Biology subjects

Singh, J.

Publications and source records attributed to Singh, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

RNA methyltransferase CMTR-1 inhibition activates a GATA transcription factor-mediated protective immune response

Organisms can activate innate immunity not only by detecting pathogens but also by sensing disturbances to essential cellular processes. Such damage-based surveillance provides a means to identify cellular disruptions that prime host immunity even in the absence of infection. To uncover pathways that enhance immunity, we perform a forward genetic screen in Caenorhabditis elegans for mutants with elevated intestinal immune activation. This screen identifies a hypomorphic allele of the conserved mRNA cap 2-O-methyltransferase cmtr-1, which strongly induces immune effector expression. Transcriptomic profiling reveals the broad activation of innate immune responses, and functional analyses demonstrate that this response requires the GATA transcription factor ELT-2. Notably, loss of cmtr-1 increases resistance to Pseudomonas aeruginosa, indicating that reduced RNA cap methylation can elicit protective immunity. Together, these findings reveal a previously unrecognized mechanism of immune surveillance and demonstrate that perturbation of CMTR-1 serves as a danger signal, triggering an ELT-2-dependent protective immune response.

immunology↗

bioRxiv2025

Deubiquitinase USP-14 controls intestinal distension-induced immune activation in Caenorhabditis elegans via Wnt/β-catenin signaling

Pathogen infections disrupt multiple host cellular processes, and hosts have consequently evolved mechanisms to detect these perturbations and initiate appropriate immune responses. In Caenorhabditis elegans, gut distension caused by bacterial colonization is known to activate innate immunity, yet the molecular mechanisms linking intestinal distension to immune activation remain poorly understood. Here, we perform a forward genetic screen to identify suppressors of intestinal distension-induced immune activation in C. elegans. This screen identifies a loss-of-function mutation in the gene encoding the deubiquitinase (DUB) USP-14 as a potent suppressor of immune activation triggered by gut distension. We show that usp-14 knockout mutants exhibit increased sensitivity to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Global transcriptomic profiling further demonstrates that USP-14 modulates innate immune responses induced by intestinal distension. Epistasis analyses establish that USP-14 functions through the Wnt/{beta}-catenin signaling pathway to regulate these immune responses. Together, our findings reveal a previously unrecognized role for the conserved DUB USP-14 in host immunity, providing a foundation for future studies investigating how USP-14 modulates immune signaling.

immunology↗

bioRxiv2025

The ID93 + GLA-3M-052-LS vaccine candidate elicits mucosal and systemic immunogenicity and protective efficacy against Mycobacterium tuberculosis challenge in BCG-primed Collaborative Cross inbred mice

New vaccine approaches are needed against tuberculosis (TB). We sought to optimize mucosal immunogenicity and protective efficacy by modulating the adjuvant component and route of immunization of a next-generation TB vaccine using the recombinant TB vaccine antigen (Ag) ID93. ID93-specific mucosal and systemic immunogenicity and protective efficacy were assessed in the Collaborative Cross 004 mouse strain, a mouse strain susceptible to Mycobacterium tuberculosis (Mtb) infection, as a suitable model of Mtb susceptible populations. Immunogenicity data from various vaccine candidates were used to select lead vaccine candidates with the most preferred immunostimulatory profiles using a pre-determined desirability index. A liposomal adjuvant system containing synthetic TLR4 and TLR7/8 ligands (GLA-3M-052-LS), administered by a heterologous intramuscular-intranasal regimen, induced an optimal comprehensive immune response profile including high levels of mucosal antibody and Th1 CD4+ T cells in the lungs. In BCG-primed mice, immunization with intramuscular followed by intranasal ID93 + GLA-3M-052-LS boosts significantly reduced Mtb burden in the lungs after challenge vs. BCG vaccinated mice alone. Thus, ID93 + GLA-3M-052-LS represents a promising next-generation TB vaccine candidate suitable for testing in additional preclinical models.

