Cell Discovery

Bifidobacteriaceae and Lactobacillaceae are key probiotic families and widely used in food production, yet a comprehensive understanding of strain functions and their gut microbial interactions based on complete genomes remain understudied. Here we constructed a complete-genome dataset of 3,300 strains from these two families, including 1,151 newly isolated from China. Compared with draft assemblies, complete genomes substantially recovered a gene functional landscape encompassing stress tolerance, surface exopolysaccharide synthesis, nutrient utilization, and mobile genetic elements. Major species from both families exhibited a prevalence >60% in the Chinese population, far higher than that in US/Dutch cohorts. Notably, as a core probiotic species with remarkable genomic plasticity and gut-adaptive potential, Lactiplantibacillus plantarum stood out in our dataset for its enriched functional profile and was particularly abundant in the Chinese population. Moreover, compared with non-Chinese genomes, our isolates of key species displayed less metabolic complementarity and stronger competition with potentially pathogenic keystone species in the gut, thereby linking strain origin to enhanced probiotic potential and ecological fitness to benefit human gut health.

Mixed-lineage leukemia translocated to 3 (MLLT3) is vital for maintaining the stemness of hematopoietic stem cells. Loss of MLLT3 in megakaryocyte (MK)-erythrocyte progenitor (MEP) cells leads to its differentiation into MKs. Despite its significance in stemness, the regulatory mechanism of MLLT3 during differentiation remains elusive. In this study, we investigate the regulatory role of histone deacetylase 6 (HDAC6) in modulating MLLT3 levels via heat shock protein 90 (Hsp90) activation during myeloid lineage differentiation into MKs, monocytes, and macrophages. We found that HDAC6 activates Hsp90 through deacetylation, enabling Hsp90 to retain MLLT3 in the cytoplasm where protein kinase C (PKC) phosphorylates MLLT3 at serine residues; leading to loss of MLLT3 during MK and macrophage differentiation but not during monocyte differentiation. This research provides valuable insights into the regulatory mechanisms underlying myeloid lineage commitment and opens new avenues for future investigations into stem cell biology and therapeutic applications.

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) deficiency is a rare genetic cause of neurodevelopmental disorders (NDDs) lacking targeted therapies. Here, we developed a transcriptomic-guided compound prioritization pipeline using Connectivity Map (CMap) analysis on multi-model transcriptomic signatures from HNRNPU-deficient human cells and mouse models. Ten compounds were selected through manual curation and functionally screened in patient-derived HNRNPU-deficient neuroepithelial stem (NES) cells with earlier observed cellular phenotypes. Two of the compounds, AS601245 and Lenalidomide, significantly reduced the elevated neural progenitor population during differentiation, and their combination further decreased primary cilia incidence, indicating partial rescue of the patient-specific cellular phenotypes. To understand the mechanisms underlying the partial rescue, we employed proteome integral solubility alteration (PISA) and expression proteomics. PISA assay identified TMEM150C and GSK3A as proximal targets of combined treatment. Additionally, we observed reversal of multiple biological pathways including downregulation of Wnt signalling and upregulation of mitochondrial pathways and transmembrane proteins. Altogether, we established a computational-experimental pipeline for transcriptomic-guided drug repurposing for a monogenic NDD, and demonstrated that the network-level modulation partially rescues the delayed neural differentiation in HNRNPU-deficient neural cells.

Human immunodeficiency virus (HIV) infection remains a global health threat. Although antiretroviral therapy (ART) has significantly transformed HIV into a manageable chronic disease, emergence of drug resistance to current ART is a continuing concern. Broadly neutralizing antibodies, as well as reagents containing both a soluble CD4 mimetic and an HIV co-receptor inhibitor--such as CD4-CD4i antibodies--are promising strategies for the prevention and treatment of HIV infection. We previously developed a CD4 D1mimetic (mD1.22) with enhanced neutralization potency, surpassing that of the clinically validated sCD4-D1D2 mimetic. We also previously identified a novel CD4i antibody (X5) that targets the conserved coreceptor binding site on the gp120 core and recognizes an epitope partially overlapping with 17b monoclonal antibody binding site. X5 binding to gp120 was augmented by CD4 and modestly enhanced by CCR5. To leverage these favorable interactions, we designed and optimized sCD4-X5 bispecific antibodies by computational structure-aided modification of antibody size and fusion linkers between the two binding moieties. The bispecific D1X5, with a (G4S)7 long linker between D1 and X5 (IgG1-LL D1X5), exhibited broad neutralization against 11 diverse HIV subtypes across B, C, G clades and AC, BC recombinants. The TZM-bl cell neutralization assay showed IgG1-LL D1X5 neutralization geometric mean IC50 and IC80 are 0.6 g/mL and 3.4 g/mL respectively, which are within the range of potent bnAbs. This work has identified a novel single domain soluble CD4 based CD4-CD4i bispecific antibody with broad HIV-1 neutralization.

