ObjectiveTo construct a nomogram prediction model for non-valvular persistent atrial fibrillation （PeAF） patients with left ventricular hypertrophy （LVH） ， followed by prognostic analysis through follow-up. MethodsThis study retrospectively enrolled 949 patients with newly diagnosed and hospitalized non-valvular PeAF. Among them， 403 patients presented with LVH. The cohort was randomly stratified into a training set （n=665） and a validation set （n=284）. Univariate and multivariate Logistic regression analyses were employed to identify independent risk factors for PeAF complicated by LVH. A nomogram prediction model was subsequently constructed and evaluated for discriminative ability， calibration， and clinical utility using receiver operating characteristic （ROC） curve analysis， calibration plots， and decision curve analysis （DCA）. ResultsSeven independent risk factors were ultimately identified and included in the prediction model： female sex， hypertension， diabetes， red blood cell distribution width-SD （RDW-SD）， body mass index （BMI）， left atrial diameter （LAD）， and left ventricular ejection fraction （LVEF）. The area under the ROC curve （AUC） in the training set was 0.862 （95% CI： 0.834-0.890）， and in the validation set， it was 0.870 （95% CI： 0.829-0.911）， demonstrating excellent predictive performance. ConclusionIndependent risk factors for LVH in PeAF patients include female， hypertension， diabetes， RDW-SD， BMI， LAD， and LVEF. The prediction model built based on this can help early identification of PeAF patients with high risk of LVH. At the same time， the incidence of major adverse cardiovascular events （MACE） is higher in PeAF patients with LVH. Patients with atrial fibrillation combined with LVH may benefit from catheter ablation.

ObjectiveAbnormal lipids in neurons can cause the accumulation of α-synuclein（α-syn）. This study aimed to explore the mechanism of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis extract （ASH） in treating Parkinson's disease （PD） mice using lipidomics combined with network pharmacology. MethodsMice were divided into the blank group， model group and ASH （45.5 mg·kg^-1） group. Motor ability was evaluated by pole climbing time and autonomous activity count； The oxidative stress indicators were detected by enzyme-linked immunosorbent assay （ELISA）. Lipid biomarkers in brain tissues were screened and identified by ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）， and metabolic pathway analysis was conducted. The key targets of ASH for PD treatment were explored using network pharmacology. The Kyoto Encyclopedia of Genes and Genomes （KEGG） database was used for pathway enrichment analysis， and the "compound-reaction-enzyme-gene" network was constructed using the MetScape plugin. The protein expression levels of glutathione S-transferase P1 （GSTP1）， glutathione S-transferase Mu 2 （GSTM2）， prostaglandin peroxide synthase 1 （PTGS1）， prostaglandin peroxide synthase 2 （PTGS2）， and prostaglandin E synthase （PTGES） were validated by Western blot. ResultsCompared with the blank group， the model group showed significantly prolonged pole climbing time and reduced autonomous activity count （P<0.01）. Compared with the model group， the ASH group demonstrated significantly faster pole climbing and increased autonomous activity count （P<0.01）. The model group exhibited significantly decreased superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） levels， and increased malondialdehyde （MDA） level in brain tissues compared with the blank group （P<0.01）. The ASH group showed increased SOD and GSH-Px levels and decreased MDA level compared with the model group （P<0.05， P<0.01）. Lipidomics analysis identified 10 differential metabolites and 8 differential metabolic pathways. Network pharmacological analysis revealed 213 intersection targets between ASH components and PD， with KEGG enrichment involving the sphingolipid signaling pathway， lipid arteriosclerosis， phosphoinositide 3-kinase/protein kinase B（PI3K/Akt） signaling pathway， mitogen-activated protein kinase（MAPK） signaling pathway， and hypoxia inducible factor-1（HIF-1） signaling pathway. Integrated lipidomics and network pharmacology analysis highlighted the central role of the arachidonic acid metabolic pathway. The Western blot results showed that ASH effectively up-regulated GSTP1， GSTM2， and PTGS1 protein expression， and down-regulated PTGS2 and PTGES protein expression. ConclusionASH can ameliorate behavioral deficits， exert antioxidant effects， regulate lipid differential metabolites and the arachidonic acid metabolic pathway， thereby exerting therapeutic effects in PD model mice.

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.

Twelve pre-processing protocols for formalin-fixed paraffin-embedded(FFPE)tissue samples were developed by orthogonal experimental design,incorporating different dewaxing buffers(Triton X-100 and xylene),lysis buffers(TFE and RapiGest),and enzyme digestion methods(iST,SP3,and FASP)to explore the optimal experimental conditions.These protocols were assessed based on protein and peptide identification depth,identification stability,and quantitative levels of protein abundance.The results indicated that Triton X-100 and xylene minimally impacted proteomics identification,whereas the TFE lysis buffer and iST digestion method significantly enhanced the proteomics analysis of FFPE samples.Considering the potential toxicity of xylene,the TTI protocol based on Triton X-100,TFE,and iST was determined to be the optimal choice.This protocol exhibited the best repeatability and stability,and a higher number of proteins associated with significant biological functions were identified.In conclusion,the established TTI protocol offered an efficient and comprehensive approach for proteomic analysis of FFPE samples,significantly enhancing the repeatability and stability of protein identification.

