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Abstract Objective To investigate the effects of paeoniflorin (Pae) on acute kidney injury ( AKI) and mouse renal tubular epithelial cell ( mRTEC) damage induced by lansoprazole (LPZ) and cisplatin (CIS) through in vivo and in vitro experiments . Methods The C57BL/6J mice or mRTECs were divided into four groups : normal control (NC) group , NC + LPZ group , CIS group , and CIS + LPZ group . Serum creatinine (CRE) and blood urea nitro- gen (BUN) levels in mice were measured , and kidney pathology was observed with HE staining. Western blot , im- munohistochemistry , and immunofluorescence were used to detect the expression levels of kidney injury molecule-1 (KIM-1) and receptor-interacting protein kinase (RIPK) 1 , RIPK3 , and mixed lineage kinase domain-like protein (MLKL) . Subsequently , C57BL/6J mice or mRTECs were divided into six groups : NC group , NC + Pae group , CIS + LPZ (M) group , and CIS + LPZ + Pae ( M + Pae) group . Serum CRE and BUN levels in each group were measured , kidney pathology was observed with HE staining , and ultrastructural changes in the kidney were ob-served with transmission electron microscopy. The KIM-1 and necroptosis-related protein expression levels were de- tected by Western blot , immunohistochemistry , and immunofluorescence . Results Compared with the NC group , CRE and BUN levels were elevated in the CIS group , and these levels were further increased after LPZ intervention (all P < 0. 001) . Compared with the CIS group , renal tubular dilation and brush border loss were evident in the CIS + LPZ group based on HE staining of kidney tissue (P < 0. 001) . Compared with the NC group , the expres- sion levels of KIM-1 , RIPK1 , RIPK3 , and MLKL in the renal tissues of mice in the CIS group increased (all P < 0. 001) , and compared with the CIS group , The expression levels of KIM-1 , RIPK1 , RIPK3 and MLKL in the re- nal tissues of mice in the CIS + LPZ group increased (all P < 0. 001) . After Pae treatment , compared with group M , the expression levels of CRE , BUN , KIM-1 , RIPK1 , RIPK3 and MLKL in each group of mice decreased sig- nificantly and in a dose-dependent manner (all P < 0. 001) . Conclusion LPZ promotes CIS-induced AKI by en- hancing necroptosis in renal tubular epithelial cells , and Pae can improve CIS and LPZ-induced AKI by inhibiting necroptosis .

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Abstract Objective To investigate the effect of loss of function of lanosterol synthase(LSS) on intestinal function in a mouse model of non-alcoholic fatty liver disease(NAFLD) induced by a high-fat diet. Methods LSS heterozygous knockout C57 mice (LSS+/-) were established using the CRISPR/CAS9 system. After being fed a high-fat diet with 60% fat content for 6 months, the fat deposition in liver tissues was detected by HE and Oil red O staining, the morphological changes of small intestine tissue were detected by HE staining. The changes in total cholesterol content in intestinal tissue were detected by kits. The gastrointestinal motility function of mice was detected by phenol red paste. The intestinal permeability was detected by Evans blue staining, and the expression of lanosterol synthase (LSS), tight junction protein (Claudin)-1, Claudin-5, platelet glycoprotein IV (CD36), and Niemann-Pick C1-like 1 (NPC1L1) proteins in small intestinal tissues were detected by Western blot. Results The results of HE and Oil red O staining of liver tissues showed that liver fat deposition in LSS knockdown mice was lower than that in wild-type mice in the high-fat diet group. The total cholesterol content in intestinal tissue of LSS gene knockout mice decreased (P<0.01), but no morphological differences were observed between the two groups of mice by HE staining of intestinal tissues. The gastrointestinal motility function of LSS gene knockout mice did not show significant changes. The intestinal permeability of LSS gene knockout mice in the high-fat diet group decreased as detected by Evans blue (P < 0.05). The expression levels of Claudin-5 protein in the intestinal tissue of LSS gene knockout mice in the high-fat diet group increased (P<0.05), while the expression of LSS in the intestinal tissues of LSS knockout mice decreased (P<0.05). Conclusion In the NAFLD model induced by a high-fat diet,LSS gene knockout reduces liver fat deposition induced by a high-fat diet and improves intestinal barrier function by regulating cholesterol metabolism in intestinal tissues and up-regulating the expression of tight junction protein-5.

