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.

Abstract Background Inherited cardiomyopathies frequently arise from rare, highly penetrant coding variants with variable clinical expressivity. Recent biobank-scale genome-wide association studies (GWAS) suggest significant polygenic contributions to cardiovascular diseases, including cardiomyopathy. Most GWAS loci map to noncoding regions, which are poorly conserved across species, requiring a human genome context for experimental validation. Methods We created engineered heart tissues (EHTs) from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and primary cardiac fibroblasts. We assayed single-cell gene expression and chromatin accessibility to generate comprehensive genome-wide regulatory maps. Open chromatin regions (OCRs) were integrated with chromatin contact information and used to functionally fine-map single nucleotide polymorphisms (SNPs) in cardiomyopathy GWAS. SNPs and their associated regulatory regions were assessed using reporter assays, genome editing, and expression profiling. Results Single-cell RNA-seq of EHTs confirmed populations recapitulating major cell types found in hearts, with advanced cardiomyocyte maturation compared to monolayer hiPSC-cardiomyocytes. More than 400,000 OCRs were resolved to cell type and assayed for canonical transcription factor footprints. Functional fine-mapping of GWAS loci prioritized 5817 variants, and reporter assays on select variants validated allele-specific enhancer activity. We identified a locus harboring significant GWAS signals from both dilated cardiomyopathy and left ventricle ejection fraction in an intergenic region at chr3p25.1. Several of these variants lie in OCRs participating in long range chromatin interactions with SLC6A6 and GRIP2 . Haplotype-resolved and synthetic reporter assays confirmed enhancer activity and narrowed candidate SNPs. CRISPR-deletion of this region reduced expression of both SLC6A6 and GRIP2 , indicating the enhancer regulates the expression of more than one gene. Conclusions EHTs derived from hiPSCs are an experimentally tractable platform for testing the function of noncoding variants as modifiers of cardiomyopathy. Variants fine-mapped from cardiomyopathies using EHT regulatory maps have functional consequences and provide a set of prioritized sites to advance the study of polygenic heart failure liability.

Abstract The efficacy and tissue specificity of RNA therapeutics are critical for clinical translation. Here, by large-scale profiling of the dynamic RNA structurome across four cell lines, we systematically characterized the impact of in vivo target RNA structure and RNA-protein interactions on CRISPR/Cas13d gRNA activity. We identified the structural patterns of high-efficacy gRNA targets and observed that structural differences can lead to variations in efficacy across different cellular contexts. By stabilizing single-stranded structure, RNA-binding proteins also enhanced gRNA efficacy. Leveraging this cell context information, along with approximately 290,000 RfxCas13d screening data, we developed SCALPEL, a deep learning model that predicts gRNA performance across various cellular environments. SCALPEL outperforms existing state-of-the-art models, and most importantly, it enables cell type-specific predictions of gRNA activity. Validation screens across multiple cell lines demonstrate that cellular context significantly influences gRNA performance, even for identical targeting sequences, underscoring the feasibility of cell type-specific knockdown by targeting structural dynamic regions. SCALPEL can also facilitate designing highly efficient virus-targeting gRNAs and gRNAs that robustly knockdown maternal transcripts essential for early zebrafish development. Our method offers a novel approach to develop context-specific gRNAs, with potential to advance tissue- or organ-specific RNA therapies.

ABSTRACT Adaptation to fluctuating nutrient supply is essential for organismal survival, but how cells sense and respond to changes in the abundance of many critical nutrients remains undefined. One such example is the amino acid cysteine, whose reactive thiol group is exploited for diverse cellular functions. Here, by characterizing the machinery required for the conditional degradation of cysteine dioxygenase type I (CDO1), the critical enzyme responsible for cysteine catabolism, we identify a Cul2 E3 ligase complex containing the uncharacterized substrate adaptor LRRC58 that is sensitive to cysteine abundance. When cysteine is replete, LRRC58 activity is restrained through auto-ubiquitination and proteasomal degradation; upon cysteine deprivation, LRRC58 is stabilized to permit CDO1 degradation. Through saturation mutagenesis stability profiling, we systematically validate a structural model of the CDO1-LRRC58 interaction and identify residues at the LRRC58 C-terminus required for cysteine-dependent instability. The LRRC58-mediated degradation of CDO1 is essential to prevent ferroptotic cell death under conditions of cysteine scarcity, and mutations in CDO1 which cause neurodevelopmental defects in humans encode dominant-active proteins refractory to LRRC58 recognition. Altogether, these data reveal the CDO1-LRRC58 axis as a critical regulator of cysteine homeostasis that safeguards neural development.

