Biology subjects

Wu, J.

Publications and source records attributed to Wu, J..

At least 289 records · Page 16Linked to original sources

bioRxiv2024

TRAIT: A Comprehensive Database for T-cell Receptor-Antigen Interactions

Comprehensive and integrated resources on interactions between T-cell receptors and antigens are still lacking for adoptive T-cell-based immunotherapies, highlighting a significant gap that must be addressed to fully comprehend the mechanisms of antigen recognition by T-cells. In this study, we present TRAIT, a comprehensive database that profiles the interactions between T-cell receptors (TCRs) and antigens. TRAIT stands out due to its comprehensive description of TCR-antigen interactions by integrating sequences, structures and affinities. It provides nearly 8 million experimentally validated TCR-antigen pairs, resulting in an exhaustive landscape of antigen-specific TCRs. Notably, TRAIT emphasizes single-cell omics as a major reliable data source for TCR-antigen interactions and includes millions of reliable non-interactive TCRs. Additionally, it thoroughly demonstrates the interactions between mutations of TCRs and antigens, thereby benefiting affinity maturation of engineered TCRs as well as vaccine design. TCRs on clinical trials were innovatively provided. With the significant efforts made towards elucidating the complex interactions between TCRs and antigens, TRAIT is expected to ultimately contribute superior algorithms and substantial advancements in the field of T-cell-based immunotherapies. TRAIT is freely accessible at https://pgx.zju.edu.cn/traitdb.

immunology↗

bioRxiv2024

Multi-stage-mixing to create a core-then-shell structure improves DNA-loaded lipid nanoparticles transfection by orders of magnitude

As they became the dominant gene therapy platform, lipid nanoparticles (LNPs) experienced nearly all their innovation in varying the structure of individual molecules in LNPs. This ignored control of the spatial arrangement of molecules, which is suboptimal because supramolecular structure determines function in biology. To control LNPs supramolecular structure, we introduce multi-stage-mixing (MSM) to successively add different molecules to LNPs. We first utilize MSM to create a core-then-shell (CTS) synthesis. CTS-LNPs display a clear core-shell structure, vastly lower frequency of LNPs containing no detectable mRNA, and improved mRNA-LNP expression. With DNA-loaded LNPs, which for decades lagged behind mRNA-LNPs due to low expression, CTS improved DNA-LNPs protein expression by 2-3 orders of magnitude, bringing it within range of mRNA-LNPs. These results show that supramolecular arrangement is critical to LNP performance and can be controlled by mixing methodology. Further, MSM/CTS have finally made DNA-LNPs into a practical platform for long-term gene expression.

bioengineering↗

bioRxiv2024

Identification of Ephrin type-B receptor 4 as a critical mediator of tissue fibrosis

Pulmonary fibrosis (PF) is a pathology associated with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). Fibrosis promotes continual secretion of extracellular matrix (ECM), producing non-functional scar tissue and causing organ failure. This study investigated the tyrosine kinase receptor Ephrin type-B receptor 4 (EphB4) as a mediator of PF. To this end, we generated mice with conditional Col1a2-driven deletion of Ephb4 and used a preclinical mouse model of PF, total and single nuclei RNA (snRNA) sequencing, NanoString, previously published single cell data, computational analysis, and functional assays of mouse and human healthy control and IPF lung fibroblasts. Col1a2-CreERT-driven Ephb4 deletion, or EphB4 inhibition via NVP-BHG712, markedly protected against bleomycin-induced PF. Total RNA-sequencing of fibroblasts isolated from Ephb4-deficient fibrotic mouse lungs exhibited reduced expression of ECM, ER Cargo, and protein trafficking-related genes. NVP-BHG712 reduced expression of these identified genes in mouse lung fibroblasts under fibrotic conditions in vitro. SnRNA sequencing of mouse lungs treated with NVP-BHG712 identified transcriptomic changes of ECM genes in specific fibroblast subpopulations. RNA sequencing, computational, and functional assays using mouse and human IPF fibroblasts identified elastin as a key mediator involved in EphB4 signaling. Combined, our data show that EphB4 is a crucial mediator of PF.

pathology↗

bioRxiv2024

Classifying Irritable Bowel Syndrome Using Spatio-Temporal Graph Convolution Networks on Brain fMRI Data

