ObjectiveThis study employs a special conformational engineering (CE) technology to construct an α-chymotrypsin-like active center, which includes a catalytic triad, an oxyanion hole, and a substrate-binding site, on silver nanoparticles (AgNPs), thereby creating an AgNP-based artificial hydrolase with high catalytic activity. This study provides a new approach for the design of highly efficient artificial enzymes and enzyme-mimicking. MethodsAgNPs were chosen as the scaffold to build the an α-chymotrypsin-like active center. A special CE procedure enables the designed peptide, Triad5, to adopt an α‑helical conformation on AgNPs, with the key catalytic residues located on one side of the α-helix forming a catalytic active center with a catalytic triad, an oxyanion hole, and a substrate-binding site. The CE procedure consists of three steps, including conformation induction via trifluoroethanol (TFE), conformation stabilization on AgNPs via Ag-S bonds, and TFE removal via lyophilization. Circular dichroism (CD) spectra were used to confirm the formation and stabilization of the α-helix conformation. Mutations of the key residues combined with stopped-flow kinetic experiments were used to demonstrate the indispensability of each key residue and the synergistic effects among the catalytic triad, the oxyanion hole, and the substrate-binding site. ResultsCD spectra show that the designed Triad5 alone is in random coil conformation; when conjugated on AgNPs, Triad5 still remains largely unstructured; but after the CE treatment, Triad5 adopts a typical α-helical conformation on AgNPs as designed, thus produces an AgNP-based artificial hydrolase, Silverzyme. Silverzyme exhibits extremely high hydrolytic activity towards p-nitrophenyl acetate (p-NPA), with an extremely high catalytic turnover number per active site of 3.5 s^-1, which is even higher than that of α-chymotrypsin. As a comparison, the AgNP-Triad5 conjugate without CE treatment shows much lower catalytic activity than Silverzyme, highlighting the important role of the right conformation of the active center for the catalytic activity and the power of the CE treatment. When the key residues of the catalytic triad of Silverzyme were mutated to alanine, the overall catalytic efficiency of this mutant dropped by about 2 orders of magnitude, unambiguously demonstrating the key role of the designed catalytic triad. Similarly, when the residues for the oxyanion hole were deleted, the mutant with the intact catalytic triad also showed significantly decreased catalytic activity, highlighting the indispensable role of the oxyanion hole for the catalytic activity. Unexpectedly, when both the catalytic triad and the oxyanion hole were kept intact, a slight change of the binding site also resulted in significantly decreased catalytic activity, indicating that the designed binding site is at the right position to align the substrate in the right orientation in the active center for catalytic hydrolysis. These results confirm the synergy among the catalytic triad, the oxyanion hole, and the substrate-binding site, indicating successful mimicking of the active center of α-chymotrypsin. Moreover, Silverzyme shows better thermal stability than α-chymotrypsin, and can even hydrolyze the tough non-activated ester diethyl phthalate, a priority pollutant by the United States Environmental Protection Agency (USEPA). ConclusionThis study successfully mimicked the complex catalytic active center of α-chymotrypsin using a conformational engineering strategy, and produced a highly active artificial hydrolase with a well-defined structure and catalytic mechanism. The findings highlight the significant potential of conformational engineering.

Background and aims The mechanism of cholestatic liver injury (CLI) is unclear, and effective therapies are lacking. While peroxisome proliferator-activated receptor alpha (PPARα) agonists show potential hepatoprotective effect and pyroptosis is implicated in hepatocellular damage, how PPARα activation mitigates lithocholic acid (LCA)-induced pyroptosis remains unknown. Methods The hepatoprotective effect of PPARα agonists was evaluated in a mouse model of intrahepatic cholestasis induced by LCA. Liver injury was assessed via serum biochemistry, hematoxylin and eosin and TUNEL staining, and electron microscopy. Pyroptosis pathways were analyzed using real-time quantitative polymerase chain reaction, Western blot, and co-immunoprecipitation. Results Combined morphological, histopathological, and biochemical analyses confirmed that PPARα activation protects against CLI. Compared with LCA treatment alone, PPARα activation significantly attenuated the elevation of serum lactate dehydrogenase (LDH), the increased TUNEL-positive cells, and the formation of hepatocyte membrane pores. Mechanistically, PPARα activation suppressed both NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis and apoptosis protease-activating factor-1 (APAF-1)/CASPASE-3/GSDME-mediated pyroptosis. Furthermore, PPARα agonist pretreatment inhibited activation of the nuclear factor-kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways. Conclusions PPARα protects against LCA-induced CLI by inhibiting both NLRP3 inflammasome-mediated pyroptosis associated with NF-κB and APAF-1/CASPASE-3/GSDME-mediated pyroptosis associated with the FOXO1 signaling pathway.

