Pseudomonas otitidis harbors an intrinsic subclass B3 metallo-β-lactamase (MBL), named POM, which contributes to reduced susceptibility to carbapenems. However, the diversity and functional implications of POM variants remain poorly studied. In this study, we investigated POM MBL in 65 strains from clinical, animal, and environmental origins from several countries, of which 19 Brazilian environmental strains were subjected to antimicrobial susceptibility testing. Sequence analysis identified the previously described POM-1 and POM-2, as well as 24 novel variants (POM-4 to POM-27). All variants displayed high amino acid identity (≥ 98.25%) and conserved key residues involved in zinc binding, indicating strong structural conservation. Whole-genome single nucleotide polymorphism analysis of P. otitidis genomes indicated that the distribution of POM variants was partly structured by strain phylogeny. Phenotypic comparisons were restricted to the 19 Brazilian environmental isolates and were descriptive only. These strains showed consistently elevated carbapenem minimum inhibitory concentrations (MIC), with meropenem and imipenem values ranging from 8 to 64 mg/L and 4 to 64 mg/L, respectively. Carbapenem MIC distributions overlapped across POM variants, with no clear genotype-phenotype pattern observed within this collection. Susceptibility to other antimicrobial classes was variable, although non-β-lactam agents generally retained activity. These findings demonstrate that POM enzymes constitute a highly conserved family of intrinsic MBL enzymes, while the relationship between POM sequence variation and carbapenem susceptibility remains unresolved within the limited phenotypic dataset. While variant-level identification has limited clinical utility, characterization of POM diversity provides insights into the evolution of intrinsic resistance.
Cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1 and cIAP2), encoded by BIRC2 and BIRC3, respectively, are important regulators of cellular survival and apoptosis. These proteins contribute to cancer cell survival by suppressing apoptotic signaling. While individual knockouts of BIRC2 or BIRC3 in cancer cells have been investigated, the consequences of simultaneously targeting both paralogs remain poorly understood. Thus, a single-gRNA CRISPR/eCas9 strategy was developed in which one gRNA targeting a shared sequence in BIRC2 and BIRC3 was used to simultaneously disrupt both genes in human colorectal cancer HCT-116 cells, and the resulting cellular and transcriptomic effects were compared with those of the individual knockouts. Individual and dual-targeting CRISPR/eCas9 constructs were experimentally validated by immunofluorescence analysis, RT-PCR, and RT-qPCR, while fluorescence microscopy and T7EI analysis were used to evaluate single-cell-derived clones. Apoptosis was evaluated using flow cytometry, and RNA sequencing was performed for differentially expressed gene (DEG) analysis. The single-gRNA dual-targeting strategy resulted in concurrent loss of cIAP1 and cIAP2 expression and produced a stronger reduction in cell survival and induction of apoptosis than targeting either gene individually. In addition, the expression levels of TNFRSF25, TRAF2, TNFSF15, and MIR663AHG were significantly upregulated following simultaneous BIRC2/BIRC3 targeting. RNA-seq analysis provided detailed insight into the molecular alterations associated with concurrent cIAP1 and cIAP2 depletion. Overall, these findings demonstrate the technical feasibility of targeting two highly homologous genes with a single gRNA and reveal that simultaneous disruption of cIAP1 and cIAP2 produces biological effects that are more pronounced than those observed following individual targeting.
Objectives Colorectal cancer (CRC) is a common malignancy of the colon and rectum, but its molecular mechanisms remain incompletely understood. Methods Gene expression omnibus (GEO) database was used to download CRC-related datasets. Weighted gene co-expression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO), support vector machine (SVM), and random forest (RF) were used to identify key CRC genes. Cell type identification by estimating relative subsets of rna transcripts (CIBERSORT) was used for immune infiltration analysis. Tumor immune estimation resource (TIMER) database was used to assess gene expression across multiple cancers. Human transcription factor target database (hTFtarget) was used to predict regulatory relationships between genes and transcription factors. Chromatin immunoprecipitation (ChIP), dual-luciferase assay, cell transfection, western blot (WB), and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to detect protein-gene binding, transcriptional activity, gene knockdown and overexpression, protein expression, and mRNA expression. JASPAR database was used to identify transcription factor binding sites. Cells were treated with a BODIPY 581/591 C11 kit to assess intracellular reactive oxygen species (ROS) levels. The assay kits were employed to determine the levels of ferrous iron (Fe2+), malondialdehyde (MDA), and superoxide dismutase (SOD). Cell counting kit-8 (CCK-8) was performed to measure cell viability. Results Matrix metallopeptidase 7 (MMP7), KIAA1199, and cadherin 3 (CDH3) were identified as potential key CRC genes. Their expression correlated with altered immune cell infiltration patterns. KIAA1199 showed the highest upregulation in CRC tissues. CCCTC-binding factor (CTCF) transcriptionally activated KIAA1199. Knockdown of CTCF decreased the expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), while increasing ROS, Fe2+, and MDA, and decreasing SOD levels. CTCF knockdown combined with KIAA1199 overexpression partially rescued these effects, modulating ferroptosis-related oxidative stress in CRC cells. Conclusion CTCF transcriptionally activates KIAA1199 and regulates ferroptosis-related oxidative stress in CRC cells, providing new insights into the molecular mechanisms of CRC.
