Background Calcium ions (Ca²⁺) serve as universal intracellular messengers regulating diverse physiological processes, while dysregulated Ca²⁺ homeostasis triggers cytotoxicity. Molecular hydrogen (H₂) exhibits protective effects against oxidative stress-related pathologies, but its mechanism of action remains incompletely understood. Transient receptor potential canonical 4 (TRPC4) channels and their associated protein TRPC4AP are critical mediators of Ca²⁺ influx ( [Ca²⁺]i), yet their role in H₂-mediated calcium signaling is unexplored. This study investigates the molecular mechanism by which H₂ modulates Ca²⁺ dynamics through the TRPC4-TRPC4AP axis, aiming to establish its therapeutic potential for calcium-related disorders. Methods The study employed heterogeneous cellular models (e.g., mesenchymal stem cells, neurons, fibroblasts) and in vivo two-photon calcium imaging in C57BL/6J mice. Techniques included CRISPR-Cas9 knockout, siRNA-mediated gene silencing, molecular docking (AlphaFold 3), and protein-protein interaction analysis. Calcium flux was quantified via fluorescence imaging, while mitochondrial integrity and cytoskeletal dynamics were assessed using JC-1 staining, ATPase activity assays, and live-cell imaging. Structural validation of TRPC4-TRPC4AP binding sites utilized mutagenesis and complementation experiments. Results H₂ selectively enhanced extracellular Ca²⁺ influx via TRPC4-TRPC4AP, with no cytotoxicity or mitochondrial dysfunction observed. Key arginine residues (730Arg-731Arg) in the TRPC4 CIRB domain formed hydrogen-bond networks essential for channel activation. In vivo, H₂ increased neuronal Ca²⁺ transient frequency and amplitude in the primary motor cortex. TRPC4AP knockout abolished H₂-induced Ca²⁺ influx, while mutagenesis of 730Arg/731Arg disrupted channel activity. H₂ also promoted cytoskeletal remodeling and cell motility, dependent on TRPC4AP-mediated Ca²⁺ signaling. Conclusions This study identifies H₂ as a novel calcium agonist that activates the TRPC4-TRPC4AP axis to regulate extracellular Ca²⁺ influx. The 730Arg-731Arg motif in TRPC4 serves as a critical H₂-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic basis for H₂-based therapies targeting calcium dysregulation in neurodegenerative, inflammatory, and metabolic diseases, while highlighting TRPC4AP as a pivotal molecular switch for gasotransmitter signaling.

ABSTRACT Streptomyces secondary metabolites account for over half of all clinically used antibiotics, as well as numerous antifungal agents, anticancer compounds, and immunosuppressants. Two-component systems, which are widespread in bacteria, are key regulators of antibiotic production in Streptomyces species, yet their activating signals remain poorly understood. CutRS was the first two-component system identified in the genus Streptomyces and deletion of cutRS in Streptomyces coelicolor was shown to enhance antibiotic production, although its CutR regulon does not include any biosynthetic genes. Here, we used Streptomyces venezuelae to further investigate CutRS function. We show that deletion of cutRS leads to an increase in growth rate and a reversal of the typical glucose-mediated carbon catabolite repression typically observed in Streptomyces species. We also demonstrate that CutR DNA binding is glucose-dependent, but CutR does not directly regulate genes involved in growth, antibiotic biosynthesis, or glucose metabolism. The only CutR targets conserved in both S. coelicolor and S. venezuelae are the foldase genes htrA3 and htrB , which are involved in the protein secretion stress response. Consistent with this, we show that CutS homologues all contain two conserved cysteine residues in their extracellular sensor domains and that changing these residues to serine constitutively activates S. venezuelae CutRS. We propose that failure of a disulfide bond to form between these cysteine residues indicates secretion stress and leads to activation of the CutRS system and the secretion stress response. IMPORTANCE Streptomyces bacteria are the primary source of clinically useful antibiotics. While many two-component systems have been linked to antibiotic biosynthesis in Streptomyces species, few have been well characterized. Here, we characterize a secretion stress sensing two-component system called CutRS and propose a model for how the sensor kinase detects extracellular protein misfolding via two highly conserved cysteine residues. Importantly, we also show that deletion of cutRS triggers antibiotic overproduction in the presence of glucose. Since glucose normally represses antibiotic biosynthesis in Streptomyces species through carbon catabolite repression, this finding reveals a simple genetic route to bypass this barrier. This has significant implications for antibiotic discovery pipelines and industrial production, where glucose-rich media are preferred for cost and scalability. Our results position CutRS as a key target for future strain improvement strategies.