The population of kisspeptin neurons located in the rostral periventricular area of the third ventricle (RP3V) is thought to have a key role in generating the GnRH surge that triggers ovulation. Using a modified GCaMP fibre photometry procedure, we have been able to record the in vivo population activity of RP3V KISS neurons across the estrous cycle of female mice. A marked increase in GCaMP activity was detected beginning on the afternoon of proestrus that lasted in total for 13±1 hours. This was comprised of slow baseline oscillations with a period of 91±4 min and associated with high frequency rapid transients. Very little oscillating baseline or transient activity was detected at other stages of the estrous cycle. Concurrent blood sampling showed that the peak of the LH surge occurred 3.5±1.1 h after the first baseline RP3V KISS neuron baseline oscillation on the afternoon of proestrus. The time of onset of RP3V KISS neuron oscillations varied between mice and across subsequent proestrous stages in the same mice. To assess the impact of estradiol on RP3V KISS neuron activity, mice were ovariectomized and given an incremental estradiol replacement regimen. Minimal patterned GCaMP activity was found in OVX mice, and this was not changed acutely by any of the estradiol treatments. However, on the afternoon of the expected LH surge, the same oscillating baseline activity with associated transients occurred for 7.1±0.5 h. These observations reveal an unexpected prolonged oscillatory pattern of RP3V KISS neuron activity that is dependent on estrogen and underlies the preovulatory LH surge as well as potentially other facets of reproductive behavior.

Abstract Lipid metabolic reprogramming in the hepatocellular carcinoma (HCC) tumor microenvironment (TME) drives immunosuppression, yet the critical mediators orchestrating tumor-immune crosstalk remain elusive. Here, we identify arachidonic acid (ARA), synthesized via the rate-limiting enzyme FADS1, as a novel oncometabolite that accumulates in TME and fuels hepatocarcinogenesis. Genetic or pharmacological inhibition of FADS1 attenuates ARA level, thereby enhancing CD8+ T cell infiltration and cytotoxicity. Using CRISPR-Cas9 screening of metabolic genes in T cell differentiation, we uncover the ARA pathway as a negative regulator of liver-resident memory CD8+ T cells (TRM). These CXCR6+ TRM exhibit antigen specificity but are impaired by FADS1 overexpression or ARA exposure, compromising anti-tumor immunity during tumor rechallenge. Mechanistically, ARA specifically disrupts IL-15-dependent metabolic fitness in CXCR6+ TRM. Therapeutically, targeting the FADS1-ARA axis synergizes with anti-PD-1 and GPC3-CAR-T therapies. Thus, our study identifies a promising target for ARA-enriched immunosuppressive TME, aiming to improve the effectiveness of immunotherapies in HCC.

Large-scale CRISPR screening in human T cells holds significant promise for identifying genetic modifications that can enhance cellular immunotherapy. However, many genetic regulators of T cell performance in solid tumors may not be readily revealed in vitro. In vivo screening in tumor-bearing mice offers greater physiological relevance, but has historically been limited by low intratumoral T cell recovery. Here, we developed a new model system that achieves significantly higher human T cell recovery from tumors, enabling genome-wide in vivo screens with small numbers of mice. Tumor-infiltrating T cells in this model exhibit hallmarks of dysfunction compared to matched splenic T cells, creating an ideal context for screening for genetic modifiers of T cell activity in the tumor microenvironment. Using this platform, we performed two genome-wide CRISPR knockout screens to identify genes regulating T cell intratumoral abundance and effector function (e.g., IFN-γ production). The intratumoral abundance screen uncovered the P2RY8-Gα13 GPCR signaling pathway as a negative regulator of human T cell infiltration into tumors. The effector function screen identified GNAS (Gαs), a central signaling mediator downstream of multiple GPCRs that sense different suppressive ligands, as a key regulator of T cell dysfunction in tumors. Targeted GNAS knockout rendered T cells resistant to multiple suppressive cues and significantly improved therapeutic performance across diverse solid tumor models. Moreover, combinatorial knockout of P2RY8 (trafficking) and GNAS (effector function) further enhanced overall tumor control, demonstrating that genetic modifications targeting distinct T cell phenotypes can be combined to improve therapeutic potency. This flexible and scalable in vivo screening platform can be adapted to diverse tumor models and pooled CRISPR libraries, enabling future discovery of genetic strategies that equip T cell therapies to overcome barriers imposed by solid tumors.

