Steroids are among the most valuable and widely used pharmaceuticals. The cholesterol side-chain cleavage enzyme (P450scc) is critical for steroid metabolism and hormone biosynthesis. While mammalian eukaryotic P450scc enzymes are well-characterized, bacterial counterparts remain underexplored despite their industrial promise and potential contributions to bacterial steroid catabolism. Here, we identify a series of CYP204 family P450 enzymes, widely distributed across diverse steroid-degrading bacterial species, that catalyze the side-chain cleavage of cholesterol, phytosterol, and cholestenone to produce pregnenolone and progesterone. Unlike mammalian enzymes, which exhibit strict cholesterol specificity, bacterial P450scc enzymes display relaxed substrate specificity, preferentially converting cholestenone to progesterone—a key precursor in steroid drug semi-synthesis. Structural and mechanistic analyses demonstrate that CYP204 enzymes employ a flexible, dual-regioselective C–H activation mechanism distinct from the sequential hydroxylation of mammalian P450scc enzymes. Iterative saturation mutagenesis identified critical residues for side-chain cleavage, improving catalytic efficiency up to 6.5-fold, and computational analyses clarified sequence–function relationships. This finding of bacterial P450scc enzymes not only underscores their potential function in bacterial steroid catabolism but also lays a foundation for promising biocatalytic strategies for pregnenolone and progesterone synthesis. Genome mining identified bacterial P450 that cleave cholesterol side-chains via dual C-H activation, differing from mammalian P450scc. Engineered CYP204A5 efficiently converts cholestenone to progesterone for biocatalysis.

Self-assembly is a fundamental property of living matter that drives the three-dimensional organization of cell collectives such as tissues and organs. Here, the co-assembly of synthetic and natural cells is leveraged to create hybrid living 3D cancer cultures. We screen a range of synthetic cell models for their ability to form augmented tumoroids with artificial but controllable micro-environments, and show that the balance of inter- and extracellular adhesion and synthetic cell surface tension are key material properties driving integrated co-assembly. We demonstrate that synthetic cells based on droplet-supported lipid bilayers can establish artificial tumor immune microenvironments (ART-TIMEs), mimicking immunogenic signals within tumoroids and eliminating the need to integrate complex living immune cells. Using the ART-TIME approach, we identify a AhR-ARNT-mediated co-signaling mechanism between PD-1 and CD2 as a driver in immune evasion of pancreatic ductal adenocarcinoma. Our study advances the field of hybrid organoid engineering, offers opportunities for the construction and modelling of artificial tumour environments, and marks a step towards the design of functional living/non-living cytomimetic materials. Synthetic cells have huge potential in model systems. Here, the authors engineer synthetic–living hybrid tumoroids that replicate tumour-immune interactions in 3D, study synthetic cells integration, and demonstrate systematic studies of immune evasion and T cell engager therapies.

Transcription factors regulate gene expression by binding specific DNA motifs, yet only a fraction of putative sites is occupied in vivo. Intrinsically disordered regions have emerged as key contributors to promoter selectivity, but the underlying mechanisms remain incompletely understood. Here, we use single-molecule optical tweezers to dissect how disordered regions influence DNA binding by Msn2, a yeast stress-response regulator. We show that these regions power a search mechanism, facilitating initial non-specific association with DNA and promoting one-dimensional scanning toward target motifs, supported by charge-mediated interactions. Remarkably, this mechanism displays sequence sensitivity, with promoter-derived sequences enhancing both initial binding and scanning rates, demonstrating that Msn2–DNA interactions alone are sufficient to confer promoter selectivity in the absence of chromatin or cofactors. Our findings provide direct mechanistic evidence for how intrinsically disordered regions tune transcription factor search dynamics for Msn2 and expand sequence recognition beyond canonical motifs, supporting promoter selectivity in complex genomic contexts. The study reveals how intrinsically disordered regions enable a yeast transcription factor to locate and selectively bind its target promoters by promoting DNA association outside its motif and sequence-dependent search dynamics.

Extraction of plant genomic DNA is a critical step for PCR-based genotyping, mapping, and breeding applications. Conventional CTAB protocols and commercial kits provide reliable DNA but are labour-intensive, costly, and generate substantial plastic waste. Simplified crude-extract methods are available, yet their performance is often compromised by PCR inhibition from salts and cellular debris. A rapid, low-cost, and high-throughput method is therefore needed for routine molecular applications. We developed a single-tube DNA extraction protocol that eliminates supernatant transfers, thereby reducing handling errors, plastic consumption, and processing time. The method consistently produces DNA of sufficient yield and purity for PCR-based assays. Validation in wheat and wheat–wild relative introgression lines demonstrated robust amplification in KASP assays. Cross-species testing in maize, Arabidopsis, and tomato using two Tris-salt extraction buffers confirmed broad applicability, supported by NanoDrop and Qubit measurements. Freeze-dried and frozen tissue produced higher yields than fresh samples, confirming their suitability for high-throughput and large-scale studies. This streamlined protocol provides a cost-effective, reliable, and scalable approach for extracting plant genomic DNA suitable for PCR-based genotyping, marker development, and diversity analysis. Its simplicity and throughput make it particularly valuable for breeding programmes, although it is not intended for applications requiring highly pure DNA, such as whole-genome resequencing.

