Summary The NLRP3 inflammasome is a major driver of immunopathology, making it a sought-after drug target. In spite of two decades of intense research, its precise activation mechanism remains elusive, impeding inhibitor design. NEK7 was reported as essential for NLRP3 activation, and several newly identified inhibitors were suggested to act by interfering with their interaction. Here we report that NEK7 accelerates, but is in principle dispensable for NLRP3 activation. The onset of inflammasome activation was unaltered in the absence of NEK7, yet the rate of cells to undergo inflammasome formation and subsequent pyroptosis was approximately 4-fold reduced. Therefore, therapeutic targeting of the NEK7-NLRP3 interaction might have an incomplete effect, which should be considered for drug development. We confirmed entrectinib as a NEK7-dependent inhibitor, while other published compounds turned out not to rely on it. Our results support two possible scenarios for the role of NEK7 in NLRP3 activation: either, NEK7 accelerates one unique pathway of NLRP3 activation, or it is essential for a fast pathway, while being dispensable for a second, slower mode of NLRP3 activation.

Quantitative cell biology often studies migration and the cell-cycle (CC) in separate assays, limiting mechanistic insights, particularly under geometric confinement. Here, we introduce a vertically integrated platform for simultaneously tracking single-cell migration and assessing CC under confinement. Our system integrates cell engineering via multiplexed sensors for cell-cycle, actin, and tubulin, as well as photopatterned engineered extracellular matrix (ECM) islands of defined sizes. It also features an automated, high-throughput pattern-aware imaging pipeline (Fab2Mic) that enables on-pattern, joint migration-CC assessment in the same live cells. Since the local microenvironment plays a critical role in metastasis by constraining cell behaviors within spatial boundaries, we used an HT1080 fibrosarcoma model as an illustrative case. Where static phenotyping yielded 40% G1 and 60% S/G2/M, with larger cell areas and tubulin spread in the S/G2/M phase, dynamic phenotyping via live-cell imaging confirmed CC-linked motility, with faster instantaneous velocities in G1, exemplifying the CC-migration correlations. These phenotypes were modulated by the spatial confinement imposed by the engineered ECM islands. Stronger confinement reduced cell area and tubulin spread and increased the frequency of abnormal CC events, particularly Long G1 states on smaller engineered ECM islands. It also induced a confinement-specific S/G2/M-G1 mitotic slippage, observed only under our confined conditions. Together, this vertically integrated system suggests that confinement may continuously tune migration–CC coupling and provides a deployable pipeline for CC-aware mechanobiology and screening. Moreover, we stress how dynamic imaging provides access to variables that are difficult or impossible to infer from static snapshots, including velocity and CC timing.

Genome editing is now available for many crops. It has increased our ability to study gene function and has changed the field of plant transgenesis. Nevertheless, the ability to regenerate plants from cell culture remains a limiting factor for many crops, and even for species with a good regeneration potential, some accessions remain recalcitrant. The physiological state of plant cells is involved in the process of plant growth and development and is closely linked to the network involving MAP-kinase signaling pathway. Some of the defense genes activated during the cellular repair process of transgenesis show high homologies with mammalian defense genes. We thus compared the percentage of transgenic plants obtained by CRISPR-Cas9 mutation in four genes involved in sugar and acid metabolism after supplementation with different mammalian growth factors and cytokines in six tomato accessions presenting a range of regeneration levels. We demonstrated, through three years of transgenesis experiments, that the use of mammalian growth factors during transgenesis improved regeneration rate of recalcitrant tomato accessions. We demonstrated that using cytokines not only improved transformation of difficult-to-transform accessions but also the production rate of stable secondary lines. Summary statement Supplementation of transformation medium with mammalian growth-regulating factors enhanced regeneration of tomato recalcitrant genotypes