immunology↗

bioRxiv2025

An acyltransferase protects Caenorhabditis elegans from thiol reductive stress through an autoinhibitory hypoxia response pathway

Cellular redox homeostasis depends on a finely tuned balance between oxidizing and reducing conditions, and disturbances in this balance lead to oxidative or reductive stress. While oxidative stress and its pathological outcomes are well studied, the molecular mechanisms underlying cellular responses to reductive stress remain poorly understood. Using Caenorhabditis elegans as a model, we investigate thiol reductive stress induced by dithiothreitol (DTT) and uncover a critical protective role for the hypoxia response pathway. We identify RHY-1, a membrane-associated acyltransferase and known negative regulator of the hypoxia-inducible factor HIF-1, as essential for survival under thiol reductive stress. Notably, rhy-1 is a direct transcriptional target of HIF-1, and overexpression of rhy-1 fully rescues the sensitivity of hif-1 loss-of-function mutants to DTT. We demonstrate that RHY-1 functions in an autoinhibitory feedback loop, where elevated RHY-1 levels suppress activation of the hypoxia response pathway even during reductive stress. Together, our findings establish RHY-1 as both a regulatory and effector component of the hypoxia response pathway that mediates cellular protection against thiol reductive stress.

physiology↗

bioRxiv2025

Mitochondrial Dysfunction in Endothelial Cells Drives Greater Vascular Impairment in Females with Diabetes-Associated Peripheral Artery Disease

BackgroundWomen with peripheral artery disease (PAD) experience poorer clinical outcomes than men, particularly in the setting of diabetes. However, the mechanistic basis for these sex- specific disparities remains unclear. MethodsHere, we investigated endothelial cell (EC) function(s) in diabetes-associated PAD, with a focus on sex differences. Limb tissues from patients with diabetes and chronic limb-threatening ischemia (CLTI) undergoing amputation, and a diabetes mouse model of hindlimb ischemia (HLI), were assessed for vasodilatory capacity, angiogenesis, oxidative stress and changes to expression of mitochondrial complex genes. ECs exposed to a hyperglycemic environment in vitro were assessed for mitochondrial function. The therapeutic potential of the mitochondrial-targeted antioxidant MitoQ was investigated. ResultsECs from females with diabetes-associated PAD have altered responses compared to males. Specifically, limb vessels and skeletal muscle from females exhibit reduced arterial relaxation, angiogenesis and increased oxidative stress in response to HLI in mice, and in tissues from patients. Single-cell RNA sequencing of murine limbs revealed marked suppression of EC mitochondrial complex genes in females with diabetes. Female human ECs exposed to high glucose had reduced respiration, reduced expression of mitochondrial genes and increased oxidative stress. Remarkably, MitoQ restored arterial relaxation and the angiogenic response in female diabetes- associated PAD. ConclusionOur findings uncover a striking sex-specific vulnerability involving oxidative stress and mitochondrial dysfunction in EC health in diabetes-associated PAD. These results highlight the need for sex-specific therapeutic strategies in diabetic PAD, which might include mitochondrial targeted antioxidant strategies.

pathology↗

bioRxiv2025

Structural basis for binding of RILPL1 to TMEM55B reveals a lysosomal platform for adaptor assembly through a conserved TBM motif

