ObjectiveTo investigate the functional role and underlying molecular mechanisms of fumarylacetoacetate hydrolase （FAH） in the progression of glioblastoma （GBM）. MethodsDifferential expression analysis was performed on the TCGA-GBM， GSE4290， and GSE116520 datasets. Weighted gene co-expression network analysis （WGCNA） was used to identify key modules， and Cox regression and risk modeling were used to screen prognostic genes. Immune infiltration analysis of prognostic genes was carried out by using single-cell RNA sequencing panels. The clinical expression signature of FAH in GBM was analyzed in the TCGA and HPA databases. The functional role of FAH was validated by in vitro and in vivo experiments， and pathway analysis was performed to explore the underlying mechanisms. ResultsA total of 152 overlapping genes were identified across the three GBM datasets （P<0.05）. WGCNA revealed that the turquoise module was most strongly associated with tumor purity， stromal score， immune score， and ESTIMATE score （P<0.001）. Compared with normal tissues， three prognostic genes （CTSD， FAH， and THBD） were upregulated in GBM and correlated with immune infiltration （P<0.05）. FAH mRNA and protein levels were elevated in GBM tissues relative to normal tissues， and its expression was significantly associated with age stratification and TP53 mutation （P<0.05）. CCK-8 assay results showed that， compared with the shNC group， the proliferative activity of GBM cells in the shFAH group was reduced （P<0.001）. Transwell migration and invasion assays demonstrated that， relative to the shNC group， the numbers of migrated and invaded cells in the shFAH group decreased （P<0.05）. Western blot analysis revealed that the protein expression levels of PI3K， p-AKT， and p-mTOR in the shFAH group decreased compared with those in the shNC group （P<0.05）. In vivo subcutaneous xenograft experiments further confirmed that tumor volume and weight significantly decreased in the shFAH group compared with the shNC group （P<0.001）. ConclusionFAH promotes GBM progression by activating the PI3K/AKT/mTOR signaling pathway and may serve as a potential therapeutic target for GBM.

Background/Aims: Early detection and effective prognosis prediction in patients with hepatocellular carcinoma (HCC) provide an avenue for survival improvement, yet more effective approaches are greatly needed. We sought to develop the detection and prognosis models with ultra-sensitivity and low cost based on fragmentomic features of circulating cell free mtDNA (ccf-mtDNA). Methods: Capture-based mtDNA sequencing was carried out in plasma cell-free DNA samples from 1168 participants, including 571 patients with HCC, 301 patients with chronic hepatitis B or liver cirrhosis (CHB/LC) and 296 healthy controls (HC). Results: The systematic analysis revealed significantly aberrant fragmentomic features of ccf-mtDNA in HCC group when compared with CHB/LC and HC groups. Moreover, we constructed a random forest algorithm-based HCC detection model by utilizing ccf-mtDNA fragmentomic features. Both internal and two external validation cohorts demonstrated the excellent capacity of our model in distinguishing early HCC patients from HC and highrisk population with CHB/LC, with AUC exceeding 0.983 and 0.981, sensitivity over 89.6% and 89.61%, and specificity over 98.20% and 95.00%, respectively, greatly surpassing the performance of alpha-fetoprotein (AFP) and mtDNA copy number. We also developed an HCC prognosis prediction model by LASSO-Cox regression to select 20 fragmentomic features, which exhibited exceptional ability in predicting 1-year, 2-year and 3-year survival (AUC=0.8333, 0.8145 and 0.7958 for validation cohort, respectively). Conclusions We have developed and validated a high-performing and low-cost approach in a large clinical cohort based on aberrant ccf-mtDNA fragmentomic features with promising clinical translational application for the early detection and prognosis prediction of HCC patients.