Synthetic biology aims to engineer or re-engineer living systems. To achieve increasingly complex functionalities, it is beneficial to use higher-level building blocks. In this study, we focus on oscillators as such building blocks, propose novel oscillator-based circuit designs and model the interactions of intracellularly coupled oscillators. We classify these oscillators on the basis of coupling strength: independent, weakly or strongly, and deeply coupled. We predict a range of fascinating dynamic behaviours to arise in these systems, such as the beat phenomenon, amplitude and frequency modulation, period doubling, higher-period oscillations, chaos, resonance, and synchronization, with the aim of guiding future experimental work in bacterial synthetic biology. Finally, we outline potential applications, including oscillator-based computing that integrates processing and memory functions, offering multistate and nonlinear processing capabilities.

The urgent need for decentralized, rapid, and affordable point-of-care (POC) diagnostic tools has been starkly highlighted by recent global health crises, underscoring their critical role in achieving health equity. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems, repurposed from their revolutionary gene-editing origins, have emerged as a transformative platform technology for molecular diagnostics, offering unparalleled programmability, specificity, and sensitivity for nucleic acid detection. The core of these diagnostics lies in the unique catalytic mechanisms of specific Cas effectors, notably the target-activated, non-specific trans-cleavage activity of Cas12 and Cas13 proteins. This collateral cleavage of reporter molecules provides a powerful intrinsic mechanism for signal amplification, enabling sensitive detection. This review critically examines the rapid evolution of CRISPR-based diagnostics, from their fundamental molecular principles to their real-world applications. Key applications in infectious disease surveillance and management are summarized, including the rapid development of assays for SARS-CoV-2, HIV, and tuberculosis, with a particular focus on adaptations for field-deployable formats such as lateral flow assays. As the technology continues to mature, overcoming remaining hurdles in sample preparation, multiplexing, and reagent stability will be paramount. Ultimately, CRISPR-based diagnostics are poised to become an indispensable and democratizing component of the global health toolkit, empowering frontline healthcare workers and transforming disease control on a global scale.

Acidocalcisomes are evolutionarily conserved acidic organelles that are rich in cations and inorganic phosphate, primarily polyphosphates. In kinetoplastid parasites, acidocalcisomes and their polyphosphate content are essential for osmoregulation and environmental adaptation during host switching. In this organelle, polyphosphate is synthesised and transported to the lumen by the vacuolar transporter chaperone (VTC) complex. Interestingly, unlike yeast VTC, which has five components, only two have been observed in kinetoplastids: Vtc1, which contains only a transmembrane domain and Vtc4, which, in addition to a transmembrane domain, also consists of SPX and catalytic domains. In this study, we used proximity-dependent biotinylation (BioID) in Leishmania tarentolae to identify proteins located close to the VTC complex. The complex was found near several known acidocalcisomal proteins, including membrane-bound pyrophosphatase (mPPase), vacuolar-type H⁺-ATPase (V-H + -ATPase), Ca²⁺-transporting P-type ATPase (Ca 2+ -ATPase), zinc transporter (ZnT), and palmitoyl acyltransferase 2 (PAT2). Importantly, this approach revealed three novel VTC binding partners (VBPs) that colocalise and interact with the complex in acidocalcisomes, as confirmed by confocal microscopy, pulldown assays, and AlphaFold 3 structural predictions. Together, our results expand the acidocalcisome interactome and suggest that the newly identified VBPs may contribute to the structural organisation and regulatory function of the VTC complex in phosphate homeostasis of kinetoplastid parasites. Author summary Protozoan parasites such as Leishmania and Trypanosoma cause serious diseases affecting millions of people worldwide. To better understand how these parasites survive environmental changes during transmission between hosts, we studied a specialised organelle called the acidocalcisome, which stores polyphosphates and helps regulate stress responses. In this work, we used the non-pathogenic Leishmania tarentolae as a safe and cost-effective model that shares key cellular features with disease-causing species. Using a combination of CRISPR-Cas9 genome editing, proximity-based labelling (BioID), confocal microscopy, pulldown assays and AlphaFold 3 structure prediction, we investigated the vacuolar transporter chaperone (VTC) complex, which synthesises and transports polyphosphate into the acidocalcisome lumen. Proximity proteomics identified several known proteins located near the VTC complex, and importantly, led us to discover three novel proteins that interact with it. These findings open new directions for exploring the organisation and regulation of the VTC complex in protozoan parasites. By revealing novel protein interactions, our study contributes to a deeper understanding of parasite biology and may help identify therapeutic targets for treating neglected tropical diseases.