2-Oxoglutarate-dependent Dioxygenases (2OGDDs) are a family of enzymes requiring molecular oxygen, 2-oxoglutarate, reduced iron, and ascorbic acid to function. This dependency renders them key sensors of the cell's metabolic state, driving crucial functions when oxygen or metabolic homeostasis is perturbed, including adaptation to low oxygen, epigenetic control of gene transcription, and the reshaping of metabolic pathways. Jumonji-C (JmjC) domain-containing protein 5 (JMJD5), a 2OGDD that alters epigenetic marks, is essential for DNA damage repair and is a key regulator of cell metabolism. Notably, JMJD5 is often lost in hepatocellular carcinoma, which correlates with poor overall survival. Despite its biological significance, the molecular functions of JMJD5 remain unresolved, and its physiological targets are elusive. Here, we identify and characterise a novel signalling pathway where JMJD5 hydroxylates an arginine residue on the protein ISY1. This modification enables ISY1 to bind to and reduce the activity of Protein Arginine N-methyltransferase 6 (PRMT6). Significantly, the inactivation of PRMT6 rescues the majority of the molecular phenotype driven by JMJD5 loss, establishing the JMJD5-ISY1-PRMT6 pathway as the principal executor of JMJD5's enzymatic function. This signalling pathway clarifies existing controversies regarding JMJD5's function and identifies PRMT6 as a potential therapeutic target for treating cancers that lack JMJD5.

Summary RH5-Interacting Protein (RIPR) is essential for the invasion of Plasmodium into host red blood cells and is currently being studied as a novel malaria vaccine candidate in Phase 1a clinical trials. To study the genetic diversity of RIPR, deep amplicon sequencing was used to identify RIPR mutations in Plasmodium falciparum clinical isolates (n=89) collected in Kédougou, a high malaria transmission region of Senegal. We identified nonsynonymous single nucleotide polymorphisms (SNPs) in 64/89 (71.9%) of the samples. In total, 26 non-synonymous SNPs were identified, of which 15 were novel. 16/26 SNPs were able to be threaded onto existing RIPR crystal structures to predict the effects of SNPs on RIPR stability. 7/16 mutations were predicted to destabilize RIPR while 2/16 increased the stability of RIPR. Additionally, we identified 3 SNPs (Q737K, T738K, V840L) in the EGF5-8 domains of RIPR where neutralizing antibodies are known to bind.

Precise cis -regulatory control of gene expression is essential for balancing plant growth and stress responses. In Arabidopsis thaliana , PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY) peptides and their receptors (PSYRs) mediate growth–stress trade-offs, yet the transcriptional regulation of these genes remains poorly understood. Here, we mapped transcription factor (TF)–promoter interactions for nine PSY and three PSYR genes by combining high-throughput yeast one-hybrid screening with DAP-seq data, uncovering 1,207 interactions that reveal both shared and gene-specific regulatory relationships. Functional analysis of 25 TF mutants identified 12 regulators that significantly influence shoot and root growth, most acting as repressors. Of these, CYTOKININ RESPONSE FACTOR 10 (CRF10) emerged as a strong growth inhibitor. We found a CRF10 binding motif in the PSYR3 promoter using DAP-seq data and validated by eY1H. Guided by these insights, we applied CRISPR/Cas9-mediated promoter editing to precisely delete functional TF-binding sites. Removal of this motif, or its surrounding region, reduced PSYR3 expression and enhanced root growth, yielding variants that retained root length comparable to the crf10 mutant but displayed reduced susceptibility to Pseudomonas syringae pv. tomato ( Pst ) DC3000. Together, our results define the global TF–promoter interaction network of the PSYR/PSY pathway and identify CRF10 as a key transcriptional regulator of PSYR3 -mediated signaling. More broadly, our work demonstrates that cis -regulatory editing can systematically fine-tune gene expression, generate predictable growth traits, and provide a TFBS-targeted framework for engineering plant growth–defense balance.

ABSTRACT Controlling Mycobacterium tuberculosis (Mtb) infection requires a precisely balanced host inflammatory response. Too little inflammation leads to uncontrolled bacterial growth but exacerbated inflammation, activated by mediators such as TNF and type I IFN, inhibits effective antibacterial responses. How these immunopathological states are established is unknown. Deeper understanding of the pathways elicited upon initial Mtb infection of the host macrophage may reveal vital regulatory mechanisms that govern the subsequent inflammatory environment and ultimate resolution of infection. To elucidate these early regulators of inflammation, we performed a genome-wide CRISPR knockout screen in macrophages to identify genes that influence the induction of TNF and iNOS upon infection with Mtb. The resulting dataset is a valuable resource that includes genes representing a wide range of unexpected regulatory mechanisms that control cytokine responses to Mtb and also cell-intrinsic resistance to infection by the bacterial pathogen Listeria monocytogenes. We show that type I IFN signaling enhances TNF production early after infection, and IRF2 acts to inhibit induction of the antibacterial state of macrophages. Our data support a model in which early production of type I IFN in response to bacterial infection serves to increase innate antibacterial resistance during the earliest stages of infection.

