Rapid, specific, and instrument-independent detection of bacterial pathogens and antibiotic resistance genes remains a major challenge in clinical diagnostics. Recombinase polymerase amplification (RPA) integrated with CRISPR/Cas12a is a promising molecular diagnostic platform, but its performance is fundamentally limited by the strict requirement for protospacer adjacent motifs (PAM) to activate Cas12a. To address this constraint, we developed a PAM-Engineered Abasic-site probe-assisted RPA-CRISPR (PEAR) assay that introduces a structurally defined artificial PAM sequence directly during the RPA process. The system incorporates an Endonuclease IV (Nfo)-cleavable probe containing an abasic site (d'Spacer) and an embedded PAM motif. Upon target-specific probe hybridization and Nfo-mediated cleavage, the PAM sequence is precisely integrated into the newly synthesized amplicon, generating PAM-containing double-stranded DNA that efficiently triggers Cas12a transcleavage activity. This design enables PAM-independent target adaptability while maintaining high analytical sensitivity. Using plasmid-derived templates, PEAR successfully detected the Klebsiella pneumoniae speciesspecific khe gene and the carbapenem resistance gene blaKPC-2, achieving a detection limit of 1 copy/mu L with 100% analytical specificity. Evaluation with 30 clinical samples yielded diagnostic accuracies of 93.3% for khe and 87.7% for blaKPC-2, demonstrating practical clinical applicability. Overall, PEAR represents a sensitive, specific, and potentially universal platform for CRISPR/Cas12a-mediated nucleic acid detection, providing a generalizable strategy to eliminate PAM dependence and expand translational applications in rapid pathogen detection and antimicrobial resistance surveillance.

Clear cell renal cell carcinoma (ccRCC), a common type of renal cortical tumor, is the most prevalent subtype of renal malignancies within the urinary system and is associated with a low survival rate. Ferroptosis plays a crucial role in the process of renal carcinogenesis and holds potential for significant applications in patient prognosis. However, the clinical prognostic relevance of ferroptosis-related genes (FRGs) for ccRCC remains unclear. The identification of FRG signatures and the development of a novel prognostic model based on FRGs demonstrate important prognostic significance for ccRCC. Univariate cox screen was performed to screen for prognostic-related genes using ccRCC data from the The Cancer Genome Atlas (TCGA) database. And then an initial screen for prognostic genes was performed by taking intersections with the differential genes of the Gene Expression Omnibus (GEO) database datasets GSE213324 and GSE66271, as well as with the FRGs, and a multigene signature was constructed using least absolute shrinkage and selection operator (LASSO) and Cox regression analysis. Subsequently, the model was evaluated using Kaplan–Meier (KM) survival curve analysis, receiver operating characteristic (ROC), nomogram, and decision curve analysis (DCA). Differences in tumor microenvironment and immune function were analyzed by single-sample gene set enrichment analysis (ssGSEA) and immune infiltration in patients in the high- and low-risk groups. The tumor immune dysfunction and exclusion (TIDE) assessed the immune checkpoint inhibitor (ICI) susceptibility in patients. The Gene Set Enrichment Analysis (GSEA) was performed for pathway enrichment analysis. Patient mutation data were downloaded and tumor mutation burden (TMB) were compared between patients in the high- and low-risk groups. ADACSB, DPEP1, KIF20A, MT1G, PVT1 and TIMP1 were utilized to establish a novel prognostic signature. The KM curve analysis revealed that patients in the high-risk group exhibited a poorer prognosis. Additionally, the ROC results demonstrated that the model displayed favorable prognostic accuracy. Independent prognostic analyses indicated that the FRGs model could serve as an independent prognostic indicator. Furthermore, calibration curve of the nomogram illustrated enhanced precision in predicting survival rates for patients at 1, 3 and 5 years. Analysis of mutation data unveiled higher tumor mutation load among patients in the high-risk group, which correlated with an increase in risk score. The FRGs model offers a novel approach for prognostic prediction of ccRCC patients and has the potential to provide personalized prognostic prediction and treatment for ccRCC patients.