Inherited mutations in VPS35 and the kinase LRRK2 lead to hyperphosphorylation of Rab GTPases and promote the formation of phospho-Rab signalling complexes. A subset of RH2 domain-containing proteins from the RILP-homology family, including RILP, RILPL1, RILPL2, JIP3, and JIP4 are Rab effectors that recognize the LRRK2-phosphorylated switch 2 threonine of phospho-Rab8A and phospho-Rab10. More recently, phospho-Rabs have been found on lysosomal membranes within multi-protein assemblies involving TMEM55B and RILPL1. TMEM55B is a 284-residue lysosomal membrane protein with no homology to known proteins. It comprises a 218-residue cytosolic N-terminal region and two predicted transmembrane -helices. Residues 80- 160, which face the cytosol, mediate binding to a C-terminal motif of RILPL1, formed after RILPL1 associates with phospho-Rab8A. Here, we report the crystal structures of TMEM55B alone and in complex with a C-terminal RILPL1 peptide, encompassing the TMEM55B interaction region, which we define as the TMEM55B Binding Motif (TBM). The cytosolic domain of TMEM55B adopts a rigid architecture of two tandem RING-like domains, each forming a Zn{superscript 2}-stabilized 40-residue {beta}-sandwich. TBM binding is mediated primarily by backbone hydrogen bonding and anchored by two glutamate residues from RILPL1. These findings support a model in which RILPL1 is recruited to phospho-Rab8A-positive lysosomes prior to TMEM55B engagement. Further co-immunoprecipitation and mutational analyses indicate that TMEM55B forms complexes independently of phospho-Rabs with proteins containing a conserved TBM, like that of RILPL1, including JIP3, JIP4, OCRL, WDR81, and TBC1D9B. Together, these findings uncover previously unrecognized regulatory networks associated with TMEM55B and lysosomal function and suggest that TMEM55B serves as a central hub for adaptor recruitment at the lysosomal membrane.

biochemistry↗

bioRxiv2025

Promoter evolution in HIV-1C establishes latent reservoirs highly resistant to reversal

Latent viral reservoirs remain a major barrier to curing HIV-1, with the long-terminal repeat (LTR) and Tat playing crucial roles in regulating viral transcription. Subtype-specific transcription factor binding site (TFBS) variations within the LTR significantly influence latency and reservoir stability. In earlier work, we identified HIV-1C LTR variants with duplicated TFBS motifs, including NF-{kappa}B, AP1, RBEIII, and TCF-1/LEF-1. Using five cell models, including Jurkat and primary CD4 T cells, we compared canonical R-LTR and variant R2-LTR strains. Across sub-genomic reporters, single-round infections, and full-length viral vectors, we found that the balance between RBEIII and NF-{kappa}B motifs governs stability of latency. The two-viruses-one-cell system that normalized confounding environmental factors further revealed that latency is primarily controlled by intrinsic transcriptional circuits rather than external stimuli. In longitudinal studies of HIV-1 individuals from acute and chronic infection phases, we observed dominant R strains during early infection and the spontaneous emergence of R2 strains in nearly half of chronic-phase subjects, a process accelerated by ART. Upon CD4 T cell activation, R strains preferentially rebounded, while R2 strains showed strong resistance to reversal, even in subjects harbouring a co-infection. Together, these findings establish the clinical significance of LTR variation in latency regulation and identify the R2 phenotype as a critical determinant of reservoir persistence. These results underscore the importance of addressing reservoir heterogeneity in cure strategies, particularly in HIV-1C-prevalent regions.

microbiology↗

bioRxiv2025

Phosphoinositide 3-kinase regulates wild-type RAS signaling to confer resistance to KRAS inhibition

Despite the availability of RAS inhibitors and the dependence of >90% of pancreatic ductal adenocarcinomas (PDAC) on oncogenic KRAS mutations, resistance to KRAS inhibition remains a serious obstacle. We show here that phosphoinositide 3-kinase (PI3K) plays a major role in this resistance through upstream activation of wild-type RAS signaling - beyond its known KRAS effector function. Combining proximity labeling, CRISPR screens, live-cell imaging, and functional assays we found that PI3K orchestrates phosphoinositide-mediated GAB1 recruitment to the plasma membrane, nucleating assembly of RAS signaling complexes that activate mitogen-activated protein kinase (MAPK) in an EGFR/SHP2/SOS1-dependent manner. We further demonstrate that inhibiting PI3K enhances sensitivity to mutant-specific KRAS inhibitors in PDAC cells, including cells with clinically identified PIK3CA mutations. Our findings refine RAS-PI3K signaling paradigms, reveal that PI3K-driven wild-type RAS activation drives resistance to KRAS inhibition, and illuminate new avenues for augmenting KRAS-targeted therapies in PDAC.

