Biology subjects

Feng, L.

Publications and source records attributed to Feng, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Fibers and Glasses are Competing Material States in FUS Protein Condensation

Dense, well-ordered material states of proteins form the amyloid fibers that are a hallmark of neurodegenerative disease in the brain. Beyond forming amyloid fibers, some of these proteins can also adopt other material states termed condensates which are initially liquid-like but evolve to a soft, glassy phase. Fiber growth requires a large supply of monomers and, thus, it is often speculated that fibers emerge from within a dense condensate as it ages and its microscopic dynamics slow into a glassy state. Here, we use the well-established model system Fused in Sarcoma (FUS) to directly observe, quantify and theoretically describe fiber growth and its interplay with condensates. We report the discovery that fibers grow overwhelmingly in the dilute phase surrounding the condensates while the condensates concurrently evolve to a glassy arrested solid. The resulting protein fibers and glassy condensates are both distinct solid-like phases that coexist but do not directly interconvert. Taken together, these findings reveal that there are two competitive aging pathways in FUS condensation that are linked through phase separation kinetics.

biophysics↗

bioRxiv2026

Predictive Modeling of Cancer Cell Growth Kinetics with Machine Learning

Quantitative characterization of cell proliferation is central to preclinical drug discovery. Here, we evaluated Random Forest (RF) regression for predicting confluence-based cell growth trends using data from human cancer cell lines and benchmarked its performance against the widely used logistic and Gompertz models. The RF model achieved higher predictive accuracy within the evaluated dataset, particularly at higher initial seeding densities. We further extended the RF framework to predict growth curves across seeding densities. Collectively, these findings highlight the potential of data-driven modeling to complement conventional mathematical approaches and support more informed cell-culture design.

bioinformatics↗

bioRxiv2026

Extracellular injection system combined with peptides for intracellular Staphylococcus aureus treatment

The inaccessibility of intracellular bacteria has long rendered the treatment of Staphylococcus aureus infections an challenge. Studies have demonstrated that the extracellular injection system PVC can accurately deliver proteins into cells, which would not need small molecules, and enables effective intracellular delivery of antimicrobial peptides for treatment. Accordingly, we selected antimicrobial peptides including Cecropin, LL37 and Indolicidin that possess potent bactericidal activity, and established the Directed Antimicrobial Assault platform (DAAT) by leveraging the intracellular delivery capacity of PVC. DAAT Cecropin, DAAT LL37 and DAAT Indolicidin inhibited intracellular bacteria in a dose-dependent manner, with DAAT LL37 reaching 86.76% inhibition; after 72 h of treatment, viable-cell numbers reduse to 66--82-fold those of the control. Tail-fibre retargeting enabled direct extracellular S. aureus killing, while combined DAAT therapy promoted wound healing in mice. These findings expand the utility of PVC-derived nanosyringes and establish DAAT as a modular platform for intracellular antimicrobial peptide therapy.

synthetic biology↗

bioRxiv2026

M1 macrophage-mediated lymphangiogenesis aggravates liver fibrosis via MDK/YAP signaling pathway

BackgroundLymphangiogenesis plays a critical role in various liver diseases, yet its function in liver fibrosis remains controversial. This study aimed to explore the role of lymphangiogenesis in liver fibrogenesis and its underlying regulatory mechanisms. MethodsLiver fibrotic mice were established by carbon tetrachloride (CCl4) or Thioacetamide (TAA)-induced injection or bile duct ligation. Lymphatic vessels were marked by podoplain (Pdpn) staining in mice and D2-40 staining in clinical samples. Lymphatic vessels area and density were measured to indicate lymphangiogenesis. Multiplexing immunohistochemistry was used to detect co-localization of proteins. ResultsIn the present study, we first verified increased lymphangiogenesis in human and murine fibrotic livers. Afterwards, we identified VEGFC rather than VEGFD as the primary driver of lymphangiogenesis in liver fibrosis. Furthermore, we demonstrated that M1 macrophages serve as the major source of VEGFC. Founctional studies revealed that VEGFC-mediated lymphangiogenesis exacerbates hepatic fibrosis, while its inhibition alleviated fibrosis. Bioinformatic analysis uncovered Midkine (MDK) as a key downstream of lymphangiogenesis. Both in vivo and in vitro studies confirmed that exogenous MDK promotes liver fibrosis via activating hepatic stellate cells (HSCs), whereas MDK inhibition counteracts the profibrotic effects of VEGFC-induced lymphangiogenesis. Importantly, we discovered that MDK activates HSCs through the Hippo/YAP signaling pathway. ConclusionsM1 macrophage-mediated lymphangiogenesis aggravates liver fibrosis via MDK secretion, which activates HSCs. These findings provide novel insights into coordinated crosstalk between macrophages, lymphatic endothelial cells and HSCs in liver fibrosis and suggest lymphangiogenesis and MDK as potential therapeutic targets for fibrotic liver diseases.