Objective To investigate the effects of sauchinone(Sch)on the growth and immune escape of liver cancer cells by regulating the Krüppel like factor 5(KLF5)-erythropoietin-producing hepatocellular receptor A2(EphA2)pathway.Methods Huh-7 cells were grouped into liver cancer group,Sch low,medium,and high-dose groups(Sch-L group,Sch-M group,Sch-H group),Sch-H+KLF5 activator negative control group(OE-NC),and Sch-H+KLF5 activator(OE-KLF5)group.5-bromo-2-deoxyuracil(EdU)staining,CCK-8,scratch assay,and Transwll were used to detect the proliferation,migration,and invasion of Huh-7 cells,respectively.Western blot was applied to detect proliferative cell nuclear antigen(PCNA),migration invasion enhancer(MIEN1),matrix metalloproteinase-2(MMP-2),programmed death receptor ligand 1(PD-L1),KLF5,and EphA2 proteins in Huh-7 cells.The Huh-7 cells in above 6 groups were co cultured with activated CD8+T cells in a 96 well plate and named as liver cancer co culture group,Sch-L co culture group,Sch-M co culture group,Sch-H co culture group,Sch-H+OE-NC co culture group,and Sch-H+OE-KLF5 co culture group.The killing rate of CD8+T cells in the co culture system and the levels of interferon-γ(IFN-γ),interleukin-4(IL-4),and tumor necrosis factor-α(TNF-α)in the supernatant were detected.Results Compared with the liver cancer group,the EdU positive rate,OD450 value,cell invasion number,and PCNA,MIEN1,MMP-2,PD-L1,KLF5,and EphA2 proteins in the Sch-L,Sch-M,and Sch-H groups reduced,the migration distance shorten(P<0.05).Compared with the Sch-H group and Sch-H+OE-NC group,the EdU positive rate,OD450 value,cell invasion number,and PCNA,MIEN1,MMP-2,PD-L1,KLF5,and EphA2 proteins in the Sch-H+OE-KLF5 group increased,the migration distance prolonged(P<0.05).Compared with the liver cancer co culture group,the killing rate of CD8+T cells on Huh-7 cells and the levels of IFN-γ,TNF-α,and IL-4 in the supernatant in the Sch-L co culture group,Sch-M co culture group,and Sch-H co culture group increased(P<0.05).Compared with the Sch-H co culture group and the Sch-H+OE-NC co culture group,the killing rate of CD8+T cells on Huh-7 cells and the levels of IFN-γ,TNF-α,and IL-4 in the supernatant in the Sch-H+OE-KLF5 co culture group decreased(P<0.05).Conclusion Sch may inhibit growth and immune escape of liver cancer cells by inhibiting the KLF5-EphA2 pathway.

Objective To investigate the effects of sauchinone(Sch)on the growth and immune escape of liver cancer cells by regulating the Krüppel like factor 5(KLF5)-erythropoietin-producing hepatocellular receptor A2(EphA2)pathway.Methods Huh-7 cells were grouped into liver cancer group,Sch low,medium,and high-dose groups(Sch-L group,Sch-M group,Sch-H group),Sch-H+KLF5 activator negative control group(OE-NC),and Sch-H+KLF5 activator(OE-KLF5)group.5-bromo-2-deoxyuracil(EdU)staining,CCK-8,scratch assay,and Transwll were used to detect the proliferation,migration,and invasion of Huh-7 cells,respectively.Western blot was applied to detect proliferative cell nuclear antigen(PCNA),migration invasion enhancer(MIEN1),matrix metalloproteinase-2(MMP-2),programmed death receptor ligand 1(PD-L1),KLF5,and EphA2 proteins in Huh-7 cells.The Huh-7 cells in above 6 groups were co cultured with activated CD8+T cells in a 96 well plate and named as liver cancer co culture group,Sch-L co culture group,Sch-M co culture group,Sch-H co culture group,Sch-H+OE-NC co culture group,and Sch-H+OE-KLF5 co culture group.The killing rate of CD8+T cells in the co culture system and the levels of interferon-γ(IFN-γ),interleukin-4(IL-4),and tumor necrosis factor-α(TNF-α)in the supernatant were detected.Results Compared with the liver cancer group,the EdU positive rate,OD450 value,cell invasion number,and PCNA,MIEN1,MMP-2,PD-L1,KLF5,and EphA2 proteins in the Sch-L,Sch-M,and Sch-H groups reduced,the migration distance shorten(P<0.05).Compared with the Sch-H group and Sch-H+OE-NC group,the EdU positive rate,OD450 value,cell invasion number,and PCNA,MIEN1,MMP-2,PD-L1,KLF5,and EphA2 proteins in the Sch-H+OE-KLF5 group increased,the migration distance prolonged(P<0.05).Compared with the liver cancer co culture group,the killing rate of CD8+T cells on Huh-7 cells and the levels of IFN-γ,TNF-α,and IL-4 in the supernatant in the Sch-L co culture group,Sch-M co culture group,and Sch-H co culture group increased(P<0.05).Compared with the Sch-H co culture group and the Sch-H+OE-NC co culture group,the killing rate of CD8+T cells on Huh-7 cells and the levels of IFN-γ,TNF-α,and IL-4 in the supernatant in the Sch-H+OE-KLF5 co culture group decreased(P<0.05).Conclusion Sch may inhibit growth and immune escape of liver cancer cells by inhibiting the KLF5-EphA2 pathway.