cancer biology↗

bioRxiv2025

Mammalian mitochondrial polyphosphate is regulated by the 5-InsP7 synthase IP6K1

Inorganic polyphosphate (polyP) is a linear polymer of varying length composed of phosphate residues linked by phosphoanhydride bonds. PolyP remains poorly understood in mammals due to its low abundance and lack of information on its synthesis and regulation. Using synthetic polyP8 as a bait, we identified polyP interacting proteins involved in RNA splicing, transcription, and mitochondrial function. We developed a DAPI fluorescence-based assay to quantify the low levels of polyP present in mammalian cell lines and tissues, and detected a higher concentration of polyP in the mitochondria compared with the nucleus and post-mitochondrial fraction. We observed that mitochondrial polyP synthesis relies on active FoF1 ATP synthase and an intact proton gradient across the inner mitochondrial membrane. Our data shows that orthophosphate (Pi) is required for the generation of mitochondrial polyP and that the presence of ATP enhances Pi-driven polyP synthesis in isolated mitochondria. We discovered that the inositol pyrophosphate 5-InsP7, synthesized by IP6K1, regulates mitochondrial polyP levels. Mice and cells deficient in IP6K1 showed a significant reduction in mitochondrial polyP synthesis compared with wild type controls. Cells lacking IP6K1 also showed impaired mitochondrial respiration and membrane potential. The expression of active IP6K1, but not its catalytically inactive form, restored mitochondrial polyP synthesis and membrane potential in IP6K1 deficient cells, but mitochondrial respiration was rescued by expression of either active or inactive IP6K1. These data show that IP6K1 regulates mitochondrial function and polyP production through both the synthesis of 5-InsP7 and via a catalytic activity-independent mechanism. Our findings uncover a link between 5-InsP7, an energy sensor, and polyP, an energy store, in the regulation of mammalian mitochondrial homeostasis.

biochemistry↗

bioRxiv2025

Bacterial metabolism of tryptophan causes toxicity in Caenorhabditis elegans

Tryptophan is an essential amino acid required not only for protein biosynthesis but also for the production of several physiologically important metabolites, including serotonin, kynurenine, and nicotinamide. Although dietary tryptophan is associated with various health benefits, excessive intake can result in adverse physiological effects. The specific tryptophan- derived metabolites responsible for such toxicity, however, remain incompletely characterized. Here, we investigate the mechanisms underlying tryptophan-induced toxicity in Caenorhabditis elegans. We observe that tryptophan concentrations of 1 mM or higher are highly toxic to C. elegans, blocking egg hatching. Notably, supplementation with various sugars, including glucose, fructose, mannose, galactose, rhamnose, and lactose, alleviates this toxicity. Genetic analyses reveal that host tryptophan metabolism is dispensable for the observed effects. Instead, bacterial metabolism, particularly the conversion of tryptophan to indole, is essential for mediating toxicity. Bacterial strains deficient in indole production abolished tryptophan-induced toxicity, and all sugars that conferred protection also suppressed bacterial indole synthesis. These findings demonstrate that tryptophan toxicity in C. elegans is primarily mediated by bacterial metabolism.

physiology↗

bioRxiv2025

Endosome transcriptomics reveal trafficking of Cajal bodies into multivesicular bodies