molecular biology↗

bioRxiv2026

The in vivo inhibitory function of the MHC-I alpha3 domain/CD8alpha interaction

The interaction between the Major Histocompatibility Complex Class I (MHC-I) 3 domain and CD8 has classically been viewed as a positive coreceptor interaction that stabilizes TCR signaling during antigen recognition. However, its physiological function in mature peripheral CD8+ T cells in vivo remains incompletely understood. Here, we identify the MHC-I 3 domain-CD8 interaction as a previously unrecognized inhibitory pathway that tonically restrains peripheral CD8+ T-cell activation and maintains T-cell tolerance in vivo. Antibody-mediated disruption of the MHC-I 3 domain-CD8 interaction induced spontaneous activation of peripheral CD8+ T cells without impairing their survival, lowered the threshold for antigen-induced activation, and enhanced responsiveness to cognate peptide stimulation. In peptide-induced OT-I T-cell anergy models, blockade of either H-2Db or H-2Kb 3 domain interactions with CD8 prevented the induction of anergy and restored responsiveness of previously anergic T cells. Notably, blockade of the H-2Kb 3 domain enhanced OT-I responses despite simultaneously disrupting the classical positive coreceptor interaction within the TCR-peptide-MHC complex, indicating that tonic inhibitory signaling mediated by the MHC-I 3 domain predominates under these conditions. Together, these findings redefine the classical MHC-I-CD8 interaction as a bidirectional pathway that not only supports antigen recognition but also imposes tonic inhibitory control over peripheral CD8+ T cells. These results identify the MHC-I 3 domain-CD8 axis as a potential target for reversing T-cell tolerance and enhancing antitumor or antiviral immunity.

immunology↗

bioRxiv2026

A multifunctional polyketide synthase in nematodes produces divergent families of signaling molecules that control different developmental arrests

To improve their chances of reproductive success, nematodes not only must arrest their development in response to adverse growth conditions but also must quickly recover if conditions improve. A polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid assembly line that is expressed in the canal-associated neurons (CANs) of Caenorhabditis elegans promotes recovery from starvation-induced larval arrest. Here, we show that in the predatory nematode Pristionchus pacificus this assembly line produces a suite of secondary metabolites, including a family of hybrid polyketide-nonribosomal peptides known as the nemamides, the related nemaketides, and a family of ascarylose-modified polyketides named ascarenes. Depending on the starter unit that is loaded onto the PKS, the assembly line can produce dramatically different downstream products. Whereas the nemamides promote recovery from starvation-induced larval arrest, the ascarenes inhibit development of the dauer larval stage and promote recovery. This dichotomy suggests that the PKS-NRPS megasynthetase serves as a signaling hub in the CANs, controlling multiple developmental events. The PKS-NRPS assembly line is highly conserved across many nematode species, and identification of these chemical signals will help to elucidate the signaling pathways that control development in the worm and lead to novel anthelmintics.