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder marked by abdominal pain and changes in stool consistency or frequency. Recent studies have explored the link between IBS and various cognitive deficits using functional MRI. Despite these efforts, an effective diagnostic or predictive model for IBS remains elusive. This shortfall is twofold: firstly, the sample sizes in these studies are typically small, and secondly, the machine learning or deep learning models currently in use fail to adequately detect the subtle and dynamic pathological changes present in fMRI data for IBS. In this study, we extracted rs-fMRI of 79 subjects with IBS and 79 healthy controls, then put them into spatio-temporal graph convolution network (ST-GCN) for classification. We also incorporated a novel interpretability module into this model to identify potential regions of interest (ROI) associated with IBS. Our model outperformed other state-of-the-art ML and DL methods with the highest average accuracy of 83.51% on our dataset. Furthermore, based on the results of our interpretability module, the Inferior Parietal Lobule (IPL.R), Inferior Frontal Orbital part (ORBinf.R), Postcentral Gyrus (PCG.R), Middle Frontal Orbital part (ORBmid.R), and Superior Medial Frontal Orbital part (ORBsupmed.L) were identified as top 5 important brain regions for distinguishing IBS patients from the control group, which are consistent with the brain regions identified in previous literature reviews. We also conducted an external data-driven experiment to further validate the effectiveness of the interpretability module. The results indicate that the selected regions significantly impact IBS.

bioengineering↗

bioRxiv2024

Brain functional connectivity as a mediator between hematological metrics and cognitive decline in children with beta-thalassemia major

Children with beta-thalassemia major ({beta} -TM) are at risk of cognitive impairment, particularly in learning and memory. While cognitive deficits in the general population have been extensively studied, brain-based predictors for {beta}-TM children remain unexplored. This study used connectome-based predictive modeling (CPM) to examine whole-brain functional connectivity in 60 participants to predict cognitive performance. {beta}-TM children showed impaired cognitive abilities, as evidenced by lower Wechsler Intelligence Scale scores compared to controls. The identified brain regions, though not typically linked to cognitive performances, exhibited functional connectivity patterns associated with {beta}-TM, as confirmed by network-based statistics. Correlations between task performance, functional connectivity identified by the CPM, and hematological metrics were also examined. Significant correlations were found between the strength of functional network identified by CPM and hematological metrics, particularly hemoglobin levels and red blood cell distribution width. The study demonstrates that hematological factors influence cognitive performance indirectly through specific functional connectivity, providing new insights into the neural underpinnings of cognitive deficits in children with beta-thalassemia major.

neuroscience↗

bioRxiv2024

GCTransNet: 3D mitochondrial instance segmentation based on Global Context Vision Transformers

Mitochondria are double membrane-bound organelles essential for generating energy in eukaryotic cells. Mitochondria can be readily visualized in 3D using Volume Electron Microscopy (vEM), and accurate image segmentation is vital for quantitative analysis of mitochondrial morphology and function. To address the challenge of segmenting small mitochondrial compartments in vEM images, we propose an automated mitochondrial segmentation method called GCTransNet. This method employs grayscale migration technology to preprocess images, effectively reducing intensity distribution differences across EM images. By utilizing 3D Global Context Vision Transformers (GC-ViT) combined with global context self-attention modules and local self-attention modules, GCTransNet precisely models long-range and short-range spatial interactions. The long-range interactions enable the model to capture the global structural relationships within the mitochondrial segmentation network, while the short-range interactions refine local details and boundaries. In our approach, the encoder of the 3D U-Net network, a classical multi-scale learning architecture that retains high-resolution features through skip connections and combines multi-scale features for precise segmentation, is replaced by a 3D GC-ViT. The GC-ViT leverages shifted window-based self-attention, capturing long-range dependencies and offering improved segmentation accuracy compared to traditional U-Net encoders. In the MitoEM mitochondrial segmentation challenge, GCTransNet achieved state-of-the-art results, demonstrating its superiority in automated mitochondrial segmentation. The code and its documentation are publicly available at https://github.com/GanLab123/GCTransNet.

cell biology↗

bioRxiv2024

Better Prior Distribution for Antibody Complementarity-Determining Regions Design