Objective:To investigate the expressions and prognostic value of lysyl oxidase like protein 2(LOXL2),chemo-kine c-c-motif ligand 18(CCL-18)and chitinase protein 40(YKL-40)in connective tissue disease with pulmonary interstitial lesions.Methods:A total of 308 patients with connective tissue disease who were treated in the First Affiliated Hospital of Hebei North Univer-sity from July 2021 to February 2022 were selected,including 108 patients with pulmonary interstitial disease(pathological group),200 patients without pulmonary interstitial disease(non pathological group),and another 35 healthy volunteers as the control group.Serum levels of LOXL2,CCL-18 and YKL-40 were detected by ELISA;Logistic regression analysis was applied to analyze the factors affecting the prognosis of patients with connective tissue disease and pulmonary interstitial disease;the predictive value of serum LOXL2,CCL-18 and YKL-40 levels on the prognosis of patients with connective tissue disease and pulmonary interstitial disease was analyzed by receiver operating characteristic(ROC)curve analysis.Results:Compared with control group,the serum levels of LOXL2,CCL-18 and YKL-40 in patients with connective tissue disease in the non pathological group and the pathological group were obviously increased(P<0.05);the serum levels of LOXL2,CCL-18 and YKL-40 in patients with connective tissue disease in the le-sion group were higher than those in the non lesion group(P<0.05);compared with the survival group,the serum levels of LOXL2,CCL-18 and YKL-40 in patients with connective tissue disease and pulmonary interstitial disease in the death group were obviously higher(P<0.05);Logistic regression analysis showed that LOXL2,CCL-18 and YKL-40 were risk factors for the prognosis of patients with connective tissue disease and pulmonary interstitial disease(P<0.05);the area under the ROC curve(AUC)of the combined detection of serum LOXL2,CCL-18 and YKL-40 in predicting the prognosis of patients with connective tissue disease and pulmonary interstitial disease was 0.967,which was better than their respective independent prediction(Zcombined detection-LOXL2=1.735,P=0.041;Zcombined detection-CCL-18=2.481,P=0.007;Zcombined detection-YKL-40=2.008,P=0.022).Conclusion:The serum levels of LOXL2,CCL-18 and YKL-40 in patients with connective tissue disease and pulmonary interstitial disease are elevated,which has certain value in the prognosis evaluation of patients with connective tissue disease combined with pulmonary interstitial disease.

