RNA secondary structure plays a critical role in gene regulation, yet existing computational and experimental tools for structure analysis are often fragmented across prediction, ensemble modeling, and functional interpretation workflows. Here, we present ShapeRNA, a user-friendly web server for integrated RNA secondary structure prediction, ensemble inference, and structure-aware regulatory annotation. ShapeRNA supports three complementary analytical workflows, including sequence-based structure prediction, reactivity-guided modeling using SHAPE or DMS data, and sequencing–guided ensemble inference from high-throughput probing experiments. The platform integrates multiple established prediction algorithms and provides standardized data processing, ensemble clustering, and visualization. In addition, ShapeRNA enables mapping of RNA modification sites, microRNA target regions, and RNA-binding protein interaction motifs onto predicted RNA structures and representative ensemble conformations. We demonstrate the utility of ShapeRNA through applications including analysis of mutation-associated structural changes in MAPT exon 10, characterization of conformational heterogeneity in the HIV-1 Rev Response Element, and regulatory annotation of the oncogenic long non-coding RNA HULC. ShapeRNA provides an accessible and extensible platform for investigating RNA structural heterogeneity and regulatory mechanisms. This website is free and open to all users, and there is no login requirement. https://shaperna.com.

Precise, site-specific insertion of large gene sequences holds great promise for the treatment of diverse genetic disorders. Although prime editing using paired guide RNAs (pegRNAs) can mediate targeted integration, insertion efficiency drops sharply for payloads exceeding 300 base pairs. Here we present a rationally designed quadruple pegRNA strategy (QuadPE) for efficient and programmable insertion of large DNA fragments. Through screening different designs, we identified that combinations of two genome-targeting pegRNAs in a PAM-out or PAM-in orientation, when paired with two donor-targeting pegRNAs in linear or circular form, yield optimal efficiency. Using QuadPE, we achieved stable integration efficiency of DNA fragments ranging from 1.6 to 26 kb, with efficiencies of around 40% at multiple loci with minimal off-target insertion activity. QuadPE substantially outperformed recombinase-mediated (PASSIGE and PASTE) and transposase-mediated (CAST) insertion systems, particularly for larger payloads, showing a 11-fold, 61-fold and 12-fold improvement for a 9.5 kb insertion, respectively. Notably, QuadPE was effective in both dividing and non-dividing primary cells such as human primary T cells and post-mitotic neurons, establishing QuadPE as a powerful and precise platform for large-fragment gene insertion without the need for double-stranded breaks or recombinases. QuadPE—a quadruple paired prime editing guide RNA strategy—enables efficient and programmable insertion of large DNA fragments.

Metagenomic sequencing has transformed virus discovery; however, downstream bioinformatic analyses for viral identification, classification, and host prediction remain fragmented across multiple tools. Here, we present PhaBOX2, a major upgrade that extends the platform from a specialized bacteriophage identification tool to a comprehensive and integrated suite for viral sequence analysis. PhaBOX2 broadens its detection, taxonomic, and host prediction scope beyond phages to enable the characterization of archaeal and eukaryotic viruses. The updated workflow incorporates rigorous quality control and quantitative analyses, automatically removes host contamination, clusters sequences into viral operational taxonomic units, and performs phylogenetic analysis based on marker genes. In contrast to traditional “black–box” deep learning approaches, PhaBOX2 combines alignment–based strategies with machine–learning models under a “glass–box” design philosophy, providing interpretable intermediate evidence alongside final predictions to improve transparency and biological interpretability. Powered by a dedicated high–performance computing infrastructure, the server delivers a fully automated, end–to–end workflow, while achieving an ~80% reduction in processing time. PhaBOX2 thus provides a robust and user–friendly ecosystem for viral metagenomic analysis and is freely available at https://phage.ee.cityu.edu.hk/.

Soils are critical reservoirs of antibiotic-resistance genes (ARGs) which are strongly shaped by microbial interactions and environmental conditions and are therefore highly sensitive to disturbance. Although climate warming is recognized as one of the most significant disturbances to microbial communities and their functions, its impacts on soil resistomes remain poorly understood. Here we investigated the effects of decade-long experimental warming on ARGs in grassland soils using integrated experimental and computational approaches. Our results revealed that ARG abundance substantially increased (23.9%) under warming—particularly glycopeptide- and rifamycin-resistance genes. Warming specifically enriched Actinomycetota hosts, including various potential plant pathogens, and enhanced ARG mobility. Large-scale unprecedented isolates-based phenotypic analyses also validated that warming increased bacterial resistance to multiple antibiotics. Further mechanistic analyses revealed that warming increased ARG abundance primarily through co-selection of resistance genes physically linked to adaptive traits (for example, thermal tolerance and nitrogen assimilation) and positive selection for thermal tolerance genes, which could be further amplified via horizontal gene transfer. Together, these findings convincingly demonstrate that climate warming substantially accelerates soil antibiotic resistance at genomic, ecological and evolutionary levels, with broad implications for public health and environmental sustainability in a warming world. Decade-long warming increased soil antibiotic-resistance genes by about 24%, enriching resistant microbes and gene transfer, driven by selection for thermal tolerance and linked traits, with implications for public health.

