild-type and mutant PETases accumulated as inclusion bodies， accounting for approximately 20% of the total bacterial protein. After solubilization in urea， the proteins were eluted at 300 mmol/L imidazole during affinity chromatography purification， with concentrations of 1.824 and 1.833 mg/mL and purities of 83.11% and 84.32%， respectively. Subsequent anion-exchange chromatography yielded highly pure enzymes in the 200 mmol/L NaCl fraction： 2.776 mg/mL （96.86% purity） for the wild type and 1.967 mg/mL （95.13% purity） for the mutant. Following refolding， the final concentrations were 0.484 mg/mL for the wild type and 0.991 mg/mL for the mutant. Hydrolysis assays revealed that the mutant released MHET and TPA at （237.67±17.00）% and （197.33±12.01）% of the wild-type levels， respectively. ConclusionThe T88H/L117D dual-catalytic-triad PETase is successfully prepared and it significantly enhanced PET-degrading activity， thus， it′s a promising biocatalyst for PET bioremediation.

Objective: To investigate the distribution of pupil diameter and its association with myopia in school age children, providing ideas into the mechanisms of the role of pupil diameter in the onset and development of myopia. Methods: Adopting a combination of stratified cluster random sampling and convenience sampling method, 3 839 children from six schools in Shandong Province were included in September 2021. Pupil diameters distribution was analyzed by age, sex, and myopic status. Pearson correlation analysis was used to assess the relationship between pupil diameter and cycloplegic spherical equivalent (SE), as well as axial length (AL) and other variables. Propensity score matching (PSM) was applied to match myopic and non myopic children at a 1∶1 ratio based on age and sex. A generalized linear model (GLM) was constructed with pupil diameter as the dependent variable to identify independent factors influencing pupil size and its association with myopia. Results: The mean pupil diameter of school age children was (5.77±0.80)mm. Pupil diameter exhibited a significant increasing trend with age ( F =49.34， P trend ＜ 0.01). Myopic children had a significantly larger mean pupil diameter [(6.10±0.73)mm] compared to non myopic children [(5.62±0.79)mm] with a statistically significant difference( t=18.10, P <0.01). Multivariable GLM analysis, adjusted for age, amplitude of accommodation, and uncorrected visual acuity, revealed a negative correlation between pupil diameter and cycloplegic SE (before PSM: β =-0.089, after PSM: β =-0.063, both P ＜0.01). Conclusions Myopic school age children exhibite larger pupil diameters than their non myopic counterparts. Pupil diameter may serve as a potential indicator for monitoring myopia development in school age children.

Objective:To analyze the correlation between near work, screen time, outdoor time and myopia in children based on objective monitoring technology and to explore the influencing factors related to myopia in children.Methods:A cross-sectional study was conducted.From October 2022 to March 2023, the purposive sampling method was used to select 596 children in Grade 2 and Grade 3 from two primary schools in Shandong Province as study subjects.Eye-Monitor technology of eye-use behavior based on artificial intelligence was used to quantify parameters of near work, screen time and outdoor time.The eye-use behavior parameters were compared within each subject and between non-myopic and myopic children on weekdays and weekends.A multivariate binary logistic regression model was constructed to analyze the influencing factors related to myopia.The study protocol was approved by the Medical Ethics Committee of the Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine (No.HEC-HY-2022023KY).Written informed consent was obtained from the legal guardian of each subject.Results:For each subject, the proportion of near work time on weekdays was greater than on weekends, the proportion of time spent looking at cell phones, computer screens, and outdoor activities was smaller, the duration of single continuous near eye use was longer, the tilt angle of the head in sitting position was greater, and the light intensity was stronger, showing statistically significant differences ( t=19.427, -9.964, -5.916, -10.470, 2.211, 2.898, 15.061; all P<0.05).During weekdays, compared with the non-myopia group, the myopia group had longer total near work duration, longer single continuous near eye use duration, shorter outdoor activity duration, closer eye use distance, larger proportion of near work time, and smaller proportion of outdoor activity time, showing statistically significant differences (all P<0.05).During weekends, compared with the non-myopia group, the myopia group had longer time spent looking at cell phones and computer screens, shorter outdoor activity time, greater proportion of time spent looking at cell phones and computer screens, and smaller proportion of outdoor activity time, with statistically significant differences (all P<0.05).During weekdays, after adjusting for confounding factors, longer single continuous near eye use duration ( OR=1.138, 95% CI: 1.086-1.192, P<0.001) was the risk factor for myopia, and longer working distance ( OR=0.906, 95% CI: 0.847-0.970, P=0.004) and longer outdoor activity time ( OR=0.127, 95% CI: 0.023-0.703, P=0.018) were protective factors for myopia.During weekends, after adjusting for confounding factors, longer time spent on looking at cell phone screens ( OR=2.437, 95% CI: 1.460-4.068, P<0.001) and longer time spent on looking at computer screens ( OR=2.260, 95% CI: 1.283-3.979, P=0.005) were risk factors for myopia, and longer outdoor activity time ( OR=0.624, 95% CI: 0.416-0.934, P=0.022) was the protective factor for myopia. Conclusions:The eyes with continuous near work, prolonged use of smartphone and computer screens, closer eye use distance, and less time spent outdoors have been confirmed to be significantly correlated with myopia based on objective monitoring data.When formulating intervention measures for myopia prevention and control in children, it is advocated to further pay attention to control the distance and duration of near work on weekdays and strengthen screen time management on weekends.

