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Abstract Background Disulfidptosis is a newly discovered type of cell death that differs from apoptosis, necrosis, ferroptosis and other death modes and is closely related to the occurrence and progression of tumors. However, the predictive potential and biological characteristics of disulfidptosis-related lncRNAs (DRGs-lncRNAs) in esophageal squamous cell carcinoma (ESCC) are unclear. Methods RNA transcriptome data, clinical information and mutation data for ESCC patients were obtained from The Cancer Genome Atlas (TCGA) database. Pearson correlation and Cox regression analyses were used to identify the DRGs-lncRNAs associated with overall survival (OS). LASSO regression analysis was used to construct the prognostic model. A nomogram was created to predict the prognosis of patients with ESCC. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were used to identify the signaling pathways associated with the model. TIMER, CIBERSORT, ESTIMATE and other methods were used to analyze immune infiltration, immune function, immune checkpoints and drug sensitivity. The tumor mutation burden (TMB) were assessed between different risk groups. Real-time polymerase chain reaction (RT‒PCR) was used to detect the expression of DRGs-lncRNAs in ESCC cell lines. Results A total of 155 lncRNAs significantly associated with disulfidptosis were identified. Through univariate Cox regression analysis, LASSO regression analysis and multivariate Cox regression analysis, 9 lncRNAs with independent prognostic significance were selected, and a prognosis model was established. Survival analysis with the prognostic model revealed that there were obvious differences in survival between the high- and low-risk groups. Further analysis revealed that the immune microenvironment, immune infiltration, immune function, immune checkpoints, and drug sensitivity significantly differed between the high-risk and low-risk groups. Patients who exhibited both high risk and high tumor mutation burden (TMB) survived shorter, while those who fell into the low risk and low TMB categories survived longer. In addition, RT‒PCR analysis revealed differential expression of DRG lncRNAs between ESCC cell lines and esophageal epithelial cell lines. Conclusions We established a DRG-lncRNA prognostic model that can be used to predict the prognosis, tumor mutation burden, immune cell infiltration, and drug sensitivity of ECSS patients. The results of this study provide valuable insights into the understanding of ESCC and provide valuable assistance for the individualized treatment of ESCC patients.

Abstract Background Carbapenemase-producing makes a great contribution to carbapenem resistance in Gram-negative bacilli. Bla AFM-1 gene was first discovered by us in Alcaligenes faecalis AN70 strain isolated in Guangzhou of China and, was submitted to NCBI on 16 November 2018. Methods Antimicrobial susceptibility testing was performed by broth microdilution assay using BD Phoenix 100. The phylogenetic tree of AFM and other B1 metallo-β-lactamases was visualized by MEGA7.0. Whole-genome sequencing technology was used to sequence carbapenem-resistant strains including the bla AFM-1 gene. Cloning and expressing of bla AFM-1 were designed to verify the function of AFM-1 to hydrolyze carbapenems and common β-lactamase substrates. Carba NP and Etest experiments were conducted to evaluate the activity of carbapenemase. Homology modeling was applied to predict the spatial structure of AFM-1. A conjugation assay was performed to test the ability of horizontal transfer of AFM-1 enzyme. The genetic context of bla AFM-1 was performed by Blast alignment. Results Alcaligenes faecalis strain AN70, Comamonas testosteroni strain NFYY023, Bordetella trematum strain E202, and Stenotrophomonas maltophilia strain NCTC10498 were identified as carrying the bla AFM-1 gene. All of these four strains were carbapenem-resistant strains. Phylogenetic analysis revealed that AFM-1 shares little nucleotide and amino acid identity with other class B carbapenemases (the highest identity (86%) with NDM-1 at the amino acid sequence level). The spatial structure of the AFM-1 enzyme was predicted to be αβ/βα sandwich structure, with two zinc atoms at its active site structure. Cloning and expressing of bla AFM-1 verified AFM-1 could hydrolyze carbapenems and common β-lactamase substrates. Carba NP test presented that the AFM-1 enzyme possesses carbapenemase activity. The successful transfer of pAN70-1(plasmid of AN70) to E.coli J53 suggested that the bla AFM-1 gene could be disseminated by the plasmid. The genetic context of bla AFM indicated that the downstream of the bla AFM gene was always adjacent to trpF and ble MBL. Comparative genome analysis revealed that bla AFM appeared to have been mobilized by an ISCR27-related mediated event. Conclusions The bla AFM-1 gene is derived from chromosome and plasmid, and the bla AFM-1 gene derived from the pAN70-1 plasmid can transfer carbapenem resistance to susceptible strains through horizontal transfer. Several bla AFM-1 -positive species have been isolated from feces in Guangzhou, China.

Abstract Background BlaAFM-1 (GenBank Accession No. 143105.1) is a new B1 subclass metallo-β-lactamase gene discovered by our group, and isolated from an Alcaligenes faecalis plasmid that renders carbapenem antibiotics ineffective. In this study, we generated a fast and reliable assay for blaAFM-1 detection. Methods We designed optimum loop-mediated isothermal amplification (LAMP) primers and constructed a recombinant plasmid AFM-1 to specifically detect blaAFM-1. Optimal LAMP primers were used to assess sensitivity of the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (simulated sputum and simulated feces). Fifty two samples, without blaAFM-1, were used to assess LAMP real-time assay specificity; these samples were verified by conventional PCR and sequencing for the absence of blaAFM-1. Three hundred clinical Gram-negative carbapenem-resistant strains were tested by LAMP assay for strains carrying blaAFM-1, which were confirmed by conventional PCR and Sanger sequencing. We calculated the sensitivity and its 95% confidence interval (95% CI), specificity and its 95% CI, and predictive values of the LAMP assay and conventional PCR/sequencing by investigating positive and negative clinical strains. Results The lowest limit of detection for the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (in both simulated sputum and simulated feces) was 101 copies/reaction. All amplification curves of the 52 blaAFM-1-free bacteria strains were negative, suggesting the LAMP assay had excellent specificity for detecting blaAFM-1. Among the 300 clinical strains, eight were positive for blaAFM-1 using LAMP. These LAMP results were consistent with conventional PCR and Sanger sequencing data. As with conventional PCR/sequencing, the LAMP method exhibits 100% sensitivity (95% CI 59.8–100%) and 100% specificity (95% CI 98.4–100%) for blaAFM-1 detection. The LAMP assay is also time-efficient (1 h) for blaAFM-1 detection. Conclusions We established a new LAMP assay with high sensitivity and specificity to detect the novel B1-β-lactamase gene, blaAFM-1.