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Abstract Objective To identify autophagy-related genes involved in pulmonary infection caused by the highly drug-resistant and virulent methicillin-resistant Staphylococcus aureus strain USA300 (USA300), and to explore the underlying molecular mechanisms, thereby providing potential targets for immunotherapy. Methods The GSE220943 dataset of a USA300-induced pulmonary infection mouse model was obtained from the GEO database. Differentially expressed genes (DEGs) were identified using the DESeq2 package. Autophagy-related genes (ARGs) were retrieved from the MSigDB and Autophagy databases. Weighted gene co-expression network analysis (WGCNA) was performed to construct gene co-expression modules. Genes overlapping among DEGs, ARGs, and WGCNA modules were identified as autophagy-related DEGs. Gene Ontology (GO) enrichment analysis was conducted using the clusterProfiler R package, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed via the Metascape platform. Immune cell infiltration was analyzed using the ImmuCellAI-mouse website. A protein–protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified through topological analysis in Cytoscape. ROC curves were plotted via the website https://www.bioinformatics.com.cn. Finally, key gene expression was validated in mouse lung tissues by real-time quantitative reverse transcription PCR (RT-qPCR).Results A total of 6 135, 4 075, 3 680, and 2 342 differentially expressed genes (DEGs) were identified at 12, 24, 48, and 96 hours post-infection, respectively. By integrating DEGs,autophagy-related genes (ARGs), and WGCNA modules, 19 autophagy-related DEGs were identified. GO and KEGG enrichment analyses indicated that these genes were mainly involved in CD4⁺ T cell activation and regulation, innate immune responses, and autophagosome membrane formation. Immune infiltration analysis revealed that innate immune cells such as neutrophils and dendritic cells predominated during the early phase of infection, while γδ T cells and M2 macrophages became more prominent in the later stages. PPI network analysis identified 12 hub autophagy-related genes, among which three upregulated key genes (Eif2ak2, Ikbke, and Nfkbiz) were further confirmed. The area under the ROC curve (AUC) for all three genes was 1.0.RT-qPCR validation demonstrated significantly elevated expression of these three genes in lung tissues at 24 hours post-infection (all P<0.05). Conclusion Eif2ak2, Ikbke, and Nfkbiz may be involved in the pulmonary infection caused by USA300 by promoting autophagy and hold promise as potential targets for immunotherapy.

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Abstract Ferroptosis is a novel form of cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Since it was first proposed in 2012, Ferroptosis has gradually attracted attention and developed rapidly. Ferroptosis plays an important role in cardiovascular diseases, malignant tumors and neurological diseases, and has become a research hotspot in the field of life science and medicine. Ferroptosis is closely related to iron overload. Iron overload and the accumulation of lipid peroxidation jointly contributes to the disruption of intervertebral disc homeostasis, leading to intervertebral disc degeneration. However, the specific mechanisms of ferroptosis in regulating intervertebral disc degeneration is not yet clear. This review discusses the relationship between ferroptosis and intervertebral disc degeneration, their molecular regulatory mechanisms, and their potential clinical applications, aiming to provide new therapeutic targets for intervertebral disc degeneration.

