RNA modifications play a fundamental role in regulating essential cellular processes, including translation fidelity and stress adaptation. While these modifications are installed post-transcriptionally by specialized enzymes, their broader functional roles remain largely unexplored. Here, we uncover an unexpected function for the Vibrio cholerae tRNA dihydrouridine synthase B (VcDusB) beyond its canonical role in tRNA dihydrouridylation. We show that deletion of dusB severely compromises V. cholerae resistance to oxidative stress, not through the loss of tRNA modification, but via disruption of an intrinsic NADPH oxidase activity. Mutational analyses reveal that DusB redox function is essential for survival under oxidative stress. Proteomic and transposon insertion sequencing analysis further linked DusB to NADPH homeostasis and metabolic reprogramming during stress adaptation. These findings redefine DusB as a bifunctional enzyme coupling tRNA modification to redox regulation, expanding the functional repertoire of RNA-modifying enzymes in stress adaptation. More broadly, this work paves the way for exploring the evolutionary versatility of tRNA-modifying enzymes, suggesting that their functions extend far beyond RNA metabolism to direct integration of translational control with cellular redox state.

Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices UMI for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platform’s performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of severe acute respiratory syndrome coronavirus 2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research.

Enzymatic food protein hydrolysates (FPHs) have emerged as innovative ingredients in food science, offering enhanced functional and bioactive properties. This review provides a critical overview of FPHs, beginning with their background and significance, followed by an exploration of enzymatic hydrolysis mechanisms, key influencing factors, and comparisons with alternative methods. Various protein sources, including animal-based, plant-based, and emerging alternatives such as insects and algae, are examined for their potential in hydrolysate production. Functional properties such as solubility, emulsifying, foaming, gelation, and antioxidant capacities in food systems are highlighted, alongside challenges in process optimization, bitterness mitigation, and regulatory hurdles. The use of precision fermentation is discussed as a promising tool for producing new peptides with structure, functionality, and sensory attributes that could accelerate innovation in the field. By addressing these opportunities and challenges, this review outlines the future potential of FPHs in sustainable food systems and functional food applications.

Recent advances in high-throughput approaches for estimating co-localization of microbes, such as SAMPL-seq, allow characterization of the biogeography of the gut microbiome longitudinally and at an unprecedented scale. However, these high-dimensional data are complex and have unique noise properties. To address these challenges, we developed MCSPACE, a probabilistic AI method that infers, from microbiome co-localization data, spatially coherent assemblages of taxa, their dynamics over time, and their responses to perturbations. To evaluate MCSPACE’s capabilities, we generated the largest longitudinal microbiome co-localization dataset to date, profiling spatial relationships of microbes in the guts of mice subjected to serial dietary perturbations over 76 days. Analyses of these data and two existing human longitudinal datasets demonstrated superior benchmarking performance of MCSPACE over existing methods and moreover yielded insights into the spatiotemporal structuring of the gut microbiome, including identifying temporally persistent and dynamic microbial assemblages in the human gut, and shifts in assemblages in the murine gut induced by specific dietary components. Our results highlight the utility of MCSPACE, which we make available to the community as an open-source software tool, for elucidating the dynamics of microbiome biogeography and gaining insights into the role of spatial relationships in host-microbial ecosystem function.

Live-cell fluorescence nanoscopy and single-molecule tracking offer profound insights into cellular biology, but their application is limited by fluorescent tags with low brightness, large size, and irreversible photobleaching. Here, we present ReTagX, a bright, regenerative, compact array tag system enabling rapid recovery of photobleached signals through reversible, fluorogenic labeling. Newly developed ReTag1 (~18 kDa) with highly fluorogenic MaP655-TMP achieves a signal-to-noise ratio up to 204-fold and allows fluorescence recovery within 1 minute in wash-free live-cell imaging. This tag extends STED imaging of mitochondrial TOM20 from fewer than 5 to over 300 frames, enabling long-term visualization of mitochondrial dynamics at 80 nm resolution for over 100 minutes. Multimeric ReTag1 arrays, such as ReTag8 (~156 kDa), increase brightness 5.5-fold and extend TNFR2 single-molecule tracking ~2.9-fold, preserving >80% of trajectories over 5 minutes. ReTagX provides a robust platform for high-resolution, long-term molecular visualization of cellular dynamics in living cells. Si, Dong, Xing and colleagues present ReTagX, a bright and compact array tag that restores photobleached signals via reversible and fluorogenic labelling, enabling long-term and high-resolution imaging of protein dynamics in living cells.