Metabonomics and proteomics were employed to investigate the mechanism of Yuzhi Zhixue Granules in treating polycystic ovary syndrome with insulin resistance(PCOS-IR). The disease model was established by feeding a high-fat diet and gavage of letrozole solution and it was then treated with different doses of Yuzhi Zhixue Granules. The therapeutic effect of Yuzhi Zhixue Granules was evaluated based on the body mass, homeostasis model assessment of insulin resistance and insulin sensitivity index, serum levels of adipokines, and histopathological changes of rats. Metabolomics and proteomics were employed to find the action pathways of Yuzhi Zhixue Granules. The results showed that Yuzhi Zhixue Granules reduced the body mass, improved the insulin sensitivity and aromatase activity, improved the levels of leptin, adiponectin and other adipokines, and alleviated insulin resistance, histopathological changes, and metabolic disorders in PCOS-IR rats. Metabolomics results revealed 14 metabolites with altered levels in the ovarian tissue, which were closely related to glutathione metabolism and pyruvate metabolism. Proteomics results showed that the therapeutic effect of Yuzhi Zhixue Granules was mainly related to the adipokine, adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK), phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt), forkhead box protein O(FoxO), and mechanistic target of rapamycin(mTOR) signaling pathways. Western blot results showed that compared with the model group, Yuzhi Zhixue Granules treatment decreased the p-AMPK/AMPK and p-FoxO1/FoxO1 levels, increased the p-mTOR/mTOR level, and up-regulated the expression level of recombinant glucose transporter 4(GLUT4). Yuzhi Zhixue Granules can balance amino acid metabolism and pyruvate metabolism by regulating the AMPK/mTOR/FoxO/GLUT pathway to maintain the homeostasis of the ovarian environment and alleviate insulin resistance, thus treating PCOS-IR.
Animals ; Female ; Insulin Resistance ; Polycystic Ovary Syndrome/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Metabolomics ; Proteomics ; Rats, Sprague-Dawley ; Humans ; Ovary/metabolism* ; Signal Transduction/drug effects*

This study aimed to explore the mechanisms and molecular targets of total flavones of Abelmoschus manihot(TFA) plus empagliflozin(EM) in attenuating diabetic tubulopathy(DT) by targeting mitochondrial homeostasis and pyroptosis-apoptosis-necroptosis(PANoptosis). In the in vivo study, the authors established the DT rat models through a combination of uninephrectomy, administration of streptozotocin via intraperitoneal injections, and exposure to a high-fat diet. Following modeling successfully, the DT rat models received either TFA, EM, TFA+EM, or saline(as a vehicle) by gavage for eight weeks, respectively. In the in vitro study, the authors subjected the NRK52E cells with or without knock-down Z-DNA binding protein 1(ZBP1) to a high-glucose(HG) environment and various treatments including TFA, EM, and TFA+EM. In the in vivo and in vitro studies, The authors investigated the relative characteristics of renal tubular injury and renal tubular epithelial cells damage induced by reactive oxygen species(ROS), analyzed the relative characteristics of renal tubular PANoptosis and ZBP1-mediatted PANoptosis in renal tubular epithelial cells, and compared the relative characteristics of the protein expression levels of marked molecules of mitochondrial fission in the kidneys and mitochondrial homeostasis in renal tubular epithelial cells, respectively. Furthermore, in the network pharmacology study, the authors predicted and screened targets of TFA and EM using HERB and SwissTargetPrediction databases; The screened chemical constituents and targets of TFA and EM were constructed the relative network using Cytoscape 3.7.2 network graphics software; The relative targets of DT were integrated using OMIM and GeneCards databases; The intersecting targets of TFA, EM, and DT were enriched and analyzed signaling pathways by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG) software using DAVID database. In vivo study results showed that TFA+EM could improve renal tubular injury, the protein expression levels and characteristics of key signaling molecules in PANoptosis pathway in the kidneys, and the protein expression levels of marked molecules of mitochondrial fission in the kidneys. And that, the ameliorative effects in vivo of TFA+EM were both superior to TFA or EM. Network pharmacology study results showed that TFA+EM treated DT by regulating the PANoptosis signaling pathway. In vitro study results showed that TFA+EM could improve ROS-induced cell injury, ZBP1-mediatted PANoptosis, and mitochondrial homeostasis in renal tubular epithelial cells under a state of HG, including the protein expression levels of marked molecules of mitochondrial fission, mitochondrial ultrastructure, and membrane potential level. And that, the ameliorative effects in vitro of TFA+EM were both superior to TFA or EM. More importantly, using the NRK52E cells with knock-down ZBP1, the authors found that, indeed, ZBP1 was mediated PANoptosis in renal tubular epithelial cells as an upstream factor. In addition, TFA+EM could regulate the protein expression levels of marked signaling molecules of PANoptosis by targeting ZBP1. In summary, this study clarified that TFA+EM, different from TFA or EM, could attenuate DT with multiple targets by ameliorating mitochondrial homeostasis and inhibiting ZBP1-mediated PANoptosis. These findings provide the clear pharmacological evidence for the clinical treatment of DT with a novel strategy of TFA+EM, which is named "coordinated traditional Chinese and western medicine".
Animals ; Rats ; Mitochondria/metabolism* ; Benzhydryl Compounds/administration & dosage* ; Glucosides/administration & dosage* ; Abelmoschus/chemistry* ; Male ; Homeostasis/drug effects* ; Flavones/administration & dosage* ; Rats, Sprague-Dawley ; Diabetic Nephropathies/physiopathology* ; Drugs, Chinese Herbal/administration & dosage* ; DNA-Binding Proteins/genetics* ; Humans ; Apoptosis/drug effects*