ATAD2 possesses a C-terminal bromodomain (BRD) that plays a critical role in recognizing and binding to acetylated lysine residues. However, because the native intracellular structure of ATAD2 remains poorly defined, the mechanisms by which the ATAD2 BRD recruits acetylated histones and the regulatory pathways involved are not yet understood. In this study, we report that the ATAD2 BRD mediates the formation of liquid-liquid phase separation (LLPS) of ATAD2 in cells. This phase separation promotes the process of histone H4 acetylation, leading to the up-regulation of C-MYC, CCND3, and ATF2 gene expression and the facilitation of chromatin remodeling. Our findings elucidate a vital function of ATAD2, wherein BRD-mediated LLPS drives histone acetylation to promote cellular chromatin remodeling.

Aberrant activation of type I interferon (IFN-I) is closely related to the development of autoimmune diseases. The metabolic regulation of cytokine signaling is essential for immune homeostasis. In this study, we characterized Urolithin A(UA), a natural gut-derived metabolite, as an inhibitor of Janus kinase (JAK) signaling. UA was found to broadly dampen JAK phosphorylation and the downstream signaling induced by cytokines such as type I interferons (IFN-I), type II interferons (IFN-II), and interleukin-6 (IL-6). UA can directly bind to JAK1 JH1 domain and treatment with UA attenuated autoimmune pathogenesis in Trex1-KO mice, IMQ-induced SLE and psoriasis models. Our findings unveil that UA is an anti-inflammatory metabolite that promotes immune homeostasis and could be used to treat inflammatory and autoimmune diseases.

The bacterial flagellar motor is an intricate nanomachine that transforms chemical energy from ion gradients into mechanical rotation, enabling bacterial movement. While stator unit architectures are conserved across species, the molecular link connecting ion translocation to rotational force generation remains elusive. In this study, we refined the cryo-EM structures of MotAB from Campylobacter jejuni (CjMotAB) and integrated a suite of approaches--including single-structure based pKa predictors and free energy perturbation (FEP) calculations, as well as standard and constant-pH molecular dynamics (CpHMD) simulations of various structural models representing the plugged, unplugged, and plug-removed states with different protonation states of D22--to dissect its rotational mechanism. Based on pKa calculations, the D22 residues in chains F and G of MotB were identified as proton carriers supporting the previous hypotheses. Importantly, we observed asymmetric hydration patterns of the two D22 residues in the MotB dimer, along with their hydrogen bonding interactions with MotA T189, which contribute to functional specialization. Our findings reveal that MotA rotation requires two essential prerequisites: plug removal and alternating D22 protonation switching, coupled with dynamic gauche-trans conformational changes in the sidechain of D22. This work clarifies how protonation dynamics and structural asymmetry synergistically regulate CjMotAB rotation, advancing our understanding of bacterial flagellar motor function and providing a foundational framework for investigating diverse ion-driven biological motors.

Bacteria have evolved multiple immune systems to resist phage invasion, however, only a small part of the defensive mechanisms have been clearly uncovered. In this study, we report a type III Druantia two-component defense system, DruH-DruE, identified from Mycobacterium smegmatis. The DruH-DruE prevents phage DNA cyclization and replication.DruE can be replaced from the defense system by either homolog in M. tuberculosis or M. smegmatis. The physical interaction between this two components is essential for fighting against phage infection. Mutations in the interaction sites led to the loss of phage-defending function of the system. The broad-spectrum antiphage ability of the defense system could be activated by the small tail protein Gp25 of phage A10ZJ24. This study fills a major gap in current knowledge of antiphage mechanism of type III Druantia defense system, expanding our understanding of the immune mechanisms in prokaryotic cells.