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Abstract Objective To explore the therapeutic effect of extracellular vesicles (EVs) of Aspergillus flavus on A. flavus infection. Methods The extracellular vesicles (EVs) of Aspergillus flavus were isolated using ultracentrifugation and detected/identified by nanoparticle tracking analysis (NTA). Quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) kits were used to assess the effect of A. flavus EVs on the polarization of bone marrow-derived macrophages (BMDMs) and the expression levels of several cytokines, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), and interleukin-10 (IL-10). The Galleria mellonella infection model was constructed. Three concentration groups of A. flavus EVs (0.2, 2, and 20 μg/larvae) were set as the experimental groups, and PBS was used as the control group for the infection model. Meanwhile, three forms of negative control groups were established, including the PBS group, the pierced controlgroup, and the negative control group. The therapeutic effect of A. flavus EVs on A. flavus infection was evaluated by the survival rate of the G. mellonella infection models. Results The particle size of A. flavus EVs ranged from 20 to 550 nm. A. flavus EVs could polarize BMDMs into both M1 and M2 phenotypes and induce the production of cytokines, including TNF-α, IFN-γ, IL-6, and IL-10. The results of the G. mellonella infection model showed that A. flavus EVs could improve the survival rate of G. mellonella after A. flavus infection. Conclusion The EVs produced by A. flavus can promote the expression of both pro-inflammatory and anti-inflammatory cytokines in BMDMs, induce M1 polarization and M2 polarization of BMDMs, and increase the survival rate of G. mellonella after A.flavus infection.

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Abstract Objective To investigate the regulatory effect of corticosterone (CORT) pretreatment on the activation of microglia by lipopolysaccharide (LPS) and the inhibitory effect of Biochanin A (Bio A) on microglia activation. Methods The MTT method was used to select the optimal concentrations for LPS, CORT and Bio A on BV2 cells; BV2 cells were divided into 5 groups: Control group, CORT (50 nmol/L) group, LPS (1 μg/mL) group, LPS (1 μg/mL) + CORT (50nmol/L) group, and LPS (1 μg/mL) + CORT (50 nmol/L) + Bio A (5 μmol/L) group; Except for the control group, each group was first incubated with CORT (50 nmol/L) for 2 h, and then each group was co-incubated with the corresponding concentrations of LPS (1 μg/mL) and BioA (5μmol/L) for 36 h; DCFH-DA probe method was used to detect reactive oxygen species (ROS) content; Western blot was used to detect the protein expression levels of inflammatory cytokines,5-hydroxytryptamine(5-HTT), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and cysteine-aspartic protease 1 (Caspase-1) Results Compared with the CORT (50 nmol/L) and LPS (1 μg/mL) groups, cells in the CORT (50 nmol/L)+ LPS (1 μg/mL) group showed increased cell viability, higher levels of ROS, and increased levels of inflammatory cytokines, NLRP3, ASC, and Caspase-1 protein expression (P<0.05);Compared with the CORT (50 nmol/L) + LPS (1 μg/mL) group, the CORT (50 nmol/L) + LPS (1 μg/mL) + Bio A (5 μmol/L) group showed decreased cell viability, decreased levels of ROS, and decreased protein expression of inflammatory cytokines, NLRP3, ASC and Caspase-1 proteins (P<0.05 ).Conclusion A low dose of CORT pretreatment reinforces LPS-induced BV2 cell activation; Bio A inhibits CORT-pretreated LPS-induced BV2 cell activation. The mechanism of which may be related to the inhibition of NLRP3 inflammasome activation.

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Abstract Objective To explore the effects and mechanisms of Kobophenol A (KPA) on lipopolysaccharide (LPS)-induced M1 macrophage polarization, and to provide a theoretical basis for the treatment of inflammatory immune diseases and the development of new drugs. Methods The M1 macrophage polarization model of RAW264.7 was established by LPS induction, and the Prdx6 knockdown model was constructed using the Prdx6 inhibitor MJ33 and Prdx6-siRNA. RAW264.7 cells, a mouse macrophage cell line, were treated with various concentrations of KPA. Cell viability was assessed using the CCK-8 assay. The expression levels of peroxiredoxin 6 (Prdx6) and M1 macrophage polarization-related proteins, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), were detected by Western blot and immunofluorescence staining. The expression levels of Prdx6 and M1 macrophage polarization-related genes iNOS, interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α), were measured by RT-qPCR. Flow cytometry was employed to detect the expression of CD86, a marker of M1 macrophages. Results Compared with the LPS-induced M1 macrophage polarization model, KPA significantly reversed the morphological changes of M1 macrophage polarization in RAW264.7 macrophages and decreased the expression of M1 macrophage polarization-related proteins iNOS, COX-2, CD86 and related genes iNOS, IL-6, TNF-α (all P<0.05). In addition, LPS significantly downregulated the expression of Prdx6 in RAW264.7 macrophages, while KPA upregulated the expression of Prdx6. Moreover, treatment with the Prdx6 inhibitor MJ33 significantly upregulated the expression of iNOS, a marker of M1 macrophage polarization, in RAW264.7 macrophages, whereas treatment with KPA significantly downregulated the expression of iNOS (all P<0.05). Conclusion KPA inhibits LPS-induced M1 polarization of RAW264.7 macrophages by upregulating the expression of Prdx6.