To investigate the molecular mechanisms underlying the mechanosensitive ion channel PI-EZO2 in osteoarthritis(OA),we developed a lentiviral vector for endogenous PIEZO2 overexpression and established a stable PIEZO2-high-expressing immortalized human primary chondrocyte line.By map-ping the open reading frame of the PIEZO2 locus and designing sequence-specific sgRNA,we employed the CRISPR/Cas9 synergistic activation mediator(SAM)system to precisely integrate transcriptional ac-tivation elements into the PIEZO2 promoter region.Lentiviral-mediated targeted genomic integration en-sured endogenous PIEZO2 overexpression,confirmed by mCherry fluorescence tracing coupled with flow cytometric sorting,which revealed membrane-specific localization of PIEZO2 protein(localization effi-ciency:78.49％).Quantitative PCR demonstrated a 17-fold upregulation of PIEZO2 mRNA,while Western blotting validated enhanced membrane-localized protein expression.Strikingly,PIEZO2-overex-pressing chondrocytes exhibited hallmark OA metabolic phenotypes compared to wild-type controls:typeⅡ collagen mRNA expression decreased to 50％of baseline levels,whereas matrix metalloproteinase 13(MMP13)mRNA surged by 20-fold.These alterations recapitulated the pathological matrix metabolic phenotype observed in biomechanical OA models induced by cyclic mechanical stress(10％strain,0.5 Hz,8 h/day for 2 consecutive days).Collectively,we successfully generated a human chondrocyte model with stable PIEZO2 overexpression,which faithfully mirrors mechanotransduction-driven OA progression.This engineered cellular system provides a robust platform for dissecting PIEZO2-mediated mechanosig-naling networks and advancing targeted therapeutic discovery.

Currently, transcatheter intervention is the preferred treatment for patients with anatomically suitable atrial septal defects. However, the use of nickel-titanium alloy occluders in interventional procedures results in lifelong presence of the implant in the body, leading to complications such as metal allergies and arrhythmias in some patients. To overcome the short-term and long-term complications associated with the presence of metal, and to avoid radiation exposure and metal toxicity, this paper reports a case of successful transcatheter closure of atrial septal defect in a pediatric patient with metal allergies using fully biodegradable occluder under ultrasound guidance, achieving excellent results by interventional therapy.

Objective To elucidate the anti-aging effect of β-sitosterol(BS),an important component in the fruits of Alpinia oxyphylla Miq.,in C.elegans and its regulatory effect on ETS-5 gene to modulate ferroptosis.Methods C.elegans treated with 10 μg/mL BS were monitored for survival time and changes in body length,motility,and reproductive function.The effect of ETS-5 gene knockdown on survival time of C.elegans was observed,and the changes in fat accumulation and lipid redox homeostasis in the transfected C.elegans were assessed using Oil Red O staining and by detecting MDA levels and the GSH/GSSG ratio.The mRNA expression levels of ferroptosis-related genes(FTN-1,GPX-1 and AAT-9)were detected using qPCR.The effects of BS treatment and ETS-5 knockdown on AAT-9 enzyme activity in C.elegans were examined.The effect of BS on nuclear localization of FEV(the human homolog of ETS-5)was validated in cultured human umbilical venous endothelial cells(HUVECs).Results Both BS treatment and ETS-5 knockdown significantly prolonged the lifespan,promoted lipid accumulation and reduced lipid peroxidation in C.elegans.ETS-5 knockdown resulted in upregulated expressions of the ferroptosis repressors GPX-1,AAT-9 and FTN-1 and increased the GSH/GSSG ratio in C.elegans.Conclusion BS inhibits ferroptosis in C.elegans by suppressing the expression of ETS-5 transcription factor and hence the activity of AAT-9 enzyme,a key gene for ferroptosis,which in turn prolongs the lifespan of C.elegans.

