Abstract Aurora Kinase A (AurA) is an essential mitotic kinase and therapeutic target in cancer. Most protein kinase inhibitors target the conserved ATP-binding pocket, often resulting in poor selectivity and off-target effects. Here, we identify and characterise small synthetic protein binders, Adhirons, as allosteric inhibitors of AurA. Using ‘phage display, we isolated Adhiron reagents that bind a previously uncharacterised site on the αG-helix of the kinase C-lobe. Structural and biochemical analyses revealed that the Adhiron inhibited AurA by modulating the activation loop via this cryptic site, which we designate the T-pocket. In cells, Adhiron expression mimics the effects of small molecule inhibitors of AurA on substrate and auto-phosphorylation, while sparing Aurora kinase B and without impairing TPX2-mediated localisation of AurA to the mitotic spindle. The AurA-inhibitory Adhirons demonstrate remarkable selectivity, potency and affinity, a highly sought-after combination of properties for kinase inhibition facilitating their use as tractable research tools for probing AurA function and as pharmacophore templates for structure-based drug design. Finally, these reagents illustrate a generalisable strategy for targeting allosteric sites across the Kinome. *Jack P Roberts & James Holder contributed equally to this work.

Summary Abiraterone acetate serves as the first-line therapeutic agent for prostate cancer (PCa) treatment. However, drug resistance frequently emerges. Employing a genome-wide CRISPR/Cas9 library screening strategy, we identified 523 long non-coding RNAs (lncRNAs) and 2183 protein-coding genes associated with abiraterone resistance. Notably, a pair of sense-antisense genes, BIRC6-AS1 / BIRC6 , was demonstrated to be a pivotal driver for abiraterone resistance. BIRC6-AS1 depletion led to a reduction in both the mRNA and protein levels of BIRC6. Moreover, depletion of either BIRC6-AS1 or BIRC6 enhanced the chemosensitivity of PCa cells to abiraterone both in vitro and in vivo settings. Further investigation revealed that BIRC6-AS1 stabilized the mRNA of BIRC6 through interaction with ILF2. Diminishing either BIRC6-AS1 or BIRC6 predominantly suppressed non-homologous end joining (NHEJ) repair activity, resulting in the disassembly of 53BP1 foci at DNA damage sites and an increased accumulation of DNA damage in PCa cells induced by abiraterone. Mechanistically, BIRC6 interacted with A20 and facilitated the K48-linked ubiquitination and subsequent degradation of A20 at the K337 residue. Additionally, A20 knockdown effectively reversed the abiraterone sensitivity induced by BIRC6-AS1 depletion. Collectively, we conducted a comprehensive screen to identify lncRNAs and protein-coding genes associated with abiraterone resistance and proposed that targeting BIRC6-AS1 /BIRC6 axis represents a promising strategy to overcome abiraterone resistance in prostate cancer.

The success of plants on land has been enabled by mutualistic intracellular associations with microbes for 450 million years (Delaux and Schornack 2021). Because of their intracellular nature, the establishment of these interactions requires tight regulation by the host plants. In particular, three genes – SYMRK, CCaMK and CYCLOPS – form the core of an ancestral common symbiosis pathway (CSP) for intracellular symbioses, and are conserved since the most recent common ancestor of land plants (Radhakrishnan et al. 2020; Delaux et al. 2015; Wang et al. 2010; Parniske 2008). Here, we describe EPP1 as a fourth gene committed to the CSP. Among land plants, EPP1 is conserved only in species able to associate with at least one type of intracellular symbiont. We found that loss-of-function epp1 mutants or EPP1 knock-down lines in four clades of land plants – legumes, Solanaceae, monocots and bryophytes – are all impaired in their ability to associate with arbuscular mycorrhizal fungi. We discovered that the plasma membrane-localized receptor-like SYMRK phosphorylates EPP1 on a conserved serine residue and that this phosphorylation is essential for symbiosis. Using a gain-of-function approach, we demonstrate that EPP1 is upstream of the nuclear kinase CCaMK. We propose that EPP1 is an ancestral component of the essential pathway that has regulated plant symbiosis for half a billion years.