The antimicrobial peptides, such as host immune system-derived human bactericidal/permeability-increasing protein (hBPI), are the potential drugs for antibiotic-resistant Gram-negative bacterial infection. However, whether the purified chimeric hBPI23-Fcγ protein has bactericidal activity against drug-resistant Gram-negative bacteria (GNB) and the relevant mechanisms have not been fully elucidated. In this study, the chimeric hBPI23-Fcγ protein, which consisting of the functional N terminus of BPI and Fcγ1, were expressed and purified in a lab-scale. The chimeric hBPI23-Fcγ protein showed longer half-life up to 148.2 min in vivo. The hBPI23-Fcγ protein also showed significant bactericidal activity against standard and clinically isolated drug-resistant Acinetobacter baumannii (A. baumannii) and Escherichia coli (E. coli). In addition, the hBPI23-Fcγ protein markedly decreased biofilm formation, neutralized bacterial lipopolysaccharides (endotoxin) and enhanced the opsonization of phagocytes, as well as significantly improved the survival rate of minimal lethal dose (MLD) of drug-resistant E. coli -infected mice. These results indicate that the BPI23-Fcγ protein protected mice from drug-resistant GNB infection not only by direct bactericidal effect, but also by promoting opsonophagocytosis of macrophages. In conclusion, the chimeric BPI23-Fcγ protein may be as a promising candidate of non-antibiotic biological agent for drug-resistant GNB infection.

Respiratory viral infection, represented by influenza virus, is easily followed by bacterial infection, the main cause of death. Clinical studies have shown that even mild influenza virus infection followed by secondary bacterial infection can mediate severe pneumonia and lung injury. In this study, mice were intranasally stimulated by polyinosinic-polycytidylic acid [poly(I:C)] followed by lipopolysaccharide (LPS) to simulate respiratory RNA virus secondary Gram-negative bacterial infection. The results demonstrated that poly(I:C) followed by LPS stimulation induced more weight loss, worse lung pathological injury, additional recruitment of neutrophils and interstitial macrophages, and elevated expression of ficolin A/B in the lung neutrophils, alveolar and interstitial macrophages. Knockout of ficolin A/B alleviated the body weight loss, the lung pathological injury, and the pulmonary inflammatory score. Mechanically, knockout of ficolin A/B was associated with reduced interstitial macrophage recruitment and alveolar macrophage exhaustion. These results suggest that ficolin A/B is a potential therapeutic target for severe pneumonia induced by respiratory RNA virus secondary Gram-negative bacterial infection.

Antibiotic resistance has become a major threat, contributing significantly to morbidity and mortality globally. Administering non-antibiotic therapy, such as antimicrobial peptides, is one potential strategy for effective treatment of multi-drug-resistant Gram-negative bacterial infections. Bactericidal/permeability-increasing protein (BPI) derived from neutrophils has bactericidal and endotoxin-neutralizing activity. However, the protective roles and mechanisms of BPI in multi-drug-resistant bacterial infections have not been fully elucidated. In this study, a chimeric BPI23-Fc gamma recombined protein comprising the functional N terminus of BPI and Fc gamma was constructed and expressed by adenovirus vector 5 (Ad5). Ad5-BPI23-Fc gamma or recombinant BPI23-Fc gamma protein significantly improved the survival of mice with pneumonia induced by a minimal lethal dose of multi-drug-resistant Acinetobacter baumannii or Klebsiella pneumoniae by ameliorating lung pathology and reducing pro-inflammatory cytokines. Transfection with Ad5-BPI23-Fc gamma significantly decreased the bacterial load and endotoxaemia, which was associated with enhanced bactericidal ability and elevated the phagocytic activity of neutrophils in vitro and in vivo. In addition, Ad5-BPI23-Fc gamma transfection significantly increased the recruitment of neutrophils to lung, increased the proportion and number of neutrophils in peripheral blood, and promoted the maturation of bone marrow (BM) neutrophils after drug-resistant A. baumannii infection. BPI23-Fc gamma and neutrophils synergistically enhanced bactericidal activity and decreased pro-inflammatory cytokines. These results demonstrated that the chimeric BPI23-Fc gamma protein protected mice from pneumonia induced by multi-drug-resistant A. baumannii infection by direct bactericidal effects and promotion of neutrophil recruitment, phagocytosis and maturation. Chimeric BPI23-Fc gamma may be a promising candidate as a non-antibiotic biological agent for multi-drug-resistant A. baumannii infection.

Supplementary Data from Sphingosine Kinase 1 Is Associated with Gastric Cancer Progression and Poor Survival of Patients

培养医学创新人才是高等医学院校的重要职责,加强本科阶段对学生创新性思维、创新能力的培养是适应国家医学创新和高精尖医学人才的必然需求.医学免疫学是基础医学学科中一门重要的主干课程,是与多学科交叉融合的桥梁课程,结合医学免疫学学科特点,在课程设置、教学模式、教学测评、实践教学等各方面融合科研创新思维和能力的训练,是兼顾新时代学生能力素养发展需求、帮助和促进低年级本科生科研创新思维和能力提升的一项重要举措.