biochemistry↗

bioRxiv2026

PlantP450Dock: an Automated Molecular Docking Pipeline of Plant Cytochrome P450s

Cytochrome P450 enzymes (CYPs) are the primary drivers of chemical diversification in plant secondary metabolism; however, fewer than 10% of the superfamily members have been functionally characterized. Computational docking provides a scalable strategy to prioritize candidates for experimental validation, yet prevailing workflows are poorly adapted to plant P450s because AlphaFold-predicted structures lack the essential heme cofactor and conventional flexible-residue selection relies on subjective geometric cutoffs. To address these limitations, we developed an automated pipeline--PlantP450Dock--that unifies heme cofactor implantation, molecular dynamics-based conformational sampling, data-driven flexible residue selection, and semi-flexible docking within a single integrated workflow. The heme is transferred from a crystallographic template to the AlphaFold model via a local coordinate transformation algorithm, achieving a positional deviation of less than 0.2 [A] relative to the experimentally determined CYP73A33 structure (PDB: 6VBY). Subsequent 100 ns molecular dynamics simulations confirmed faithful preservation of the Fe-S coordination geometry (2.61 {+/-} 0.08 [A]) across all trajectory frames. A singular value decomposition-based heme-plane filtering strategy objectively identified distal active-site residues for flexible treatment, eliminating user-dependent subjectivity. Cross-family validation against four phylogenetically distinct P450s (CYP73, CYP711, CYP706, and CYP701) generated catalytically competent binding poses with substrate-to-heme-iron distances of 2.8-4.4 [A] without enzyme-specific parameterization. Released as an open-source tool, this pipeline furnishes the plant science community with a standardized, reproducible computational framework to accelerate functional annotation of the largely unexplored plant P450 families.

bioinformatics↗

bioRxiv2026

Fungal Hyphae as Distributed Vapor Sinks

Hygroscopic surfaces act as local vapor sinks that reshape the condensation field around them, but whether distributed biological structures do the same has not been investigated. We have established that hyphae of fungal colonies functionally behave as vapor sinks, creating a dry region of width{delta} around themselves when placed on a cooled substrate. In addition, the radial distribution of droplet sizes steepens during condensation, and the rate at which droplets evaporate locally after chamber drying increases. In order to quantify this behavior, we employed a combination of time-resolved imaging and survival analysis to determine how long individual droplets persist on the surface surrounding the colony. These data were used to derive three quantitative measures of the vapor-sink effect. Each measure was found to be directly proportional to the vapor-sink strength of the substrate, as calibrated against NaCl-agar hydrogels of known water activity (LOOCV RMSE = 0.031 for recovered aw). These findings were consistent across three fungal genera (35 experiments), and all species fell along calibration lines defined by the hydrogel standards. This result is consistent with a diffusion-limited vapor-depletion framework. The measured genus-level{delta} ratios agreed to within 6% of predictions from structural absorbing capacity, and field measurements on Gymnosporangium-infected apple leaves were consistent with the same signatures under natural conditions. These results establish a non-contact method for inferring the material properties of thin hygroscopic biological surfaces from their condensation patterns.

biophysics↗

bioRxiv2026

Genuine Directed Evolution In Test Tube (GENie)

Directed evolution has long been constrained by complex screening hardware and labor-intensive workflows. Here, we report the first genuine test-tube screening platform that uses His6-tagged peptide-functionalized magnetic beads and Fe3+-decorated E. coli cells to establish a phenotype-genotype linkage, thereby decoupling ultrahigh-throughput screening from specialized instrumentation and democratizing directed evolution. The platform demonstrated a screening throughput of > 108 events s-1 and an enrichment factor of up to 63-fold. Using galactose oxidase as a model, we identified variants with up to a 26-fold increase in catalytic efficiency. Extensions to D-amino acid oxidase and alcohol oxidase yielded variants with up to 5383-fold and 25-fold improvements over their respective wildtypes after a single round of screening. These results highlight the platforms capacity to rapidly engineer H2O2-generating oxidases and to advance AI-driven enzyme design through rapid data generation.

bioengineering↗

bioRxiv2026

Long-term maintenance of H3K27me3 in postmitotic neurons is dispensable for gene expression regulation