Ultrasound technology has been applied in the biomedical field,particularly in drug delivery and cell processing.In this study,the effects of different ultrasound power levels(40 W to 100 W)and time durations(1 min,5 min,or 5 min discontinuously)on the morphology of human red blood cells(hRBCs)membranes were systematically investigated using cryo-electron tomography(Cryo-ET).The hRBCs membranes were firstly subjected to ultrasound at power levels of 40 W and 60 W for 5 min each.Cryo-ET observations revealed minimal morphological changes in the hRBCs membranes following the 40 W treatment,with the membrane structure remaining relatively intact and only minor undulations appearing on the membrane surface.These undulations might result from the mild mechanical stress induced by ultrasound,which was insufficient to disrupt the overall membrane structure.At power of 60 W,the hRBCs membranes largely preserved their structural integrity.When the ultrasonic power was increased to 80 W,the structural damage to the hRBCs membranes became more severe.Cryo-ET images showed irregular ruptures and larger pores on the membrane surface,indicating a significant compromise in membrane integrity.At ultrasound power of 100 W,the hRBCs membranes were completely disrupted,resulting in the formation of numerous membrane fragments,and a complete loss of membrane continuity.To further explore the effects of ultrasound duration on erythrocyte membrane morphology,the ultrasonic power was fixed at 100 W and the impacts of varying treatment durations(1 min,5 min,and intermittent ultrasound)on the membrane structure were systematically investigated.After 1 min of ultrasonic treatment,Cryo-ET images showed minimal changes in erythrocyte membrane morphology.Although some small pores and undulations appeared on the membrane surface,the overall structure remained relatively intact.As the ultrasound duration extended to 5 min,the degree of membrane damage increased significantly.Cryo-ET images revealed extensive rupture and detachment of the membrane,with continuity being severely compromised.As to treatment alternating 1 min of ultrasound with 1 min of rest,for a total of 5 min of ultrasound exposure,Cryo-ET observations showed the integrity of the membrane-cytoskeleton attachment remained.Under intermittent ultrasound treatment,although some pores and ruptures were observed on the membrane surface,the overall structure remained more intact compared to continuous ultrasonic treatment.This preservation might be due to the intermittent treatment providing buffer periods for the membrane,allowing partial recovery after mechanical stress,thereby reducing the cumulative damage caused by continuous ultrasound.This work provided experimental basis for further understanding of mechanism of ultrasound induced change of cell membrane and cytoskeleton.

Non-cystic fibrosis bronchiectasis(referred to as bronchiectasis)is a common chronic airway disease.Neutrophils have traditionally been considered to play a dominant role in the airway inflammation of bronchiectasis.However,it has been discovered that approximately 20％to 30％of bronchiectasis patients exhibited elevated eosinophil counts(Sputum eosinophil ratio＞3％or blood eosinophil count＞300/μl)in their sputum and blood samples,referred to as eosinophilic bronchiectasis(EB).Based on this novel discovery of the inflammatory phenotype,some scholars have advocated treating bronchiectasis as an"inflammatory disease"rather than an"infectious disease"in a novel therapeutic approach.This article reviews the discovery and the latest research advancements of EB,exploring its pathophysiological basis,clinical characteristics,and treatment,with the aim to facilitate the understanding and management of EB.