Objective:To analyze the correlation between near work, screen time, outdoor time and myopia in children based on objective monitoring technology and to explore the influencing factors related to myopia in children.Methods:A cross-sectional study was conducted.From October 2022 to March 2023, the purposive sampling method was used to select 596 children in Grade 2 and Grade 3 from two primary schools in Shandong Province as study subjects.Eye-Monitor technology of eye-use behavior based on artificial intelligence was used to quantify parameters of near work, screen time and outdoor time.The eye-use behavior parameters were compared within each subject and between non-myopic and myopic children on weekdays and weekends.A multivariate binary logistic regression model was constructed to analyze the influencing factors related to myopia.The study protocol was approved by the Medical Ethics Committee of the Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine (No.HEC-HY-2022023KY).Written informed consent was obtained from the legal guardian of each subject.Results:For each subject, the proportion of near work time on weekdays was greater than on weekends, the proportion of time spent looking at cell phones, computer screens, and outdoor activities was smaller, the duration of single continuous near eye use was longer, the tilt angle of the head in sitting position was greater, and the light intensity was stronger, showing statistically significant differences ( t=19.427, -9.964, -5.916, -10.470, 2.211, 2.898, 15.061; all P<0.05).During weekdays, compared with the non-myopia group, the myopia group had longer total near work duration, longer single continuous near eye use duration, shorter outdoor activity duration, closer eye use distance, larger proportion of near work time, and smaller proportion of outdoor activity time, showing statistically significant differences (all P<0.05).During weekends, compared with the non-myopia group, the myopia group had longer time spent looking at cell phones and computer screens, shorter outdoor activity time, greater proportion of time spent looking at cell phones and computer screens, and smaller proportion of outdoor activity time, with statistically significant differences (all P<0.05).During weekdays, after adjusting for confounding factors, longer single continuous near eye use duration ( OR=1.138, 95% CI: 1.086-1.192, P<0.001) was the risk factor for myopia, and longer working distance ( OR=0.906, 95% CI: 0.847-0.970, P=0.004) and longer outdoor activity time ( OR=0.127, 95% CI: 0.023-0.703, P=0.018) were protective factors for myopia.During weekends, after adjusting for confounding factors, longer time spent on looking at cell phone screens ( OR=2.437, 95% CI: 1.460-4.068, P<0.001) and longer time spent on looking at computer screens ( OR=2.260, 95% CI: 1.283-3.979, P=0.005) were risk factors for myopia, and longer outdoor activity time ( OR=0.624, 95% CI: 0.416-0.934, P=0.022) was the protective factor for myopia. Conclusions:The eyes with continuous near work, prolonged use of smartphone and computer screens, closer eye use distance, and less time spent outdoors have been confirmed to be significantly correlated with myopia based on objective monitoring data.When formulating intervention measures for myopia prevention and control in children, it is advocated to further pay attention to control the distance and duration of near work on weekdays and strengthen screen time management on weekends.