Indoor vertical farming (VF) offers several practical advantages for the cultivation of plant protein bio-factories, including plant uniformity, product consistency, water/nutrient recycling and production cycles on a year-round basis. Much progress has been achieved in recent years toward the development of innovative systems for artificial lighting, automated irrigation, plant handling, environment control and space use optimization in VF systems. Here, we used a CRISPR-Cas9 gene editing approach to generate mutant lines of transient protein expression host Nicotiana benthamiana presenting a compact, space-efficient phenotype compared to the so-called LAB strain commonly used for protein production. Our strategy consisted of altering apical dominance by suppressing the biosynthesis of strigolactone, a negative regulator of axillary bud outgrowth-promoting cytokinins. Strigolactone-depleted lines were generated by knocking-down the expression of either Carotenoid cleavage dioxygenase 7 (CCD7) or Carotenoid cleavage dioxygenase 8 (CCD8), two key enzymes of the metabolic pathway leading to strigolactone synthesis. Knocking-down the genes of either enzyme had no impact on the overall growth rate of the plant but drastically influenced its leaf proteome, auxin/cytokinin ratio and overall architecture. More specifically, the ΔCCD mutants exhibited altered glycolytic and malate-processing enzyme fluxes driving the production of pyruvate and cytokinins in leaf tissue, an axillary growth-oriented development pattern and, most importantly, a spatial footprint reduced by 45% to 50% compared to the LAB strain. Most importantly, recombinant protein yields per plant were maintained in the mutant lines, as here illustrated for the model protein GFP and for rituximab, a chimeric monoclonal antibody of confirmed clinical value in humans. Our data demonstrate the usefulness of ΔCCD7 and ΔCCD8 knockout leading to strigolactone depletion for the generation of compact, space-efficient N. benthamiana lines well suited to VF systems intended for biopharmaceutical production.

Emerging evidence that circulating levels of key metabolic intermediates are sensed by a range of G-Protein Coupled receptors (GPCRs) is providing critical new insights into the control of systemic metabolic homeostasis, and how disturbances in such sensing may contribute to metabolic disease. The hydroxycarboxylic acid receptors for lactate (HCAR1), β-hydroxybutyrate (HCAR2), and octanoate (HCAR3) are encoded by three closely homologous GPCR genes co-located in a region where common genetic variation has been reportedly associated with lipid levels and body fat distribution. By resolving sequence homology in this region, we were able to refine this signal to a coding variant (R311C) in HCAR2. Using corrected genotypes from ∼500K participants from UK Biobank and direct genotyping of four other studies, we found that carriage of the HCAR2 p.R311C variant was significantly associated with type 2 diabetes risk, reduced gynoid fat mass, increased waist-hip ratio, higher circulating triglycerides, glucose and alanine aminotransferase levels, lower levels of HDL cholesterol and adiponectin and impaired suppression of circulating levels of non-esterified fatty acids after oral glucose. Adipose tissue explants from mice engineered to express the equivalent mutation variant (p.R308C) in the mouse ortholog showed increased lipolytic activity, basally and after β-hydroxybutyrate (BHB) treatment. In vivo, the mice were insulin resistant and had increased liver fat and impaired post-prandial suppression of NEFAs. The variant alters an amino acid located in the intracellular C-terminal tail of HCAR2, increasing recruitment of β-arrestin and resulting in enhanced internalisation and reduced cell surface expression. In conclusion, a common variant in the human ketone body receptor results in impaired control of adipocyte lipolysis and adversely impacts systemic lipid and glucose metabolism. These findings highlight the importance of anti-lipolytic ketone body signalling in adipocytes for the maintenance of metabolic health Graphical Abstract

Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods using transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we developed CRISPR-Cas9 engineered HEK293T cell lines with stable integration of mCherry-C1C2 fusion proteins at the AAVS1 locus for continuous production of surface-modified EVs. The engineered cell lines demonstrated significantly higher surface display efficiency compared to transient transfection, with reduced batch-to-batch variability. EVs maintained native characteristics including size distribution (120-130 nm) and marker expression while showing efficient cellular uptake. The platform maintained consistent production of uniformly modified EVs with stable transgene expression over at least 25 passages (~3 months), eliminating the need for repeated transfections and reducing batch-to-batch variability inherent to transient expression systems.