Designing antibodies with desired binding specificity and affinity is essential for pharmaceutical research. While diffusion-based models have advanced the co-design of the Complementarity-Determining Regions (CDRs) sequences and structures, challenges remain, including non-informative prior distribution, incompatibility with discrete amino acid types, and impractical computational cost in large-scale sampling. To address these, we proposed FlowDesign, a sequence-structure co-design approach based on Flow Matching, offering: (1) Flexible selection of prior distributions; (2) Direct matching of discrete distributions; (3) Enhanced computational efficiency for large-scale sampling. By leveraging various priors, data-driven structural models proved the most informative. FlowDesign outperformed baselines in Amino Acid Recovery (AAR), RMSD, and Rosetta energy. We also applied FlowDesign to design antibodies targeting the HIV-1 receptor CD4. FlowDesign yielded antibodies with improved binding affinity and neutralizing potency compared to the antibody Ibalizumab across multiple HIV mutants, validated by Biolayer Interferometry (BLI) and pseudovirus neutralization. This highlights FlowDesigns potential in antibody and protein design. A record of this papers Transparent Peer Review process is included in the Supplemental Information.

bioinformatics↗

bioRxiv2024

Evaluation of T Cell Receptor Construction Methods from scRNA-Seq Data

T cell receptors (TCRs) serve pivotal roles in the adaptive immune system by enabling recognition and response to pathogens and irregular cells. Various methods exist for TCR construction from single-cell RNA sequencing (scRNA-seq) datasets, each with its unique characteristics regarding accuracy, sensitivity, adaptability, usability, time, and memory consumption. Yet, a comprehensive understanding of their relative strengths and weaknesses for different applications remains elusive. In our research, we implemented a benchmark analysis utilizing experimental single-cell immune profiling datasets encompassing paired scRNA-seq as input and scTCR-seq datasets as ground truth reference from human and mouse. Additionally, we introduced a novel simulator, YASIM-scTCR (Yet Another Simulator for single-cell TCR), capable of generating scTCR-seq reads containing a diverse array of TCR- derived sequences under different sequencing depths and read lengths. Our results consistently showed that TRUST4 outperformed others across multiple datasets, while MiXCR and DeRR also demonstrated considerable accuracy. We also discovered that the sequencing depth inherently imposes a critical constraint on successful TCR construction from scRNA-seq data. In summary, we present a benchmark study to aid researchers in choosing the most appropriate methods for reconstructing TCR from scRNA-seq data.

bioinformatics↗

bioRxiv2024

Structural basis of pseudoGTPase-mediated protein-protein interactions

GTPases regulate various cellular processes through conformational changes triggered by GTP or GDP binding. Recently, pseudoGTPases, the catalytically inactive counterparts of GTPases, have been identified across species from bacteria to human, although their functions and mechanisms remain unexplored. In this study, we demonstrate that the N-terminal region of the assembly chaperone AAGAB is a type i pseudoGTPase using biochemistry and X-ray crystallography. Furthermore, we discovered that the AAGAB pseudoGTPase domain (psGD) interacts with the {sigma} subunits of AP1 and AP2 adaptor complexes, heterotetrameric complexes involved in clathrin-mediated membrane trafficking. AAGAB psGD engages the {sigma} subunits via a unique interface distinct from the conventional GTPase interacting regions. Further biochemical and cell-based assays confirmed the crucial role of the newly identified interface in binding and membrane trafficking. Collectively, our results establish AAGAB pseudoGTPase domain as a critical protein-protein interaction module. These findings offer new insight into the structural basis and molecular mechanisms of pseudoGTPases. HighlightsO_LIAAGAB N-terminal region is a pseudoGTPase C_LIO_LIAAGAB pseudoGTPase domain interacts with and stabilizes AP1 and AP2 {sigma} subunits C_LIO_LIThe crystal structure of AAGAB psGD:AP1{sigma}3 is the first reported psGD complex structure, revealing a unique interface independent of guanine nucleotide regulation C_LIO_LIThe psGD:AP1{sigma}3 structure offers mechanistic insights into {sigma} subunits stabilization and protection through adaptor complex assembly C_LI

biochemistry↗

bioRxiv2024

Genome-wide investigation of outer membrane protein families under mosaic evolution in Escherichia coli

