Objective: This study aimed to investigate the impact of vasculogenic mimicry (VM) and postoperative adjuvant therapy on the prognosis and survival of patients with esophageal squamous cell carcinoma (ESCC), as well as to assess whether VM affects the clinical benefit of postoperative adjuvant therapy. Methods: A single-center retrospective analysis was conducted on patients who underwent radical surgery for squamous esophageal cancer, documented in the medical record system. The presence or absence of VM in surgical specimens was determined using double staining with PAS/CD31. Stratification was applied based on adjuvant therapy and VM status. Survival curves and COX modelling were used to analyze the impact of the presence or absence of VM on the benefit of adjuvant therapy and survival prognosis of patients. Results: VM-positive patients were more prone to postoperative recurrence and metastasis. VM was identified as an independent risk factor for progression-free survival (p<0.001, 95% CI:1.809-3.852) and overall survival (p<0.001, 95% CI:1.603-2.786) in postoperative squamous esophageal cancer. Postoperative adjuvant therapy significantly prolonged progression-free survival (p=0.008) and overall survival time (p<0.001) in patients with stage II and III esophageal cancer, with concurrent chemoradiotherapy being the most effective. However, the presence of VM significantly reduced the benefit of postoperative adjuvant therapy (p＝0.152). Conclusion: VM negatively impacts the prognosis of postoperative ESCC patients and reduces the efficacy of postoperative adjuvant therapy.

Based on multiple bioinformatics methods and machine learning techniques, this study was designed to explore potential hub genes of gastric cancer with a diagnostic value. The novel biomarkers were detected through multiple databases of gastric cancer-related genes. The NCBI Gene Expression Omnibus (GEO) database was used to obtain gene expression files. Three hub genes (ESRRG, ATP4A, and ATP4B) were detected through a combination of weighted gene co-expression network analysis (WGCNA), gene-gene interaction network analysis, and supervised feature selection method. GEPIA2 was used to verify the differences in the expression levels of the hub genes in normal and cancer tissues in the RNA-seq levels of Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases. The objectivity of potential hub genes was also verified by immunohistochemistry in the Human Protein Atlas (HPA) database and transcription factor-hub gene regulatory network. Machine learning (ML) methods including data pre-processing, model selection and cross-validation, and performance evaluation were examined on the hub-gene expression profiles in five Gene Expression Omnibus datasets and verified on a GEO external validation (EV) dataset. Six supervised learning models (support vector machine, random forest, k-nearest neighbors, neural network, decision tree, and eXtreme Gradient Boosting) and one semi-supervised learning model (label spreading) were established to evaluate the diagnostic value of biomarkers. Among the six supervised models, the support vector machine (SVM) algorithm was the most effective one according to calculated performance metrics, including 0.93 and 0.99 area under the curve (AUC) scores on the test and external validation datasets, respectively. Furthermore, the semi-supervised model could also successfully learn and predict sample types, achieving a 0.986 AUC score on the EV dataset, even when 10% samples in the five GEO datasets were labeled. In conclusion, three hub genes (ATP4A, ATP4B, and ESRRG) closely related to gastric cancer were mined, based on which the ML diagnostic model of gastric cancer was conducted.

Mycoplasma synoviae (M. synoviae) is an important avian pathogen that causes arthritis and airsacculitis in young chickens and turkeys. Infection by M. synoviae results in considerable economic losses to the poultry industry worldwide. Cytoadherence is a crucial stage during mycoplasma infection. Dihydrolipoamide dehydrogenase (PdhD) is a flavin-dependent enzyme that is critical for energy metabolism and redox balance. To date, its role in cytoadherence is poorly understood. In this study, recombinant PdhD from M. synoviae (rMSPdhD) was expressed in the supernatant component of E. coli BL21 and rabbit anti-rMSPdhD serum was prepared. rMSPdhD was shown to be an immunogenic protein by immunoblot assays, while the mycoplasmacidal assay revealed that the rabbit anti-rMSPdhD serum had a high complement-dependent mycoplasmacidal rate (88.5 %). Using a suspension immunofluorescence assay and subcellular localization analysis, MSPdhD was shown to be a surface-localized protein distributed in both the cytoplasm and cell membrane of M. synoviae. The enzymatic activity of rMSPdhD was determined by measuring its ability to reduce lipoamide to dihydrolipoamide and convert NADH to NAD+. Using an indirect immunofluorescence assay, rMSPdhD was shown to adhere to DF-1 chicken embryo fibroblast cells. Furthermore, the attachment of M. synoviae to DF-1 cells was significantly inhibited by rabbit anti-rMSPdhD serum. Western blot and ELISA binding assays confirmed that rMSPdhD also bound to fibronectin (Fn) and plasminogen (Plg) in a dose-dependent manner. In conclusion, our data show that MSPdhD is not only a biological enzyme, but also an immunogenic surface-exposed protein that can bind to Fn and Plg as well as adhere to host cells. In addition, we show that rabbit anti-rMSPdhD serum can inhibit the adhesion of M. synoviae to DF-1 cells and has a significant complement-dependent bactericidal activity. Our findings suggest that MSPdhD may be involved in the pathogenesis of M. synoviae.

