Animal venoms constitute a rich source of bioactive peptides and proteins with high target specificity, representing valuable scaffolds for therapeutic development. However, the biotechnological exploitation of venom-derived toxins is limited by challenges in achieving efficient, scalable, and reproducible production. Native venom extraction is constrained by low yields and biological variability, making recombinant platforms essential. Yet, most venom toxins are cysteine-rich peptides with complex disulfide bond architectures and stringent structure–function relationships, posing significant challenges to heterologous expression. Inefficient folding, proteolysis, and secretion bottlenecks frequently compromise functional yield. Among microbial hosts, Komagataella phaffii has emerged as a robust system combining eukaryotic protein processing with high cell-density fermentation and cost-effective cultivation. Its oxidative secretory pathway, strong and regulatable promoters, and suitability for strain engineering make it particularly attractive for producing disulfide-rich toxins. This review provides a critical analysis of recombinant venom toxin production in K. phaffii, focusing on molecular and bioprocess determinants of expression performance. We discuss post-translational modifications, yields, and bioactivity, as well as promoter selection and secretion signal optimization. By integrating data across toxin families, we identify recurring technical bottlenecks and highlight engineering approaches to enhance venom biomanufacturing within microbial biotechnology frameworks.

The human gut microbiome is shaped by diverse selective forces that originate from host and environmental factors and it substantially influences health and disease. Whereas the association of microbial lineages with various health conditions has been shown at different taxonomic levels, the extent to which unifying adaptive mechanisms sort microbial lineages into ecologically differentiated populations remains poorly understood. Here we show that genome-wide selective sweeps are a pervasive mechanism that differentiates bacteria in the microbiome. This mechanism leads to population structures akin to global epidemics across geographically and ethnically diverse human populations. Such sweeps arise when an adaptation allows a clone to outcompete others in its niche followed by rediversification, and they manifest as clusters of closely related genomes on long branches in phylogenetic trees. This structure is revealed by excluding recombination events that mask the clonal descent of the genomes. Indeed, we show that genome-wide sweeps originate under a wide range of recombination rates in at least 66 taxa from 25 bacterial families. Estimated ages of divergence suggest that sweep clusters can spread globally within decades and that this process has occurred throughout human history. Sweep clusters are associated with different host conditions—such as age, colorectal cancer, inflammatory bowel diseases and type 2 diabetes—as an indication of their ecological differentiation. Our results reveal an evolutionary mechanism for the observation of stably inherited strains with differential associations and provide a theoretical foundation for analysing adaptation among microbial populations. Genome-wide selective sweeps commonly occur in the human gut microbiome and can spread across the world within decades to produce epidemic-like population structures.

Urea, the world’s predominant nitrogen fertilizer, has supported human population growth for the past 60 years, yet its effects on freshwater ecosystems are largely unknown. Here urea additions at ecologically relevant rates tripled summer phytoplankton abundance in replicate agricultural reservoirs with significant responses by most eukaryotic algae, but not cyanobacteria or their toxins. Mass budgets reveal that fertilized reservoirs did not become limited by phosphorus due to its continuous release from sediments. Further, most added nitrogen did not accumulate in reservoirs but was lost to the atmosphere, probably as NH3. Sub-continental spatial analysis shows that study reservoirs are characteristic of shallow water bodies within Canada’s largest agricultural region, where >40% of surface waters are vulnerable to degradation by urea. Similar degrees of water quality loss by urea are expected in other global agricultural regions (for example, China, India, North America) where elevated urea use interacts with phosphorus-rich surface waters to induce extreme eutrophication. Urea, the most widely used nitrogen fertilizer, has unknown impacts on freshwater ecosystems. This study demonstrates that urea additions in Canadian prairie agricultural reservoirs triple phytoplankton abundance without increasing cyanobacterial toxins, revealing considerable nitrogen loss to the atmosphere and highlighting potential global water quality degradation in phosphorus-rich agricultural regions.