All eukaryotic cells secrete exosomes, a type of extracellular vesicles (EVs) derived from the endocytic compartments known as multivesicular bodies (MVBs), or late endosomes (LEs). Exosomes contain a diverse range of cargo such as nucleic acids, proteins, lipids and small molecules but whether these contents have a biological function remains an area of intense investigation. Over the last decade, numerous studies have described the transcriptome of exosomes but very little is known about the RNA content of the MVBs, the source compartment for exosome biogenesis. Here we determine the small-RNA transcriptome of highly purified MVBs and report that various classes of nuclear small regulatory RNAs such as small-Cajal body associated RNAs (scaRNAs), small-nucleolar RNAs (snoRNAs) and small-nuclear RNAs (snRNAs) traffic to MVBs. We show that this RNA-trafficking requires the function of ESCRT machinery but is independent of canonical LC3 lipidation mediated selective autophagy. Furthermore, blocking the activity of a PI3K Class 3 enzyme, VPS34, required for recruitment of the ESCRT machinery to the endosome, prevents the turnover of these nuclear RNAs in MVBs. Our results provide a mechanism for targeting nuclear ribonucleoprotein complexes (RNPs), such as Cajal bodies, for degradation and turnover by the cytoplasmic endo-lysosomal pathway. Significance StatementEndosomes are cytoplasmic, membrane-bound subcellular organelles that are sites for biogenesis of exosomes, a class of extracellular vesicles, thought to mediate intercellular communication via their packaged cargo such as RNA. Previous studies have focused on the transcriptome of exosomes however very little is known about the identity of RNAs and mechanisms by which they are sorted into endosomes. Here we report a comprehensive endosome transcriptome and provide evidence that several nuclear RNA-protein complexes (RNPs) sort into endosomes, a previously unappreciated phenomenon. We show that this process requires the activity of endosomal sorting complexes and phospholipids characteristic of cellular endocytic compartments. Our study provides a mechanism for recycling and disposal of unwanted nuclear RNPs by the cytoplasmic endolysosomal pathway.

biochemistry↗

bioRxiv2025

Accelerate the discovery of genetic variants in mitochondrial diseases with VIOLA: Variant PrIOritization using Latent space

Interpreting variants from whole-exome sequencing remains a major challenge, particularly for heterogeneous disorders such as mitochondrial diseases (MD). To help finding a diagnosis for complex cases, we have developed VIOLA (Variant prIoritizatiOn using Latent spAce). The pipeline includes a variational autoencoder to embed functional annotations into a low-dimensional space, followed by DBSCAN-based outlier detection. To prioritize potential pathogenic variants, filtering steps and phenotype integration via HPO terms are then applied. Two scores are associated to the selected variants: the VIOLA score (Vscore), which combines variant features, transcriptomic co-expression data, and MD-specific annotations, and the VIOLA Aggregated score (VAscore) that merges Vscore with Exomisers pathogenicity score. Finally, we also provide the ARrank, specifically designed for variants compatible with autosomal recessive inheritance. We illustrate the functionalities of VIOLA on a novel cohort of patients with suspected MD with complex phenotypes. VIOLA systematically ranked causal variants among the top, outperforming existing methods. Overall, VIOLA is a patient-specific approach to help to discover novel variants in complex MD.

bioinformatics↗

bioRxiv2025

Development of an Intranasally- and Intramuscularly-Administrable Replicon Vaccine Efficacious Against H5N1 Influenza Virus

The risk of a respiratory viral pandemic is significant, including from the now widespread panzootic H5N1 influenza virus, highlighting the need for effective, stable, and inexpensive vaccine technologies that elicit strongly protective immunity. Intranasal vaccines can stimulate local immune responses at the site of natural respiratory viral infection, a key characteristic that can not only reduce morbidity and mortality caused by respiratory viruses but also potentially reduce viral transmissibility to limit outbreaks. Nucleic acid vaccines are now a valuable tool in pandemic responses, with high potency and rapid adaptability to target circulating or emerging viral strains; however, data are limited on which vaccine attributes are needed for efficient transmucosal delivery and immune stimulation following intranasal delivery. To demonstrate proof of concept, here we have developed a replicon vaccine expressing an H5 influenza antigen that uses a nanostructured lipid carrier (NLC) delivery system. A relationship was established between the molar ratio of positive charges on the NLC to the negative charges on the nucleic acid (N:P ratio) and the immunogenicity of the vaccine formulations, with higher N:P ratios resulting in an increase in vaccine immunogenicity. We demonstrated the ability of this replicon vaccine to be administered via intramuscular and intranasal routes with a singular vaccine formulation. The vaccine induced systemic immunity when dosed intramuscularly or intranasally in an immunocompetent mouse model, whereas intranasal dosing uniquely stimulated a strong mucosal immune response. Moreover, a mixed intramuscular/intranasal dosing strategy using this unified formulation stimulated a balanced systemic and mucosal immune response. Finally, we demonstrated the protective efficacy of this intranasally and intramuscularly/intranasally delivered H5 replicon-NLC vaccine against morbidity and mortality in a lethal H5N1 influenza challenge ferret model. This work establishes the replicon-NLC vaccine platform as a potential novel intranasal technology for rapid pandemic response.