Protein misfolding involving changes in non-covalent lasso entanglement (NCLE) status has been proposed based on simulations and biochemical assays of a small number of proteins. Here, we detect hallmarks of these misfolded states across hundreds of proteins by integrating E. coli proteome-wide limited-proteolysis mass spectrometry data with structural datasets of protein native structures. Proteins containing native NCLEs are twice as likely to misfold, predominantly in regions where these NCLEs naturally occur. Surprisingly, the chaperones DnaK and GroEL do not typically correct this misfolding, except in the case of essential proteins. Statistical analysis links this differential rescue activity to weaker loop-closing contacts in the NCLEs of essential proteins, suggesting misfolding involving these loops is easier to rectify by chaperones. Molecular simulations indicate a mechanism where premature NCLE loop closure, prior to proper placement of the threading segment, leads to persistent misfolded states. This mechanism can explain why, in this mass spectrometry dataset, proteins with NCLEs are more likely to misfold and misfold in NCLE regions. These results suggest the potential for widespread NCLE misfolding, that such misfolded states in non-essential proteins could bypass the refolding action of chaperones, and that some protein sequences may have evolved to allow chaperone rescue from this class of misfolding. Non-covalent lasso entanglements are pervasive structural features of increasing importance. Here, the authors show that these entanglements are associated with in vitro misfolding across the E. coli proteome and that chaperones preferentially rescue such misfolding in essential proteins.

The recent expansion of prokaryotic genomes reveals many ortholog groups (OGs) whose function cannot be inferred from conventional, sequence similarity-based annotation methods, especially in metagenome-assembled genomes. Phylogenetic profiling is one of the promising methods to annotate these OGs, by identifying functional relationships of OGs using co- or anti-occurrence of OG distributions, not sequence similarity. Here, we proposed two new phylogenetic methods for large-scale data, Ancestral State Adjustment (ASA) and Simultaneous EVolution test (SEV), which consider the ancestral state of OG presence/absence. In evaluations using three distinct prokaryotic datasets, ASA and SEV showed better or comparable performance to both established and recently proposed methods for large-scale data. We compared the functionally related OGs detected by each method and found that SEV and its predecessor can identify slowly evolving OGs, such as housekeeping genes. In contrast, ASA and its predecessors can detect functionally related OGs that tend to be gained or lost in a fixed order, indicating a strong evolutionary constraint that provides clues for functional prediction. Using matrix multiplication, we also showed that SEV is scalable in the latest genome databases.

Bacterial genomes encompass numerous small open reading frames (smORFs), some of which encode functional microproteins or perform noncoding regulatory roles. The evolution of microproteins remains poorly understood, largely due to challenges in homology detection for these short sequences. To address this challenge, we constructed 36 957 orthologous groups of microproteins (microOGs) across 5668 Enterobacteriaceae genomes. Our pipeline identified dozens of novel, widely distributed microprotein families and refined conservation patterns for known ones. However, 86% of the microOGs are genus-specific and functionally uncharacterized, suggesting that enterobacteria harbor a pool of evolutionarily young, de novo-originated small genes. Nevertheless, the microprotein-encoding smORFs in the microOGs are preferentially adjacent to membrane transporter genes suggesting a role in regulating transport processes. MicroOGs formed closed pangenomes, indicative of a limited contribution to the noncore genome of enterobacteria, likely due to the limitations on the size of intergenic regions where microproteins could arise de novo and frequent loss of microprotein-encoding smORFs during bacterial evolution. Overall, we identified 4838 microOGs with clear signatures of de novo origin from noncoding sequences. Many of the microprotein-encoding smORFs overlap transcriptional regulatory signals or repetitive elements suggesting that the origin of microproteins is tied to selection for maintenance of regulatory sequences.

CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show leaky Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we have established transgenic platforms that minimize Cas9 functionality in the OFF-state along with maximized and uncompromised ON-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we have developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (ON vs. OFF-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we have created a Branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we have constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions. Achieving tight Cas9 regulation without sacrificing activity remains difficult. Here, the authors design multi-level circuits combining anti-CRISPRs, splice sites, chemical induction, and degron control to enable ultra-high dynamic range and precise, on-demand genome editing across contexts.

Both endophytes, microorganisms that reside within plants, and methylotrophs, which grow using methanol produced from plant leaves, play key roles in protecting plants against biotic and abiotic stresses. However, the source of endophytes and the mechanisms of their selection in plants are poorly understood. Therefore, experiments were carried out to identify wheat seed methylotrophic endophytes and evaluate their partitioning in root, stem and leaf of aseptic-controlled plants cultivated from surface-sterilized seeds.  The counts of endophytic methanol utilizers were higher in leaf tissue than in stem, root and seed, as estimated using viable counts and qPCR targeting rrn gene. The methanol dehydrogenase subunit mxaF was PCR-detected in all pink-colored isolates that grew using methanol or succinate. These pink-pigmented facultative methylotrophs (PPFM) were dominant in shoot tissue. Using mass spectrometry for alkaloid content analysis, peganine was detected as a peak 16.6% higher in root than shoot. Root extracts and peganine alone inhibited the growth of PPFM.  PPFM transmitted from seed are more abundant in shoot than root. How plant compounds such as peganine are involved in the methylotrophic endo-phytomicrobiome dynamics remains to be better characterized.

Ammonia monooxygenase (AMO) oxidizes ammonia to hydroxylamine. Limited knowledge of the structural information of AMO hinders our understanding of the molecular mechanism underlying ammonia oxidation, impacting the mitigation of greenhouse gas emissions and enhancing agricultural productivity using ammonium as a nitrogen source. Herein, we report the cryo-electron microscopy structure of the AMO complex from an isolated strain of ammonia-oxidizing bacteria (AOB). AMO is a cylinder-shaped homotrimeric assembly composed of five subunits. A single-transmembrane protein and a soluble protein are potentially crucial in signal transduction during ammonia oxidation and mediating interactions with the outer membrane protein assembly machinery. Three modeled coppers, along with an adjacent water-mediated hydrogen-bond network, may facilitate an efficient proton transfer pathway from the periplasmic CuB to the active site CuD within the inner membrane, where CuC and CuD will act in concert to catalyze substrate reaction. The distinctive surface charge characteristics of AMO provide valuable insights into the structural features that govern ammonium assimilation and material transport during ammonia oxidation. These findings shed light on the molecular complexities of AMO and provides a structural foundation for elucidating the catalytic mechanism of ammonia oxidation. This study presents the high-resolution cryo-EM structure of the ammonia monooxygenase (AMO) complex, revealing a homotrimeric assembly with five subunits, a dinuclear copper active site, a putative proton transport pathway with distinct electrostatic features for substrate handling.

Glycans are branched, structurally diverse, and highly flexible biomolecules. These characteristics make glycoanalytics and structural characterization challenging, resulting in often unclear structure-to-function relationships. GlycoShape, currently the largest open-access database of glycan 3D structures from molecular dynamics (MD) simulations, provides an opportunity to fill this information gap. Here, we present GlyContact, an open-source Python package designed and developed to retrieve, process, and analyze glycan 3D structures, from MD, NMR, or X-ray crystallography. We demonstrate that GlyContact can (i) unveil the impact of sequence context on glycan motif structure, (ii) yield a predictive understanding of motif flexibility and surface accessibility on lectin-glycan binding, which improved lectin-binding prediction by ~ 7%, and (iii) accurately predict torsion angle distribution between disaccharides using von Mises graph neural networks. We envision that GlyContact will allow researchers to explore glycan structures within their 3D space, obtaining insights into their biological functions. GlyContact is available open-access at https://github.com/lthomes/glycontact. GlyContact enables systematic analysis of glycan 3D structures, revealing how structural properties like flexibility and surface accessibility determine lectin binding and introducing AI models to predict structural features directly from sequences.