Neuronal maturation is associated with extensive changes in gene expression and chromatin organization. However, the molecular mechanisms that control the epigenetic landscape in terminally differentiated neurons remain poorly understood. Here, we show that maturing cerebellar granule cells undergo a striking and specific increase in the levels of the repressive histone modification H3K27me3 across different genomic regions, including individual genes, broad intergenic regions, and gene clusters. The accumulation of H3K27me3 coincides with a developmental switch from EZH2 to EZH1 and colocalizes with H3K36me2 and DNA non-CpG methylation. Using mice with a conditional deletion in the catalytic domain of EZH1, we demonstrate that the maintenance of H3K27me3 in mature neurons depends on EZH1. Unexpectedly, an almost complete loss of H3K27me3 in postmitotic GCs induces minimal changes in gene expression and chromatin accessibility at 7 months of age. Using single-nucleus RNA sequencing (snRNAseq) from the mouse neocortex, we show that, similarly to GCs, the loss of EZH1-mediated H3K27me3 also has a minimal impact on cortical neuron gene expression. The amino acid composition of EZH1 suggests reduced sensitivity to H3K36 methylation, providing a potential basis for its activity in chromatin contexts that are not permissive for EZH2. Together, our results show that a postmitotic switch from EZH2 to EZH1 establishes novel chromatin domains in neurons with a minimal role in transcriptional maintenance.

molecular biology↗

bioRxiv2026

Multiomic screening platform uncovers the impact of histone mutations on chromatin and cell fate

Somatic missense mutations in histone genes, often referred to as oncohistones, have been identified in diverse types of human cancers. The functional and mechanistic impact of most oncohistones remains unknown. To address this gap, we developed CHANCLA, a modular platform for high-throughput functional screening of oncohistones using multiomic phenotypic readouts. We used CHANCLA to systematically measure the impact of 303 human oncohistones on cellular proliferation, differentiation, histone-specific post-translational modifications, and chromatin accessibility. Integrative multiomic analyses revealed discrete oncohistone molecular classes that promote proliferation, block lineage-specific differentiation, and physically remodel the chromatin landscape by altering specific histone modifications and reducing nucleosome stability. Structural mapping and computational modeling studies uncovered that functionally convergent mutations are clustered at key nucleosome interfaces, particularly H2B-H4, and that chromatin accessibility-promoting mutations are linked to mono-nucleosome destabilization. Leveraging this multiomic resource, we discovered that the H3.3-Q5H mutant histone is a bona fide human oncohistone that accelerates lung adenocarcinoma growth in vivo. Mechanistically, we found that H3.3-Q5H expression leads to suppression of promoter-associated H3K4me3 and expansion of repressive H3K27me3 domains, resulting in increased KRAS signaling and gene expression programs associated with epithelial-to-mesenchymal transition. Together, this work provides a multiomic functional atlas of cancer-associated histone mutations, identifies structural and mechanistic principles governing chromatin reprogramming by oncohistones, and establishes CHANCLA as a modular platform for systematic discovery of mechanisms and vulnerabilities associated with these genetic lesions.

cancer biology↗

bioRxiv2026

Using machine learning to overcome mosquito collections missing data for malaria modeling

Entomological surveillance plays a crucial role in areas where malaria remains endemic, yet gathering data on mosquito populations is often expensive and complicated, particularly in remote locations with challenging logistics and inconsistent sampling schedules. Access to extensive time series data on mosquito species at specific sites would greatly enhance insights into seasonal trends and the biting habits of vectors of malaria parasites. Gaps in mosquito count records pose a significant challenge for researchers and public health officials seeking to establish early warning systems and effective vector control programs. In this study, we apply quantitative machine learning techniques to address missing data in estimates of mosquito abundance collected from 2009 to 2016 in Bolivar State, Venezuela. We evaluated Linear Regression, Stochastic Linear Regression, K Nearest-Neighbor, and Gradient Boosting methods for imputing missing counts of Anopheles mosquitoes, employing a leave-one-out cross-validation strategy. Additionally, we developed a predictive malaria transmission model incorporating mosquito abundance and climate variables (El Nino 3.4 Index, rainfall, and mean air temperature) as covariates. Our generalized time series model forecasts malaria incidence of Plasmodium vivax and Plasmodium falciparum based on climate dynamics and imputed mosquito data. Model performance was assessed using root mean square error, mean absolute error, and mean absolute percentage error. The final results demonstrated that machine learning imputation significantly improved the accuracy and reliability of P. vivax malaria incidence predictions but failed to predict P. falciparum incidence. The study demonstrates that method choice significantly influences the reconstruction of seasonal abundance patterns and the performance of malaria incidence models. Nevertheless, the proposed models strengthen the foundation for targeted interventions and surveillance in endemic regions. Despite limitations in data continuity and coverage, the findings highlight the value of combining multiyear entomological data sets with robust imputation and sensitivity analyses to improve predictive modeling in resource-constrained, malaria-endemic settings.