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Abstract Objective To construct Csf1rCre Rosa26YFP mice, and to detect the efficiency of Csf1rCre -mediated yellow fluorescent protein (YFP) labeling monocytes and tissue-resident macrophages in different tissues. Methods The Rosa26YFP mutant mice had a loxP-flanked STOP sequence, followed by a yellow fluorescent protein gene (YFP) inserted into the Rosa26 locus. When crossed with mice expressing the Csf1rCre recombinase, the STOP sequence was deleted, and yellow fluorescent protein (YFP) expression was observed in the tissues of double-mutant offspring (Csf1rCre Rosa26YFP). Csf1rCre mice were mated with Rosa26YFP mice, and genotype of Csf1rCre Rosa26YFP mice were identified by PCR. Blood and bone marrow monocytes, liver macrophages, kidney macrophages, alveolar macrophages and spleen macrophages of adult Csf1rCre Rosa26YFP mice were isolated, labeled with flow cytometry antibodies, and the recombination efficiency of Csf1rCre-mediated YFP labeling tissue macrophages was analyzed by flow cytometry. Results In Csf1rCre Rosa26YFP reporter mice, the median percentage of YFP+ was 93.25% in renal macrophages, 92.45% in liver macrophages, and 91.10% in spleen macrophages. The percentage of YFP+ was 94.7% in alveolar macrophages, 98.2% in blood monocytes, and 93.9% (2.4%) in bone marrow monocytes. Conclusion Csf1rCre can be used to trace tissue-resident macrophages as well as bone marrow and blood monocytes. At the same time, Csf1rCre can target these cells to prepare conditional gene knockout mice.

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Abstract Objective To explore the mechanism of hesperidin on hindlimb motor function in mice with spinal cord injury (SCI). Methods Oxygen glucose deprivation (OGD) was used to induce SCI in PC12 neuronal cells. CCK-8 assay determined the optimal hesperidin concentration (0, 10, 20, 30, 40, 50, 60 mmol/L) for subsequent experiments after 24h treatment. PC12 cells were divided into Control group, OGD group and Hesperidin (30 mmol/L)+OGD groups. Hoechst staining assessed apoptosis; Western blot (WB), qPCR and immunofluorescence (IF) detected the expression of iNOS, CD206 and TNF-α . Flow cytometry measured apoptosis rates, and WB examined the impact of hesperidin on PI3K, p-PI3K, AKT, p-AKT, and NF-κB expression. Forty-five healthy male C57BL/6 mice were randomly divided into Sham group, Sci group and Hesperidin groups. Sham and Sci groups received 0.1 mL PBS, while the Hesperidin group received 0.1 mL hesperidin. After modeling, iNOS, CD206, and TNF-α expression levels, spinal cord cavitation, and hindlimb motor function were evaluated. Results Hesperidin at 10–30 mmol/L for 24h did not significantly affect PC12 cell activity. Hoechst staining showed reduced apoptosis in PC12 cells after 24h of 30 mmol/L hesperidin treatment. Compared to the OGD group, Hesperidin+OGD group had decreased mRNA and protein expression of inflammatory factors (iNOS and TNF-α) and increased expression of the anti-inflammatory factor (CD206). Hesperidin treatment inhibited PI3K/AKT/NF-κB pathway activation, with similar results in mouse experiments. Compared to Sci group, Hesperidin group had reduced spinal cord cavitation area and improved hindlimb motor function. Conclusion Hesperidin may improve hindlimb motor function in mice with SCI by downregulating the phosphorylation level of the PI3K/AKT/NF-κB pathway, inhibiting inflammatory factor release, promoting anti-inflammatory factor release, regulating the inflammatory microenvironment after SCI, and suppressing the acute inflammatory response.