G-quadruplexes (G4s) are critical nucleic acid secondary structures that play pivotal roles in regulating gene expression. In this study, we conducted a proteome-wide in silico analysis across multiple viruses causing hemorrhagic fevers to identify candidate proteins containing a conserved G4-binding motif. Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. We selected the NS3 protease domain of Yellow Fever virus for further validation. Biochemical assays demonstrated that the NS3 protease domain binds G4 structures with high specificity and affinity, particularly favoring the parallel conformation. Molecular docking and simulations further revealed that the NS3 protease domain interacts with the terminal G-tetrads and loop regions of G4 via key residues, including PHE40, adopting an insertion and stacking composite binding mode. These findings expand our understanding of virus - G4 interactions and offer novel potential targets for G4-based antiviral strategies. Bullet points- We screened viruses causing hemorrhagic fevers for potential G4-binding peptides. - Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. - Biochemical assays demonstrated that the NS3 protease domain of YFV binds G4 structures with high specificity and affinity.

The inositol monophosphatase (IMPase) orthologue is pivotal for virulence, pathogenesis, and biofilm regulation, and is therefore considered a potential drug target in Pseudomonas aeruginosa and other bacterial pathogens. The mammalian IMPase orthologue is an established drug target for bipolar disorder. The precise catalytic mechanism in this class of enzymes remains obscure despite five to six decades of extensive efforts and detailed studies of substrate, transition-state analogue, and product-bound structures. Here, we have solved the crystal structures of the IMPase orthologue from Pseudomonas aeruginosa (PaIMPase), capturing pre- and post-catalytic snapshots of metal-substrate- and metal-product-mimic-bound states. Moreover, we solved the metal-substrate transition-state-analogue-bound crystal structure of the enzyme. Critical evaluation of these high-resolution crystal structures of PaIMPase complexed with substrate, transition-state analogue, and product mimic (myo-inositol and phosphate) supports three Mg2+-dependent catalytic mechanisms of PaIMPase. The structural snapshots indicate that, at the enzyme active site, a metal (Mg2+)-coordinating water molecule, activated by two bound Mg2+ ions and the active-site-proximal Threonine/Aspartate dyad, attacks the central phosphorus atom of the bound substrate, leading to formation of a trigonal bipyramidal transition state. Following that, the immediate breakdown of the P-O bond results in the formation of inositolate and phosphate ions. The second water molecule, activated by another Mg2+ dyad, facilitates the departure of myo-inositol and phosphate from the active site. The detailed mechanistic insights gained from this work may offer a foundation for the rational design of precise inhibitors against PaIMPase.

Human milk oligosaccharides (HMOs) are abundant and structurally diverse glycans that shape the development of infant gut microbiota. Yet, how individual HMOs and bacterial genes drive the community assembly remain elusive. Here, we reconstructed an eight-member infant Bacterial Community (iBaCo) from representing dominant taxa in human infant feces. When individual HMOs were the sole carbohydrate source, they showed deterministic effects on the iBaCo composition and metabolic output. Notably, the tetramer HMO lacto-N-tetraose (LNT), in spite of its identical monomer composition as lacto-N-neotetraose (LNnT), showed a strong effect on maintaining Bifidobacterium breve abundance in iBaCo, whereas LNnT did not. Monoculture growth profiling, proteomics, enzymatic kinetic assay, and molecular docking revealed that {beta}-galactosidase D4BMY8 and the relevant downstream pathways are induced by LNT and that D4BMY8 has substrate preference on LNT over LNnT, enabling a faster growth of Bi. breve and accumulation of acetate and lactate in LNT compared to LNnT. Metabolic flux analysis indicated that the substrate-preference of {beta}-galactosidase D4BMY8 drives the skewed energy cost toward lactate/acetate metabolic output. Finally, the D4BMY8-encoding gene lacZ5 is widely spread in all isolated Bi. breve genomes, but divergently distributed in infant metagenome-assembled Bi. breve genomes. Together, we demonstrated that a single enzyme-substrate interaction could orchestrate the composition and metabolic function of an infant bacterial community, which may contribute to the assembly of dynamic infant gut microbiota. Our integrative approach provides a mechanistic framework for understanding the interaction between diet, microbial community, and infant gut health.

