To construct a recombinant fowl adenovirus 4(FAdV-4)expressing the Cap protein of goose astrovirus genotype 2(GoAstV-2),the expression cassette of Cap gene was inserted into the natural 1 966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cm-FAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line(LMH)to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rF Ad V4-Cap/GoAstV-2.After 15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GoAstV-2 infected LMH cells as template.LMH cells were in-fected with 15th passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15th passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant vi-rus rF AdV4-Cap/Go Ast V-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-in-fection of FAdV-4 and GoAstV-2 in goose.

Objective:To summarize and analyze the clinical efficacy and experience of ultrasound-guided radiofrequency ablation (RFA) in the treatment of Kasabach-Merritt syndrome (KMS).Methods:A retrospective analysis was conducted on the data of pediatric patients with KMS who underwent ultrasound-guided RFA in Department of Hemangioma Surgery, the Third Affiliated Hospital of Zhengzhou University, between March 2018 and March 2024. Preoperative laboratory tests and imageological examination were performed. Under general anesthesia, the working tip of the RFA electrode needle was precisely reached the bottom of the lesion under ultrasound guidance. The electrode needle was then gradually withdrawn until the entire lesion area was covered by hyperechoic signals, indicating complete ablation. Postoperative symptomatic and supportive treatments, such as ice pack application and dressing changes, were administered to the surgical area. Platelet detection was performed immediately after the operation. Complications were closely monitored and regular follow-ups were carried out.Results:A total of 30 pediatric patients were included, comprising 14 males and 16 females, from 10 min to 5 months and 29 d after birth, with a median time of 6 d. Lesions were located in the limbs and trunk in 27 cases, and head and neck region in 3 cases, with lesion volumes ranged from 2.4 cm×2.3 cm×1.2 cm to 14.4 cm×9.3 cm×3.3 cm. The mean preoperative platelet count was 43×10 9/L, among them, the platelet values of 11 cases were (10-30) ×10 9/L, and those of 6 cases were lower than 10×10 9/L, other 13 cases with progressive thrombocytopenia. All patients successfully underwent RFA, achieving complete lesion ablation and normalization of platelet counts postoperatively. Platelet counts recovered to above 300×10 9/L in 15 patients, with no severe complications observed. The RFA area became slightly hardened within 7 d postoperatively but gradually returned to normal after consistent dressing changes for 2 weeks. During the follow-up period of 6 months to 2 years, complete lesion ablation was confirmed, with disappearance of the mass, no recurrence, good local function, mild local scar formation, and satisfactory cosmetic appearance. Conclusion:Ultrasound-guided RFA for KMS has advantages of favorable therapeutic outcomes, minimal tissue damage, no significant complications, and satisfactory cosmetic result.

The Notch signaling pathway is a highly conserved cell signaling system. Delta-like ligand 4 (Dll4), the only Notch ligand specifically expressed in endothelial cells, plays a critical role in the formation of abnormal vascular lumens under physiological and pathological conditions. The Dll4-Notch signaling pathway is essential for the regulation of gene transcription. Venous malformation, the most common type of vascular malformation, is a vascular disease characterized by progressive destruction of multiple tissues and organs; however, the mechanisms underlying its invasive progression remain unclear. This article summarizes the biological characteristics of the Dll4-Notch signaling pathway and its roles in vasculogenesis, venous malformations, and other vascular anomalies. It has been found that the Dll4-Notch signaling pathway can influence venous lumen structure and may contribute to the persistent abnormal development of malformed veins, leading to local invasion. Current research on the Dll4-Notch signaling pathway in venous malformations is still in its preliminary stages, and further in-depth experimental studies are required to untangle the relationship between them.