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Abstract Objective To investigate the impacts of remifentanil on the proliferation, apoptosis and Wnt/β-catenin pathway of breast cancer MDA-MB-231 cells. Methods Different concentrations of remifentanil (0, 0.5, 2.5, 5.0, 10.0, 20.0, 40.0 µg/mL) were used to treat human normal breast MCF-10A cells and breast cancer MDA-MB-231 cells to screen for experimental concentrations of remifentanil. MDA-MB-231 cells were divided into control group, remifentanil low (5.0 µg/mL), medium (10.0 µg/mL), and high (20.0 µg/mL) concentration groups, and remifentanil+SKL2001 (Wnt/β-catenin pathway agonist) group. MTT assay and colony formation assay were applied to detect cell proliferation ability. Flow cytometry was applied to detect cell apoptosis and cell cycle changes. Western blot was applied to detect protein expression related to cell proliferation, apoptosis, and the Wnt/β-catenin signaling pathway. Results Remifentanil at concentrations of 5.0, 10.0, and 20.0 µg/mL could reduce the viability of MDA-MB-231 cells and had no prominent toxicity to MCF-10A cells. Compared with the control group, the optical density value of cell proliferation, colony formation number, proportion of S-phase cells, and the protein levels of c-Myc, Cyclin D1, B lymphoblastoma 2 (Bcl-2), precursor of cysteine aspartate protease-3 (pro-caspase-3) and β-catenin were lower in the low, medium, and high concentration remifentanil groups (P<0.05), while the proportion of G0/G1 phase cells, apoptosis rate, early apoptosis rate, the protein levels of Bcl-2 associated X protein (Bax) and cleaved cysteine aspartate protease-3 (Cleaved caspase-3), and glycogen synthase kinase-3β (GSK-3β) were higher (P<0.05). SKL2001 could weaken the effects of remifentanil on the proliferation and apoptosis of MDA-MB-231 cells. Conclusion Remifentanil inhibits the proliferation of MDA-MB-231 cells, induces apoptosis and cell cycle arrest, and its mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway activation.

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Abstract Objective To investigate the therapeutic effect of bone marrow mesenchymal stem cells (BMSCs) modified by bone morphogenetic protein (BMP)-2 gene combined with nano-hydroxyapatite (nHA)/polyamide 66 (PA66) on femoral nonunion in rats. Methods Rat BMSCs were isolated and divided into the stent+BMSCs group, stent+BMSCs+rhBMP-2 group,and stent+BMSCs+Ad-BMP-2 group; human recombinant BMP-2 (rhBMP-2) or adenovirus-infected BMP-2 (Ad-BMP-2) vector was transfected into BMSCs and loaded onto nHA/PA66 stent materials. Flow cytometry was used to detect that BMSCs expressed specific surface markers. Cell proliferation was detected by MTT assay, related bioactive factors [platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), osteocalcin (OCN), osteonectin (ON)] were detected by ELISA, alkaline phosphatase (ALP) activity was detected, and cell growth and adhesion were observed by scanning electron microscopy (SEM). In the atrophic nonunion model of SD rats, the stent+BMSCs+rhBMP-2 or stent+BMSCs+Ad-BMP-2 complex was implanted into the defect area, which was divided into the rhBMP-2 group and the Ad-BMP-2 group, and the therapeutic effect was evaluated by X-ray and Micro CT.