Invasive fungal respiratory infections (IFRIs) remain a major cause of morbidity and mortality among immunocompromised patients, yet diagnosis continues to be hindered by nonspecific clinical features, limited sample accessibility, and the poor sensitivity or specificity of conventional tests. Microfluidic and microelectromechanical systems (MEMS)-based biosensing platforms have emerged as promising alternatives, enabling rapid, minimally invasive, and highly specific detection of fungal pathogens and host responses. Microfluidic nucleic acid and antigen assays allow on-chip amplification and immunodetection with reduced sample volumes and turnaround times, while CRISPR-enhanced systems further improve analytical sensitivity. Parallel advances in host-response profiling—including transcriptomic, proteomic, and cytokine-based signatures—have demonstrated feasibility for integration into lab-on-a-chip platforms. MEMS-based technologies extend this potential by facilitating real-time analysis of exhaled volatile organic compounds, mechanical biosensing of fungal DNA and antigens, and in situ monitoring of device-associated biofilms. Translational studies highlight potential applications across intensive care, hematology–oncology, and transplant settings, as well as in outpatient monitoring of high-risk populations. However, several challenges remain, including limited multicenter validation, matrix-related biofouling effects, and lack of standardization in fungal biomarker panels. Future directions include AI-driven interpretation of multianalyte data, multiplexed integration of host and pathogen markers, and development of fully cartridge-based systems for near-patient deployment. Collectively, these innovations may shift fungal diagnostics toward earlier, more precise, and patient-tailored interventions, improving outcomes in vulnerable populations.

Proteases are enzymes that catalyse the hydrolysis of peptide bonds in proteins for their functional, modification or degradation. Members of the Dipeptidyl Peptidase IV (DPPIV) family are exopeptidases that cleave dipeptides off the N-termini of their substrate peptides, typically after proline or alanine. Recently, we showed that human DPP4 and Caenorhabditis elegans DPF-3 have a larger target repertoire in vitro , permitting cleavage after additional amino acids. Here, we use TAILS (Terminal Amine Isotopic Labelling of Substrates) to identify DPF-3 targets in vivo and observe cleavage of MEP-1 after threonine, confirming a broader substrate specificity of DPF-3 also in vivo . Demonstrating physiological relevance, we show that rendering MEP-1 resistant to cleavage disrupts its stability, leading to developmental abnormalities such as defective gonadal migration and reproductive issues. Collectively, our findings highlight a previously unappreciated complexity in the substrate specificity of DPPIV family proteases and suggest that their physiological roles may extend beyond what is currently known. IMPORTANT Manuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. Upon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. The identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. The identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. GUIDELINES For reviewers: https://www.reviewcommons.org/reviewers For authors: https://www.reviewcommons.org/authors CONTACT The Review Commons office can be contacted directly at: office@reviewcommons.org

SUMMARY Transcriptional regulation is a key central mechanism of cell fate determination in developing tissues. The homeobox transcription factor NKX2.2 is an essential regulator of mouse and human pancreatic endocrine development, however its precise molecular role in a human system has not been previously investigated. In this study we generated NKX2.2 null (NKX2.2KO) human embryonic stem cell (hESC) lines using CRISPR/Cas9 technologies and differentiated them towards a pancreatic β cell fate using a stem cell-derived β cell differentiation protocol. Functional and transcriptomic analyses of the hESC-derived pancreatic endocrine cells lacking NKX2.2 revealed similarities and differences compared to the molecular functions of NKX2.2 in mice. In the absence of NKX2.2, the β cell differentiations result in reduced numbers of insulin-producing cells, and the differentiations become skewed towards polyhormonal fates, including cells co-expressing insulin, ghrelin and somatostatin. Deletion of NKX2.2 also eliminates the off-target formation of enterochromaffin cells. Single cell transcriptome analysis of the early endocrine cell population revealed a marked disruption of metabolic pathways that was confirmed by comparative metabolite tracing, providing novel insights into the regulation of early endocrine lineage decisions. Furthermore, NKX2.2 directly regulates several genes in the WNT signaling pathway, suggesting this is a key molecular mechanism through which NKX2.2 regulates these islet cell fate decisions in the human system.