The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction with IgaA, but the precise activation mechanisms remain unclear. In this study, we determined the structures of IgaA and the IgaA/RcsF complex using Cryo-electron microscopy (Cryo-EM). We also determined the structures of RcsC and RcsD, which both form homodimers stabilized by hydrophobic interactions, creating ladder-like structures. Combining the Cryo-EM structures, AlphaFold3 structure predictions of IgaA/RcsD and RcsF/IgaA/RcsD, and genetic studies, we describe a model for how RcsF modifies the IgaA/RcsD interaction, lifting negative regulation and activating the Rcs phosphorelay. Our findings provide a high-resolution depiction of the Rcs stress response system and suggest potential targets for small molecule inhibitors.

Highly pathogenic avian influenza viruses (HPAIVs) continue to cause substantial disease in birds and mammals, with repeated H5N1 spillovers highlighting the need for broadly protective antiviral strategies. Here we develop a programmable RNA-targeting antiviral platform based on RfxCas13d and evaluate its activity in avian cells. Screening of five Cas13 orthologs in chicken DF1 fibroblasts revealed RfxCas13d as the most potent and well tolerated effector. Virus-specific CRISPR RNAs (crRNAs) targeting conserved regions of positive- and negative-sense influenza RNA were tested against A/WSN/033[H1N1] and multiple HPAIV isolates, including a member of clade 2.3.4.4b H5N1. Targeting positive-sense RNA conferred superior influenza inhibitory activity and further enhanced by multiplexed crRNA expression. These findings establish RfxCas13d as a versatile RNA-guided antiviral platform and provide a route for broad-spectrum influenza control through conserved RNA targeting.

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected illness with outdated treatments. Bromodomain factors (BDFs) are essential proteins that recognize acetylated lysines and have strong therapeutic potential. They form part of epigenetic complexes that regulate chromatin accessibility and, therefore, gene expression. However, little is known about their structure in trypanosomatids. Here, we used a combination of experimental and bioinformatic approaches to infer the stoichiometry and structure of T. cruzi bromodomain-containing complexes. By reconstructing the proximity networks of five BDFs using TurboID-directed proximity labeling, we identified highly interconnected components that assemble into the CRKT and NuA4 complexes. Using novel structure prediction strategies that systematically explore the stoichiometric space, we inferred that CRKT assembles into three distinct modules and NuA4 in two, with different degrees of interaction dynamics. The core module of CRKT contains two copies of each component, including BDF3, BDF5, and BDF8, arranged in a subcomplex with central symmetry. The catalytic module of CRKT has three subunits, including the histone acetyltransferase 2 (HAT2), while the BET (bromodomain and extra-terminal) module has one unit of both BDF4 and BDF1. The catalytic module of NuA4 closely resembles the yeast piccolo-NuA4 module and contains HAT1, while the TINTIN module associates with the catalytic module via the C-terminal domain of BDF6. These insights shed light on the structure and composition of epigenetic complexes in trypanosomatids, opening new avenues for rational drug design aimed at disrupting their function.

The developmental program governing meibomian gland (MG) morphogenesis and proliferation remains poorly understood, largely due to the lack of physiologically relevant model systems. Here, we established a novel high-fidelity, three-dimensional organoids model derived from mouse meibomian gland (mMGO) epithelium. Transcriptomic and phenotypic analyses demonstrated that mMGOs faithfully recapitulate postnatal gland development in vivo, including dynamic transcription program, branching morphogenesis, lineage differentiation, and functional lipid accumulation. Leveraging this model, we identified the Hippo-YAP pathway as a pivotal regulator of MG epithelial proliferation and homeostasis for the first time. YAP inhibition severely impaired organoids growth, while pharmacological inhibition of Hippo pathway with XMU-MP-1 enhanced proliferation and progenitor clonogenicity. Crucially, in inflammation-induced atrophic organoids, XMU-MP-1 treatment rescued YAP nuclear localization and stimulated regrowth and functional restoration. Our study provided new mechanistic insights and a robust organoids platform for MG development research, and nominated targeted Hippo pathway inhibition as a promising strategy to reverse glandular atrophy in meibomian gland dysfunction.