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Abstract Objective To investigate the diagnostic value of combined detection of folate receptor-positive circulating tumor cells (FR+-CTCs) and hemostatic function indicators in improving the diagnosis of gastrointestinal tumors (GITs) metastasis. Methods A retrospective analysis was conducted on the clinical data of 186 patients aged 18 to 80 years who were diagnosed with gastrointestinal tumors via pathology and received treatment, including data on FR+-CTCs, hemostatic function indicators, and pathological staging. The collected data encompassed FR+-CTCs levels, coagulation parameters, and pathological staging. Statistical analysis included t-tests, chi-square tests, fisher’s exact test, logistic regression analysis, and receiver operating characteristic (ROC) curves to assess the diagnostic value of combined FR+-CTCs and coagulation parameters in detecting tumor metastasis. Results FR+-CTCs levels and positive rates demonstrated significant associations with clinicopathological characteristics (gender, histological type, N staging) in GITs patients (P<0.05). In patients with metastasis, elevated fibrinogen levels were observed, while both thrombin time and platelet counts showed significant increases in N1 ~ N3 stages (P<0.05). Logistic regression analysis showed that PLT and antithrombin III (AT-III) were independent risk factors for GITs metastasis (P<0.05). The areas under the ROC curves for predicting GITs metastasis were 0.678 (95%CI: 0.540-0.816) and 0.664 (95%CI: 0.512~0.815), respectively. When combining multiple factors, including FR⁺-CTCs, PLT, AT-III, pathological type, FIB, TT, and gender, for the diagnosis of GITs metastasis, the AUC increased to 0.757 (95%CI: 0.621-0.893), indicating higher sensitivity and specificity compared to using each indicator alone. Conclusion The combined detection of FR+-CTCs and anticoagulation function indicators has a higher diagnostic value for the diagnosis of GITs, providing a valuable basis for the early diagnosis of GITs, especially in metastasis surveillance.

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Abstract Objective To establish and identify hepatocyte-specific transmembrane protein 121 (Tmem121) knockout mice. Methods The hepatocyte-specific Tmem121 knockout mice (Tmem121flox/flox/Cre, Tmem121ΔHep) were obtained by crossbreeding of Tmem121flox/+/Cre and Tmem121flox/flox mice, which were generated using the CRISPR/Cas9 and Cre/Loxp systems. The genotype was verified by PCR using genomic DNA extracted from mouse tails as template. The growth, reproduction and organ development of both control and knockout mice were observed and analyzed. PCR and Western blot methods were performed to assess the knockout efficiency of Tmem121 in mouse primary hepatocytes. CellMask™ Deep Red plasma membrane staining was employed to compare the morphological differences in primary hepatocytes between control and knockout mice. Results Tmem121flox/flox/Cre mice were successfully obtained according to genotype identification analysis, and there were no significant differences between control and knockout mice in body mass, reproductive ability, growth and development of liver. The specific knockout of Tmem121 gene in primary hepatocytes did not significantly affect the morphological structure or pathological characteristics of liver tissue. However, compared to the control group, the levels of Tmem121 mRNA and protein in the primary hepatocytes of the knockout group were significantly reduced (P<0.01). CellMask™ Deep Red plasma membrane staining indicated that the proportion of binucleated hepatocytes in Tmem121-deficient mice significantly increased (P<0.05), while the cell area was significantly reduced (P<0.001). Conclusion Hepatocyte-specific Tmem121 knockout mice are successfully constructed, which provides an animal model for further exploration of the function and mechanism of Tmem121 gene in liver diseases.

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Abstract Objective To perform transient transfection of recombinant human Factor X (rhFX) into HEK293 cells, to optimize transfection parameters, to develop a high-yield in vitro expression system for rhFX production, and to assess the biological activity of expressed rhFX. Methods The eukaryotic expression vector pcDNA3.1-EGFP-FX was constructed by inserting the FX gene into pcDNA3.1-EGFP and subsequently transfected into HEK293 cells to validate the transfection system efficiency. The recombinant expression vector pcDNA3.1-FX was generated through ligation of the FX gene fragment with pcDNA3.1, followed by liposome-mediated transfection into HEK293 cells. The expression of rhFX in the cell supernatant was analyzed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Transfection conditions were systematically optimized, and rhFX concentration was quantified by enzyme-linked immunosorbent assay (ELISA). The coagulation bioactivity of rhFX was evaluated through prothrombin time (PT) assay and chromogenic substrate method. Results Recombinant human Factor X (rhFX) was successfully expressed in the supernatant ofHEK293 cells. Optimal expression was achieved at 80% cell confluence with a ratio of plasmid DNA to transfection reagent as 1:2. Peak rhFX production was observed on day 3 post-transfection. Triplicate ELISA measurements demonstrated a maximum rhFX concentration of 5.20 ng/mL in the supernatant. The prothrombin time (PT) of rhFX-containing supernatant was significantly shorter (P < 0.0001) compared to control supernatant from pcDNA3.1 empty vector-transfected cells. The half maximal inhibitory concentration (IC50) of etoxaban was determined to be 1.449 nmol/L using the chromogenic substrate method based on rhFX, which was in the same order of magnitude as the reported 0.78 nmol/L in the literature. Conclusion HEK293 cells successfully express biologically active recombinant human Factor X (rhFX) protein, laying a foundation for advancing the development of rhFX-based therapeutics.