Hepatocellular carcinoma (HCC) is a malignant tumor that has been associated with dysbiosis of the gut microbiota. However, how the gut microbiota plays an oncogenic role in HCC remains largely unknown. Here, we show that Enterococcus faecalis (E. faecalis) is highly enriched in liver tumor tissues and is positively correlated with pathogenesis of HCC. E. faecalis promotes liver cancer cell proliferation, protein translation, cell migration and tumorigenesis. Mechanistically, we found that EF-derived extracellular vesicles (EF-EVs) deliver EF-Obg GTPase to activate host mTOR, thereby promoting liver cancer progression. Intriguingly, the EF-Obg protein exerts its influence on the mTOR pathway via a Ras-like G domain involved in GTP binding. EF-Obg exhibits a striking homology with the G1 site of the G domain of Rheb, a key positive regulator of mTOR. Obg gene is critical for E. faecalis to activate mTOR to promote hepatocarcinogenesis based on an engineered obg knockdown strain by CRISPR interference experiment. Clinically, abundant EF-Obg protein expression is correlated with enhanced activation of mTOR, leading to poor overall survival in HCC patients. Significantly, treatment of mTOR inhibitor Everolimus confers effectiveness in EF-colonized liver cancer orthotopic model, suggesting that Everolimus therapeutic approach can be effective for liver cancer patients with enriched E. faecalis . Taken together, we provide mechanistic and functional evidence to verify a direct causal relationship between tumor-resident E. faecalis enrichment and liver carcinogenesis, revealing that EF-Obg functions as a previously unidentified cross-kingdom activator of mTOR to promote liver tumorigenesis.

Single-cell perturbation sequencing technologies (e.g., Perturb-seq, CROP-seq), which integrate CRISPR-based gene editing with single-cell transcriptome profiling, have revolutionized the analysis of transcriptomic changes induced by genetic perturbations at single-cell resolution. These technologies serve as a powerful tool for identifying key genes that inhibit tumor growth or reverse cancer cell phenotypes. However, they face two major challenges: data explosion with high experimental costs, and data complexity characterized by high dimensionality, noise, sparsity, and heterogeneity. To address these challenges, we developed the single-cell Rank-based Genetic Perturbation predictor (scRGP), the first deep learning framework leveraging gene expression rank-order information for this task. scRGP demonstrates superior performance in terms of robustness, cross-cell-line perturbation prediction, and high-throughput screening. Specifically, scRGP achieves an approximately 10-16 percentage points improvement in Pearson correlation coefficient (PCC) over state-of-the-art methods (e.g., GEARS and scFoundation) for single- and double-gene perturbation predictions, while also extending prediction capability to triple-gene perturbations. Furthermore, it outperforms these methods by approximately 5-9 percentage points in cross-cell-line predictions. These advancements promise to shift the paradigm of single-cell perturbation studies from experiment-driven to computation-driven approaches, providing new support for functional genomics and precision medicine.

CRISPR–Cas systems are central to prokaryotic adaptive immunity, widely harnessed for biotechnology. Yet, their vast and uncharacterized diversity, especially non-canonical variants, impedes full exploitation. Here we present BioPrinCRISPR, a class-agnostic computational framework leveraging gene co-conservation, protein domain co-occurrence, and embedding similarity to identify and characterize CRISPR–Cas systems across prokaryotic genomes. Applying BioPrinCRISPR to over one million bacterial genomes, we uncovered extensive canonical and uncharacterized systems, revealing a rich landscape of atypical Cas proteins and novel domain architectures. Notably, we identified recurrent fusion proteins with unique enzymatic combinations, suggesting roles in regulatory control or nucleic acid remodeling. Experimental validation of two divergent Cas13an-like effectors demonstrated RNA knockdown capacity in human cells, confirming our framework’s predictive power. These findings expand the functional repertoire of CRISPR-associated proteins and highlight unexplored modes of microbial immunity. BioPrinCRISPR thus stands as a powerful tool for comprehensively mapping CRISPR–Cas diversity, offering new insights into prokaryotic defense and facilitating discovery of novel candidates for next-generation genome engineering. An accompanying interactive web platform was also developed to facilitate data exploration.