bioinformatics↗

bioRxiv2026

A mitochondrial program encodes brain vascular reserve

Brain resilience depends on collateral vessels whose geometries preserve blood flow when primary arteries are perturbed. How these protective vascular architectures are developmentally established remains unknown. Using longitudinal in vivo imaging in zebrafish, we show that mitochondrial state in embryonic angiogenic tip cells encodes the topology of basal and surface brain collateral networks. Mechanistically, microRNA-125a establishes the bioenergetic and redox balance of endothelial tip-cell mitochondria through conserved repression of the metabolic regulator PGC1a. Disruption of the microRNA-125a-PGC1a axis uncouples mitochondrial capacity from redox buffering in developing brain tip cells, redirecting their migration toward sparse, incompletely connected collateral network topologies and increasing adult cerebrovascular vulnerability. Consistent with this mechanism, humans with subclinical cerebrovascular injury exhibit reduced circulating microRNA-125a levels associated with incomplete basal collateral configurations. Together, these findings identify mitochondrial state as an instructive and conserved developmental program that encodes brain vascular reserve.

developmental biology↗

bioRxiv2026

A synonymous mutation in MSMEG_4729 occurs at a high frequency in spontaneous D29-resistant mutants of Mycobacterium smegmatis

Compassionate use of mycobacteriophage therapy highlights the promising potential of phage therapy as an alternative treatment option for antibiotic-resistant infections when conventional treatments fail. However, realizing the full potential of phage therapy requires addressing key challenges, including host immune responses, the limited arsenal of therapeutically-useful mycobacteriophages, and the emergence of phage resistance. Dissecting the mechanisms of phage resistance is critical for ensuring the effectiveness and sustainability of phage therapy. In this study, we demonstrate that exposure to the lytic mycobacteriophage D29 triggers diverse genetic changes in Mycobacterium smegmatis. A synonymous mutation in MSMEG_4729 arises frequently but is insufficient to confer D29 resistance on its own. Instead, we identified possible Lsr2-independent activation of the lipooligosaccharide (LOS) biosynthesis cluster in a D29-resistant mutant harboring this mutation. We have also detected the possible activity of MSMEG_3213, a type II methyltransferase associated with m6A modifications in M. smegmatis. Finally, we isolated defense escape mutants (DEMs) of D29 capable of overcoming resistance in a strain with the MSMEG_4729 synonymous mutation. This profiling of M. smegmatiss likely defensive arsenal against the therapeutically-useful mycobacteriophage D29 provides a roadmap for further investigations and rational engineering of next-generation mycobacteriophages to combat drug-resistant mycobacterial infections. Impact statementInterest in phage therapy has been gaining traction recently, which is largely due to the serious threat of antimicrobial resistance. However, the efficacy and sustainability of phage therapy is threatened by certain challenges, which includes the ever existent threat of phage resistance. In this study, we identified several likely factors involved in D29 interaction with the model mycobacterium M. smegmatis. These findings set a roadmap for future investigations that would guide rational phage engineering to expand the currently limited arsenal of therapeutically useful mycobacteriophages as well as improve the efficiency of existing ones.

microbiology↗

bioRxiv2026

Patchouli alcohol suppresses gastric cancer growth and immune evasion via inhibition of the NF-κB/PD-L1 axis