Targeted gene editing can be achieved using CRISPR/Cas9-assisted recombineering. However, high-efficiency editing requires careful optimization for each locus to be modified which can be tedious and time-consuming. In this work, we developed a simple, fast and cheap method for the E diting and A ssembly of SY nthetic operons using CRISPR/Cas9-assisted recombineering (EASY-CRISPR) in Escherichia coli . Highly efficient editing of the different constitutive elements of the operons can be achieved by using a set of optimized guide RNAs and single- or double-stranded DNA repair templates carrying relatively short homology arms. This facilitates the construction of multiple genetic tools, including mutant libraries or reporter genes. EASY-CRISPR is also highly modular, as we provide alternative and complementary versions of the operon inserted in three loci which can be edited iteratively and easily combined. As a proof of concept, we report the construction of several fusions with reporter genes confirming known post-transcriptional regulation mechanisms and the construction of saturated and unbiased mutant libraries. In summary, the EASY-CRISPR system provides a flexible genomic expression platform that can be used both for the understanding of biological processes and as a tool for bioengineering applications.

Abstract Saffold virus (SAFV), a member of the species Cardiovirus saffoldi within the Picornaviridae family, causes acute respiratory and gastrointestinal illnesses, as well as hand, foot, and mouth diseases. It is also suspected to be associated with neuronal disorders such as encephalitis and meningitis in severe cases. Despite its clinical significance, the virus-host interactions underlying SAFV pathogenicity remain largely unknown. Using a genome-wide CRISPR-Cas9 knockout screen, we identified receptors for SAFV infection: sulfated glycosaminoglycans (GAGs) and integrin aVb8. Single knockouts of SLC35B2 , an essential gene for sulfated GAG synthesis, or the integrin genes, ITGAV or ITGB8 partially reduced SAFV-3 susceptibility in HeLa cells, and double knockout conferred complete resistance. Furthermore, we demonstrated that SAFV-3 virions bind directly to sulfated GAGs and integrin aVb8. Based on these findings, we propose a model of SAFV infection, in which sulfated GAGs and integrin aVb8 function in parallel pathways during viral entry.

Abstract Duchenne muscular dystrophy (DMD) is a severe X-linked disorder caused by mutations in the DMD gene, with a global prevalence of 3.6 per 100,000 people. Despite its well-documented genetic basis, no previous studies have characterised DMD in Guatemala. We analysed 33 genetically confirmed cases to estimate prevalence, describe the mutation spectrum, and assess clinical features. Prevalence was 0.61 per 100,000 men under 30. Symptoms began before age 5 in 85% of cases, yet 60% were diagnosed after age 6, highlighting significant diagnostic delays. Deletions were the most common mutation (55%), followed by point mutations (30%) and duplications (15%), with two novel variants identified. Most deletions clustered in the exon 45–55 hotspot. Nearly half of the cases were eligible for exon-skipping therapies. These findings reveal genetic heterogeneity in the Guatemalan population, substantial delays in diagnosis, and the need for improved access to genetic testing, targeted treatments, and a national DMD registry.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels and the main mediators of synaptic neurotransmission in the insect brain. In insects, nAChRs are pivotal for sensory processing, cognition and motor control, and are the primary target of neonicotinoid insecticides. Neonicotinoids are potent neurotoxins and pollinators such as honey bees are more sensitive and affected by extremely low sub-lethal doses. The pentameric nAChR channel is made up either of five α-subunits constituting five ligand-binding sites or a mixture of two to three α and β subunits constitute two to three ligand-binding sites. Of particular note, the honey bee nAChRα8 subunit is converted into a β subunit (nAChRβ2) in Drosophila , raising the question whether this α to β conversion makes flies less sensitive to neonicotinoids. To investigate species-specific aspects of neonicotinoid toxicity we CRISPR-Cas9 engineered a cross-species chimeric nAChR subunit by swapping the ligand-binding domain in Drosophila of nAChRβ2 with honey bee nAChRα8. Toxicity assessment by neonicotinoid thiamethoxam revealed significantly impaired motor functions in climbing and flight assays when comparing the α8/β2 chimeric channel to wild type or a β2 knock-out. However, both the α8/β2 chimeric channel and the β2 knock-out showed the same increased survival after neonicotinoid exposure compared to wild type flies. Combinatorial exposure to neonicotinoids also did not reveal differences. These findings highlight the critical role of nAChR subunit composition in motor control and demonstrate how subtle structural differences can profoundly impact motor function and pesticide response, offering new insights into the molecular mechanisms of neurotoxicity across species.