Interleukin (IL)-1α is a pro-inflammatory member of the IL-1 cytokine superfamily and is important for inflammatory responses to infection and injury. Unlike pro-IL-1β, pro-IL-1α is mainly localised to the nucleus upon expression. This is mediated by a nuclear localisation sequence (NLS) responsible for its importin-dependent transport into the nucleus. This nuclear localisation and the presence of histone acetyl transferase (HAT)-binding domains within the pro-domain suggest a role of this cytokine in gene transcription regulation. In addition, nuclear trafficking of pro-IL-1α is proposed to regulate its secretion. To-date, studies on the nuclear role of pro-IL-1α have used overexpression systems. Here, we generated a mouse where the endogenous Il1a gene was edited with CRISPR to disrupt the NLS (mNLS). Using an in vitro approach with murine macrophages we found that this NLS mutation did not affect pro-IL-1α RNA expression levels in response to LPS but increased its protein expression levels. Moreover, we found that the transcriptional signature induced by LPS was not altered between WT and mNLS macrophages. Release of IL-1α in response to different stimuli such as ionomycin was not negatively impacted by disrupted nuclear localisation, although higher levels of IL-1α release were detected, potentially due to increased levels of pro-IL-1α. Inflammatory responses in an in vivo model of peritonitis and an influenza infection model were comparable between WT and mNLS mice. Thus, we have established a mouse model in which pro-IL-1α nuclear localisation is disrupted, although future research is required to reveal the importance of this nuclear localisation for IL-1α function.

Abstract In most solid tumors, hypoxia is a critical physical attribute that reprograms malignant cells into a highly metastatic state. Specifically, hypoxia is a well-established inducer of cellular plasticity, which is associated with treatment resistance and metastasis. Furthermore, hypoxia exacerbates chromosomal instability (CIN), a hallmark of cancer that can be initiated by the loss of Trp53 and a key contributor to metastasis. Despite this, the mechanisms by which malignant cells concurrently co-opt these elements of hypoxic adaptation to promote metastasis remains unknown. Here we report that hypoxia promotes metastasis by suppressing the JmjC-containing histone lysine demethylase Kdm8. CRISPR/Cas9-mediated targeting of Kdm8 in a Kras;Trp53-driven mouse model of pancreatic ductal adenocarcinoma robustly rewires the malignant cell transcriptomic programs, leading to a profound loss of the epithelial morphology and widespread metastatic disease. Mechanistically, Kdm8 suppression in normoxia recapitulates major aspects of the global epigenetic changes and the transcriptomic rewiring induced by hypoxia. Moreover, Kdm8 deficiency leads to mitotic defects, increased micronuclei formation, Kras copy number gains, and enhanced CIN. Of note, disruption of Kdm8’s demethylase function phenocopies the effects of Kdm8 loss, whereas expression of hypermorphic Kdm8 variants that are resistant to hypoxic suppression reduces metastasis beyond the levels achieved by the wildtype counterpart. Through the suppression of Kdm8 demethylase activity, hypoxia unleashes a potent metastatic program by simultaneously advancing cellular plasticity and CIN.

Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. While condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, additional mechanisms—particularly those involving phosphorylation and deacetylation of specific histone residues—have been proposed to contribute a further 2- to 4-fold reduction in mitotic chromatin volume. In this study, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing a highly synchronous mitotic entry at 2.5-minute resolution. Our analysis reveals three different programmes of histone H3 phosphorylation on T3, S10 and S28. These modifications are consistent with methyl-phos switches regulating the association of readers with chromatin other than at promoters. Mass spectrometry and quantitative ChIP-Seq reveal that H3 T3 phosphorylation is a general marker of heterochromatin and not specifically centromeres as previously suggested. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, previously reported reductions in acetylation associated with chromosome formation were apparently attributable to delays in mitotic exit used as part of mitotic synchronisation protocols. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.