immunology↗

bioRxiv2025

Biological polyethylene deconstruction initiated by oxidation from DyP peroxidases

Polyethylene (PE) is the most commonly used plastic on Earth due to its favorable material properties such as high ductility, mechanical strength, and bond homogeneity that make the material resistant to deconstruction. However, the lack of robust recycling infrastructure for PE end-of-life management is leading to an estimated 4 million tons of environmental accumulation annually, with implications for human and environmental health. Biological deconstruction and upcycling could potentially aid in PE waste management by allowing for high-yield conversion of waste plastics to high value products, although such processes are not yet possible. In this work, we mined the gut of low-density PE (LDPE) fed mealworms that can reduce LDPE molecular weight by >40% and discovered dye decolorizing peroxidases (DyPs) that oxidized LDPE, initiating biological deconstruction. A plastic-active DyP is characterized by a hydrophobic loop near its active site that helps mediate binding and tunes activity. LDPE oxidation is driven by surface exposed residues proximal to the active site enabling activity on polymeric substrates. Our work provides robust evidence for enzymatic LDPE deconstruction and identifies molecular targets for further development to realize scalable biological LDPE upcycling. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=41 SRC="FIGDIR/small/640435v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@146b8fborg.highwire.dtl.DTLVardef@1a72adorg.highwire.dtl.DTLVardef@5ece5forg.highwire.dtl.DTLVardef@3ee20a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Iron-deplete diet enhances Caenorhabditis elegans lifespan via oxidative stress response pathways

Gut microbes play a crucial role in modulating host lifespan. However, the microbial factors that influence host longevity and their mechanisms of action remain poorly understood. Using the expression of Caenorhabditis elegans FAT-7, a stearoyl-CoA 9-desaturase, as a proxy for lifespan modulation, we conduct a genome-wide bacterial mutant screen and identify 26 Escherichia coli mutants that enhance host lifespan. Transcriptomic and biochemical analyses reveal that these mutant diets induce oxidative stress and activate the mitochondrial unfolded protein response (UPRmt). Antioxidant supplementation abolishes lifespan extension, confirming that oxidative stress drives these effects. The extension of lifespan requires the oxidative stress response regulators SKN-1, SEK-1, and HLH-30. Mechanistically, these effects are linked to reduced iron availability, as iron supplementation restores FAT-7 expression, suppresses UPRmt activation, and abolishes lifespan extension. Iron chelation mimics the pro-longevity effects of the mutant diets, highlighting dietary iron as a key modulator of aging. Our findings reveal a bacterial-host metabolic axis that links oxidative stress, iron homeostasis, and longevity in C. elegans.