Objective The objective of this study was to understand the role of bactericidal permeability increasing protein (BPI) in the pathogenesis of experimental murine colitis. Methods We used the Cre-LoxP system to generate BPI knockout (BPI KO) mice. Acute colitis was induced in BPI KO mice and wild-type (WT) mice by subjecting the mice to 5% dextran sulfate sodium (DSS). Mice were observed for symptoms of experimental colitis. The survival of BPI KO mice to infection with Acinetobacter baumannii, a gram-negative bacterium, was also assessed. Results Southern blot, RT-PCR, and western blot results showed that the 2nd and 3rd exons of the murine Bpi gene were knocked out systemically, confirming successful construction of the BPI KO mouse. BPI KO mice subjected to DSS showed increased symptoms of experimental colitis, increased colonic mucosal damage, increased epithelial permeability, elevated levels of serum LPS, and a disrupted fecal microbiome as compared with WT mice. Furthermore, BPI KO mice challenged intraperitoneally with A. baumannii died sooner than WT mice, and the total number of bacteria in the abdominal cavity, spleen, and liver was increased in BPI KO mice as compared to WT mice. Conclusions We successfully generated BPI KO mice. The BPI KO mice developed worse colitis than WT mice by increased colitis symptoms and colonic mucosal damage, elevated levels of serum LPS, and a disrupted microbiome. BPI could be a potential target for treatment of ulcerative colitis in humans.

Objective To investigate the role of endoplasmic reticulum stress in skeletal muscle in surgery-induced insulin resistance in rats. Methods The rat models of open-chest surgery were created. Twenty-four rats were randomly divided into 4 groups (n=6):control group, 4-phenyl butyric acid (4-PBA) group, operation group and 4-PBA+operation group. Rats in each group were treated with hyperinsulinemic-euglycemic clamp test to assess insulin sensitivity 1 day after operation. Western blot assay was used for detecting the expression of binding immunoglobulin protein (BiP) in skeletal muscles. Results Compared with the control group, 4-PBA group and 4-PBA+operation group, the plasma free fatty acid level of the operation groups was elevated, while glucose infusion rate (GIR) was decreased, BiP was significantly increased in rat soleus samples (P<0.05). Conclusions Endoplasmic reticulum stress in skeletal muscles is involved in surgery-induced insulin resistance. 4-PBA, endoplasmic reticulum stress inhibitor, ameliorates surgery-induced insulin resistance in rats.

Objective To construct and identify a gene mutation library of the N-terminal fragments of human bactericidal/ permeability increasing proteins (BPI23) for enhancing its activity binding lipopolysaccharides (LPS) by error-prone polymerase chain reaction (PCR) and DNA shuffling method.Methods Error-prone PCR was used to obtain random mutant fragments of BPI600 by adding different concentrations of Mg2+ and Mn2+.Random mutation rate was calculated by DNA sequencing and genetic comparison with wildtype BPI600.The product of error-prone PCR was further mutated via DNA shuffling.The reassembled product was cloned into pYD1 vector and transformed into E.coli XL10-Gold.Ten clones were randomly picked out,digested and identified by restriction enzyme Hind Ⅲ and Xho Ⅰ.The positive clones were identified by DNA sequencing.Results DNA sequencing result showed that the random mutation rate was 2.3％ by error-prone PCR.After DNA shuffling,6 positive clones including BPI600 were identified in the 10 random clones selected from LB culture medium with ampicillin.Among them,4 had 6,9,1 1 and 14 mutations,respectively;1 had 10 mutations and 1 deletion determined by DNA sequencing.By amino acid comparison,only 2 DNA mutants were able to encode a full-length mutant BPI23 protein,with 4 and 14 amino acid mutations respectively.Conclusion A library of BPI23 mutants was constructed with a size of 2 × 105.The results laid the foundation for the study of its activity binding LPS.