Bacterial pathogens adapt rapidly to clinical and within-host selective pressures. Insertion sequences (IS) are transposable elements that can contribute to pathogenic adaptation, but their activity and consequences in contemporary clinical populations are not well characterized. Here, combining large-scale genomic surveys with long-read sequencing of clinical isolates and longitudinal gut metagenomes, we quantify pathogen IS dynamics from global patterns to within-host evolution. Across 19,485 publicly available high-contiguity ESKAPEE pathogen genomes, Enterococcus faecium genomes are the most IS dense, dominated by replicative ISL3 family elements, which have proliferated in clinical lineages over the past 30 years. We find extensive chromosomal structural variation, largely involving ISL3, within a new single-hospital collection of bloodstream isolates. Long-read metagenomic sequencing of 28 longitudinal stool samples from 12 haematopoietic cell transplantation (HCT) recipients demonstrates within-host IS dynamics and their regulatory consequences. In one patient, an ISL3 insertion upstream of a folate transporter formed a strong promoter, increasing transcription and improving relative fitness under folate limitation. Enhanced folate scavenging may enable E. faecium to thrive in the setting of microbiome collapse, which is common in HCT and other critically ill patients. Together, these results show that a recent ISL3 expansion is driving rapid evolution in healthcare-associated E. faecium, with consequences for its metabolic fitness that may help explain its increasing clinical burden. Several other pathogens also show elevated IS loads in our survey, which suggests that IS expansion-mediated evolution might be more broadly relevant. Over three decades, rapid expansion of the transposable element ISL3 has reshaped Enterococcus faecium, which helps to explain this pathogen’s growing clinical threat.

Phytoplankton are responsible for approximately half of Earth’s net primary production and, together with heterotrophic bacteria—the main consumers of organic matter—play a pivotal role in biogeochemical cycles. Their key ecological importance has led to growing interest in the interactions between these two groups. Yet, our understanding of the microscale mechanisms driving these interactions remains limited. Recent work highlighted the contribution of bacterial motility and chemotaxis to promoting encounters and nutrient exchange between bacteria and phytoplankton. In contrast, the ecological role of bacterial attachment—an important adaptation enabling bacteria to establish the closest contact with their phytoplankton host and retain it over extended periods of time—remains less explored. Here we describe the current evidence and understanding of bacterial attachment to phytoplankton and highlight recent insights from single-cell studies. Motivated by the implications for large-scale ecosystem processes, we discuss promising research avenues to further unveil the ecological relevance of bacterial attachment to phytoplankton. Here the authors review current understanding of the mechanistic marine microbial interactions that underpin large-scale ecosystem processes and biogeochemical cycling.

Mutualism in the symbiosis between arbuscular mycorrhizal fungi and plants is based upon the exchange of carbon for soil minerals, with phosphate being of central importance. The exchange of nutrients occurs when the fungus transiently colonizes root cells, producing hyphal structures called arbuscules. The movement of phosphate from fungus to plant is well established, however its coordination and regulation at the ephemeral arbuscules remains elusive. Here, non-invasive imaging captures the complete growth and collapse of the arbuscules in unprecedented resolution, revealing heterogeneity in arbuscule development. Tracking the dynamics of rice PHosphate Transporter 1;11 (OsPHT1;11/ PT11) as a proxy for symbiotic phosphate transport shows consistent localization across diverse arbuscules. However, we uncover phosphate-responsive variability in PT11 abundance, representing an essential, cellular-level layer of nutrient regulation. Such plasticity in arbuscule phosphate uptake capacity evidences uncoupling of arbuscule presence and arbuscule function, thereby demonstrating that arbuscules are not identical units of nutrient exchange. This study uncovers the previously-hidden dynamics of nutrient exchange structures central to the symbiosis between plants and arbuscular mycorrhizal fungi, revealing highly variable development, lifespans and phosphate transport capacities.