Nuclear pore complexes, critical for nucleocytoplasmic transport, are composed of nucleoporins (Nups). Recent studies have uncovered roles for different Nups in processes like cellular differentiation, contributing richly to organismal development. Intriguingly, the Nup107 complex member, Nup43, is linked with premature ovarian insufficiency (POI) in humans. We report that Nup43 is integral to the maintenance of Drosophila fertility. Nup43 null mutant ( Nup43 KO ) generated by CRISPR-Ca9 causes sterility in both females and males. While oocyte development is halted at the first division stage, the Nup43 KO males are sterile due to defective spermatogenesis, which is arrested at the canoe stage of development in Nup43 KO mutants. The nuclear elongation, shaping, and actin cone formation steps of individualization complex (IC) formation are adversely affected, suspending sperm maturation. All these defects were rescued by the expression of the Nup43 transgene in null mutants, suggesting the criticality of Nup43 function in spermatogenesis. Myosin VI (jar), an actin cone modulator, and Nup43 interactor can partially rescue the actin cone formation but not the sterility defects. We propose that Nup43 facilitates sperm individualization along with jar by promoting actin cone formation during spermatogenesis. These observations uncover a novel yet critical function for Nup43 in Drosophila gonad development and spermatogenesis.

Phage defense systems in bacteria exhibit high degrees of modularity, with sensing, signal transmission, and effector enzymes frequently being exchanged among phage defense gene clusters. In this study, we capitalized on this modularity to discover phage defense systems by searching for defense-associated modules in new gene contexts. This approach revealed a large and interconnected network of modular components distributed across diverse gene clusters. From over 500 candidate defense systems, we selected nine for experimental testing and validated three: Dionysus, a TerB-encoding system that disrupts early phage infection vesicle formation by Jumbo phages; Ophion, a Radical SAM-containing system that prevents the formation of the Jumbo phage nucleus; and Ambrosia, a tightly regulated RM-like system. Collectively, we demonstrate that leveraging the modular architecture of phage defense systems is an effective approach to their discovery.

ABSTRACT Self-renewal and differentiation are at the basis of hematopoiesis. While it is known that tight regulation of translation is vital for hematopoietic stem cells’ (HSCs) biology, the mechanisms underlying translation regulation across the hematopoietic system remain obscure. Here we reveal a novel mechanism of translation regulation in the hematopoietic hierarchy, which is mediated by ribosomal RNA (rRNA) methylation dynamics. Using ultra-low input ribosome-profiling, we characterized cell-type-specific translation capacity during erythroid differentiation. We found that translation efficiency changes progressively with differentiation and can distinguish between discrete cell populations as well as to define differentiation trajectories. To reveal the underlying mechanism, we performed comprehensive mapping of the most abundant rRNA modification - 2’-O-methyl (2’OMe). We found that, like translation efficiency, 2’OMe dynamics followed a distinct trajectory during erythroid differentiation. Genetic perturbation of individual 2’OMe sites demonstrated their distinct roles in modulating proliferation and differentiation. By combining CRISPR screening, molecular and functional analyses, we identified a specific methylation site, 28S-Gm4588, which is progressively lost during differentiation, as a key regulator of HSC self-renewal. We showed that low methylation at this site led to translational skewing, mediated mainly by codon frequency, which promoted differentiation. Functionally, HSCs with diminished 28S-Gm4588 methylation exhibited impaired self-renewal capacity ex-vivo , and loss of fitness in-vivo in bone marrow transplantations. Extending our findings beyond the hematopoietic system, we also found distinct dynamics of 2’OMe profiles during differentiation of non-hematopoietic stem cells. Our findings reveal rRNA methylation dynamics as a general mechanism for cell-type-specific translation, required for cell function and differentiation. KEY POINTS Hematopoietic differentiation is associated with rRNA methylation dynamics to control cell-type-specific translation. Translation efficiency can distinguish discrete cell types and define differentiation trajectories. HSC fitness is regulated by a single rRNA methylation.