Transcription factors (TFs) regulate gene expression by binding to specific DNA sites on genome, making accurate TF binding site prediction critical for understanding gene regulation and downstream phenotypes. Almost all current deep learning methods use only DNA-related information to predict TF binding sites, ignoring the fact that different TF protein sequences and structures recognize distinct DNA patterns. Not leveraging TF information not only limits prediction accuracy but also makes the methods not generalizable to predicting binding sites of new TFs that do not exist in the training data. Here, we present TransBind, a protein-aware deep learning architecture that integrates DNA sequence information with protein embeddings containing both sequence and structural information derived from a protein language model pretrained on DNA-binding proteins, to improve TF binding site prediction. Through the cross-attention, a TF embedding selectively attends to genomic regions according to its unique binding properties. Evaluated on the data of 690 ChIP-seq experiments spanning 161 TFs across 91 human cell types, TransBind achieves an AUROC of 0.9508 and AUPR of 0.3741—representing a 11.8% relative AUPR improvement over state-of-the-art methods including TBiNet, EPBDXDNABERT-2, DanQ, and DeepSEA. The model outperformed existing methods in 98% of TF–cell type combinations. It also recovered 160 known TF binding motifs in the JASPAR database, providing the biological interpretability of the model. Moreover, the approach enables label-zero-shot prediction for unseen TFs, demonstrating its potential of generalizing to new, poorly characterized TFs. The source code of TransBind is available at https://github.com/jianlin-cheng/TransBind. The version used in this work is archived at https://doi.org/10.5281/zenodo.19462292.

Optogenetic tools—light-responsive proteins that enable to regulate specific cellular activities, study biological processes, and develop new therapies—are attractive approaches for achieving endogenous gene regulation under minimally invasive conditions. Our first step in constructing an optogenetic system to regulate endogenous Drosophila gene expression was to identify inhibitory anti-CRISPR (Acr) proteins that block CRISPRa-mediated activation. Next, we inserted optogenetic protein LOV2 into these Acrs, tested for their ability to optogenetically modulate endogenous gene upregulation through the CRISPRa-based flySAM system in Drosophila, and found that the photoswitchability of these prototypes was weak. We therefore engineered an optimized Acr–LOV2 fusion module by refining length of intrinsically disordered and ordered regions (IDR and IOR) of Acrs. This optimization yielded a variant with significantly greater sensitivity to blue-light-induced endogenous gene upregulation than the prototypes, leading to new in vivo discoveries. In addition, this work provides insights for in vivo functional characterization of the IDR and the IOR of these small-sized proteins. Together, these findings establish a robust optogenetic toolbox for precise, light-controlled endogenous gene regulation in Drosophila.

Selectively eradicating target cells on the basis of their genetic or transcriptional identity remains important in basic research, medicine, biotechnology and agriculture. For applications involving bacteria, CRISPR nucleases offer promising options due to their ability to enact RNA-guided counterselection; however, using these same nucleases for counterselection in eukaryotes has proven much more restrictive. Here we show that Cas12a2, a recently discovered type V CRISPR nuclease, exhibits RNA-triggered DNA shredding and enables programmable and sequence-specific elimination of yeast and human cells expressing a target transcript. Triggering Cas12a2 elicits rampant double-stranded DNA breaks in trans, leading to cell death. Cell killing can be activated by a wide range of target transcripts, with no observed off-target activation. Leveraging thist approach, we selectively eliminate cells that harbor human papillomavirus, cells that failed to undergo gene editing, or cells that encode a prevalent oncogenic point mutation in KRAS. These findings expand the CRISPR toolbox to allow the selective elimination of eukaryotic cells on the basis of their transcriptional profile. Cas12a2 enables RNA-triggered, sequence-specific killing of eukaryotic cells via widespread DNA shredding, allowing selective elimination of cells on the basis of gene expression, including virus-infected or mutation-bearing cells.

Identifying genomic regions bound by individual proteins such as transcription factors is essential to understanding bacterial gene regulation; however, comprehensive understanding of the effect of protein occupancy on gene regulation would be prohibitively laborious and expensive to achieve using methods such as chromatin immunoprecipitation with sequencing (ChIP-seq) and ChIP with exonuclease treatment (ChIP-exo) for every protein and condition of interest. Here we describe a protocol for performing in vivo protein occupancy display–high resolution (IPOD-HR), a powerful method for genome-wide profiling of protein-bound DNA in prokaryotic systems. Although assay for transposase-accessible chromatin with sequencing (ATAC-seq) is the method of choice for assaying general protein occupancy in eukaryotic systems, bacterial nucleoid-associated proteins can affect ATAC-seq, rendering it unsuitable for use in bacteria. In contrast, IPOD-HR can be used to identify regions of bacterial genomes that are highly bound by proteins, regardless of the identity of the proteins bound, allowing the identification of condition- and genotype-dependent changes in protein occupancy associated with changes in gene regulation. The technique is coupled to RNA polymerase ChIP, followed by sequencing of the extracted samples and downstream analysis using open-source, automated software that we provide and actively maintain. Once cross-linked samples are obtained, the core DNA selection portion of the IPOD-HR protocol takes 3 calendar days to perform. The resulting DNA extracts are subjected to high-throughput sequencing, resulting in sequencing data that are analyzed, which typically requires a few additional days, depending on the number of samples and computing resources. The IPOD-HR experimental method requires familiarity with standard molecular biology techniques suitable for preparing Illumina sequencing inputs, and the computational post-processing pipeline requires basic knowledge of the Linux command line environment. This protocol presents IPOD-HR, a high-resolution approach for profiling genome-wide protein occupancy in bacteria, supported by a computational pipeline for streamlined downstream analysis.