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Abstract Objective To construct and to validate a nomogram prediction model based on Lipoprotein-associated phospholipase A2 (Lp-PLA2) and Lipoprotein(a) [LP(a)] for predicting the risk of major adverse cardiovascular events (MACE) in patients with coronary heart disease (CHD). Methods A retrospective analysis was conducted on the clinical data of 442 patients with coronary heart disease (CHD). Among them, 411 patients who completed follow-up were randomly divided into a training set (288 cases) and a validation set (123 cases) at a 7:3 ratio. Independent risk factors for major adverse cardiovascular events (MACE) in CHD patients were screened through Lasso regression analysis and Cox regression analysis, and a nomogram prediction model was constructed. The predictive performance of the model was evaluated using time-dependent receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis. Results Variables were screened through Lasso regression and Cox regression analysis. The final model included nine independent predictors, namely age, smoking history, clinical phenotype of CHD, the number of coronary artery lesions, Gensini score, BNP, Lp-PLA2, LP(a), and the history of statin use. The area under the ROC curve in the training set was 0.897, 0.885, and 0.909 at 1, 2, and 3 years, respectively; The area under the ROC curve in the validation set was 0.885, 0.881, and 0.923 at 1, 2, and 3 years, respectively. These results demonstrated that the model had excellent discriminatory power.The calibration curves and decision curves demonstrated that the model had high clinical practicality in predicting the occurrence of MACE in CHD patients. Conclusion The nomogram prediction model based on LP-PLA2, LP(a) and other risk factors provides an effective tool for the prognosis assessment of CHD patients, facilitating the early identification of high-risk patients and enabling individualized intervention.

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Abstract Objective To isolate a lytic phage targeting K. pneumoniae type K39 from untreated sewage and systematically analyze its biological characteristics and genomic information. Methods The bacteriophage ofK. pneumoniae K39 Kp1000 was identified based on the sequence of the capsular polysaccharide gene. It was isolated and purified using the double agar plate method. The morphology of the phage was observed through negative staining and transmission electron microscopy, and its bacteriophage spectrum was evaluated by phagocytosis. The biological characteristics of the bacteriophage were assessed by determining the optimal multiplicity of infection (MOI), constructing a one-step growth curve, and conducting temperature and pH tolerance tests. Finally, the phage DNA was extracted, and its whole genome was sequenced using the Illumina HiSeq2000 sequencing platform. The sequencing results were then annotated and analyzed. Results A lytic phage specifically targeting K. pneumoniae type K39 was successfully isolated and named IME330. Transmission electron microscopy showed that the head diameter of the phage was (75±1) nm and the tail length was (185±1) nm. The lytic spectrum analysis revealed that IME330 lysed K. pneumoniae type K39, demonstrating a narrow host range and high specificity. The optimal multiplicity of infection (MOI) was 0.1, The lysis amount reached 168 PFU/cell. Physicochemical experiments indicated that IME330 exhibited strong tolerance to high temperatures and a broad pH range (pH 4-10). Genomic analysis demonstrated that the IME330 genome had a length of 144,245 bp, a molecular weight of 46,463 MDa, a G+C content of 44.8%, and encoded 320 open reading frames (ORFs). Conclusion IME330 is a novel lytic K. pneumoniae phage belonging to the order Caudovirales. This phage exhibits strong tolerance to physicochemical factors (e.g., temperature, acidic/alkaline conditions) and demonstrates high lytic activity, while its lytic spectrum remains narrow with strict host specificity.