Reactive oxygen species (ROS) pose a significant threat to biological molecules, prompting organisms to develop systems that buffer oxidative stress and contain iron, which otherwise amplifies ROS production. Understanding oxidative stress responses requires identifying the key proteins involved and their cellular organization. Here, we combined proteomics and cryo-EM to investigate the response of the anaerobic hyperthermophilic archaeon Pyrococcus furiosus to oxygen exposure. Proteome analysis revealed a significant upregulation of the oxidoreductase Rubrerythrin (Rbr) under oxidative stress. Cryo-electron tomograms showed the formation of prominent oxidative stress-induced tubules (OSITs). Single-particle cryo-EM and mass spectrometry of enriched OSITs identified them as stacked rings of Rbr homotetramers. The 3.3 Å structure demonstrates that rubredoxin-like domains mediate homotetramer assembly, suggesting that their oxidation drives OSIT formation. Within OSITs, we discovered virus-like particles formed by a ferritin-like/encapsulin fusion protein with iron hydroxide cores, uncovering a sophisticated organelle that protects P. furiosus from ROS through advanced compartmentalization.

SUMMARY Natural killer (NK) cells, a type of potent cytotoxic lymphocytes, are particularly promising for the treatment of cancers that lose or downregulate major histocompatibility complex class I (MHC-I) expression to evade T cell-mediated immunotherapy. However, the hostile and immune suppressive tumor microenvironment (TME) greatly hinders the function of tumor-infiltrating NK cells limiting the therapeutic efficacy against solid tumors. Here, we show that a fusion protein of interleukin-21 (IL-21−Fc), as a direct in vivo intervention, can safely and effectively reprogram NK cell metabolism and enhance their effector function in the TME. Our research demonstrates that combining IL-21−Fc with IL-15 superagonist (IL-15SA) or NK cell transfer leads to the eradication of MHC-I-deficient tumors and confers durable protection in syngeneic and xenograft tumor models. Mechanistically, we uncover that IL-21−Fc enhances NK cell effector function by upregulating glycolysis in a lactate dehydrogenase A (LDHA)-dependent manner. These findings not only underscore the considerable potential of IL-21−Fc as an in vivo therapeutic intervention to bolster NK cell-based immunotherapy, but also unveil an innovative strategy of metabolic reprogramming for NK cell rejuvenation within tumors. GRAPHICAL ABSTRACT HIGHLIGHTS NK cells display functional exhaustion in MHC-I deficient solid tumors IL-21−Fc as an in vivo-applicable and safe immunotherapy reinvigorates intratumoral NK cells for enhanced effector function IL-21−Fc enhances NK cell function by elevating glycolysis in a LDHA-dependent manner Combining IL-21−Fc with low-dose IL-15SA or NK cell transfer eradicates syngeneic and xenografted solid tumors

Abstract RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ, a recently identified miniature type V effector encoded in phage genomes, has emerged as a promising candidate for genome editing due to its nuclease activity in mammalian and plant cells. However, the detailed molecular mechanisms of Casλ family of enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. The structures revealed that, unlike other type V CRISPR-Cas effectors, the REC2 domain directly interacts with the substrate DNA within the RuvC active site to facilitate the target DNA cleavage. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.