Several genes in Gram-negative bacteria encoding outer membrane proteins (OMPs) have been reported to show patterns of mosaic evolution featured by mixture of negative selection and local recombination. Here, we proposed a strategy, and applied it to screen OMPs under mosaic evolution in Escherichia coli in the genome level. In total, 21 OMP families, including 16 new ones, were detected with the typical patterns of mosaic evolution. An abosolute majority of the protein families are conserved in E. coli for the composition, genomic loci and the overall structures. Highly Variable Regions (HVRs) can be recognized, which are frequently located extracellularly within the protruding loops. There are only limited number of major HVR sequence types, within which positively selected sites can be detected ocassionally. Based on the simulated results of multiple models, the OMPs under mosaic evolution are often with good antigenicity, with HVRs of various sequence types coinciding with the B-cell epitopes of the strongest immunogenicity. The study futher broadened our understanding of the characteristics of mosaic evolution and the functions of OMPs in Gram-negative bacteria, laying an important foundation for their potential translational applications. ImportanceIt is important to understand the evolutionary mechanisms of bacterial OMP-encoding genes, which would facilitate the development of anti-bacterial reagents. This study made the first genome-wide screening of bacterial OMPs under mosaic evolution, and increased the list of candidate OMP families by 3 folds in E. coli, far more than we expected. The study further confirmed the hypothesis about the evolutionary, micro-evolutionary and structural features of these OMPs, and facilitated the functional theory of mosaic evolution. Moreover, the findings of limited HVR sequence types and strong immunogenicity of HVRs paved important foundation for application of these OMPs and their HVRs in development of antibodies or other anti-bacterial treatment.

microbiology↗

bioRxiv2024

A Structural Mechanism for Noncanonical GPCRSignal Transduction in the Hedgehog Pathway

SUMMARY PARAGRAPHThe Hedgehog (Hh) signaling pathway is fundamental to embryogenesis, tissue homeostasis, and cancer. Hh signals are transduced via an unusual mechanism: upon agonist-induced phosphorylation, the noncanonical G protein-coupled receptor SMOOTHENED (SMO) binds the catalytic subunit of protein kinase A (PKA-C) and physically blocks its enzymatic activity. By combining computational structural approaches with biochemical and functional studies, we show that SMO mimics strategies prevalent in canonical GPCR and PKA signaling complexes, despite little sequence or secondary structural homology. An intrinsically disordered region of SMO binds the PKA-C active site, resembling the PKA regulatory subunit (PKA-R) / PKA-C holoenzyme, while the SMO transmembrane domain binds a conserved PKA-C interaction hub, similar to other GPCR-effector complexes. In contrast with prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize key structural elements within the SMO cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural mechanism for a central step in the Hh cascade and defines a paradigm for disordered GPCR domains to transmit signals intracellularly.

biochemistry↗

bioRxiv2024

Shared Transcriptomic Signatures Reveal Synaptic Pruning as a Link Between Alzheimer's Disease and Epilepsy

Alzheimers disease (AD) and epilepsy (EP) share a complex bidirectional relationship, yet the molecular mechanisms underlying their comorbidity remain insufficiently explored. To identify potential transcriptional programs across animal models and human patients with AD and EP, we conducted a comprehensive genome-wide transcriptomic analysis. Our investigation included mouse models of temporal lobe epilepsy (pilocarpine- and kainic acid-induced; n = 280), AD transgenic models (7 transgenic models expressing human tau or amyloid pathology; n = 257), and performed cross-species validation in human cohorts (EP: n = 182; AD: n = 301). We identified a highly conserved immune-related module across all models and patient cohorts. The hub consensus signatures of this module were centered around a microglial synaptic pruning pathway involving TYROBP, TREM2, and C1Q complement components. Gene regulatory network analysis identified TYROBP as the key upstream hub signature. These signatures showed consistent up-regulation in both conditions and strong diagnostic potential. Differential expression analyses revealed their predominant expression in specific microglial subpopulations associated with complement-mediated synaptic pruning and immune activation. Neural circuit modeling further demonstrates the asymmetric sensitivity of synaptic pruning to network dynamics. Loss of inhibitory synapses has a disproportionately significant impact on neural network excitation/inhibition balance and synchronization. Our findings support microglial complement-mediated synaptic pruning as a conserved central pathway linking neurodegeneration to epileptogenesis, suggesting a promising therapeutic target for AD and EP comorbidity. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/618752v12_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@179aa28org.highwire.dtl.DTLVardef@922dfaorg.highwire.dtl.DTLVardef@108a99eorg.highwire.dtl.DTLVardef@1b7d2f6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGenome-wide transcriptomic analysis across epilepsy (EP) and Alzheimers disease (AD) models and patients identified a conserved immune module. C_LIO_LIThe TYROBP-TREM2-C1Q microglial synaptic pruning pathway was identified as a central consensus signature across model and patient cohorts. C_LIO_LIConsensus signatures possess potential diagnostic utility in AD and EP patients, with predominant expression in specific microglial subpopulations C_LIO_LINeural circuit modeling demonstrates the asymmetric effects of synaptic pruning, whereby loss of inhibitory synapses disproportionately disrupts the E/I balance, leading to increased network synchronization. C_LI