Objective To explore the anti-inflammatory properties of the ethyl acetate extract(ETCB)derived from Trollius chinensis Bge.using in vitro RAW264.7 cells stimulated with lipopolysaccharide and an in vivo mouse auricle model induced by xylene.Utilizing UHPLC-Q-TOF-MS(LC-MS)and network pharmacology,the components of ETCB were analyzed,and its anti-inflammatory mechanisms were preliminarily explored.Methods The anti-inflammatory activity of various solvent extracts of Trollius chinensis Bge.was assessed through the Griess assay.The impact of ETCB on the production of TNF-α and IL-6 in RAW264.7 cells induced by lipopolysaccharide was evaluated using ELISA.Real-time qPCR was conducted to determine the effect of ETCB on the expression levels of inflammatory factors such as TNF-α,IL-6,and iNOS in cells.The anti-inflammatory efficacy was further validated in a xylene-induced ear inflammation mouse model by measuring ear swelling and tissue levels of IL-6 and TNF-α.The composition of ETCB was analyzed using LC-MS.Network pharmacology was employed to screen for effective components,targets,and pathways involved in the anti-inflammatory effects of Trollius chinensis Bge.,followed by molecular docking verification between core components and targets.Results ETCB demonstrated the most potent inhibitory effect on NO production in RAW264.7 cells stimulated by lipopolysaccharide,indicating its primary role in the anti-inflammatory activity of Trollius chinensis Bge..ETCB significantly reduced TNF-α and IL-6 levels in inflammatory cells(P<0.01)and inhibited the mRNA expression of TNF-α,IL-6,and iNOS.In the xylene-induced mouse ear inflammation model,ETCB effectively alleviated ear swelling and decreased tissue levels of TNF-α and IL-6.LC-MS analysis identified 30 chemical components in ETCB,including 21 flavonoids,7 organic acids,1 polysaccharide,and 1 anthocyanin.Network pharmacology prediction and screening revealed TNF,Akt1,PTGS2,EGFR,SRC,and MMP9 as core targets,with hydroxyquercetin,lignin from fragrant leaves,zeaxanthin from willows,plantain,thistle,and sophora flavins as key anti-inflammatory active ingredients.The molecular docking analysis revealed positive interactions,characterized by favorable binding energy,between the active components and key targets.Conclusion ETCB demonstrates strong anti-inflammatory properties both inside and outside the body,functioning through various targets and pathways.This establishes a basis for deeper understanding of the anti-inflammatory mechanism of Trollius chinensis Bge.

Objective:To utilize single-nucleus RNA sequencing(snRNA-seq) technology to investigate the primary cell subpopulations in human limb venous malformations (VMs) tissue and the role of the key gene RhoJ.Methods:Surgical resection specimens of VMs tissues and surrounding normal vein tissues were collected from 100 clinically diagnosed and screened patients with limb VMs at the Department of Hemangioma Surgery of the Third Affiliated Hospital of Zhengzhou University from January 2021 to December 2023. (1) Transcriptome analysis: Three patient samples were randomly selected for snRNA-seq studies, with the surgically removed VMs tissue serving as the experimental group and the surrounding normal vein tissue as the control group. A gene expression matrix for cell nuclei was established, followed by data quality control, dimensionality reduction, clustering, and cell type annotation. Cell-to-cell communication analysis was performed using the R language CellChat package to identify dominant cell subpopulations. The FindMarkers function was utilized to screen for differentially expressed genes (DEGs) between the dominant cell subpopulations of the experimental and control groups, and functional enrichment analysis was conducted. (2) Tissue experiments: An additional 35 patient samples from both the experimental and control groups were randomly selected. The mRNA and protein expression levels of the RhoJ gene were measured using real-time quantitative PCR (RT-qPCR) and Western blotting, respectively. (3) Validation experiments with human umbilical vein endothelial cells (HUVECs): HUVECs were transfected with pcDNA3.1-NC (blank control) and pcDNA3.1-RhoJ (plasmid expression vector carrying the RhoJ gene), respectively. The biological behavior differences between the two groups of cells were examined using the CCK-8 cell proliferation assay, Transwell invasion assay, and Matrigel angiogenesis assay. Measurement data conforming to a normal distribution were expressed as Mean±SD, and comparisons between the two groups were performed using an independent samples t-test. Results:Through CellChat intercellular communication analysis, it was discovered that endothelial cells were the predominant cell subpopulation in both the experimental and control groups, exhibiting strong communication links with other cell subpopulations. In the analysis of DEGs, it was found that the RhoJ gene in endothelial cells was significantly involved in the biological processes of angiogenesis and regulation. In tissue experiments, RT-qPCR and Western bloting results indicated that the relative expression levels of RhoJ mRNA (4.48±1.29 vs. 1.01±0.17) and protein (1.22±0.03 vs. 0.51±0.20) in the experimental group were significantly higher than those in the control group, with statistically significant differences ( P<0.01 for both). The results of the HUVECs validation experiment showed that the cell proliferation, invasion, and angiogenesis abilities of the pcDNA3.1-RhoJ group were significantly enhanced compared to the pcDNA3.1-NC group. Conclusion:Endothelial cells represent the dominant cell subpopulation during the occurrence and locally invasive progression of VMs, playing a crucial role in this process. The RhoJ gene is significant in regulating the biological behavior of VMs endothelial cells.