Stone fruits (Prunus spp.) occupy a pivotal position in global fruit production due to their significant nutritional profile and distinctive organoleptic characteristics. Contemporary orchard systems are undergoing transformation through innovative cultivation approaches, notably high-density dwarfing systems, greenhouse cultivation, agri-tech integration, and simplified management. As a crucial agronomic component in modern stone fruit cultivation, rootstock systems confer multi-benefits including enhanced environmental resilience, improved scion productivity, superior fruit quality, controlled vigor and dwarfing capacity. While the majority of european apple orchards have transitioned to dwarfing rootstock systems, achieving substantial gains in productivity and profitability, stone fruits cultivation lag significantly due to the key gaps in prunus rootstock development include genetic complexity, extended evaluation cycles, clonal propagation barriers and limited research programs. Urgent innovation is required to address these challenges in rootstock breeding to meet the demand of sustainable stone fruits production. This review systematically examines strategic breeding objectives and innovative molecular methodologies in Prunus rootstock development, with particular emphasis on marker-assisted selection and genomic prediction technologies. We provide a comprehensive synthesis of breeding achievements across major commercial rootstock cultivars, while proposing forward-looking research strategies incorporating CRISPR-based genome editing and multi-omics approaches. The synthesized insights establish a theoretical pathway for advancing rootstock genetic improvement and sustainable orchard management practices in stone fruit cultivation systems.

Sotorasib (AMG510) and adagrasib (MRTX849) have shown significant efficacy in KRAS G12C mutant NSCLC, but acquired resistance occurs within 6–12 months. While some resistance arises from new mutations, over half of the resistant cases lack identifiable genomic alterations. We hypothesize that resistance is driven by signaling network rewiring, creating new therapeutic vulnerabilities. To investigate acquired resistance (AR) mechanisms, multiple AR models, including cell lines (H23AR & H358AR), PDXs (TC303AR & TC314AR), CDXs (H358AR CDX), and PDXOs (PDXO303AR & PDXO314AR) were developed. H23AR and H358AR cells displayed >600-fold and 200-fold and PDXO303AR and PDXO314AR, exhibited >300-fold and >100-fold resistance to sotorasib, respectively compared to their parental counterparts, however, no additional mutations in KRAS or other potential genetic alterations were identified. The AR cells and PDXOs also showed comparable resistance to adagrasib. Proteomic and phosphoproteomic analyses in TC303AR & TC314AR PDXs identified distinct protein signatures associated with KRAS reactivation, mTORC1 signaling upregulation, and PI3K/AKT/mTOR pathway activation. PI3K protein levels were significantly elevated in AR PDXs, H23AR, and H358AR cells. Pharmacological inhibition of PI3K with copanlisib or genetic knockout via CRISPR-Cas9 restored sotorasib sensitivity, suppressed colony formation, and inhibited downstream effectors, including p-AKT, p-mTOR, p-S6, p70S6K, p-GSK3β, and p-PRAS40 in AR cells. copanlisib also sensitized both acquired and primary resistant PDXOs and synergized with sotorasib in restoring drug sensitivity. We found that p4E-BP1 was significantly upregulated in H23AR and H358AR cells, and copanlisib suppressed its expression. The level of p4E-BP1 expression was correlated with Sotorasib sensitivity in PI3K knockout clones, where the most sensitive clone displayed reduced or no p4E-BP1 expression. CRISPR-Cas9-mediated knockout of 4E-BP1, either alone or in combination with PI3K knockout, dramatically restored sotorasib sensitivity to levels comparable to parental cells. Suppression of 4E-BP1 hyperphosphorylation required dual inhibition of mTORC1 and mTORC2, and treatment with AZD8055 or sapanisertib (mTORC1/2 dual inhibitors) significantly dephosphorylated 4E-BP1 and restored sotorasib sensitivity in resistant cells and PDXOs. In contrast, everolimus (a mTORC1-selective inhibitor) did not restore sotorasib sensitivity. In PDX, CDX, and xenograft models in vivo, the combination of sotorasib with either copanlisib or sapanisertib resulted in robust, synergistic, and durable tumor regression at well-tolerated doses. These findings showed the critical role of PI3K/mTOR signaling as a bypass mechanism of resistance to KRAS G12C inhibitors. We conclude that mTORC1/2 mediated inhibition of p4E-BP1 and combination strategies targeting this pathway effectively overcomes acquired resistance to KRAS G12C inhibitors in NSCLC.