bioinformatics↗

bioRxiv2024

Bystander base editing interferes with visual function restoration in Leber congenital amaurosis

Base editors (BEs) have emerged as a powerful tool for gene correction with high activity. However, bystander base editing, a byproduct of BEs, presents challenges for precise editing. Here, we investigated the effects of bystander edits on phenotypic restoration in the context of Leber congenital amaurosis (LCA), a hereditary retinal disorder, as a therapeutic model. We observed that in rd12 of LCA model mice, the highest editing activity version of an adenine base editors (ABEs), ABE8e, generated substantial bystander editing, resulting in missense mutations despite RPE65 expression, preventing restoration of visual function. Through AlphaFold-based mutational scanning and molecular dynamics simulations, we identified that the ABE8e-driven L43P mutation disrupts RPE65 structure and function. Our findings underscore the need for more stringent requirements in developing precise BEs for future clinical applications.

genetics↗

bioRxiv2024

IGF2 mediates Hippo signaling to control liver size

The Hippo pathway is a central mechanism in organ size control, but the mediator for its function remains elusive. Here, we show that the expression of insulin-like growth factor II (IGF2) is directly induced by YAP/TAZ transcription cofactors of the Hippo pathway in a developmental stage- and cell type-specific manner. In mouse livers, Igf2 expression is sustained by YAP/TAZ in hepatoblasts and immature hepatocytes at fetal and neonatal stages, coupling with rapid cell proliferation and liver size expansion, whereas turned off in matured hepatocytes following YAP/TAZ inactivation to prevent liver overgrowth. In contrast, YAP/TAZ fails to regulate Igf2 expression in cholangiocytes and diverse liver mesenchymal cells where epigenetic barriers, including DNA and histone methylation, are implanted near Igf2 promoters. Furthermore, IGF2 activates IGF1R signaling and promotes normal and neoplastic liver growth, while the inactivation of IGF2 or IGF1R effectively blocks YAP/TAZ-induced hepatomegaly and hepatoblastoma. In conclusion, our findings reveal that IGF2 is a bona fide mediator of the Hippo pathway, indicating a fundamental role of the Hippo-IGF2-IGF1R signaling axis in organ size regulation and malignancies.

cell biology↗

bioRxiv2024

Dysregulation of FicD AMPylation causes diabetes by disrupting pancreatic endocrine homeostasis

Bi-functional enzyme FicD regulates the endoplasmic reticulum chaperone BiP using AMPylation and deAMPylation during ER homeostasis and stress, respectively. Human FicD with an arginine-to-serine mutation disrupts FicD deAMPylation activity resulting in severe neonatal diabetes. We generated the FicDR371S mutation in mice to create a pre-clinical murine model for neonatal diabetes. We observed elevated BiP AMPylation levels across multiple tissues and signature markers for diabetes including glucose intolerance and reduced serum insulin levels. While the pancreas of FicDR371S mice appeared normal at birth, adult FicDR371S mice displayed disturbed pancreatic islet organization that progressed with age. FicDR371S mice provide a preclinical mouse model for the study of UPR associated diabetes and demonstrate the essentiality of FicD for tissue resilience.

physiology↗

bioRxiv2024

Substrate tunnel regulates substrate specificity switching in hyaluronan synthase