ObjectiveThis study aimed to investigate the anti-gastric cancer effect of Patchouli alcohol (PA), especially its influence on PD-L1-mediated immune evasion, and to elucidate the underlying molecular mechanisms. MethodsA CCK-8 assay was used to evaluate the effects of PA on the viability of the gastric cancer cell lines HGC-27 and MKN-45. RT-qPCR and western blotting were performed to analyze the mRNA and protein levels of NF-{kappa}B and PD-L1, respectively. In a coculture system of gastric cancer cells and peripheral blood mononuclear cells (PBMCs), the effect of PA pretreatment on the PBMC-induced apoptosis of cancer cells was analyzed by flow cytometry, and the cytotoxic activity of the PBMCs was assessed by a lactate dehydrogenase (LDH) release assay. Flow cytometry was also used to determine the proportions of CD3CD8 T cells and IFN-{gamma}CD8 T cells. ELISA was used to measure the levels of IFN-{gamma}, TNF-, and granzyme B in the coculture supernatants. Immunofluorescence staining was conducted to assess NF-{kappa}B nuclear translocation. In a mouse xenograft model, tumor volume and weight were measured after 14 days of PA treatment. Histopathological changes and apoptosis were analyzed by HE and TUNEL staining. A luciferase reporter assay was used to examine the transcriptional regulation of PD-L1 by NF-{kappa}B. ResultsPA inhibited the viability of HGC-27 and MKN-45 cells in a dose- and time-dependent manner and significantly downregulated the expression of NF-{kappa}B and PD-L1 at both the mRNA and protein levels. In a PBMC coculture model, PA pretreatment enhanced the ability of PBMCs to induce apoptosis and directly kill gastric cancer cells. Furthermore, PA pretreatment increased the proportions of CD3CD8 T cells and IFN-{gamma}CD8 T cells in a dose-dependent manner. Consistent with this immunostimulatory effect, PA increased the levels of IFN-{gamma}, TNF-, and granzyme B in the coculture supernatants. Mechanistically, western blotting analysis demonstrated that PA significantly reduced the protein levels of AKT, NF-{kappa}B, and PD-L1 in gastric cancer cells. Immunofluorescence staining further indicated that PA suppressed the nuclear translocation of NF-{kappa}B. In a mouse xenograft model, PA treatment significantly inhibited tumor growth, induced apoptosis, and downregulated NF-{kappa}B and PD-L1 protein expression in tumor tissues. Flow cytometry of tumor-infiltrating lymphocytes revealed increased proportions of CD3CD8 and IFN-{gamma}CD8 T cells following PA treatment. Finally, luciferase reporter assays demonstrated that NF-{kappa}B directly regulates PD-L1 transcription by binding to its promoter region. ConclusionPA exerts antitumor effects in gastric cancer by suppressing the NF-{kappa}B/PD-L1 axis, thereby enhancing CD8 T-cell-mediated cytotoxicity and inhibiting immune evasion.

pharmacology and toxicology↗

bioRxiv2026

Likely role of promoter reconstitution in Mpr-mediated D29 resistance by Mycobacterium smegmatis

The multi-copy phage resistance gene (mpr) of M. smegmatis is a major factor in resistance to the lytic mycobacteriophage D29. Mpr is a membrane-bound exonuclease that cleaves phage DNA post injection, hence blocking downstream stages in the phage infection cycle. The mechanism of resistance allows for adsorption, is non-abortive and independent of any mutation in the gene. Rather, it depends on overexpression of a wild type copy of the gene. However, the underlying factor behind mpr overexpression in spontaneous D29-resistant mutants of M. smegmatis remained elusive. Here, we report that D29 infection triggers insertion sequence (IS) rearrangements, including the transposition and integration of IS6120 directly upstream of mpr. Mutants with IS6120 integration upstream of mpr show highly elevated Mpr expression. Whole genome sequence analysis reveals that IS6120 introduced a putative transcription factor-binding site and a canonical -35 promoter element at the integration site, hence reconstituting a fuller promoter (rcp) than the original promoter (wtp) at the site. Promoter reporter assays suggest that rcp is a far stronger promoter than wtp, implying that elevated mpr expression in D29-resistant mutants with this transposition event could be due to promoter reconstitution. While strains with this transposition event appear to grow normally, rcp-driven, vector-borne Mpr overexpression appears to be toxic as it barely allows for colony formation on agar plates. This study reports a previously unknown factor likely behind mpr regulation in M. smegmatis, adding to the existing knowledge of mycobacterial anti-phage defense mechanisms and guiding rational phage engineering efforts for therapeutic applications.