[目的]研究滑液支原体(Mycoplasma synoviae,MS)脂蛋白P80的免疫反应性及其在MS血清抗体ELISA检测中的应用.[方法]对MS P80的氨基酸序列进行生物信息学分析、原核表达和纯化,并用免疫印迹法分析其与6种不同MS分离株阳性血清的免疫反应性以及与其他禽病原血清的交叉反应性;运用纯化的MS P80表达蛋白作为包被抗原建立了MS血清抗体的间接ELISA检测方法,对其敏感性和重复性进行检测;比较检测了与美国爱德士检测试剂盒对50份临床血清样品的阳性符合率.[结果]生物信息学分析预测MS P80蛋白为脂蛋白且含有信号肽,其在MS种内同源性高达98％-100％,与其他种属P80蛋白同源性在25％-34％之间,成功表达和纯化了MS P80重组蛋白(rMS P80);Western blotting分析表明纯化的rMS P80具有良好的免疫反应性和特异性;运用rMS P80建立的MS血清ELISA抗体检测方法可对不同株MS阳性血清进行抗体效价检测,而对其他禽病原阳性血清均无交叉反应性;该检测方法的批内变异系数小于5％,批间变异系数小于10％,重复性良好;与美国IDEXX检测试剂盒比较,本文建立的ELISA抗体检测方法敏感性更高,阳性符合率为75％,阴性符合率为89.47％,总样本符合率为86％.[结论]MS P80具有较好的免疫反应性、种内保守性和种间特异,并且可用作MS抗体检测的靶标抗原.

[背景]许多研究表明,支原体的NADH氧化酶(NADH oxidase,NOX)不仅在胞浆中发挥生物酶学功能,也存在于细胞膜上发挥黏附宿主细胞功能.[目的]对滑液支原体(Mycoplasma synoviae,MS)的NOX进行酶学活性及亚细胞定位研究,分析其在MS致病过程中的潜在作用.[方法]对MS的NOX蛋白进行原核表达、纯化,然后对重组MSNOX (rMSNOX)的酶学活性及影响酶活的条件进行研究,测定其酶比活力、米氏常数及最大反应速率,接着用MS阳性血清及制备的rMSNOX兔多克隆抗体,分别与rMSNOX蛋白及MS全菌、膜蛋白和胞浆蛋白进行Western blotting 反应,鉴定rMSNOX的免疫原性及其在MS中的分布情况.[结果]在大肠杆菌BL21(DE3)中成功表达rMSNOX蛋白,相对分子质量约为53kD,并获得纯化的rMSNOX蛋白;酶活测定显示rMSNOX蛋白的酶比活力为14.17 IU/mg,最适酶促温度为37℃,最适pH为7.5,双倒数法求得rMSNOX的最大反应速率Vmax为21.8 μmol/(L·min),米氏常数Km(NADH)为244.0 μmol/L;rMSNOX蛋白与MS阳性血清特异性结合,证明具有良好的免疫原性;亚细胞定位研究表明NOX蛋白主要存在于MS的胞浆中,细胞膜上有少量的分布.[结论]首次证实MS的NOX不仅具有NADH氧化酶活性,也是MS具有免疫原性的膜蛋白,为进一步探索NOX在MS致病过程中的作用提供了分子基础.

Mycoplasma gallisepticum is a causative agent of chronic respiratory disease in chickens, typically causing great economic losses. Cytoadherence is the critical stage for mycoplasma infection, and the associated proteins are important for mycoplasma pathogenesis. Many glycolytic enzymes are localized on the cell surface and can bind the extracellular matrix of host cells. In this study, the M. gallisepticum pyruvate dehydrogenase E1 alpha subunit (PDHA) and beta subunit (PDHB) were expressed in Escherichia coli, and their enzymatic activities were identified based on 2,6-dichlorophenol indophenol reduction. When recombinant PDHA (rPDHA) and recombinant PDHB (rPDHB) were mixed at a 1:1 molar ratio, they exhibited strong enzymatic activity. Alone, rPDHA and rPDHB exhibited no or weak enzymatic activity. Further experiments indicated that both PDHA and PDHB were surface-exposed immunogenic proteins of M. gallisepticum. Bactericidal assays showed that the mouse anti-rPDHA and anti-rPDHB sera killed 48.0% and 75.1% of mycoplasmas respectively. A combination of rPDHA and rPDHB antisera had a mean bactericidal rate of 65.2%, indicating that rPDHA and rPDHB were protective antigens, and combining the two sera did not interfere with bactericidal activity. Indirect immunofluorescence and surface display assays showed that both PDHA and PDHB adhered to DF-1 chicken embryo fibroblast cells and adherence was significantly inhibited by antisera against PDHA and PDHB. Adherence inhibition of M. gallisepticum to DF-1 chicken embryo fibroblast cells was 30.2% for mouse anti-rPDHA serum, 45.1% for mouse anti-rPDHB serum and 72.5% for a combination of rPDHA and rPDHB antisera, suggesting that rPDHA and rPDHB antisera may have synergistically interfered with M. gallisepticum cytoadherence. Plasminogen (Plg)-binding assays further demonstrated that both PDHA and PDHB were Plg-binding proteins, which may have contributed to bacterial colonization. Our results clarified the enzymatic activity of M. gallisepticum PDHA and PDHB and demonstrated these compounds as Plg-binding proteins involved in cytoadherence.