Preclinical cancer research requires robust model systems, especially for poor prognosis entities like acute myeloid leukemia (AML), a highly aggressive blood cancer. Here, primary tumor cells from 137 AML patients of all age groups were transplanted into immune compromised mice to generate patient-derived xenografts (PDX). From these, 23 models enable robust, virtually endless serial re-transplantation and are amenable to lentiviral genetic engineering ( * PDX AML models). These models primarily originate from patients with highly aggressive, relapsed disease. Comprehensive genomic, transcriptomic, and epigenomic analyses confirmed that they replicate primary AML biology more faithfully than conventional cell lines. Notably, * PDX AML models include AML subgroups that are underrepresented or absent in existing model systems, such as cytogenetically normal or IDH1/2 -mutant AML. They withstand freeze-thaw cycles, making them suitable for broad distribution and reproducibility across research institutions. Luciferase-based in vivo imaging enables real-time monitoring of tumor progression and treatment responses in preclinical trials. Surprisingly, long-term treatment, including repeated cytarabine therapy over a period of one year, showed a gradual reduction in leukemia cell proliferation, which decreased continuously after each treatment block. Collectively, our * PDX models represent a robust, versatile, and relevant platform that holds great promise to accelerate translational research for the benefit of cancer patients. Visual Abstract Key Points We present new robust AML PDX models covering subgroups for which no cell lines exist for use in various ex vivo and in vivo applications. * PDX models enable serial transplantation, genetic engineering and better representation of primary AML biology than cell lines. One-year in vivo trials mimicking clinical chemotherapy showed surprising gradual decline in leukemia growth after each treatment block.

ABSTRACT The emergence of CRISPR-Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13’s heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first dual-guide RNA system for Cas13 that enhances mismatch discrimination and improves target specificity. This system employs two distinct RNAs—dcrRNA and dtracrRNA—which hybridise to refine target recognition and activation. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, demonstrating its potential for pathogen diagnostics, and successfully discriminated between KRAS G12D and G12C mutations—clinically relevant single-nucleotide variants in cancer diagnosis. These results highlight the dual-guide Cas13 platform’s potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a significant advance over conventional Cas13 systems, offering improved specificity without compromising sensitivity. Its versatility makes it a promising tool for next-generation molecular diagnostics and precision gene editing applications. GRAPHICAL ABSTRACT

African swine fever (ASF), induced by the African swine fever virus (ASFV), is an acute hemorrhagic disease characterized by high fever, systemic hemorrhages, and elevated mortality. Current diagnostic techniques including PCR and ELISA present limitations in field applications due to requirements for specialized equipment and prolonged processing duration. Therefore, rapid and accurate detection of ASFV has become a key link in ASF prevention and control. This study established a rapid and precise visual diagnostic approach by integrating the CRISPR/AapCas12b system with lateral flow strip (LFS) technology, specifically targeting the B646L gene encoding the major capsid protein p72. The CRISPR/AapCas12b-LFS platform achieved a sensitivity threshold of 6 copies/µL for B646L gene detection, completing analysis within an hour. Validation study confirmed exceptional specificity against common porcine pathogens including PRRSV, CSFV, PRV, PPV4 and PCV3. The developed assay demonstrated complete concordance with real-time PCR results when analyzing 34 clinical specimens for ASFV detection. Overall, this method is sensitive, specific, and practicable onsite for the ASFV detection, showing a great application potential for monitoring the ASFV in the field.

Abstract Background: Hemifacial Microsomia (HFM) is a genetically complex craniofacial disorder. While GWAS and family studies have identified multiple candidate genes, functional validation rates remain low (<10%). Methods: We established a high-throughput zebrafish CRISPR-Cas9 platform to functionally validate 16 prioritized genes (12 literature-derived, 4 bioinformatically predicted). Tg(col2a1a:EGFP) embryos underwent F0 knockout with ≥70% editing efficiency. Mandibular development was quantitatively analyzed using six morphometric parameters at 5 dpf. Results: We identified three high-confidence pathogenic genes: EDNRB knockout caused pan-mandibular hypoplasia (Meckel's cartilage ↓21%, p<0.0001); FGF3 deficiency led to selective arch defects (ceratohyal length ↓28%, p<0.0001); EPAS1 ablation resulted in unilateral dysgenesis (cranial length ↓24%, p<0.0001). PAX1 knockout induced lethal pan-craniofacial defects. Conclusion: This study establishes EDNRB-FGF3-EPAS1 as a core pathogenic axis and validates environmental susceptibility genes (TP53/ESR2). These findings enable: 1) OMENS+ molecular subtyping, 2) gene-targeted therapeutic strategies, and 3) prenatal risk assessment for environmental exposures.