Microbial translation arrest peptides monitor intracellular environments and feedback-regulate downstream gene expression. Previous studies have identified a class of bacterial arrest peptides with C-terminal RAPP-like sequences, encoded upstream of genes involved in protein localization. In this study, we found that among RAPP-like sequences, RAPP (Arg-Ala-Pro-Pro) and RGPP (Arg-Gly-Pro-Pro) could more readily evolve into translation-impeding sequences with a particularly robust arrest that is refractory to EF-P. RAPP-like motifs were found to be strongly excluded from bacterial proteomes, likely reflecting the risk of disrupting the cellular translation system. Meanwhile, these motifs tended to occur near the C-terminus of relatively small secretory and membrane proteins. Notably, they were encoded upstream of genes with diverse functions beyond protein localization. Indeed, we identified seven RAPP/RGPP-containing arrest peptides from Streptomyces lividans encoded upstream of genes with diverse functions. These findings illustrate the bidirectional evolution of RAPP-containing proteins: their elimination from bacterial proteomes and their adaptation into arrest peptides with various regulatory roles.

Drug-resistant bacterial infections, exacerbated by antibiotic resistance and biofilm resilience, disrupt tissue repair through dysregulated inflammation and impaired regeneration. Neutrophil extracellular traps (NETs) play a crucial role in endogenous immunity by entrapping and eliminating pathogens, inspiring the development of synthetic biomaterials that replicate this function. However, current synthetic NETs face challenges in complexity, biocompatibility, structural integrity and effectiveness. Here, we present a NETs-mimicking hydrogel composed of reversible lysozyme amyloid flexible nanofibrils (FFs) enabling pathogen elimination and tissue regeneration. The FFs therein self-assemble from natural egg-white lysozyme endowing these nanoNETs with bioactivity against pathogens, and when duly labeled to respond to near-infrared irradiation, they disassemble into unfolded lysozyme monomers with antimicrobial activity. Notably, the hydrogel disassembly is followed by the controlled release of pre-dissolved Mg²⁺ ions, reprogramming macrophages toward a pro-regenerative phenotype and mitigating inflammation. In both murine and porcine models, these biocompatible nanoNETs demonstrate excellent antibacterial performance, accelerating healing of wounds infected by methicillin-resistant Staphylococcus aureus (MRSA). Moreover, these nanoNETs boost in-vivo healing of MRSA-infected periprosthetic joints, preserving osteogenic and regenerative microenvironments. These results build on the reversible nature of flexible amyloids to introduce stimuli-responsive biocompatible nanoNETs with significant potential for antimicrobial and regenerative therapies in bacterial-resistant infections. Drug-resistant bacterial infections hinder tissue repair and regeneration. Here, authors present a lysozyme nanofibril-based hydrogel that mimics neutrophil extracellular traps, enabling pathogen elimination and promoting tissue regeneration.

DNA methylation is a crucial epigenetic mechanism that regulates gene expression. Precise editing of DNA methylation has emerged as a promising tool for dissecting its biological function. However, challenges in delivery have limited most applications of DNA methylation editing to in vitro systems. Here, we develop two transgenic mouse lines harboring an inducible dCas9-DNMT3A or dCas9-TET1 editor to enable tissue-specific DNA methylation editing in vivo. We demonstrate that targeted methylation of the Psck9 promoter in the liver of dCas9-DNMT3A mice results in decreased Pcsk9 expression and a subsequent reduction in serum low-density lipoprotein cholesterol level. Targeted demethylation of the Mecp2 promoter in dCas9-TET1 mice reactivates Mecp2 expression from the inactive X chromosome and rescues neuronal nuclear size in Mecp2+/- mice. Genome-wide sequencing analyses reveal minimal transcriptional off-targets, demonstrating the specificity of the system. These results demonstrate the feasibility and versatility of methylation editing, to functionally interrogate DNA methylation in vivo. Precise editing of DNA methylation has emerged as a promising tool in disease biology but most applications are limited to in vitro systems. Here, we develop two transgenic mouse lines harboring an inducible dCas9-DNMT3A or dCas9-TET1 editor to enable tissue-specific DNA methylation editing in vivo.

The incidence of cardiometabolic diseases is increasing globally, and both poor diet and the human gut microbiome have been implicated. However, the field lacks large-scale, comprehensive studies exploring these links in diverse populations. Here, in over 34,000 US and UK participants with metagenomic, diet, anthropometric and host health data, we identified known and yet-to-be-cultured gut microbiome species associated significantly with different diets and risk factors. We developed a ranking of species most favorably and unfavorably associated with human health markers, called the ‘ZOE Microbiome Health Ranking 2025’. This system showed strong and reproducible associations between the ranking of microbial species and both body mass index and host disease conditions on more than 7,800 additional public samples. In an additional 746 people from two dietary interventional clinical trials, favorably ranked species increased in abundance and prevalence, and unfavorably ranked species reduced over time. In conclusion, these analyses provide strong support for the association of both diet and microbiome with health markers, and the summary system can be used to inform the basis for future causal and mechanistic studies. It should be emphasized, however, that causal inference is not possible without prospective cohort studies and interventional clinical trials. Comprehensive large-scale studies of multi-national populations identified microbiome species consistently associated with favorable and unfavorable health markers, informing future studies of the human gut microbiome and its association with diet and cardiometabolic conditions.

