Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri (HRK), caspase-7 and death receptor 4 (DR4) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.

This study conducted a comparative genomic investigation of 40 strains of Lentilactobacillus buchneri isolated from various environments—including fermented foods, silage, cattle rumen, and the nasopharynx—to identify species-level diversity and assess their CRISPR immunity. An average genome size of 2.55 ± 0.07 Mb, a GC content of 44.18 ± 0.15%, and 2444 ± 83 coding sequences were identified. Prophages were found in all strains except for two, while 17 strains contained plasmids. No genes associated with bacteriocins were identified. CRISPR analysis revealed the presence of 42 Type II-A and 45 Type I-E systems, with each strain having at least one Type II-A system (~ 2 systems per strain). Among the 33 tested strains, 29 encoded complete LbCas9 proteins, consisting of 1371 amino acids. In-silico analysis of PAM in Type II-A systems revealed a 5’-DNAWDHV-3’ motif, with a noted preference for 5’-AAAA-3’ at positions 3–6. The spacers found in CRISPR arrays targeted proteins involved in plasmid mobilization as well as components of phage tails, indicating their roles in inhibiting horizontal gene transfer and providing defense against phages. Remarkably, 27 spacers from 24 strains were found to match phages associated with human gut microbiomes, with several showing the ability to cross-target phages from livestock, kefir, and wastewater. This research expands the genomic understanding of L. buchneri from 10 to 40 genomes, uncovering the dynamics of CRISPR-phage co-evolution. The defined PAM preferences of the identified CRISPR systems, together with the broad predicted target range of their spacers, highlight their potential for biotechnological applications—most notably targeted CRISPRization of L. buchneri strains and in-silico-guided phage control during fermentation. These findings deepen our understanding of the ecological adaptability of L. buchneri and provide a foundation for future industrial exploitation of its native CRISPR immunity.

Several strains of Bifidobacterium animalis subsp. lactis are blockbusters of commercial dietary supplement cocktails, widely recognized for their probiotic properties and found in various ecological niches. The present study aimed to perform an in-depth comparative genomic analysis on 71 B. animalis subsp. lactis strains isolated from diverse sources, including human and animal feces, breast milk, fermented foods, and commercial dietary supplements, to better elucidate the strain level diversity and biotechnological potential of this species. The average genome size was found to be 1.93 ± 0.05 Mb, with a GC content of 60.45% ± 0.2, an average of 1562 ± 41.3 coding sequences (CDS), and 53.4 ± 1.6 tRNA genes. A comparative genomic analysis revealed significant genetic diversity among the strains, with a core genome analysis showing that 34.7% of the total genes were conserved, while the pan-genome remained open, indicating ongoing gene acquisition. Functional annotation through EggNOG-Mapper and CAZYme clustering highlighted diverse metabolic capabilities, particularly in carbohydrate metabolism. Nearly all (70 of 71) Bifidobacterium animalis subsp. lactis strains were found to harbor CRISPR-Cas adaptive immune systems (predominantly of the Type I-E subtype), underscoring the ubiquity of this phage defense mechanism in the species. A comparative analysis of spacer sequences revealed distinct strain-specific CRISPR profiles, with certain strains sharing identical spacers that correlate with common phylogenetic clades or similar isolation sources—an indication of exposure to the same phage populations and shared selective pressures. These findings highlight a dynamic co-evolution between B. lactis and its bacteriophages across diverse ecological niches and point to the potential of leveraging its native CRISPR-Cas systems for future biotechnological applications. Our findings enhance our understanding of the genetic and functional diversity of B. animalis subsp. lactis, providing valuable insights for its use in probiotics and functional foods.