Neutrophils are the major populations of white blood cells and have been reported to facilitate cancer metastasis. Meanwhile, emerging evidence has recently suggested the anti-cancer role of neutrophils. Our previous study revealed that CB-839 and 5-FU-treated colorectal cancer (CRC) tumors recruited neutrophils and induced neutrophil extracellular traps (NETs). Cathepsin G (CTSG), which is released during NET formation, enters CRC cells through the receptor for advanced glycation end products (RAGE) and cleaves 14-3-3ε to promote apoptosis. However, the detailed mechanism underlying CTSG’s anti-tumor function remains less studied. In this study, we report that CTSG enters CRC cells through RAGE-mediated endocytosis. Knocking out RAGE or inhibiting endocytosis blocks CTSG from entering CRC cells and attenuates CTSG-induced apoptosis. Furthermore, the clathrin coat assembly complex and SNARE proteins were enriched in an arrayed CRISPR/Cas9 screening targeting human membrane trafficking genes. Knocking out SNARE protein STX1A prevents the spread of CTSG in CRC cells and the induction of cleaved PARP. A pooled genome-wide CRISPR/Cas9 screening further identifies the role of CDK1 in the NET-induced killing of CRC cells. Inhibiting CDK1 protected CRC cells from killing by CTSG. Our study reveals novel mechanisms by which CTSG enters and kills CRC cells.

ABSTRACT Patients with T-cell lymphomas and leukemias have overall poor outcomes due to the lack of targeted and effective treatments, particularly in the relapsed and refractory settings. Development of chimeric antigen receptor (CAR) T-cells against T-cell neoplasms is limited by a lack of discriminating T-cell antigens that allow for effective anti-tumor responses while preventing CAR T-cell fratricide. We hypothesized that targeting CD2, a pan-T-cell antigen, using anti-CD2 CAR T-cells engineered without CD2 expression (CART2), would support CAR T-cell manufacturability and preclinical efficacy. Optimized CD2-knockout CART2, generated using CRISPR-Cas9, eradicated primary patient-derived CD2+ hematological neoplasms in vitro and in vivo, secreted effector cytokines, and exhibited adequate proliferative capacity. Nevertheless, CD2 has a key costimulatory function, and its deletion could lead to CAR T-cell dysfunction. Therefore, we tested the role of the CD2:CD58 axis in CAR T-cells, using the anti-CD19 CART models. We demonstrate that CD2 loss attenuates CART19 efficacy by reducing avidity for tumor antigen, co-stimulation, and ultimately in vivo activity. Analogously, we show that tumor CD58 loss reduces CART19 efficacy. To overcome this issue, we developed a novel PD-1:CD2 switch receptor that rescues intracellular CD2 signaling, particularly when PD-L1 is engaged, resulting in improved in vivo outcomes. Collectively, we studied the role of CD2 both as a target for CAR T cell therapy and as a critical costimulatory protein, whose signaling can be rescued using the PD-1:CD2 switch receptor. This receptor can be incorporated into CAR T-cells and provides an effective strategy to overcome CD2-signaling deficiencies.