Bactericidal/permeability increasing (BPI) is an antibiotic protein which kills Gram-negative bacteria and neutralizes endotoxin. We have previously developed a recombinant adeno-associated virus which contains human BPI amino acid residues 1-199 and Fc fragment of human IgG1 gene (AAV-hBPI-Fc) and shown that the recombinant virus can protect mice from lethal endotoxemia. However, whether AAV-hBPI-Fc can be used in vivo for the long term remains unclear. To address this, we established an adeno-associated virus-containing mouse BPI and Fc fragment genes (muBPI-Fc) and compared antigenicity of these recombinant proteins in murine models. Immunohistochemistry showed the expression of both fusion proteins at injected sites. ELISA and Western blotting showed that the muBPI-Fc protein was detected in serum up to 8 weeks after injection, without generation of autoantibodies against muBPI-Fc. In contrast, expressed hBPI-Fc protein was only detected on the 2nd week, whereas the autoantibody against hBPI-Fc protein occurred in serum from the 4th week to the end of study. muBPI-Fc also reduced production of proinflammatory cytokines and protected mice from endotoxemia and bacteremia. Our data showed that AAV-muBPI-Fc has potential long-term efficacy as an anti-endotoxin and has anti-bacterial activity in mice, suggesting the potential clinical application of AAV-hBPI-Fc, such as in endotoxin shock.

Case-based learning has gradually become popular and is considered to be the direction of the successful education model for the future. In the present paper, the authors used a case for teaching medical immunology based on years of teaching experience and questionnaires analysis. Hope this paper can provide methodological guidance and ideas for teaching with case.

Bactericidal/permeability increasing (BPI) is an antibiotic protein which kills Gram-negative bacteria and neutralizes endotoxin. We have previously developed a recombinant adeno-associated virus which contains human BPI amino acid residues 1–199 and Fc fragment of human IgG1 gene (AAV-hBPI-Fc) and shown that the recombinant virus can protect mice from lethal endotoxemia. However, whether AAV-hBPI-Fc can be used in vivo for the long term remains unclear. To address this, we established an adeno-associated virus-containing mouse BPI and Fc fragment genes (muBPI-Fc) and compared antigenicity of these recombinant proteins in murine models. Immunohistochemistry showed the expression of both fusion proteins at injected sites. ELISA and Western blotting showed that the muBPI-Fc protein was detected in serum up to 8 weeks after injection, without generation of autoantibodies against muBPI-Fc. In contrast, expressed hBPI-Fc protein was only detected on the 2nd week, whereas the autoantibody against hBPI-Fc protein occurred in serum from the 4th week to the end of study. muBPI-Fc also reduced production of proinflammatory cytokines and protected mice from endotoxemia and bacteremia. Our data showed that AAV-muBPI-Fc has potential long-term efficacy as an anti-endotoxin and has anti-bacterial activity in mice, suggesting the potential clinical application of AAV-hBPI-Fc, such as in endotoxin shock.

大学生综合素质培养是深化教育改革的一项重要内容，而科研实践是培养本科生综合能力的重要手段，对课外科研活动在医学本科生综合素质中的积极作用作了初步探讨。

Objective To explore the changes and mechanism of colonic epithelial resistance and ion transport, with adeno-associated virus 2 (AAV2)-bactericidal permeability increasing protein 700 (BPI700)-fragment crystallizable gamma one 700 (Fcγ1700) chimeric gene-transferred mice were infected by the minimal lethal dose of E.coli..Methods Gene transfection, the short circuit current(Isc) record, ELISA and other methods were used to determine the colonic ion transport information.Results The basal membrane resistance and Isc of colonic mucosa in gene-transferred mice were significantly increased, but the apical addition of amiloride, a blocker of epithelial sodium channels, significantly inhibited the ISC-5-HT significantly increased the change in colonic ISC, which was much stronger in gene-transferred mice.The ISC was significantly inhibited by both the apical addition of diphenylamine-2, 2'-dicarboxylicacid (DPC), a blocker of the Cl-channel, and the basolateral application of bumetanide, an inhibitor of Na+-K+-2Cl-co-transporter (NKCC).Furthermore, the 5-HT-induced increased in intracellular cAMP levels was higher in gene-transferred mice.Pretreatment with 5-HT4 receptor antagonists GRI 13808 significantly inhibited 5-HT induced cAMP increases in transgenic mice.Expression of 5-HT4 receptor in gene-transferred mice was significantly higher than that in the control mice.Conclusions The study demonstrates that 5-HT enhances cAMP-dependent Cl-secretion of colonic mucosa via 5-HT4 receptor in AAV2-BPI700-Fcγ1700 transgenic mice.