Objective To construct a mammography image classification assistant system suitable for Chinese population,and explore the potential of artificial intelligence technology to assist early screening of breast cancer in China.Methods Curated breast imaging subset of digital database for screening mammography(CBIS-DDSM),Mammographic image analysis society database(MIAS)and other international open datasets were used to conduct model training respectively in order to reproduce the mainstream in-depth learning methods in the current literature.The model was also tested on the Chinese breast mammography database(CBMD)provided by Huajiao Technology Co.,Ltd,and the performance was compared.Aiming at the problem that the Chinese population data are not ideal in the performance test of the open dataset training model,an optimization strategy based on the sliding window adjustment mechanism was implemented in combination with the characteristics of Chinese population data.Then a two-stage migration learning method was designed to improve the overall performance of the model,and then development of our system was carried out.Results With the sliding window adjustment mechanism and the CBMD training model after two-stage transfer learning,the accuracy of our developed system was improved from 0.50 of the open datasets to 0.80,precision from 0.54 to 0.82,sensitivity from 0.52 to 0.80,F1 value from 0.52 to 0.80,and AUC value from 0.51 to 0.89 based on the Chinese population dataset as the test set.Conclusion Through the introduction of sliding window adjustment mechanism and two-stage migration learning strategy,the performance of the breast molybdenum target image classification model has been significantly improved in the Chinese population dataset,and our system primarily achieves the purpose of assisting the classification of breast molybdenum target images for the Chinese population.

Objective:This study aimed to identify key genes in endothelial cell (EC) associated with the pathogenesis and progression of human venous malformations (VMs) through bioinformatics analysis, providing potential biomarkers for early screening and targeted therapy of VMs.Methods:A case-control study was conducted using surgically resected tissue specimens from VMs patients at the Third Affiliated Hospital of Zhengzhou University (from September 2021 to September 2023), with malformed venous tissues as the experimental group and distal normal venous tissues as controls. Single-nucleus RNA sequencing (snRNA-seq) was performed on paired experimental and control samples from four VM patients. High-dimensional weighted gene co-expression network analysis (hdWGCNA), combined with gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein-protein interaction (PPI) network analysis, identified critical genes. Validation experiments included 15 additional VM cases and controls using reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry (IHC), and Western blot.Results:A total of 55 430 nuclei were captured using snRNA-seq, with 30 391 nuclei from the experimental group and 25 039 nuclei from the control group. Cluster analysis identified 22 distinct cell populations, which were annotated into 8 cell types. hdWGCNA revealed four modules associated with invasion, which were enriched in angiogenesis, integrin signaling, and cell adhesion according to GO analysis. KEGG pathway analysis indicated that the PI3K-AKT signaling pathway and focal adhesion are key regulatory mechanisms. PPI network analysis combined with cytoscape identified EGFL7, TEK, and FLT1 as key genes. RT-qPCR results demonstrated that the relative mRNA expression levels of these three genes in the experimental group (6.66±2.31, 1.86±0.62, 3.49±0.58) were significantly higher than those in the control group (1.05±0.14, 1.00±0.14, 1.06±0.25), with statistically significant differences ( t=9.37, 4.27, 11.20, P<0.05). Immunohistochemical analysis showed that the relative protein expression levels of these three genes in the cytoplasm of the experimental group (0.84±0.15, 0.68±0.14, 0.85±0.12) were also significantly higher than those in the control group (0.19±0.05, 0.23±0.06, 0.30±0.05), with statistically significant differences ( t=16.62, 5.93, 11.68, P<0.05). Western blot analysis confirmed that the relative protein expression levels of these three genes in the experimental group (0.35±0.04, 0.36±0.09, 0.31±0.04) were significantly higher than those in the control group (0.19±0.01, 0.13±0.02, 0.14±0.04), with statistically significant differences ( t=7.05, 4.61, 5.93, P<0.05). Conclusion:EGFL7, FLT1, and TEK in EC may play crucial roles in the occurrence and invasion of VMs.