The clinical success of gene therapy depends critically on the development of delivery platforms capable of achieving precise, efficient and tissue-specific delivery of genetic payloads in vivo. A diverse array of carriers, including viral, non-viral, synthetic and natural vectors, have been explored to address this challenge. Among them, adeno-associated viruses, lipid nanoparticles and extracellular vesicles have emerged as leading candidates, each offering distinct advantages and translational hurdles. Here we provide a comparative analysis of these delivery modalities, highlighting their respective design principles, targeting capabilities, immunogenicity profiles and clinical progress. We survey preclinical and clinically adopted delivery strategies and explore how the three delivery platforms can be tailored for gene therapeutics in different diseases. Finally, we discuss emerging strategies to overcome current limitations and outline future directions for the rational design of next-generation gene delivery platforms that combine safety, scalability and functional precision. This Review offers a comparative analysis of platforms for the tissue-specific delivery of genetic payloads, discusses how carriers such as adeno-associated viruses, lipid nanoparticles and extracellular vesicles can be tailored for use in different diseases, and charts a path for engineering improved platforms for clinical use.

Nanoplastics (NPs), as persistent and unregulated aquatic contaminants, evade conventional removal technologies, posing severe health risks and urgently demanding innovative, effective solutions. Here we introduce recyclable magnetic biohybrid nanonets (LAF-IONPs), engineered through in situ growth of iron oxide nanoparticles (IONPs) on lysozyme amyloid fibrils (LAFs). Stabilized by synergistic interfacial interactions, LAF-IONPs efficiently capture NPs over a wide range of sizes (30–1,000 nm) and chemical compositions across environmentally relevant concentrations and conditions (pH 7–9, high salinity and co-existing pollutants). The superior removal efficiency arises from magnetic active motion and abundant fibril binding sites. Remarkably, LAF-IONPs achieved 98.0–99.9% NPs removal from various real water samples and maintained >95% efficiency over 100 recycling cycles when using a custom alternating magnetic field system. Critically, LAF-IONPs treatment reduced in vivo NPs bioaccumulation by 91.5%. This work establishes a blueprint for designing recyclable biohybrid adsorbents for active, efficient and sustainable removal of NPs and potentially other emerging contaminants. Nanoplastics are persistent water contaminants that evade conventional removal methods. Magnetic biohybrid nanonets are found to capture up to 99.9% of them, remain highly recyclable and greatly reduce in vivo accumulation.

Elucidating protein–ligand interactions is pivotal for understanding biological mechanisms and accelerating drug discovery. Blind docking, which identifies binding sites without prior knowledge, has become an indispensable computational strategy for analyzing the surge of protein structures generated by Cryo-EM and AI-based prediction tools like AlphaFold3. Our previous server, CB-Dock2, has been widely adopted by the global research community, averaging over 1000 daily submissions since July 2022 due to its accuracy and user-friendliness. Building on this foundation and incorporating extensive user feedback, we present CB-Dock3, a substantially enhanced platform. Key upgrades include a refined docking engine, an expanded template library, and support for diverse file formats. Benchmark evaluations on CASF-2016 demonstrate that CB-Dock3 achieves a success rate of 67.4% (RMSD ≤ 2.0 Å), representing a 10.6 percentage-point absolute improvement over its predecessor and outperforming other popular blind docking tools. Additionally, CB-Dock3 introduces critical new features driven by community needs: support for user-defined docking regions to handle large complexes, and a metal-aware protocol that explicitly retains essential metal ions and cofactors during simulation.