Background The advent of CRISPR-Cas9 genome editing has brought about a paradigm shift in molecular biology and gene therapy. However, the persistent challenge of off-target effects continues to hinder its therapeutic applications. Unintended genomic alterations can lead to significant genomic damage, thereby compromising the safety and efficacy of CRISPR-based therapies. Although in-silico prediction tools have made substantial progress, they are not sufficient for capturing the complexity of genomic alterations and experimental validation remains crucial for accurate identification and quantification of off-target effects. In this context, Genome-wide Unbiased Identification of Double-strand breaks Enabled by Sequencing (GUIDE-Seq) has emerged as a gold standard method for the experimental detection of off-target sites and assessment of their prevalence by introducing short double-stranded oligonucleotides (dsODNs) at the break sites created by the nuclease. The bioinformatic analysis of GUIDE-Seq data plays a pivotal yet challenging role in accurately mapping and interpreting editing sites and current pipelines suffer limitations we aim to address in this work. Results In this study, we present a rapid and versatile single-command pipeline designed for the comprehensive analysis of GuideSeq and similar techniques of sequencing. Our pipeline is capable of simultaneously processing multiplexed libraries from different organisms, PCR orientations, and Cas with different PAM specificities in a single run, all based on user-specified sample information. To ensure reproducibility, the pipeline operates within a closed environment and incorporates a suite of well-established bioinformatics tools. Key novel features include the ability to manage bulges in gDNA/gRNA interaction and multi-hit reads, and a built-in tool for off-target site prediction. The pipeline generates a detailed report that consolidates quality control metrics and provides a curated list of off-target candidates along with their corresponding gRNA alignments. Conclusions Our pipeline has been tested and successfully applied to analyze samples under a variety of experimental conditions, including different source organisms, PAM motifs, dsODN sequences and PCR orientations. The robustness and flexibility of our pipeline make it a valuable tool for researchers in the field of genome editing. The source code and comprehensive documentation are freely accessible on our GitHub repository: https://github.com/gcorre/GNT_GuideSeq .

The CRISPR/Cas9 system deployed through crosses of transgenic lines expressing Cas9 and gRNA facilitates efficient mutagenesis. However, its application in non-model insects remains limited, primarily due to a lack of well-characterized promoters capable of driving robust and stable expression of Cas9 and gRNA . In the malaria mosquito Anopheles sinensis , we evaluated several ovary-biased promoters— Asvasa2, Aszpg , and Asnanos —for driving Cas9 expression. Notably, the Asvasa2 promoter mediated mutagenesis in nearly 60% of G 0 individuals following microinjection of gRNA Aswhite . Among four RNA polymerase III promoters derived from AsU6 genes, AsU6 -1 yielded the highest gRNA transcriptional output, enabling 62% editing efficiency in G 0 offspring. In addition, hybrid crosses between established transgenic lines demonstrated that the Asvasa2-Cas9 and AsU6 -1- gRNA combination enabled complete germline editing penetrance, where all F 2 progeny inherited the intended mutations. This work provides a essential genetic toolkit for synthetic biology applications in Anopheles mosquitoes and a scalable framework for engineering other non-model insects.

Summary Humans and animals are ubiquitously colonized by Enterobacteriaceae , a bacterial family that contains both commensals and clinically significant pathogens. Here, we report Enterobacteriaceae megaplasmids of up to 1.58 Mbp in length in infant and adult guts, and other microbiomes. Of the 19 complete plasmid genomes, one was reconstructed from an Escherichia coli isolate; others were linked to species of Citrobacter and Enterobacter via analysis of genome modification patterns. The detection of related plasmids in different Enterobacteriaceae , conjugation machinery, and more diverse modified motifs in certain plasmids compared to hosts suggests that these elements are self-transmissible, with a broad host range. The plasmids encode multi-drug efflux systems and potential secreted effectors. Up to 208 tRNAs are encoded, and include sequence variants that may counter tRNA-centric defense mechanisms. Overall, the vast megaplasmid coding capacity may broaden host range, increase competitiveness, control invasion by other elements, and counter programmed cell death.