Objective:To explore the safety and efficacy of intraosseous regional administration (IORA) of vancomycin for preventing infection in primary total knee arthroplasty (TKA).Methods:A total of 124 patients with knee osteoarthritis undergoing TKA between February 2024 and May 2024 at nine hospitals were enrolled. Preoperative infection prophylaxis involved either IORA (0.5 g vancomycin administered via intraosseous regional infusion before incision) or intravenous infusion (1 g vancomycin via peripheral vein). The IORA group included 15 males and 47 females with a median age of 66.5 years (range, 60.0-70.0 years), while the intravenous group included 14 males and 48 females with a median age of 66.0 years (range, 61.8-70.3 years) years. Intraoperative samples were collected including fat and synovium tissues after incision, before prosthesis placement, and after tourniquet release; distal femoral cancellous bone during femoral osteotomy; proximal tibial cancellous bone during tibial osteotomy; proximal intercondylar cancellous bone before prosthesis placement; and peripheral blood from non-infused arms at surgery initiation and after tourniquet release. Vancomycin concentrations were measured using liquid chromatography-tandem mass spectrometry. Vital sign changes were recorded from admission to 5~10 minutes post-IORA (IORA group) or post-incision (intravenous group). Follow-ups were conducted on postoperative day 1 and 3, and at 1 and 3 months, to document complications including IORA-related adverse events, periprosthetic joint infections, surgical site infections, red man syndrome, acute kidney injury, deep vein thrombosis and so on.Results:Vancomycin concentrations in bone, fat, and synovial tissue samples were significantly higher in the IORA group than in the intravenous group ( P<0.05), while vancomycin concentrations in blood samples were significantly lower in the IORA group than in the intravenous group ( P<0.05). Only 7.3%(41/558) of tissue samples in the IORA group had vancomycin concentrations below 2.0 μg/g (the minimum inhibitory concentration of vancomycin against coagulase-negative staphylococcus), compared to 59.3%(331/558) in the intravenous group (χ 2=11.285, P<0.001). In the intravenous group, 16.9%(21/124) of blood samples had vancomycin concentrations exceeding 15.0 mg/L (the threshold associated with a significantly increased risk of nephrotoxicity), while all concentrations in the IORA group were below this threshold, the difference was statistically significant (χ 2=22.943, P<0.001). There were no statistically significant difference ( P>0.05) in vital signs changes before and after vancomycin administration between the two groups. Two patients in the intravenous group experienced incision exudate, while no other related complications occurred in either group. Conclusions:Compared to the traditional intravenous infusion of 1 g vancomycin, intraosseous injection of a low dose (0.5 g) of vancomycin achieves higher local tissue concentrations in the knee joint with a lower incidence of adverse reactions and is safe for infection prophylaxis. Despite guidelines not recommending the routine use of vancomycin for preventing infection after primary TKA, intraosseous injection of 0.5 g vancomycin may be considered intraoperatively for primary TKA in the following scenarios: patients in medical institutions with a high prevalence of methicillin-resistant staphylococcus aureus (MRSA) infections, patients with potential preoperative MRSA colonization, or patients with cephalosporin allergy.

Background/Aims: Early detection and effective prognosis prediction in patients with hepatocellular carcinoma (HCC) provide an avenue for survival improvement, yet more effective approaches are greatly needed. We sought to develop the detection and prognosis models with ultra-sensitivity and low cost based on fragmentomic features of circulating cell free mtDNA (ccf-mtDNA). Methods: Capture-based mtDNA sequencing was carried out in plasma cell-free DNA samples from 1168 participants, including 571 patients with HCC, 301 patients with chronic hepatitis B or liver cirrhosis (CHB/LC) and 296 healthy controls (HC). Results: The systematic analysis revealed significantly aberrant fragmentomic features of ccf-mtDNA in HCC group when compared with CHB/LC and HC groups. Moreover, we constructed a random forest algorithm-based HCC detection model by utilizing ccf-mtDNA fragmentomic features. Both internal and two external validation cohorts demonstrated the excellent capacity of our model in distinguishing early HCC patients from HC and highrisk population with CHB/LC, with AUC exceeding 0.983 and 0.981, sensitivity over 89.6% and 89.61%, and specificity over 98.20% and 95.00%, respectively, greatly surpassing the performance of alpha-fetoprotein (AFP) and mtDNA copy number. We also developed an HCC prognosis prediction model by LASSO-Cox regression to select 20 fragmentomic features, which exhibited exceptional ability in predicting 1-year, 2-year and 3-year survival (AUC=0.8333, 0.8145 and 0.7958 for validation cohort, respectively). Conclusions We have developed and validated a high-performing and low-cost approach in a large clinical cohort based on aberrant ccf-mtDNA fragmentomic features with promising clinical translational application for the early detection and prognosis prediction of HCC patients.