The CRISPR-Cas system constitutes an adaptive immune mechanism in prokaryotes that defends against mobile genetic elements. Within the perpetual co-evolutionary arms race between bacteria and their viral predators, bacteriophages encode anti-CRISPR (Acr) proteins that use sophisticated molecular strategies to sabotage CRISPR-Cas function. While canonical Acr proteins rely on steric blockade of Cas effectors, recent discoveries reveal unprecedented noncanonical mechanisms spanning CRISPR immunity stages. This review synthesizes recent mechanistic advances in this field since 2023, highlighting the expansion of noncanonical inhibition mechanisms beyond type I to include types II, V, and VI, as well as novel Acr interventions targeting multiple functional stages, such as spacer acquisition, translation-coupled inhibition, complex assembly/disassembly, and R-loop DNA binding. Structural insights demonstrate how Acr proteins achieve substoichiometric inhibition via conformational hijacking, catalytic repurposing, and molecular mimicry. Forged by the intense selective pressure of the phage–host conflict, these molecular innovations represent both remarkable evolutionary adaptations and versatile precision tools. They enable spatiotemporal control of CRISPR technologies, from engineered off-switches to diagnostic reset mechanisms, while posing critical challenges for therapeutic safety and microbiome management.

Infectious disease diagnostics has been transformed by metagenomic next-generation sequencing (mNGS), an unbiased approach that detects bacteria, viruses, fungi, and parasites in a single assay. By sequencing all nucleic acids in a sample, mNGS overcomes the narrow detection scope and slow turnaround of conventional tests, substantially improving pathogen detection. In conditions such as meningitis/encephalitis, sepsis, and pneumonia, mNGS frequently identifies etiologies missed by routine diagnostic tests, thereby facilitating earlier pathogen-directed therapy and, in selected settings, improving clinical management and outcomes. This approach is particularly valuable for immunocompromised, pediatric, and intensive care unit (ICU) patients with atypical infections. Currently, clinical mNGS workflows primarily rely on short-read sequencing platforms (e.g., Illumina), whereas long-read platforms (e.g., Nanopore, PacBio) offer advantages for rapid or high-resolution applications. Optimized bioinformatics and stringent quality control are essential for reliable results. Beyond clinical diagnostics, mNGS provides valuable genetic data on antimicrobial resistance (AMR) and pathogen phylogeny, supporting public health and outbreak surveillance (e.g., wastewater monitoring and variant tracking). Current challenges include distinguishing colonization from infection, interpreting sequencing data quantitatively, and reducing cost and turnaround time. Looking ahead, emerging strategies such as targeted panels, rapid automated workflows, and host‑response integration are expected to further shorten time‑to‑result and improve diagnostic specificity. Parallel progress in ethical and regulatory frameworks remains essential to ensure responsible implementation. To support clinical adoption, a standardized framework for clinical interpretation of mNGS results, together with associated training, has been developed and implemented. Overall, mNGS is likely to become an increasingly important component of infectious disease diagnostics, with ongoing innovations expected to broaden its clinical and epidemiological impact.

Mycosporine-like amino acids (MAAs) are natural sunscreens synthesized by a wide range of organisms. Although the induction of MAA production by ultraviolet radiation is well established, the signaling pathways involved and specific functions of MAAs under other stress conditions remain poorly understood. We demonstrated that MAAs serve as effective osmoprotectants for desiccation tolerance in the desert cyanobacterium Nostoc flagelliforme. Genetic disruption of genes encoding MAA biosynthetic enzymes eliminated MAA production, resulting in elevated oxidative damage, increased lipid peroxidation, and impaired photosynthesis under dehydration. Biochemical assays revealed that MAAs stabilize proteins and scavenge reactive oxygen species, indicating dual roles as osmolytes and antioxidants. Furthermore, we identified a signaling pathway Dsp1–OrrA that mediates the osmotic induction of MAA biosynthesis. Genetic disruption of either gene of Dsp1 and OrrA abolished osmotic induction and severely reduced desiccation tolerance. Phylogenomic analysis suggests that MAA biosynthesis is an ancient trait conserved in desiccation-tolerant cyanobacteria. This work deepens our understanding of microbial adaptation to extreme environments and provides a foundation for synthetic biology applications of MAAs.

Bacterial persisters show tolerance to bactericidal antibiotics and play essential roles in chronic infections; however, the general mechanisms underlying persister formation and antibiotic tolerance remain insufficiently characterized. In this study, the Escherichia coli Keio library was used to identify genes involved in ciprofloxacin tolerance by culturing each mutant to the late stationary phase (to induce persistence via starvation), followed by dilution into fresh medium for antibiotic exposure. This two-step, genome-wide screening approach enabled the identification of 37 ciprofloxacin-sensitive mutants with diverse biological functions and 11 ciprofloxacin-tolerant mutants related to amino acid and β-nicotinamide adenine dinucleotide (NAD⁺) biosynthesis, with 25 genes being identified as persister-related genes for the first time. Notably, sensitive mutants (ΔatpC, ΔatpF, ΔruvC, and Δrnr) were specifically sensitive to quinolone antibiotics, whereas tolerant mutants (ΔmetR, ΔleuB, and ΔnadB) showed tolerance to ampicillin and gentamicin. Importantly, adenosine triphosphate (ATP) levels were downregulated in ciprofloxacin-tolerant mutants and upregulated in ciprofloxacin-sensitive mutants, implying a negative correlation between ATP levels and ciprofloxacin tolerance among these genetically distinct persisters. This negative correlation was further observed when ATP levels in different mutants were chemically modulated using specific metabolites, nutrients, and respiration inhibitors. In addition, ciprofloxacin persistence across different mutants was found to correlate closely with antibiotic uptake and reactive oxygen species (ROS) levels. Collectively, these findings establish a universal role for ATP in the ciprofloxacin tolerance of genetically diverse persisters under varying resuscitation conditions, conceivably through the modulation of antibiotic uptake and ROS accumulation, and it is implied that the provision of abundant nutrients is potentially beneficial for anti-persister chemotherapy in clinic settings.