Objective To evaluate the bioequivalency of single administration of metformin hydrochloride sustained-release tablets(Ⅲ)and double administration of metformin hydrochloride tablet by self-cross test in healthy subjects.Methods A two-cycle,open,self-controlled cross-control trial was conducted in which 10 cases healthy subjects first received test prepatration metformin hydrochloride sustained-release tablet(Ⅲ)2.0 g and then received reference preparation(metformin hydrochloride tablet)1.0 g twice daily after a 7-day washout period.The plasma concentrations of metformin at different time points after administration were measured by liquid chromatography tandem mass spectrometry(LC-MS/MS).The pharmacokinetic parameters were calculated by Analyst 1.6.3,and the bioequivalency of the two drugs was evaluated.A double unilateral t-test and 90％confidence interval were used to assess the equivalence.A non-parametric test statistical analysis was used to evaluate the equivalence of the secondary parameter tmax.Results The pharmacokinetic parameters of test prepatration and reference preparation were AUC0-24h were(16.43±4.46)and(18.69±4.68)μg·h·mL-1,AUCINF-obs were(17.60±4.56)and(20.09±5.78)μg·h·mL-1,Cmax were(1 893.97±585.88)and(1 437.35±281.21)ng·mL-1,tmax were(7.60±1.58)and(4.70±2.41)h;t1/2 were(10.95±2.58)and(3.33±0.62)h.Conclusion The peak time and half-life of metformin hydrochloride sustained-release tablet(Ⅲ)after single administration in Chinese healthy subjects are longer than that of metformin hydrochloride tablet,and the bioequivalency is comparable to that of metformin hydrochloride tablet after two administration.

Objective To evaluate the bioequivalency of single administration of metformin hydrochloride sustained-release tablets(Ⅲ)and double administration of metformin hydrochloride tablet by self-cross test in healthy subjects.Methods A two-cycle,open,self-controlled cross-control trial was conducted in which 10 cases healthy subjects first received test prepatration metformin hydrochloride sustained-release tablet(Ⅲ)2.0 g and then received reference preparation(metformin hydrochloride tablet)1.0 g twice daily after a 7-day washout period.The plasma concentrations of metformin at different time points after administration were measured by liquid chromatography tandem mass spectrometry(LC-MS/MS).The pharmacokinetic parameters were calculated by Analyst 1.6.3,and the bioequivalency of the two drugs was evaluated.A double unilateral t-test and 90％confidence interval were used to assess the equivalence.A non-parametric test statistical analysis was used to evaluate the equivalence of the secondary parameter tmax.Results The pharmacokinetic parameters of test prepatration and reference preparation were AUC0-24h were(16.43±4.46)and(18.69±4.68)μg·h·mL-1,AUCINF-obs were(17.60±4.56)and(20.09±5.78)μg·h·mL-1,Cmax were(1 893.97±585.88)and(1 437.35±281.21)ng·mL-1,tmax were(7.60±1.58)and(4.70±2.41)h;t1/2 were(10.95±2.58)and(3.33±0.62)h.Conclusion The peak time and half-life of metformin hydrochloride sustained-release tablet(Ⅲ)after single administration in Chinese healthy subjects are longer than that of metformin hydrochloride tablet,and the bioequivalency is comparable to that of metformin hydrochloride tablet after two administration.

This study aims to explore the mechanism of Qingkailing(QKL) Oral Preparation's heat-clearing, detoxifying, mind-tranquilizing effects based on "component-target-efficacy" network. To be specific, the potential targets of the 23 major components in QKL Oral Preparation were predicted by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction. The target genes were obtained based on UniProt. OmicsBean and STRING 10 were used for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the targets. Cytoscape 3.8.2 was employed for visualization and construction of "component-target-pathway-pharmacological effect-efficacy" network, followed by molecular docking between the 23 main active components and 15 key targets. Finally, the lipopolysaccharide(LPS)-induced RAW264.7 cells were adopted to verify the anti-inflammatory effect of six monomer components in QKL Oral Preparation. It was found that the 23 compounds affected 33 key signaling pathways through 236 related targets, such as arachidonic acid metabolism, tumor necrosis factor α(TNF-α) signaling pathway, inflammatory mediator regulation of TRP channels, cAMP signaling pathway, cGMP-PKG signaling pathway, Th17 cell differentiation, interleukin-17(IL-17) signaling pathway, neuroactive ligand-receptor intera-ction, calcium signaling pathway, and GABAergic synapse. They were involved in the anti-inflammation, immune regulation, antipyretic effect, and anti-convulsion of the prescription. The "component-target-pathway-pharmacological effect-efficacy" network of QKL Oral Preparation was constructed. Molecular docking showed that the main active components had high binding affinity to the key targets. In vitro cell experiment indicated that the six components in the prescription(hyodeoxycholic acid, baicalin, chlorogenic acid, isochlorogenic acid C, epigoitrin, geniposide) can reduce the expression of nitric oxide(NO), TNF-α, and interleukin-6(IL-6) in cell supernatant(P<0.05). Thus, the above six components may be the key pharmacodynamic substances of QKL Oral Preparation. The major components in QKL Oral Prescription, including hyodeoxycholic acid, baicalin, chlorogenic acid, isochlorogenic acid C, epigoitrin, geniposide, cholic acid, isochlorogenic acid A, and γ-aminobutyric acid, may interfere with multiple biological processes related to inflammation, immune regulation, fever, and convulsion by acting on the key protein targets such as IL-6, TNF, prostaglandin-endoperoxide synthase 2(PTGS2), arachidonate 5-lipoxygenase(ALOX5), vascular cell adhesion molecule 1(VCAM1), nitric oxide synthase 2(NOS2), prostaglandin E2 receptor EP2 subtype(PTGER2), gamma-aminobutyric acid receptor subunit alpha(GABRA), gamma-aminobutyric acid type B receptor subunit 1(GABBR1), and 4-aminobutyrate aminotransferase(ABAT). This study reveals the effective components and mechanism of QKL Oral Prescription.
Chlorogenic Acid ; Drugs, Chinese Herbal/pharmacology* ; gamma-Aminobutyric Acid ; Interleukin-6 ; Medicine, Chinese Traditional ; Molecular Docking Simulation ; Tumor Necrosis Factor-alpha/genetics* ; Animals ; Mice ; RAW 264.7 Cells