Objective We used the murine orthotopic tracheal transplantation model to study the role of IL-17A in posttransplant airway epithelium.Methods Pathogen-free male C57BL/6 (n =50) and BALB/c (n =20) weighing 19-21 g.Weight-matched mice were randomly assigned to 5 groups.Control group:normal C57BL/6 (n =10) ; Group A:C57BL/6 (n =10) → C57BL/6(n =10),Group B:BALB/c(n =10) → C57BL/6(n =10),Group C:BALB/c(n =5) → C57BL/6 (n =5) and receive anti-IL-17 A neutralizing antibody injections,Group D:BALB/c (n =5) → C57 BL/6 (n =5) and receive IgG antibody injections.Group A and group B were randomly divided into 3 days and 14 days two subgroups(n =5).The expression level of IL-17A mRNA in tracheal of group A and group B was detected by real time quantitative PCR at 3 days after transplantation,and the frequencies of γδ T cells in draining lymph nodes of tracheal were analyzed by flow cytometry.Histopathological changes of tracheal were observed by HE staining and Masson staining at 14 days after transplantation.Results The mortality was 0 after tracheal transplantation.At the 3rd day,the IL-17A mRNA level of group B increased significantly as compared with group A(P ＜ 0.05),and the percent of γδ T cells among CD3 + T cells of group B was higher than group A(P ＜ 0.05).At the 14th day,the airway epithelia of group A maintained normal pseudostratified epithelium,and proliferation of fibroblast in submucosa tissue was not observed.The airway pseudostratifie epithelia of group B and D changed into flat epithelium,and proliferation of fibroblast was observed.The airway epithelia of group C preserved a pseudostratified epithelium,and proliferation of fibroblast was observed.The percentage of luminal obliteration of 4 groups was (31.21 ± 2.82) ％、(42.45 ± 2.21) ％、(39.04 ± 1.98) ％、(40.67 ±1.54) ％ respectively.The percentage of luminal obliteration of group B was higher than group A(P ＜ 0.05).Conclusion IL-17A plays an important role at the early stage of tracheal allograft rejection and mediates airway epithelial lesion.

目的：杀菌通透性增加蛋白嵌合基因(BPI700-Fcγ1700)转染的小鼠，给予感染最小致死剂量(MLD)的大肠杆菌，观察和探究结肠上皮电阻和离子转运的变化和机制。方法利用基因转染、短路电流记录、ELISA测定技术，记录转基因小鼠结肠粘膜的基础电流、电压以及给予促分泌物对上皮离子转运的影响。结果基因转染小鼠与对照组相比，结肠上皮基础电阻和短路电流明显增加，在上皮的顶膜侧加入钠离子通道阻断剂阿米洛利后，则可以明显抑制增加的电阻；应用上皮离子促分泌物forskolin后，可以明显增加结肠上皮的短路电流，这种短路电流可以被氯离子通道阻断剂DPC、Na+-K+-2Cl-共转运体抑制剂bumetanide明显抑制；并且forskolin作用结肠粘膜后上皮cAMP的含量明显高于对照组。结论基因转染小鼠结肠上皮阴离子转运明显增加，从而促进了肠道排毒反应，这可能是转基因小鼠对致死性大肠杆菌抵抗力增加的原因之一。本研究为革兰氏阴性菌感染的研究和治疗提供了理论基础。

Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

Cytokine-like 1 (CYTL1) was first identified in CD34(+) cells derived from bone marrow and cord blood. The biological functions of CYTL1 remain largely unknown. Here, we reveal a relationship between CYTL1 expression and the biological characteristics of neuroblastoma (NB). The expression of CYTL1 was detected in 10 human tumor cell lines and human NB tissues by RT-PCR and real-time PCR. The inhibitory effect of CYTL1 knockdown on the proliferation, migration and invasion of SH-SY5Y human neuroblastoma cells was studied using the CCK-8 assay and Transwell chamber assays. Among the 10 human tumor cell lines that we examined, CYTL1 was expressed only in SH-SY5Y human neuroblastoma cells. Furthermore, we also observed high levels of CYTL1 expression in human NB tissues. When CYTL1 expression was blocked by siRNA, SH-SY5Y cells showed decreased proliferation, migration and invasion activities. Taken together, our results showed the first evidence of CYTL1 expression in SH-SY5Y neuroblastoma cells and human NB tissues, revealed a possible link between CYTL1 and NB development, and suggested CYTL1 as a potential therapeutic target and diagnosis biomarker for NB.