The development of safe and effective radioprotective agents with minimal side effects, particularly for high-dose exposure, remains a global priority. E0703, a novel steroidal compound structurally derived from estradiol, has shown promising radioprotective efficacy with limited estrogenic activity in prior pharmacodynamic studies. In this study, E0703 was found to significantly increase the abundance of Akkermansia muciniphila (AKK) in the intestines of both irradiated and non-irradiated mice. Co-administration of E0703 and AKK markedly improved the 7-day survival rate of mice exposed to a lethal 8.5 Gy dose of radiation. E0703 induced beneficial transcriptional changes in AKK, with enrichment in metabolic pathways such as amino acid biosynthesis, aminoacyl-tRNA biosynthesis, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis. These alterations supported the production of glucosamine 6-phosphate (GlcN-6-P) by AKK, which contributed to intestinal tissue regeneration following irradiation. Single-cell transcriptomic analysis revealed that E0703 significantly increased the proportion of intestinal stem cells and goblet cells by Day 5 post irradiation. Mechanistically, E0703 modulated the oxidative phosphorylation pathway in these cell types, including regulation of Muc2 production. E0703 also enhanced AKK abundance in irradiated mice, particularly in the presence of mucin, thereby elevating the availability of GlcN-6-P—a critical substrate for intestinal organoid repair. These findings indicate that E0703 exerts direct effects on goblet cells and AKK, promoting host–microbe interactions that facilitate intestinal regeneration and improve survival following radiation exposure.

Central carbon metabolism is thought to link reactive oxygen species (ROS) with antibiotic-mediated bacterial death. During enrichment screening of Escherichia coli with the first-generation quinolone oxolinic acid, unstable antibiotic-tolerant mutants containing deficiencies in purB were obtained. Examination of a stable deletion mutant of purA, a gene functionally related to purB, revealed reduced lethality of oxolinic acid and ciprofloxacin. This deletion mutation had little effect on the minimal inhibitory concentration (MIC) of quinolones, thereby demonstrating that the observed protection from killing was attributable to antibiotic tolerance. AMP synthesis was blocked by the ΔpurA mutation, and ciprofloxacin tolerance was reversed by exogenous AMP supplementation. Because AMP is a precursor of ATP, interference with ATP synthesis occurs in the ΔpurA mutant. RNA-Seq analysis showed that, prior to antibiotic stress, transcript levels of NADH:quinone oxidoreductase genes were reduced by the purA deficiency, thereby predisposing E. coli to antibiotic tolerance through reduced respiration. During ciprofloxacin exposure, the purA deficiency also suppressed the surge in expression of tricarboxylic acid (TCA) cycle and ATP synthesis genes, as well as the accumulation of intracellular ATP and ROS. Thus, wild-type PurA, and by extension the downstream enzyme PurB, directs AMP toward an antibiotic-mediated, ROS-dependent death pathway. Overall, defects in PurA/PurB-mediated adenosine ribonucleotides de novo biosynthesis reveal a novel quinolone tolerance mechanism that is initiated outside central carbon metabolism; tolerance is likely attributable to a limited supply of AMP, resulting in reduced ATP synthesis and suppression of ROS accumulation.

Rapid identification of bacterial species from patient samples is crucial for clinical decision-making. In severe infections, such as bloodstream infections, the early start of an effective treatment is directly associated with reduced mortality rates. Current rapid species identification methods, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) or multiplex PCR, require specialized hardware and extensive technical support that prevents application in resource-limited settings. Here, we present a staining and imaging procedure for bacterial smears using fluorescent dyes directed against intracellular structures and cell wall components. Data on relevant features were extracted from segmented images and used to train a machine learning (ML) model for species classification. The method was tested on clinical isolates from 126 patients. For the seven most common bacteria, the classification performance, indicated by area under the receiver operating characteristic (ROC) curve, ranged from 0.8 (Klebsiella pneumoniae) to 1 (Pseudomonas aeruginosa). Species that were not part of the training dataset, were reliably classified as unknown species. These results hold promise for the identification of further species, particularly Enterobacterales, and clinical application.

Histone modifications and chromatin-binding proteins play crucial roles in regulating gene expression in eukaryotes, with significant implications for fungal pathogenicity and development. However, profiling these modifications or proteins across the genome in fungi remains challenging due to the technical limitations of the traditional, widely used Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) method. Here, we present an optimized fungal Cleavage Under Targets and Tagmentation-Sequencing (fCUT&Tag-Seq) protocol specifically designed for filamentous fungi and dimorphic fungi. Our approach involves the preparation of protoplasts and nuclear extraction to enhance antibody accessibility, along with formaldehyde crosslinking to improve protein-DNA binding efficiency. We then successfully applied fCUT&Tag-Seq to accurately profile multiple histone modifications like H3K9me3, H3K27me3, H3K4me3, and H3K18ac, across different plant pathogenic or model fungal species, including Verticillium dahliae, Neurospora crassa, Fusarium graminearum, and Sporisorium scitamineum, showing good signal-to-noise ratios, reproducibility, and detection sensitivity. Furthermore, we extended this method to profile chromatin-binding proteins, such as the histone acetyltransferase Gcn5. This study establishes fCUT&Tag-Seq as a robust and useful tool for fungal epigenetic research, enabling detailed exploration of chromatin dynamics and advancing our understanding of fungal gene regulation, development, and pathogenicity.