Objective:This study aimed to identify key genes in endothelial cell (EC) associated with the pathogenesis and progression of human venous malformations (VMs) through bioinformatics analysis, providing potential biomarkers for early screening and targeted therapy of VMs.Methods:A case-control study was conducted using surgically resected tissue specimens from VMs patients at the Third Affiliated Hospital of Zhengzhou University (from September 2021 to September 2023), with malformed venous tissues as the experimental group and distal normal venous tissues as controls. Single-nucleus RNA sequencing (snRNA-seq) was performed on paired experimental and control samples from four VM patients. High-dimensional weighted gene co-expression network analysis (hdWGCNA), combined with gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein-protein interaction (PPI) network analysis, identified critical genes. Validation experiments included 15 additional VM cases and controls using reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry (IHC), and Western blot.Results:A total of 55 430 nuclei were captured using snRNA-seq, with 30 391 nuclei from the experimental group and 25 039 nuclei from the control group. Cluster analysis identified 22 distinct cell populations, which were annotated into 8 cell types. hdWGCNA revealed four modules associated with invasion, which were enriched in angiogenesis, integrin signaling, and cell adhesion according to GO analysis. KEGG pathway analysis indicated that the PI3K-AKT signaling pathway and focal adhesion are key regulatory mechanisms. PPI network analysis combined with cytoscape identified EGFL7, TEK, and FLT1 as key genes. RT-qPCR results demonstrated that the relative mRNA expression levels of these three genes in the experimental group (6.66±2.31, 1.86±0.62, 3.49±0.58) were significantly higher than those in the control group (1.05±0.14, 1.00±0.14, 1.06±0.25), with statistically significant differences ( t=9.37, 4.27, 11.20, P<0.05). Immunohistochemical analysis showed that the relative protein expression levels of these three genes in the cytoplasm of the experimental group (0.84±0.15, 0.68±0.14, 0.85±0.12) were also significantly higher than those in the control group (0.19±0.05, 0.23±0.06, 0.30±0.05), with statistically significant differences ( t=16.62, 5.93, 11.68, P<0.05). Western blot analysis confirmed that the relative protein expression levels of these three genes in the experimental group (0.35±0.04, 0.36±0.09, 0.31±0.04) were significantly higher than those in the control group (0.19±0.01, 0.13±0.02, 0.14±0.04), with statistically significant differences ( t=7.05, 4.61, 5.93, P<0.05). Conclusion:EGFL7, FLT1, and TEK in EC may play crucial roles in the occurrence and invasion of VMs.

To construct a recombinant fowl adenovirus 4(FAdV-4)expressing the Cap protein of goose astrovirus genotype 2(GoAstV-2),the expression cassette of Cap gene was inserted into the natural 1 966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cm-FAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line(LMH)to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rF Ad V4-Cap/GoAstV-2.After 15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GoAstV-2 infected LMH cells as template.LMH cells were in-fected with 15th passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15th passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant vi-rus rF AdV4-Cap/Go Ast V-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-in-fection of FAdV-4 and GoAstV-2 in goose.

Objective To explore the anti-inflammatory properties of the ethyl acetate extract(ETCB)derived from Trollius chinensis Bge.using in vitro RAW264.7 cells stimulated with lipopolysaccharide and an in vivo mouse auricle model induced by xylene.Utilizing UHPLC-Q-TOF-MS(LC-MS)and network pharmacology,the components of ETCB were analyzed,and its anti-inflammatory mechanisms were preliminarily explored.Methods The anti-inflammatory activity of various solvent extracts of Trollius chinensis Bge.was assessed through the Griess assay.The impact of ETCB on the production of TNF-α and IL-6 in RAW264.7 cells induced by lipopolysaccharide was evaluated using ELISA.Real-time qPCR was conducted to determine the effect of ETCB on the expression levels of inflammatory factors such as TNF-α,IL-6,and iNOS in cells.The anti-inflammatory efficacy was further validated in a xylene-induced ear inflammation mouse model by measuring ear swelling and tissue levels of IL-6 and TNF-α.The composition of ETCB was analyzed using LC-MS.Network pharmacology was employed to screen for effective components,targets,and pathways involved in the anti-inflammatory effects of Trollius chinensis Bge.,followed by molecular docking verification between core components and targets.Results ETCB demonstrated the most potent inhibitory effect on NO production in RAW264.7 cells stimulated by lipopolysaccharide,indicating its primary role in the anti-inflammatory activity of Trollius chinensis Bge..ETCB significantly reduced TNF-α and IL-6 levels in inflammatory cells(P<0.01)and inhibited the mRNA expression of TNF-α,IL-6,and iNOS.In the xylene-induced mouse ear inflammation model,ETCB effectively alleviated ear swelling and decreased tissue levels of TNF-α and IL-6.LC-MS analysis identified 30 chemical components in ETCB,including 21 flavonoids,7 organic acids,1 polysaccharide,and 1 anthocyanin.Network pharmacology prediction and screening revealed TNF,Akt1,PTGS2,EGFR,SRC,and MMP9 as core targets,with hydroxyquercetin,lignin from fragrant leaves,zeaxanthin from willows,plantain,thistle,and sophora flavins as key anti-inflammatory active ingredients.The molecular docking analysis revealed positive interactions,characterized by favorable binding energy,between the active components and key targets.Conclusion ETCB demonstrates strong anti-inflammatory properties both inside and outside the body,functioning through various targets and pathways.This establishes a basis for deeper understanding of the anti-inflammatory mechanism of Trollius chinensis Bge.