Foliar application of double-stranded RNA (dsRNA) as RNA interference (RNAi)-based bio-pesticides represents a sustainable alternative to chemical-based crop protection strategies. A key feature of RNAi in plants is its ability to act non-cell autonomously, a process that plays a critical role in plant development and protection against pathogens. Whether RNAi induced by foliar dsRNA application acts non-cell autonomously remains debated, with the mechanisms and implications of this movement largely unexplored. We show that upon foliar application, dsRNA enters the leaf vasculature and moves to vegetative, reproductive, and belowground tissues in multiple plant species. Unprocessed mobile dsRNA was detected in the apoplast, being maintained in new growth, indicating apoplastic rather than symplastic transport. Mobile dsRNA could transfer to infecting fungi, where it was processed and loaded by the fungal RNAi machinery to elicit gene silencing. Using a novel biochemical purification technique and small RNA sequencing, we detected functional small interfering RNA species derived from foliar-applied dsRNA that elicit effective silencing in both the applied and distal tissue types. Our mechanistic dissection of the uptake and movement of dsRNA provides crucial insights into the mode of action of RNAi biopesticides and stands to add significant benefit to this emerging field of plant protection.

The composition of the vaginal microenvironment has significant implications for gynecologic and obstetric outcomes. Where a Lactobacillus-dominated microenvironment is considered optimal, a polymicrobial environment is associated with increased risk for female reproductive diseases. Recent work examined bacteria-derived extracellular vesicles (bEVs) as an important mode of microbe-host communication that may influence reproductive outcomes. However, in order to communicate with female reproductive tissues, bEVs must penetrate the protective cervicovaginal mucus barrier. We demonstrate increased diffusion of bEVs compared to whole bacteria. Additionally, we evaluate the uptake of bEVs by, and the resulting effects on, human vaginal epithelial, endometrial, and placental cells, highlighting potential mechanisms of action by which vaginal dysbiosis contributes to gynecologic and obstetric diseases. Taken together, our work demonstrates the ability of bEVs to mediate female reproductive outcomes and highlights their potential as therapeutic modalities for treating dysbiosis and dysbiosis-associated diseases in the female reproductive tract.

RNA capped with dinucleoside polyphosphates has been discovered in bacteria and eukaryotes only recently. The likely mechanism of this specific capping involves direct incorporation of dinucleoside polyphosphates by RNA polymerase as noncanonical initiating nucleotides. However, how these compounds bind into the active site of RNA polymerase during transcription initiation is unknown. Here, we explored transcription initiation in vitro, using a series of DNA templates in combination with dinucleoside polyphosphates and model RNA polymerase from Thermus thermophilus. We observed that the transcription start site can vary on the basis of the compatibility of the specific template and dinucleoside polyphosphate. Cryo-electron microscopy structures of transcription initiation complexes with dinucleoside polyphosphates revealed that both nucleobase moieties can pair with the DNA template. The first encoded nucleotide pairs in a canonical Watson–Crick manner, whereas the second nucleobase pairs noncanonically in a reverse Watson–Crick manner. Our work provides a structural explanation of how dinucleoside polyphosphates initiate RNA transcription. Using cryo-electron microscopy technologies, Serianni and Škerlová et al. reveal how NpnNs initiate bacterial transcription as noncanonical RNA caps by showing one nucleobase pairing with the template in canonical mode while the other pairs in reverse Watson–Crick mode.

Nucleotide sequences in the FASTQ or BAM format are widely shared, yet derived from platform-specific raw data outputs that differ across sequencing platforms. In Oxford Nanopore Technologies (ONT) sequencing, raw signal data contain valuable biological information and enable basecaller optimization and modification detection. These raw signals also underpin algorithms that could improve ONT device portability and enhance target enrichment efficiency through adaptive sampling. Nevertheless, the storage and sharing of raw nanopore data remain limited due to technical constraints and the lack of standardized and centralized infrastructure. To address this challenge, we developed SquiDBase (https://squidbase.org), a dedicated repository for raw microbial nanopore sequencing data with linked processed data and metadata. To maximize immediate utility, we built SquiDPipe, a Nextflow pipeline for the automated removal of human reads from raw nanopore data, sequenced 24 clinically relevant viruses and incorporated them into SquiDBase, and added publicly available reference datasets and new community contributions. By offering a centralized, open-access raw data collection platform, SquiDBase facilitates data sharing, enhances reproducibility, and supports the development and benchmarking of computational tools, reinforcing open science in nanopore sequencing.