Abstract Secondary lymphoid tissues develop specialized reticular networks to facilitate immune cell communication and efficient activation of adaptive immunity. This stromal network architecture is robust, maintaining topology throughout extensive remodelling and tissue expansion in response to immune challenge. We have previously reported that cytoskeletal mechanics of the fibroblastic reticular cell (FRC) networks determine tissue tension, and that increased tension initiates stromal proliferation for lymph node growth. However, it is not known how FRCs maintain stromal network connectivity and what cellular mechanisms reinforce stromal cell-cell and cell-matrix interactions. Here, we present a signalling mechanism which coordinates reduced FRC contractility and induction of stromal cell protrusions. RhoA/C GTPase activity is blocked in FRCs to inhibit actomyosin contractility through contact with dendritic cells (DCs) and binding between podoplanin and the C-type lectin CLEC-2. We now find that an additional Rho GTPase target, the PKC family kinase PKN2, regulates the function of myristoylated alanine-rich protein kinase C-substrate (MARCKS). FRCs generate cell protrusions via MARCKS in response to DC contact, which reinforces stromal cell connectivity. In vivo, we found that PKN2 knock-out lymph nodes are unable to regulate MARCKS and show severely disrupted stromal architecture. These results reveal a mechanism of stromal/immune cell crosstalk which actively induces stromal protrusions – an essential component of lymph node remodelling to maintain tissue integrity during an adaptive immune response.

The intestinal mucus layer is essential for the integrity of the intestinal barrier. It is produced by goblet cells, whose depletion is common in colonic inflammation but remains poorly understood. Here, we show that goblet cell survival relies on a reciprocal dependence with newly discovered BEST4/CA7 + cells. We developed a method to follow BEST4/CA7 + and goblet cells in time from birth to death in human colon organoids. Notably, goblet cells induce BEST4/CA7 + fates in sister cells and other neighbors, using DLL1-mediated lateral activation of Notch-signaling. BEST4/CA7 + cells in turn promote goblet survival, with the latter depleting rapidly after differentiation in absence of BEST4/CA7 + cells. This apoptosis inhibition does not require direct cell-cell contact and instead depends on their shared lumen. Such differentiation and survival interdependencies may be relevant beyond the maintenance of mucosal homeostasis.

ABSTRACT Agroinfiltration of Nicotiana benthamiana is frequently used to produce recombinant proteins, both for plant science and for molecular pharming. Here, we introduce two genome-edited lines of N. benthamiana lacking two polyphenol oxidases (PPOs). These double ppo knockout lines grow slightly faster than wild-type and show similar levels of transient GFP expression. However, leaf extracts produced in native buffers stay greener and show much less native crosslinking of Rubisco and other proteins, demonstrating that PPO depletion reduces enzymatic browning and protein crosslinking in leaf extracts. Transient PPO1 expression in the ppo mutant restores browning and crosslinking in leaf extracts. These ppo mutants offer tremendous opportunities to increase yield and purity of recombinant proteins and study protein complexes, as illustrated with a nearly 4-fold increase in purification yield and a substantial improvement of protein purity upon purification of transiently expressed His-tagged tomato immune protease P69B from total leaf extracts.

ABSTRACT Imps are a highly conserved family of RNA-binding proteins involved in embryonic development, cancer progression, and neurogenesis. However, the molecular pathways and RNAs regulated by Imp to control these processes remain poorly understood. Embryos derived from Imp mutant germline clones arrest development, and transcriptome analysis revealed significant dysregulation of genes involved in cell growth, differentiation, tube morphogenesis, neuronal projection development, and RNA metabolism, along with de-repression of transposable element (TE) RNAs. Consistent with these findings, Imp mutant embryos display TE-overexpression phenotypes, are smaller in size, and exhibit defective organ development, including impaired tracheal branching and gastrulation. Reduced levels of Imp at the larval neuromuscular junction (NMJ) impair synaptic bouton formation and decrease adult longevity. RIP-seq experiments showed that Imp-associated RNAs are enriched for TE RNAs. Proteomic analyses confirmed that several TE-encoded proteins are upregulated in Imp mutant embryos. Specifically, the Ty1 family retrotransposon Copia was derepressed. Consistent with recent findings that Copia is a potent inhibitor of synaptogenesis, its upregulation likely contributes to the impaired NMJ formation and broader embryonic defects observed in Imp mutants. Moreover, Imp associates with piRNA pathway proteins, ensures Piwi nuclear localization, and—like piwi mutants—its loss disrupts TE silencing and causes position-effect variegation (PEV) defects. The analysis of Imp complexes further points to potential mechanisms by which Imp may regulate TE expression. Overall, these results indicate that Imp maintains genome stability and ensures proper developmental progression and neuronal activity by regulating post-transcriptional processes and suppressing transposons.