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer lacking estrogen receptor, progesterone receptor, and HER2 expression. Due to the absence of actionable molecular targets, patients rely heavily on chemotherapy, often facingearly recurrence and poor prognosis. There is an urgent need for novel therapeutic strategies that utilize alternative cell death mechanisms beyond conventional apoptosis. Methods: To identify metabolic vulnerabilities specific to TNBC, we employed an integrative strategy combining genome-scale metabolic modeling based on patient transcriptomic data with CRISPR-Cas9 dependency datasets. Riboflavin kinase (RFK), an enzyme that converts riboflavin into FMN and FAD, was identified as a top-ranked candidate target. Functional validation was conducted via genetic knockdown and pharmacological inhibition using roseoflavin. Cellular proliferation was assessed by WST assay and crystal violet staining. Apoptosis and ferroptosis were evaluated by Annexin V/PI flow cytometry, western blotting, JC-1 and C11-BODIPY fluorescence, ROS and MDA assays, and glutathione quantification. In vivo efficacy was tested in orthotopic xenograft models using RFK-silenced TNBC cells or roseoflavin-treated mice. Immunohistochemical analyses (Ki67, 4-HNE, TUNEL) were used to assess tumor proliferation, ferroptosis, and apoptosis, respectively. Results: RFK suppression significantly inhibited TNBC cell proliferation in vitro and in vivo . Mechanistically, RFK loss reduced glutathione levels, increased intracellular ROS accumulation,and enhanced lipid peroxidation, resulting in mitochondrial dysfunction and concurrent induction of ferroptosis and apoptosis. In TNBC xenograft models, RFK knockdown or roseoflavin treatment markedly reduced tumor growth, enhanced lipid peroxidation, and increased cell death. Transcriptomic analyses suggest that TNBC tumors, exhibiting heightened ferroptosis susceptibility, may engage in metabolic reprogramming,characterized by upregulation of genes involved in riboflavin uptake, flavin cofactor biosynthesis, and glutathione synthesis, as a compensatory adaptation toenhance redox buffering capacity and resist ferroptotic stress. Conclusions: Our study identifiedRFK as a TNBC-specific metabolic vulnerability, regulatingredox homeostasis and cell death pathways. Targeting RFK represents a promising therapeutic strategy for TNBC, as it induced both ferroptosis and apoptosis. These findings underscore the potential of exploiting the riboflavin–FMN/FAD–glutathione axis as a redox metabolic checkpoint in ferroptosis-prone TNBC.

Abstract Petroleum contamination presents a significant environmental challenge, contributing to soil and water pollution. Bioremediation provides a sustainable and cost-effective approach. In this study, we isolated and characterized a novel petroleum-degrading strain, Rhodococcus indonesiensis SARSHI1. Whole-genome sequencing of SARSHI1 was conducted using a hybrid sequencing approach, integrating Oxford Nanopore Technologies (ONT) (PromethION) and Illumina (NovaSeq 6000) platforms. The complete genome of SARSHI1 comprises 5.7 Mbp, along with a plasmid of 159,118 bp, encoding a total of 5,150 coding sequences (CDS). The genome consists of 5,695,289 base pairs, with 5,220 identified genes comprising 5,094 protein-coding genes. Additionally, it contains 12 ribosomal RNA (rRNA) genes, 55 transfer RNA (tRNA) genes, one non-coding RNA, one CRISPR array, 56 pseudogenes, and 243 hypothetical proteins. The raw reads obtained were 13,900,477 from Illumina and 2,539,063 from ONT, with processed reads of 13,169,190 and 1,567,736, respectively. Genome assembly achieved 100% completeness, confirming the reconstruction of a fully intact genome without missing sequences. A total of 570 single-copy marker genes were identified, resulting in a coding density of 91.4%. Functional annotation and comparative genomic analysis revealed key genes associated with hydrocarbon degradation, including alkB , ahyA , and almA (Group I) families for long-chain alkane degradation, as well as bph , ben , and xylC clusters for aromatic hydrocarbon degradation under aerobic conditions. Additionally, multiple antibiotic resistance genes, including those conferring resistance to beta-lactams, were identified. Secondary metabolite analysis identified 19 distinct biosynthetic gene clusters (BGCs), encoding variants of known compounds, highlighting the genomic potential for diverse secondary metabolite production. The complete genome sequence has been deposited in GenBank under accession numbers CP180630 (chromosome) and CP180631 (plasmid). The raw sequencing reads have been submitted to the Sequence Read Archive (SRA), NCBI, under accession numbers SRX27520007 (Illumina) and SRX27520006 (ONT).

ABSTRACT Dishevelled is a pivotal cytoplasmic hub protein that transmits Wnt signals to various cytoplasmic effectors to specify cell fates and behaviors during animal development. The molecular mechanisms by which Dishevelled directs Wnt outputs towards β-catenin or other non-canonical effectors remain unclear. Its PDZ domain is dispensable for signaling to β-catenin but essential for multiple non-canonical Wnt responses in Drosophila and vertebrate systems. None of its functionally relevant binding partners are known even though a broad range of PDZ-binding ligands have been identified. Here, we combined proximity labeling with structural and biophysical analysis to discover that Daple and its Girdin-L paralog bear unique extended C-terminal PDZ-binding motifs that bind to the PDZ domain of the main human Dishevelled paralog DVL2 with exceptionally high affinity. Assays in HEK293T cells revealed that deletions of these motifs or their cognate PDZ domain of DVL2 resulted in elongated primary cilia and rendered these cilia unresponsive to Wnt5a-stimulated disassembly following serum starvation. We conclude that an unprecedented molecular interaction between Dishevelled and Daple or Girdin-L underpins the disassembly of these ciliary organelles with universal links to signaling and cell cycle progression. One-sentence summary The PDZ domain of Dishevelled engages in unique interactions with the C-termini of Daple or Girdin-L to mediate the disassembly of primary cilia in response to Wnt5a.