Fluorescent pseudomonads catabolize purines via uric acid and allantoin, a pathway whose end-product is glyoxylate. In this work, we show that in Pseudomonas aeruginosa strain PAO1, the ORFs PA1498–PA1502 encode a pathway that converts the resulting glyoxylate into pyruvate. The expression of this cluster of ORFs was stimulated in the presence of allantoin, and mutants containing transposon insertions in the cluster were unable to grow on allantoin as a sole carbon source. The likely operonic structure of the cluster is elucidated. We also show that the purified proteins encoded by PA1502 and PA1500 have glyoxylate carboligase (Gcl) and tartronate semialdehyde (TSA) reductase (GlxR) activity, respectively, in vitro. Gcl condenses two molecules of glyoxylate to yield TSA, which is then reduced by GlxR to yield d-glycerate. GlxR displayed much greater specificity (kcat/KM) for Gcl-derived TSA than it did for the TSA tautomer, hydroxypyruvate. This is relevant because TSA can potentially spontaneously tautomerize to yield hydroxypyruvate at neutral pH. However, kinetic and [1H]-NMR evidence indicate that PA1501 (which encodes a putative hydroxypyruvate isomerase, Hyi) increases the rate of the Gcl-catalysed reaction, possibly by minimizing the impact of this unwanted tautomerization. Finally, we use X-ray crystallography to show that apo-GlxR is a configurationally flexible enzyme that can adopt two distinct tetrameric assemblies in vitro.

Protoplasts, which are plant cells devoid of cell walls, are valuable tools in plant biotechnology. However, they are highly sensitive to mechanical and osmotic stress during isolation and early culture, often leading to significant loss of viability. Reliable and efficient methods for monitoring protoplast quality are essential for downstream applications. We applied impedance flow cytometry to assess the viability, cell size, and early division of freshly isolated protoplasts from Arabidopsis thaliana, Brassica napus, and Beta vulgaris. This label-free technique enables fast, objective, and high-throughput assessment of individual protoplasts, allowing reliable monitoring of viability and early division in large populations. Importantly, IFC-derived viability metrics strongly correlated with microcallus formation, demonstrating their predictive value for culture competence. Impedance flow cytometry provides a robust, efficient and reproducible method for characterizing protoplast cultures. It enables rapid assessment of viability and growth potential, supporting quality control and optimization in plant cell culture workflows.

CRISPR-Cas9 tools have revolutionized genetic engineering, yet the efficient precise integration of DNA cargos, particularly for large DNA payloads (>1 kilobase, kb), remains a technical bottleneck. Herein, we develop a Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 approach, referred to as RED-CRISPR, which offers a versatile yet robust homology-directed repair (HDR) strategy enabling efficient and precise kb-scale DNA insertion across various cell types, including immortalized and primary cells of variable origins. RED-CRISPR significantly enhances HDR efficiencies by 2- to 5-fold change across diverse loci and further elevates HDR rates by 1.5- to 2.5-fold when synergizing with other HDR-enhancing strategies. We achieved up to 45% knock-in efficiency for CAR-T cell manufacturing, and attained 43% knock-in rate for generation of genetically modified mice using an 8-kb DNA cargo. Through a head-to-head comparison, RED-CRISPR profoundly mitigates off-target mutational burden and chromosomal translocations. We envision RED-CRISPR as a powerful genome-editing tool with broad biomedical and therapeutic applications. Insertion of a long DNA sequence into the host genome is challenging in mammalian cells. Here, the authors develop a recombinase (Redα/β)-enhanced DNA integration approach, which enables efficient and precise kilobase-scale DNA insertion in both primary cells and mouse embryos.

Human immune protection against bacteria critically depends on activation of the complement system. The direct bacteriolytic activity of complement molecules against Gram-negative bacteria acts via the formation of Membrane Attack Complex (MAC) pores. Bactericidal MAC pores damage the bacterial outer membrane, leading to destabilization of the inner membrane. Although it is well-established that inner membrane damage is crucial for bacterial cell death, the critical event causing MAC-mediated inner membrane damage remains elusive. Here we question whether the bacterial cell envelope possesses vulnerable spots for MAC pores to insert. By following the localization of MAC pores on E. coli over time using fluorescence microscopy, we elucidate that MAC deposition initiates at the new bacterial pole, which induces inner membrane damage and halts bacterial division. MAC components C8 and C9 preferentially localize at new bacterial poles, while C3b localizes randomly on the bacterial surface. This suggests that preferential MAC localization is determined by one of the initial steps of MAC formation. These findings provide valuable information about the interplay between immune components and the Gram-negative cell envelope.