Hyaluronic acid (HA), a ubiquitous linear polysaccharide in the extracellular matrix of vertebrates and the capsule of certain pathogenic bacteria, is characterized by its unique structure of alternating {beta}-1,3-N-acetylglucosamine and {beta}-1,4-glucuronic acid units. HA is synthesized by Class I hyaluronan synthase (HAS) in the majority of organisms, but the molecular mechanisms determining the alternating sequence of HA remain unclear. Here, we demonstrated that HA synthesized in vitro using Streptococcus equisimilis HAS maintained its alternating sequence, regardless of substrate ratios and concentrations. While the active pocket lacks intrinsic selectivity, a distal allosteric regulation from the transmembrane tunnel is revealed. The lingering HA terminals in the active pocket ensure lower binding energies for alternating substrates through Coulombic repulsion and competitive binding. Moreover, the dynamic C-terminal loop at the substrate tunnel entrance reduces transport distance for alternating substrates. Our findings indicate that HA-substrate interactions and enzyme dynamics in the substrate tunnel collectively govern the alternating substrate specificity of HAS. This study provides the first molecular insight into precise substrate specificity control within a single active pocket during template-independent polymer synthesis.

biophysics↗

bioRxiv2024

Establishment and Molecular Characterization of Patient-Derived Organoids for Primary Central Nervous System Lymphoma

Primary central nervous system lymphoma (PCNSL) exhibits substantial heterogeneity, both intra-tumoral and intertumoral, posing challenges in developing effective treatment methods. Existing in vitro models fail to simulate the inherent microenvironment and the cellular and mutational diversity of native tumors and require a prolonged generation time. To address this concern, we described an organoid culture method for patient-derived PCNSL organoids (CLOs) and evaluated them through extensive molecular characterization. These CLOs accurately mimicked the histological attributes, gene expression landscapes and mutational profiles of their original tumors through rigorous histopathological analysis, RNA sequencing and whole-exome sequencing. Notably, CLOs were generated within 2 weeks, demonstrating rapid development and reliability. Furthermore, therapeutic profiling was performed on three selected CLOs using four standard treatment drugs. High concordance was observed between the drug responses of patients and those observed in the CLOs, with specific sensitivity to ibrutinib and methotrexate and resistance to dexamethasone and rituximab. Taken together, these results emphasize that CLOs can effectively emulate the key characteristics of PCNSL, enhancing the understanding of the genetic landscape of this complex disease. CLOs provide a rapid and reliable platform for exploring individualized treatment strategies, potentially accelerating the transition of research findings to clinical practice.

cancer biology↗

bioRxiv2024

Medicinal chemistry of benzoisoxazole-4,7-dione analogues identifies the ribosomal small subunit as a target for specific mycobacterial translational fidelity

A central dogma of molecular biology is the "speed-accuracy trade-off," where ribosomes must slow down to ensure accurate protein synthesis. In mycobacteria, a high basal level of mistranslation at glutamine and asparagine codons, caused by an indirect tRNA aminoacylation pathway, promotes tolerance to the antibiotic rifampicin. While pharmacologically increasing translational fidelity is a promising strategy to combat antibiotic tolerance, the underlying mechanisms remain poorly understood. Here, we screened 9,000 synthetic compounds and identified benzo[d]isoxazole-4,7-diones as a novel chemical class that reduces mycobacterial mistranslation. Medicinal chemistry optimization yielded a lead compound, 9787, with superior potency in decreasing mistranslation and reversing rifampicin tolerance. Using competitive chemical proteomics, we identified the 30S ribosomal protein S5 (RpS5) as the specific cellular target. Remarkably, compound 9787 enhances translational fidelity at concentrations that do not measurably impact the overall rate of protein synthesis. Our findings challenge the universality of the speed-accuracy trade-off, demonstrating that fidelity can be improved independently of translation speed. This work reveals that the ribosomal small subunit is a druggable target for modulating translational quality control and introduces a new strategy for combating antibiotic-tolerant bacteria without the associated fitness cost of slowed translation. ImportanceA fundamental principle in molecular biology holds that ribosomes face a trade-off between translation speed and accuracy: going faster means making more errors, while maintaining high fidelity requires slowing down. This study challenges that paradigm by identifying a small molecule that increases translational accuracy in mycobacteria without affecting the rate of protein synthesis. The compound targets ribosomal protein S5 and specifically reduce errors arising from physiologically mischarged tRNAs - a quality control problem distinct from the well-studied codon*anticodon mismatches. This form of mistranslation contributes to antibiotic tolerance in tuberculosis, making it a potential therapeutic target. Our findings reveal unexpected flexibility in how ribosomes maintain translation quality and suggest that pharmacologically increasing fidelity without the fitness cost of slowed protein synthesis may be an attractive strategy for combating antibiotic-tolerant bacteria.

microbiology↗