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Abstract Objective To investigate the high-risk human papillomavirus (HR-HPV) infection and its relationship with vaginal infectious diseases among women in the region of the Fourth Division of Xinjiang Production and Construction Corps, and to provide a basis for the prevention and control of cervical cancer among women in this region. Methods Uterine exfoliative cells and vaginal secretions were collected from women aged 25-64 years in the resident population (≥6 months of residence) who participated in cervical cancer screening in the Fourth Division of Xinjiang Corps from 2023-2024, and human papillomavirus typing and vaginal microecology tests were performed to compare the status of HPV infections in different age groups. The study subjects were divided into HR-HPV-positive and HR-HPV-negative groups according to whether HR-HPV infection was detected or not, and multifactorial logistic regression analysis was used to explore the relationship between HR-HPV and vaginal infectious diseases. Results The HR-HPV infection rate of women in the Fourth Division of Xinjiang Production and Construction Corps was 10.43%, and the HR-HPV infection rate of each age group was 14.34% from 25 to 34 years old, 10.16% from 35 to 44 years old, 8.70% from 45 to 54 years old, and 11.89% from 55 to 64 years old, and there were differences in the infection rate of HR-HPV among different age groups (P<0.05). Among HR-HPV, HPV52, HPV16, HPV58, HPV51, HPV68 were the most common, with 76.69% of single infections, 18.02% of dual infections, 4.46% of triple infections, and 0.83% of quadruple infections. HR-HPV single infections and multiple infections were prevalent in the age group of 25-34 years old and 55-64 years old.HR-HPV mono-infection and multi-infection rates were statistically different between age groups (P<0.05). The infection rates of bacterial vaginosis (BV) and trichomoniasis vaginitis (TV) were higher in HR-HPV-positive patients (P<0.05). Logistic regression analysis showed that the infection rates of BV (OR=1.560, 95% CI: 1.195~2.035), TV (OR=2.208, 95% CI: 1.221~3.993) were risk factors for HR-HPV infection (P<0.05). Conclusion In the screened population of the Fourth Division of Xinjiang Production and Construction Corps, HR-HPV mono-infection is the most prevalent type. Women aged 25-34 and 55-64 years old show higher rates of HR-HPV infection. The most common HR-HPV genotypes, in descending order, are 52, 16, 58, 51, and 68. BV and TV are the two major risk factors closely associated with HR-HPV infection.

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Abstract Objective To investigate the effect and mechanism of methyltransferase-like3 (METTL3) , which func- tions as m6 A modification methyltransferase , on the cellular proliferation , clone formation and migration of non- small cell lung cancer (NSCLC) cells . Methods The METTL3 level and patient overall survival were analyzed by bioinformatics . In the transfected A549 cells , the expression of METTL3 was detected by RT-PCR and Western blot assays , the m6 A level was detected by m6 A RNA Methylation Assay kit , the cellular viability and growth were de- tected by CCK8 assay , the apoptosis was detected by immunofluorescent assay , the colony growth was detected by clone formation assay , the cell migration growth was detected by scratch . Results Bioinformatics showed that METTL3 was highly expressed in NSCLC and negatively correlated with patient overall survival . The RT-PCR and Western blot data showed that the METTL3 level was higher in NSCLC cells . Moreover , higher METTL3 signifi- cantly promoted m6 A level , cell proliferation , colony formation , migration , inhibited cell apoptosis , increased the mRNA and protein expressions of EMT-related marker Vimentin but inhibited apoptosis and decreased the mRNA and protein expressions of EMT-related marker E-cadherin in the A549 cells . Furthermore , the contrasting results were obtained in the A549 cells with the knockdown of METTL3 . Conclusion The over-expressed METTL3 in- creases the m6 A levels in NSCLC cells and promotes cellular proliferation , colony formation , and migration growth , thereby laying a theoretical foundation for future research on early diagnosis and targeted drug development for NSCLC by targeting METTL3 .