Neutrophils are abundant innate effector cells that drive mucosal inflammation, yet the mechanisms by which they contribute to chronic inflammatory diseases across distinct tissues remain incompletely understood. Here, by reanalyzing single-cell RNA-seq datasets from patients with inflammatory bowel disease (IBD) and chronic obstructive pulmonary disease (COPD), we identify a shared neutrophil activation program enriched for type I interferon (IFN) signaling, nuclear factor-κB (NF-κB) and AP-1 transcriptional regulators, and effector pathways including NETosis, degranulation, and leukocyte trafficking. To interrogate these signatures, we established a CRISPR-compatible neutrophil differentiation platform from adult CD34⁺ progenitors, which yielded cells closely resembling primary neutrophils at transcriptomic, proteomic, and functional levels. A targeted CRISPR-Cas9 screen revealed a central role for the mitochondrial iron transporter mitoferrin-1 (SLC25A37) in coordinating neutrophil oxidative phosphorylation, NET formation, and type I IFN production downstream of TLR9. Mechanistically, we show that NET-derived citrullinated histones activate an autocrine IFNα–IFNAR1 loop, amplifying neutrophil inflammatory functions without impairing phagocytosis. Disruption of this loop, through IFNAR1 depletion or blockade, dampened neutrophil-driven tissue damage in human intestinal and alveolar organoid co-cultures as well as in murine models of colitis and cigarette smoke–induced lung inflammation. These findings uncover a conserved IFN-driven metabolic circuit in neutrophils that underpins pathology across chronic mucosal diseases and identify IFNAR1 as a therapeutic node to selectively disarm neutrophil-mediated tissue injury.

The FLOWERING LOCUS T ( FT ) gene is a central integrator of floral induction in Arabidopsis thaliana , with expression tightly regulated by complex transcriptional networks. Using CRISPR/Cas9 genome editing, we dissected the functional architecture of the FT downstream region and reveal that a 2.3-kb region immediately downstream of the FT coding sequence containing the Block E enhancer is essential for proper FT expression and flowering. Fine-scale deletions revealed a 63-bp core module with adjacent CCAAT- and G-boxes, whereas other conserved motifs had minor, context-dependent effects. We also uncovered a cryptic CCAAT-box module that becomes active when repositioned, coinciding with increased transcription factor binding and local chromatin accessibility, indicating that enhancer function is governed by local chromatin and motif context. The cis -regulatory logic revealed here provides insights into manipulating gene expression through the architecture and spatial arrangement of enhancer elements, potentially applicable beyond flowering genes or plant species.

The mitochondrial translocator protein (TSPO) was once proposed to mediate mitochondrial cholesterol import for steroid hormone biosynthesis, but genetic deletion studies in multiple models have refuted this role. Nevertheless, the idea that pharmacological ligands of TSPO can modulate steroid output continues to be invoked. One such compound, 19-Atriol (androst-5-ene-3β,17β,19-triol), was reported to inhibit progesterone synthesis via TSPO binding in MA-10 Leydig cells. To evaluate this proposed mechanism, we used CRISPR/Cas9-generated Tspo -deleted MA-10 cells to study 19-Atriol activity. We found that 19-Atriol inhibited Bt 2 -cAMP-stimulated steroid output independent of TSPO expression; it acted as a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), blocking the conversion of pregnenolone to progesterone. Mass spectrometry revealed that 19-Atriol is also a substrate for 3β-HSD, yielding 19-hydroxytestosterone (19-OHT), which itself inhibits 3β-HSD activity. In addition to this effect, both 19-Atriol and 19-OHT decreased cholesterol-to-pregnenolone conversion during stimulation. Partial inhibition of 22R-hydroxycholesterol metabolism by CYP11A1 was observed with 19-Atriol, but not 19-OHT, suggesting direct or indirect effects on this upstream step, potentially involving the steroidogenic acute regulatory protein (STAR). These findings decisively exclude TSPO as a functional mediator of 19-Atriol activity and instead identify direct enzymatic targets within the de novo steroidogenic pathway. By resolving a key mechanistic misattribution, this study underscores the importance of rigorous target validation, particularly for compounds previously assumed to act via TSPO.