MicroRNAs are small non-coding RNAs that mediate post-transcriptional silencing of most mammalian genes. They are generated in a multi-step process initiated by the Microprocessor, a protein complex composed of DROSHA and DGCR8. Recent studies have described the phenomenon of “cluster assistance”, in which a prototypic primary miRNA hairpin can license the Microprocessor-mediated processing of a clustered suboptimal hairpin in cis . Genetic screening and mechanistic analyses led to the identification of two critical factors for this process, SAFB2 (scaffold attachment factor B2) and ERH (enhancer of rudimentary homolog), which have been shown to associate with the N-termini of DROSHA and DGCR8, respectively, but also form a complex with each other. However, it remains unclear how SAFB2 and ERH can alter the Microprocessor substrate specificity, and whether the described protein-protein interactions are required for cluster assistance. In this study, we focused on the role of ERH and show that its loss largely phenocopies the effect of SAFB1/2 deletion on the miRNA transcriptome, suggesting that both factors are involved in the same processes of primary miRNA biogenesis. In this context, our data demonstrate that both SAFB1/2 and ERH are required for efficient Microprocessor feedback regulation via processing of pri-miR-1306, uncovering a clear physiological function of cluster assistance. Mechanistically, our data show that ERH-mediated cluster assistance depends neither on its direct association with SAFB2 nor on its described interaction with DGCR8. In contrast, disrupting the ERH binding site within DGCR8 drives the processing of a subset of cluster assistance-unrelated pri-miRNAs. Thus, this study reveals dual roles of ERH in primary miRNA biogenesis, a largely suppressive one driven by its direct binding to DGCR8, and the other in cluster assistance that does not require DGCR8 binding. Highlights Loss of SAFB1/2 and ERH, respectively, induces overlapping, but not identical defects in primary miRNA biogenesis. Both SAFB2 and ERH are involved in Microprocessor feedback regulation. ERH-mediated cluster assistance functions independent of its binding to the DGCR8 N-terminus. Binding of ERH to the DGCR8 N-terminus confers a largely inhibitory function for primary miRNA biogenesis

SUMMARY AMPK (5′-AMP-activated protein kinase) is an energetic sensor for metabolic regulation and integration. Here, we employed CRISPR/Cas9 to generate non-activatable Ampkα knock-in (KI) mice with mutation of threonine 172 phosphorylation site to alanine, circumventing the limitations of previous genetic interventions that disrupt the protein stoichiometry. KI mice of Ampkα2, but not Ampkα1, demonstrated phenotypic changes with increased fat-to-lean mass, impaired endurance exercise capacity, and diminished mitochondrial maximal respiration and conductance in skeletal muscle. Integrated temporal multi-omic analysis (proteomics/phosphoproteomics/metabolomics) in skeletal muscle at rest and during exercise establishes a pleiotropic yet imperative role of Ampkα2 T172 activation for glycolytic and oxidative metabolism, mitochondrial respiration, and contractile function. Importantly, there is a significant overlap of skeletal muscle proteomic changes in Ampkα2 T172A KI mice with that of type 2 diabetic patients. Our findings suggest that Ampkα2 T172 activation is critical for exercise performance and energy transduction in skeletal muscle and may serve as a therapeutic target for type 2 diabetes.

Abstract Genomic imprinting secures parent-specific gene expression through differential DNA methylation at imprinted control regions (ICRs). However, how unmethylated imprinted alleles resist de novo methylation remains unclear. Using an allelic methylation reporter at the Dlk1-Dio3 ICR and a genome-wide loss-of-function screen, we identify that the zinc finger protein GZF1 specifically binds the unmethylated maternal ICR and protects it from de novo methylation in mouse embryonic stem cells, early development, and oocytes. We further show that this protection occurs independently of Tet-mediated demethylation. A regulatory element containing GZF1 and ZFP57 motifs mediates mutually exclusive, methylation-dependent binding. Loss of either factor causes reciprocal imprinting failure: Gzf1 loss induces maternal allele methylation, H3K4me3 depletion, and silencing of maternal transcripts, whereas Zfp57 loss erases paternal methylation, causing locus maternalization. Gzf1-null mice exhibit postnatal growth retardation, contextualizing cis-deletions of this region and illustrating how imprinting phenotypes may differ when studied through epigenetic perturbation versus genetic deletion. Together, our findings define a reciprocal mechanism by which sequence- and methylation-sensitive factors actively memorize parental epigenetic asymmetry through opposing waves of developmental reprogramming.