ABSTRACT Multidrug transporters, including multidrug resistance-1 (MDR1), are recognized chiefly for effluxing chemotherapeutic drugs out of tumor cells. However, they are also expressed in many normal cells and tissues, including lymphocytes, but their physiological role is less well-understood. Here, we investigated the role of MDR1 in tumor-specific CD8 T cells (TST), which are critical in antitumor immunity and key targets of immunotherapies. Using a clinically-relevant genetic liver cancer mouse model, we investigated the efflux dynamics of TST as they underwent activation, proliferation, and differentiation to dysfunctional states in tumor-bearing hosts. Surprisingly, we found that late-stage/terminally dysfunctional TST had the highest efflux capacity in both murine and human liver tumors. TST upregulated transcription of Abcb1a , encoding MDR1. We used CRISPR/Cas9 to generate MDR1-deficient TST, which persisted poorly in tumor-bearing mice as compared to MDR1-sufficient TST. MDR1 expression improved TST viability and reduced reactive oxygen species accumulation. Loss of MDR1 made T cells more susceptible to cytotoxic chemotherapy-induced cell death. Our findings demonstrate a role for MDR1 in regulating TST persistence and oxidative stress, with implications for antitumor T cell therapies in patients and immune regulation following cytotoxic chemotherapy.

Nitric oxide (NO) is an important signaling molecule in flowering plant immunity. It rapidly accumulates in response to pathogen perception. In addition to it’s direct response to microbes, NO controls a range of defence responses primarily through S -nitrosylation. This process is a redox-dependent modification where a NO group attaches to the thiol of a cysteine residue, creating an S -nitrosothiol (SNO). To explore the role of S -nitrosylation more broadly, we characterised the single-copy S - nitrosoglutathione reductase 1 (Mp GSNOR1 ) gene in the liverwort Marchantia polymorpha (Marchantia), a representative of a lineage widely diverged from flowering plants. We generated loss-of-function alleles using CRISPR/Cas9 genome editing. Disrupting Mp GSNOR1 resulted in pronounced morphological alterations, highlighting the role of GSNOR1 in the structural development of Marchantia. Additionally, we show that Mp GSNOR1 is essential for SNO homeostasis and immune function. Our results suggest that GSNOR was part of the tool kit of the ancestral land plant and functioned in immunity and development. Highlight First evidence from a Liverwort shows GSNOR controls immunity and development via S -nitrosylation, revealing these regulatory roles as ancient traits of land plants.

Cellular senescence is a hallmark of aging and a promising target for extending human healthspan. Senescence is often accompanied by upregulation of the key senescence marker gene CDKN2A , yet the mechanism underlying its transcriptional activation remains unclear due to complex cis -regulations within the 9p21.3 locus. Here, we performed complementary CRISPR activation and interference screens in human mesenchymal stromal cells (MSCs) to systematically map non-coding cis -regulatory elements (CREs) at this locus that epigenetically regulate senescence. This approach revealed senescence-regulating CREs (SenReg-CREs) that bidirectionally modulate senescence through P16 INK4a and P15 INK4b . Notably, we identified a primate-specific short interspersed nuclear element (SINE) MIR3 embedded within the most potent distal SenReg-CRE. Deletion of this SINE:MIR3 accelerated senescence, revealing its potential insulator function in restraining CDKN2A/CDKN2B activation. Collectively, these findings reveal novel mechanisms underlying senescence-associated transcriptional activation of CDKN2A/CDKN2B and demonstrate that senescence is malleable through manipulation of regulatory element activity, highlighting the potential of epigenetically targeting these SenReg-CREs for senomorphic interventions.