Symbiotic relationships have an important role in most life forms, but the molecular and cellular processes that establish and maintain these harmonious interactions remain largely unknown. The relationship between leguminous plants and rhizobial bacteria is a classic example of symbiosis, where the bacterium converts atmospheric nitrogen to plant-usable ammonia in exchange for fixed carbon and nutrients. Some legumes such as Medicago truncatula has evolved a set of small peptides that exploit this relationship, turning its bacterial partner, Sinorhizobium meliloti , into a terminally differentiated bacterium that loses its capability to survive outside the host. However, the mechanisms of how this transformation happens remain elusive due to the absence of high-throughput tools for targeted gene knockdowns in the bacterium. To overcome these limitations in the plant-rhizobia field, we developed an inducible CRISPR-interference knockdown system which can reversibly block the transcription of a target gene through the combined action of a deactivated-Cas9 (dCas9) and single-guide RNAs (sgRNAs). We used a taurine-inducible promoter to achieve fine-tunable expression levels of dCas9 in free-living S. meliloti and demonstrated that this tool is suitable for the study of essential genes that could be involved in the symbiotic process, including hemH, dnaN and ctrA . Our cost-effective inducible CRISPRi strategy will contribute to understanding the molecular mechanisms underlying legume-rhizobia symbiosis, ultimately allowing soil improvement and reducing chemical fertilizers usage while meeting global food demands.

MicroRNAs (miRNAs) serve critical regulatory roles in gene expression and are valuable biomarkers for early disease detection. However, their inherent low concentration in biological fluids poses significant detection challenges. Although traditional methods like real-time quantitative PCR (RT-qPCR) are highly sensitive, they require thermal cycling, limiting their application in point-of-care testing (POCT). Here, we present an isothermal amplification-based miRNA detection system integrating Three-Way Junction (TWJ) formation, Multistep Low-Temperature Amplification (L-TEAM), and CRISPR-Cas3-mediated signal amplification. The integration of the Multistep L-TEAM with the TWJ method achieves high sensitivity, detecting miRNA at concentrations as low as 10 femtomolar within 50 minutes, and effectively distinguishes single-nucleotide mismatches. When CRISPR-Cas3-mediated reaction was integrated, it still proved effective for confirming the presence of the target, but its quantitative reliability requires further optimization. We developed a predictive model using machine learning to facilitate rational optimization of experimental conditions through contribution analysis and to establish a methodology for designing more favorable sequences. The modular nature of our method permits adaptation to diverse miRNA targets without modifications to the fundamental amplification mechanism.

Abstract Background: Esophageal carcinoma (ESCA) is a highly aggressive malignancy with poor prognosis. The apelin gene (APLN) encodes a secreted peptide involved in various physiological processes, but its role in ESCA progression and chemoresistance remains unclear. Methods: We integrated transcriptomic data from TCGA and GEO databases with CRISPR screening to identify key oncogenes in ESCA. ALPN was identified as a key gene. Functional assays in vitro and in vivo were performed to investigate the biological role of APLN. Mechanistic studies explored the involvement of APLN in autophagy regulation and chemoresistance. Furthermore, we developed an exosome-based siRNA delivery system targeting APLN and constructed a prognostic nomogram incorporating APLN expression. Results: APLN was significantly overexpressed in ESCA tissues and correlated with poor patient prognosis. DNA hypomethylation contributed to APLN upregulation. Functional experiments demonstrated that APLN knockdown suppressed tumor cell proliferation, induced apoptosis, and enhanced sensitivity to cisplatin. Mechanistically, APLN promoted autophagic flux, which mediated chemoresistance in ESCA cells. Exosome-mediated delivery of APLN siRNA effectively inhibited tumor growth in vivo without systemic toxicity. Additionally, a nomogram combining APLN expression with clinical stage accurately predicted patient survival, providing a practical tool for individualized prognosis. Conclusions: Our study identifies APLN as a novel driver of ESCA progression and chemoresistance through autophagy regulation. Targeting APLN via exosome-based siRNA delivery offers a promising therapeutic strategy. Moreover, the APLN-based prognostic nomogram holds potential for guiding personalized treatment decisions in ESCA patients.