Objective To explore the characteristics of traditional Chinese medicine(TCM)syndrome,syndrome elements and their combination,and the distribution of TCM syndrome types in advanced non-small cell lung cancer(NSCLC)patients suffering from hypothyroidism after treatment with immune checkpoint inhibitors(ICIs).Methods The analysis was conducted on 168 patients with NSCLC at stage ⅢB-ⅣB confirmed by pathological findings,whose epidermal growth factor receptor/anaplastic lymphoma kinase(EGFR/ALK)was negative,and then suffering from hypothyroidism after treatment with ICIs from January 2020 to June 2023,who admitted to Zhongshan Hospital of Chinese Medicine Affiliated to Guangzhou University of Chinese Medicine.The patients'information collected by four diagnostic methods of TCM was analyzed,and then cluster analysis was used to explore the characteristics of TCM syndrome and the distribution of TCM syndrome types of immunotherapy-induced hypothyroidism in advanced NSCLC.Moreover,the distribution of TCM syndrome types in the patients with different genders,age groups,and hypothyroidism grades was analyzed.Results(1)The TCM syndrome of hypothyroidism appearing in 168 patients with advanced NSCLC after immunotherapy was characterized by deficiency type,which manifested as follows:cough,fatigue and weakness,amnesia,lusterless complexion,spontaneous sweating,dry skin,white sputum,unwilling to talk,dizziness,nocturnal polyuria,blurred vision,emaciation,poor skin elasticity,poor appetite or even anorexia,somnolence,long-term poor appetite,edema,insomnia,low voice,dull pain,light white color of fingernails,spitting,frequently intolerance of cold,thirst,bright pale complexion,preference of warmth and aversion to cold,thirst with preference of hot drink,dyspnea,frequent constipation,dry stools,puffiness of face and eyelid,dreaminess,abdominal fullness,lumbar pain,and weakness in defecation.The tongue manifestation and pulse condition were characterized by white and thin coating,pale-red tongue,tongue with tooth-marks,pale and enlarged tongue,pale tongue,deep pulse,slippery pulse,feeble pulse,weak cubital pulse,and thready pulse.(2)The disease-location syndrome elements usually involved in the lung,spleen,and kidney,and the disease-nature syndrome elements usually involved in qi deficiency,yang deficiency,blood deficiency,and water retention.(3)The cluster analysis yielded three syndrome types,and they were lung and spleen qi deficiency syndrome,kidney yang deficiency syndrome,and qi deficiency and water retention syndrome in decreasing sequence of occurrence frequency.(4)Statistically significant difference of the distribution of TCM syndrome types was presented in the patients with various age groups(P<0.01).Lung and spleen qi deficiency syndrome was the main syndrome type in the patients aged 60-69 years old,kidney yang deficiency syndrome was frequently seen in the patients being or over 70 years old,and qi deficiency and water retention syndrome was frequently seen in the patients less than 50 years old.No statistically significant difference of the distribution of TCM syndrome types was presented in the patients with various genders and in the patients with various grades of hypothyroidism(P>0.05).Conclusion The immunotherapy-induced hypothyroidism in patients with advanced NSCLC is usually differentiated as the TCM syndrome types of lung-qi and spleen-qi deficiency,kidney yang deficiency,and qi deficiency and water retention.Deficiency of healthy qi contributes to the fundamental pathogenesis of the development and progression of the disease.Clinicians should pay attention to the changes of symptoms in time and monitor the thyroid function indicators of the patients,thus to avoid serious immunotherapy-related adverse events(irAEs).

The p21-activated kinase 4 (PAK4), a key regulator of malignancy, is negatively correlated with immune infiltration and has become an emergent drug target of cancer therapy. Given the lack of high efficacy PAK4 inhibitors, we herein reported the identification of a novel inhibitor 13 bearing a tetrahydrobenzofuro[2,3-c]pyridine tricyclic core and possessing high potency against MIA PaCa-2 and Pan02 cell lines with IC₅₀ values of 0.38 and 0.50 μmol/L, respectively. This compound directly binds to PAK4 in a non-ATP competitive manner. In the mouse Pan02 model, compound 13 exhibited significant tumor growth inhibition at a dose of 100 mg/kg, accompanied by reduced levels of PAK4 and its phosphorylation together with immune infiltration in mice tumor tissue. Overall, compound 13 is a novel allosteric PAK4 inhibitor with a unique tricyclic structural feature and high potency both in vitro and in vivo, thus making it worthy of further exploration.

OBJECTIVES: To elucidate the anti-aging effect of β-sitosterol (BS), an important component in the fruits of Alpinia oxyphylla Miq., in C. elegans and its regulatory effect on ETS-5 gene to modulate ferroptosis. METHODS: C. elegans treated with 10 µg/mL BS were monitored for survival time and changes in body length, motility, and reproductive function. The effect of ETS-5 gene knockdown on survival time of C. elegans was observed, and the changes in fat accumulation and lipid redox homeostasis in the transfected C. elegans were assessed using Oil Red O staining and by detecting MDA levels and the GSH/GSSG ratio. The mRNA expression levels of ferroptosis-related genes (FTN-1, GPX-1 and AAT-9) were detected using qPCR. The effects of BS treatment and ETS-5 knockdown on AAT-9 enzyme activity in C. elegans were examined. The effect of BS on nuclear localization of FEV (the human homolog of ETS-5) was validated in cultured human umbilical venous endothelial cells (HUVECs). RESULTS: Both BS treatment and ETS-5 knockdown significantly prolonged the lifespan, promoted lipid accumulation and reduced lipid peroxidation in C. elegans. ETS-5 knockdown resulted in upregulated expressions of the ferroptosis repressors GPX-1, AAT-9 and FTN-1 and increased the GSH/GSSG ratio in C. elegans. CONCLUSIONS BS inhibits ferroptosis in C. elegans by suppressing the expression of ETS-5 transcription factor and hence the activity of AAT-9 enzyme, a key gene for ferroptosis, which in turn prolongs the lifespan of C. elegans.
Animals ; Caenorhabditis elegans/physiology* ; Ferroptosis/drug effects* ; Alpinia/chemistry* ; Sitosterols/pharmacology* ; Longevity/drug effects* ; Fruit/chemistry* ; Humans