microbiology↗

bioRxiv2026

A specialized ARGONAUTE enables trans-species RNA interference in plant immunity

Trans-species RNA interference (tsRNAi), in which plants produce small RNAs (sRNAs) to silence target genes in pathogens, has emerged as a promising strategy for disease control. However, whether tsRNAi constitutes an endogenous, regulated immune response remains unclear. Here, we show that ARGONAUTE10 (AGO10) plays a critical role in pathogen-induced tsRNAi. Loss of AGO10 in Arabidopsis abolished pathogen gene silencing during infection, leading to hypersusceptibility to oomycete and fungal pathogens. Importantly, AGO10 rapidly responds to pathogen infection through increased protein accumulation and re-location into discrete cytoplasmic condensates, thus promoting the production of trans-species sRNAs at the pathogen infection sites. This immune responsiveness relies on the N terminal intrinsically disordered region (IDR) of AGO10, which is responsible for sensing and responding to immune activation. Specific features in the IDR partitions AGO10 into two deeply diverged subgroups, AGO10a and AGO10b, with the immune responsiveness and defense function evolutionarily conserved in AGO10a but not AGO10b. Together, these findings establish tsRNAi as a bona fide, evolutionarily conserved immune response and position AGO10 as a signal-responsive hub linking pathogen perception to tsRNAi-based defense.

plant biology↗

bioRxiv2026

Identification and characterization of ethR and ethA genes impacting the sensitivity of Mycobacterium abscessus to ethionamide

Mycobacterium abscessus is a rapidly growing non-tuberculous mycobacterium with rising global incidence. This pathogen exhibits intrinsic resistance to most antibiotics, presenting a major public health threat. Ethionamide (ETH) requires bioactivation by monooxygenase EthA to form the active ETH-NAD adduct. We previously identified MAB_3513 (NudC) as a phosphohydrolase that confers intrinsic resistance to M. abscessus by hydrolyzing this adduct. However, deletion of nudC results in only partial susceptibility to ETH, indicating the existence of additional resistance mechanisms. This study identified MAB_0984 as the EthR regulator in M. abscessus. Deletion of ethR in a nudC knockout background ({Delta}{Delta}ethR) significantly enhanced ETH-NAD adduct accumulation, leading to hypersusceptibility to ETH. Notably, the {Delta}ethR mutant exhibited higher susceptibility than {Delta}nudC, demonstrating that EthR is a more dominant mediator of ETH resistance than NudC. Furthermore, MAB_0985 (EthA1) and MAB_0103 (EthA2) were identified as the primary EthAs in M. abscessus. Deletion of either gene alone or in combination in the {Delta}nudC reduced adduct formation and increased resistance, while the triple mutant {Delta}{Delta}ethA1{Delta}ethA2 restored wild-type resistance. Using an intergenic region-eGFP reporter system and quantitative reverse transcription-PCR, we demonstrated that EthR confers resistance by specifically suppressing ethA1 expression in M. abscessus. The Mycobacterium tuberculosis EthR inhibitor BDM31343 could boost the efficacy of ETH against M. abscessus by inhibiting EthR. Collectively, this study identified the ethR and ethA genes in M. abscessus for the first time and elucidated their role in mediating resistance to ETH. Therefore, EthR is a promising target for potentiating the efficacy of ETH against M. abscessus. Impact statementMycobacterium abscessus constitutes an escalating global health threat, primarily due to its intrinsic resistance to most antibiotics. This study identifies MAB_0984 (EthR) as a dominant resistance determinant that exerts a more profound impact on ETH susceptibility than the previously characterized NudC. We demonstrate that EthR mediates resistance by specifically repressing the expression of EthA1 (MAB_0985), one of the primary monooxygenases responsible for ETH bioactivation. The EthRMtb inhibitor BDM31343 potentiated the activity of ETH against M. abscessus through inhibition of the EthR. These findings elucidated the mechanism of ETH resistance in M. abscessus, identifying EthR as a promising target for boosting the efficacy of ETH against M. abscessus.

microbiology↗