physiology↗

bioRxiv2025

A bivalent self-amplifying RNA vaccine against yellow fever and Zika viruses

IntroductionYellow fever (YFV) and Zika (ZIKV) viruses cause significant morbidity and mortality, despite the existence of an approved YFV vaccine and the development of multiple ZIKV vaccine candidates to date. New technologies may improve access to vaccines against these pathogens. We previously described a nanostructured lipid carrier (NLC)-delivered self-amplifying RNA (saRNA) vaccine platform with excellent thermostability and immunogenicity, appropriate for prevention of tropical infectious diseases. MethodsYFV and ZIKV prM-E antigen-expressing saRNA constructs were created using a TC-83 strain Venezuelan equine encephalitis virus-based replicon and complexed with NLC by simple mixing. Monovalent and bivalent vaccine formulations were injected intramuscularly into C57BL/6 mice and Syrian golden hamsters, and the magnitude, durability, and protective efficacy of the resulting immune responses were then characterized. Results and discussionMonovalent vaccines established durable neutralizing antibody responses to their respective flaviviral targets, with little evidence of cross-neutralization. Both vaccines additionally elicited robust antigen-reactive CD4+ and CD8+ T cell populations. Notably, humoral responses to YFV saRNA-NLC vaccination were comparable to those in YF-17D-vaccinated animals. Bivalent formulations established humoral and cellular responses against both viral targets, commensurate to those established by monovalent vaccines, without evidence of saRNA interference or immune competition. Finally, both monovalent and bivalent vaccines completely protected mice and hamsters against lethal ZIKV and YFV challenge. We present a bivalent saRNA-NLC vaccine against YFV and ZIKV capable of inducing robust and efficacious neutralizing antibody and cellular immune responses against both viruses. These data support the development of other multivalent saRNA-based vaccines against infectious diseases.

immunology↗

bioRxiv2025

A highly efficient CRISPR-Cas9-based gene editing system in oat (Avena sativa)

Cultivated oat (Avena sativa) is an emerging cereal for healthy lives owing to its unique characteristics, such as high {beta}-glucan and oil content, distinctive fatty acid composition, and gluten-free nature. The recent unravelling of the 12.5 Gb hexaploid oat genome underlined breeding barriers caused by ancestral translocations and inversions, leading to recombination suppression and pseudo-linkage further hindering conventional trait introgression. Over the past decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has been extensively used for crop improvement and functional genomics in all other cereals except oats. Its large repetitive genome with three sub-genomes, lack of efficient transformation, recalcitrant nature, and complex molecular screening due to gene redundancy have been major obstacles to gene editing success in oat. We report the first successful CRISPR-Cas9-based gene editing in oat in three genes -- AsTLP8, AsVRN3 and AsVRN3D with gene-editing efficiency of up to 41.1%. The gene-edited plants for all the genes carried deletions and/or one base insertion. Further analysis of VRN3 T1 and T2 mutants revealed bent leaves in heterozygous knockouts (AACCdD), while an extended vegetative growth phase was seen in the T1 homozygous and biallelic mutants (aaccdd), accentuating the important role of VRN3 in oat development. We are confident that this highly efficient oat gene editing system will pave the way for a deeper molecular understanding of this healthy cereal, deciphering oats functional genomics, and creating genetic diversity at the cold spots of recombination in oat.

plant biology↗

bioRxiv2025

JNK2 pathway in colonocytes enhances gut barrier integrity in response to microbial acetate.

Weaning involves a nutritional shift from fat-rich milk to carbohydrate-based solid food, reshaping metabolism, microbiota, and gut immune tolerance. While dairy remains a component of the human diet beyond weaning, the impact of continued milk supplementation on gut epithelial homeostasis remains poorly understood. Here, using a mouse model, we show that continued milk-based feeding post-weaning promotes intestinal barrier function by enriching the commensal bacterium Dubosiella newyorkensis, which produces acetate to activate epithelial JNK2 signaling. This pathway enhances barrier integrity and suppresses inflammation induced by mild Dextran Sodium Sulphate (DSS) treatment. In contrast, feeding a lard-based high-fat diet or transient pharmacologic inhibition of JNK2 induces epithelial P38 activation, resulting in barrier disruption and inflammation. Importantly, the beneficial effects of milk were observed only if initiated during the weaning period, when the microbiome is in a metastable transitional state. Initiation of the same intervention two weeks after weaning led to P38 activation and inflammatory responses. These findings highlight the microbiome-dependent, stage-specific effects of diet and underscore the importance of early-life nutritional interventions for long-term gut health.

physiology↗