Owing to the increasing incidence and prevalence of inflammatory bowel disease (IBD) worldwide, effective and safe treatments for IBD are urgently needed. Hydrogen sulfide (H₂S) is an endogenous gasotransmitter and plays an important role in inflammation. To date, H₂S-releasing agents are viewed as potential anti-inflammatory drugs. The slow-releasing H₂S donor 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), known as a potent therapeutic with chemopreventive and cytoprotective properties, has received attention recently. Here, we reported its anti-inflammatory effects on dextran sodium sulfate (DSS)-induced acute (7 days) and chronic (30 days) colitis. We found that ADT-OH effectively reduced the DSS-colitis clinical score and reversed the inflammation-induced shortening of colon length. Moreover, ADT-OH reduced intestinal inflammation by suppressing the nuclear factor kappa-B pathway. In vivo and in vitro results showed that ADT-OH decreased intestinal permeability by increasing the expression of zonula occludens-1 and occludin and blocking increases in myosin II regulatory light chain phosphorylation and epithelial myosin light chain kinase protein expression levels. In addition, ADT-OH restored intestinal microbiota dysbiosis characterized by the significantly increased abundance of Muribaculaceae and Alistipes and markedly decreased abundance of Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio. Transplanting ADT-OH-modulated microbiota can alleviate DSS-induced colitis and negatively regulate the expression of local and systemic proinflammatory cytokines. Collectively, ADT-OH is safe without any short-term (5 days) or long-term (30 days) toxicological adverse effects and can be used as an alternative therapeutic agent for IBD treatment.
Humans ; Mice ; Animals ; Gastrointestinal Microbiome ; Intestinal Barrier Function ; Mice, Inbred C57BL ; Colitis/metabolism* ; Inflammatory Bowel Diseases/drug therapy* ; Inflammation ; Anti-Inflammatory Agents/pharmacology* ; Disease Models, Animal

Bitter almonds (Semen Armeniacae Amarum) are prone to oil deterioration during storage, so they often require mashing prior to clinical use. To confirm the medical value of bitter almonds "being mashed when used" and to determine the optimal storage conditions for bitter almonds, UPLC-MS/MS was used to perform a comparative study of the chemical composition of bitter almonds in different storage states (mashed and unmashed), storage times (0, 2 and 4 weeks), and storage temperatures (25 ℃ and 4 ℃). A total of 58 substances were identified in bitter almond extracts through literature review, this group's previous work, and a Compound Discoverer software search. Statistically significant differences were found in the chemical composition and content of bitter almonds in different storage states, storage times, and storage temperatures. The results show that the chemical composition of bitter almonds stored unmashed was more stable than that of bitter almonds stored mashed; the chemical composition of bitter almonds stored at 4 ℃ was more stable than that of bitter almonds stored at 25 ℃; and the shorter the storage time, the less the chemical composition changed. Amygdalin, the main medicinal component of bitter almonds, showed statistically significant differences in content under the above three storage conditions, which can be used as a potential quality marker for bitter almonds.