Objective To explore the effectiveness of drawing-based method in medical courses and their impact on students' learning habits,academic performance,and comprehensive competencies,in order to meet the demand for high-quality,interdisciplinary,and innovative talent,and provide theoretical support for integrating aesthetic education into medical training.Methods A questionnaire survey was conducted among medical students(n=310)at Dalian Medical University,covering the frequency of using drawing method and their effects on learning outcomes,innovation ability,and humanistic qualities.Data were analyzed using the Chi-square test and Fisher's exact test(P＜0.05).Results Totally 93.6％of students approved of using drawing method for learning medical courses,with 70.8％having developed a habit of drawing-based learning.Students with stronger drawing skills were more inclined to use drawing method and supported their application in teaching.The frequency of drawing-based learning was positively correlated with anatomy scores(P＜0.05).Students generally agreed that drawing method enhanced knowledge comprehension,learning interest,long-term memory,innovation ability,critical thinking,and humanistic qualities.However,students with weaker drawing skills perceived drawing method as potentially increasing learning burdens and being less efficient,but this perception significantly decreased with increased drawing frequency(P＜0.05).Conclusion Drawing methods are widely used in medical courses and effectively improve learning outcomes and comprehensive competencies.Drawing proficiency and frequency are key factors influencing students' acceptance and learning effectiveness.Future efforts should focus on promoting drawing method,strengthening students' drawing skills,and optimizing learning processes to deepen the integration of aesthetic education in medical training.

AIM To study the chemical constituents from the buds of Aralia chinensis L.var.nuda Nakai and their in vitro anti-inflammatory activities.METHODS The 70％ethanol extract from the buds of A.chinensis var.nuda was isolated and purified by silica gel,Sephadex LH-20,ODS and semi-preparative HPLC,then the structures of compounds were identified by physicochemical properties and spectral data.Their anti-inflammatory activities in vitro were evaluated by RAW264.7 model.RESULTS Sixteen compounds were isolated and identified as 4-(2,2-dibutoxyethyl)phenol(1),trans-linalool-3,7-oxide-6-O-β-D-glucopyranoside(2),2'-O-(9Z,12Z,15Z-octadecatrienoyl)glyceryl β-D-galactopyranoside(3),quercetin-3-O-β-D-glucopyranoside(3'→ O-3''')quercetin-3-O-β-D-galactopyranoside(4),syringaresinol-4'-O-β-D-glucopyranoside(5),p-hydroxybenzaldehyde(6),7α-hydroxystigmasterol 3-O-β-D-glucopyranoside(7),trans-p-hydroxy cinnamic acid methyl ester(8),funingensin A(9),3,4-dihydroxy-acetophenone(10),N-acetyltyramine(11),3,4-di-O-caffeoyl quinic acid(12),chlorogenic acid(13),aralia cerebroside(14),caffeic acid methyl ester(15),tetradecanoic acid(16).The IC50values of compounds 8,10,12 and 13 were(22.19±1.59),(35.25±1.30),(13.38±0.72),(15.73±1.16)μmol/L,respectively.CONCLUSION Compound 1 is a new compound,2-13 are isolated from genus Aralia for the first time.Compounds 8,10,12,13 exhibit significant in vitro anti-inflammatory activities.

Cervical spinal cord injury without fracture-dislocation (CSCIWFD) is referred to as a special type of cervical spinal cord injury characterized by traumatic spinal cord dysfunction and no significant bony structural abnormalities on imagines. Duo to the high risk of missed diagnosis during the initial consultation, CSCIWFD may lead to progressive neurological deterioration or even complete paralysis, severely impacting patients′ prognosis. Currently, there are no established consensuses over the diagnosis and treatment of CSCIWFD, such as the lack of evidence-based standards for indications of non-surgical treatment and risk of secondary neurological injury, as well as debates over the optimal timing for surgical intervention and indications for different surgical approaches. To address these issues, the Spine Trauma Group of the Orthopedic Branch of the Chinese Medical Doctor Association organized experts in the relevant fields to formulate Diagnosis and treatment guideline for acute cervical spinal cord injury without fracture- dislocation in adults ( version 2025) . Based on evidence-based medicine and the principles of scientific rigor and clinical applicability, the guidelines proposed 11 recommendations covering terminology, diagnosis, evaluation treatment, and rehabilitation, etc., aiming to standardize the management of CSCIWFD.