Hantaviruses are zoonotic negative-sense RNA viruses that cause two severe diseases; haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) for which no approved antiviral therapies are available. To identify host-directed modulators of hantavirus infection in the available annotated drug space, we performed a drug repurposing screen in A549 cells and HUVECs, using live Puumala virus (PUUV). We identified and validated 70 drugs with antiviral activity across these 2 different cell systems. Functional clustering confirmed the known infection-inhibitory effect of several group of compounds, including inhibitors of heat shock proteins, mTOR pathway or nucleotide synthesis. In addition, we also identified compounds yet unexplored as antivirals against Hantaviruses, such as certain antibiotics. This dataset provides a systematic map of host pathways influencing PUUV infection and highlights candidate compounds and cellular processes that warrant further investigation.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease and strongly linked to obesity and insulin resistance. We previously reported that the common nuclear envelope variant rs6461378 (g.842031C>T; SUN1 H118Y) associated with MASLD and related traits including insulin resistance. To gain insight into how wild-type (WT) and H118Y SUN1 might differentially impact insulin signaling, we performed affinity purification-mass spectrometry (AP-MS) in human liver-derived cells stably expressing WT or H118Y SUN1. Unbiased AP-MS revealed a novel SUN1-CUL3 interaction, with comparative analysis showing that WT SUN1 interacted robustly with CUL3, while CUL3 interaction was markedly diminished with H118Y SUN1. Cells in which SUN1 was silenced via siRNA, or in which H118Y SUN1 was ectopically expressed, showed increased CUL3 neddylation, which is required for cullin RING ligase (CRL)-mediated ubiquitination of insulin receptor substrate (IRS) proteins. Inhibition of neddylation restored IRS-1 levels and insulin signaling in H118Y SUN1-expressing cells. Together, our findings provide a potential mechanism of H118Y SUN1-driven insulin resistance and a viable therapeutic approach for its reversal.

Human coronavirus OC43 (HCoV-OC43) constitutes one of the most common causes of the seasonal cold but can also cause severe disease among elderly and immuno-compromised. Currently, there are no approved antiviral drugs to combat HCoV-OC43 infection. Coronaviruses are positive single-stranded RNA (+ssRNA) viruses and utilize -1 programmed ribosomal frameshifting (-1 PRF) to obtain the correct stoichiometry of viral protein components. As such, the ribosomal frameshifting stimulation element (FSE) is a promising target for antiviral drug discovery, due to its high conservation. By repurposing available drugs, we identified a group of anthracycline compounds that can reduce -1 PRF of HCoV-OC43 and reduce viral infection of cells. Furthermore, we show that anthracyclines that reduce infection also bind the FSE and reduce frameshift frequency. We also show that the selected anthracyclines reduce SARS-CoV-2 infection, but without affecting -1 PRF frequency. All together, we demonstrate that a subset of anthracyclines selectively binds and inhibit the HCoV-OC43 FSE and could thus serve as a robust framework when developing new antivirals targeting coronaviruses.

The maintenance of protein homeostasis is vital for all cells. Alteration in protein handling underlies several diseases. The small molecule sephin1 is a promising clinical candidate against proteostasis disruption, but its mechanism of action is still uncertain. Our experimental evidence shows that sephin1 binds G-actin and drives actin cytoskeleton misfolding, and eventually, Golgi disintegration. At first, sephin1 impairs the autophagic flux and elicits the phosphorylation of the subunit of eIF2 and the ER-stress independent expression of CHOP via GCN2 kinase. Sephin1 also inhibits the mammalian target of rapamycin (mTORC1), activates the transcription Factor EB (TFEB), drives the expression of TFEB-direct target genes, and eventually stimulates the autophagy lysosomal pathway. Our results reveal that the actin cytoskeleton may regulate autophagy via mTORC1-TFEB complemented with the GCN2-eIF2-CHOP signaling pathway.

O_LIWD40 is a highly conserved protein domain in eukaryotes, playing a critical role in various cellular process. C_LIO_LIWe conducted genome-wide functional analysis of WD40 genes in Fusarium graminearum--a phytopathogenic fungus that causes severe yield loss and mycotoxin contamination in major cereal crops. C_LIO_LIComprehensive phenome analysis of 119 WD40 gene deletion mutants across 22 distinct phenotypic traits revealed phenotypic divergence within the phenome, establishing a strong correlation between virulence and sexual reproduction. Notably, 21 "core WD40 genes" were identified, offering valuable insights into divergent biological processes. C_LIO_LIPilot interactome studies of Fgwd101 and Fgwd133 provided further insights into their potential pathobiological functions. Our investigation contributes to broadening our knowledge of the biological mechanisms underlying fungal pathogenesis and may assist in the identification of targets for antifungal agents. C_LI