The trans cleavage activity of Cas12a has been extensively used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions in the RuvC lid and substitutions to introduce positively charged residues in the Nuc could enhance both cis and trans cleavage. These Cas12a variants improved DNA detection and genome editing efficacy. Overall, this study provides a blueprint for rationally engineering the DNase activities of Cas12a.

Engineering synthetic intrinsically disordered proteins (synIDPs) enables regulation of biomolecular condensation and protein solubility. However, limited understanding of how sequence-dependent interaction cooperativity relates to the fitness impacts of synIDPs on endogenous cellular processes constrains our design capability. Here, to circumvent this design challenge, we present a systematic directed evolution method for the evolution of synIDPs capable of mediating diverse phase behaviors in living cells. The selection methods allow us to evolve a toolbox of synIDPs with distinct phase behaviors and thermoresponsive features in living cells, leading to the evolution of synthetic condensates. The reverse-selection method further allows us to select synIDPs as solubility tags. We demonstrate the applications of the evolved synIDPs in protein circuits to (1) regulate intracellular protein activity and (2) reverse antibiotic resistance. Our systematic evolution and selection strategies provide a versatile platform for developing synIDPs for broad applications in synthetic biology and biotechnology. Ma et al. developed a directed evolution method with various selection strategies for the evolution of synthetic intrinsically disordered proteins capable of forming condensates with desired properties in living cells.

While the degradation of plastics into molecules and micro- and nanoplastics (MNPs) through abiotic processes is increasingly well documented, bacterial-driven biodegradation remains poorly understood. Yet, as microbial strategies to mitigate plastic pollution gain traction, identifying the full spectrum of degradation products, from soluble molecules to microplastics and nanoplastics, is essential for elucidating both toxicological impacts and fundamental degradation mechanisms. In this study, we developed an integrated analytical method combining thermodesorption and pyrolysis gas chromatography–mass spectrometry (TDS/Py-GC-MS). A tailored liquid–liquid extraction protocol was optimized for both polymers and applied to culture media containing microbial communities in contact with solid plastic samples. This approach enabled the identification of specific depolymerization products from polystyrene (PS) and polyvinyl chloride (PVC), including known markers (e.g., phthalic anhydride, 2-ethylhexan-1-ol, acenaphthene) and novel aromatic compounds likely associated with microbial activity, such as (1E)-1-benzylideneindene, 2-ethylcyclopentan-1-one, and benzene in samples from superworms intestinal microbiota cultivated with PS or PVC film at different temperature (n = 1). In terms of fragmentation, PS particles were recovered in varying quantities, while no PVC fragments were detected. This study provides a robust analytical framework to characterize plastic degradation and identify molecular markers of degradation.

Aspergillus niger serves as a cell factory for efficient enzyme production. The lipase derived from Thermomyces lanuginosus (TLL) is known for its remarkable thermal stability and is extensively utilized in various industrial fields. In this study, a heterologous expression strain, ΔAnTll-11, of TLL was successfully constructed in A. niger. Through fermentation optimization, the lipase activity was enhanced 8.7-fold to 4547.95 U/mL, compared with the initial value of 520 U/mL. Enzymatic characterization indicated that the recombinant lipase exhibited optimum activity at pH 9.5 and 45 °C, and its activity was positively influenced by Ca2⁺, Ag⁺, Mg2⁺, and Cu2⁺. The ΔAnTll-11 strain exhibited enhanced tolerance to high osmolarity, oxidative stress, and thermal shock. Furthermore, transcriptomic analysis of the ΔAnTll-11 strain revealed that half of the total annotated genes (7199 genes) were differentially expressed genes (DEGs). According to protein‒protein interaction network and weighted gene coexpression network analyses, genes involved in ribosome function, amino acid metabolism, glycosylation, energy metabolism, and the MAPK signalling pathway may affect the expression of lipase. The transcriptomic findings elucidated the regulatory mechanisms by which A. niger expresses foreign proteins and enzymes, establishing a groundwork for further enhancing A. niger as a cell factory for efficient enzyme and protein production.