Accurate diagnostics are essential for disease control and elimination efforts. However, access to diagnostics for neglected tropical diseases (NTDs) is hindered by limited healthcare infrastructure in many NTD-endemic regions, as well as by reliance on time- and labor-intensive diagnostic methods, such as smear microscopy. New diagnostic tools that are portable, rapid, low-cost, and meet World Health Organization (WHO) sensitivity and specificity targets are urgently needed to accelerate NTD control and elimination programs. Here, we introduce the NTDscope, a portable microscopy platform that enables point-of-care imaging and automated detection of parasites and other pathogens in patient samples. The NTDscope builds on and extends the capabilities of the LoaScope, a device that turned the camera of a mobile phone into a microscope and used on-board image processing to automatically quantify Loa loa microfilariae burden in whole blood samples. The NTDscope replaces the mobile phone of the LoaScope with a system-on-module (SOM) that enables the integration of multiple imaging modalities in a single package designed to improve robustness and expand applications. In this work, we demonstrate use of the NTDscope as a portable brightfield, darkfield, and fluorescence microscope for samples including microfilariae and helminth eggs. We also show that the device can be used to quantify molecular assays, such as a lateral flow test and a CRISPR-Cas13a-based assay. The ability to combine diagnostic capabilities of conventional microscopy with molecular assays and machine learning in a single device could expand access to diagnostics for populations in NTD-endemic areas and beyond. Author summary Neglected tropical diseases (NTDs) impact one billion of the world’s most vulnerable individuals. Diagnostics are a necessary part of NTD disease control and elimination efforts, but identifying infected individuals remains a challenge. Here we present the NTDscope, a portable multi-contrast microscope designed to diagnose multiple NTDs at the point-of-care. We show that the NTDscope can be used to detect the parasitic worm Loa loa in videos of whole blood samples and parasitic eggs in images of urine and stool samples. The NTDscope can also be used to image thick blood smears in disposable capillaries and serves as a lateral flow assay reader. In addition to brightfield and darkfield imaging, fluorescence imaging on the device enables molecular assays based on CRISPR-Cas enzymes. This portable (<1 kg), field-friendly device—tested in Cameroon, Gabon, Côte d’Ivoire, and Bangladesh—has the potential to become a platform technology that addresses diagnostic needs for multiple NTDs and could serve as a key element of decentralized healthcare in the future.

Autophagy is a vital cellular quality control pathway that enables plants to adapt to changing environments. By degrading damaged or unwanted components, autophagy maintains cellular homeostasis. While the organismal phenotypes of autophagy-deficient plants under stress are well-characterized, the contribution of cell-type-specific autophagy responses to whole-plant homeostasis remains poorly understood. Here, we show that root hair-forming cells (trichoblasts) of Arabidopsis thaliana exhibit higher autophagic flux than adjacent non-hair cells (atrichoblasts). This differential autophagy is genetically linked to cell fate determination during early development. Mutants disrupting trichoblast or atrichoblast identity lose the autophagy distinction between these cell types. Functional analyses reveal that elevated autophagy in trichoblasts is essential for sodium ion sequestration in vacuoles—a key mechanism for salt stress tolerance. Disrupting autophagy specifically in trichoblasts impairs sodium accumulation and reduces plant survival under salt stress. Conversely, cell-type-specific complementation restores both sodium sequestration and stress tolerance. Our findings uncover a cell-type-specific autophagy program in root hairs and demonstrate how developmental cues shape autophagy to enhance stress resilience. This work establishes a direct link between cell identity, autophagy, and environmental adaptation in plants.

Abstract Introduction: Thyroid cancer, exhibits distinct histopathological and molecular profiles that dictate clinical behavior. Advances in next-generation sequencing have elucidated subtype-specific genomic and transcriptomic alterations, enabling the classification of papillary (PTC), follicular (FTC), medullary (MTC), and anaplastic thyroid carcinoma (ATC). Despite progress, a significant gap remains in systematically integrating transcriptomic signatures with clinically actionable outcomes across all subtypes, particularly in resolving intra-tumoral heterogeneity and linking molecular profiles to therapeutic responses. Objective : To harness AI-driven clustering to identify subtype-specific transcriptomic signatures using large-scale datasets, such as The Cancer Genome Atlas (TCGA). Method : Transcriptomic datasets from TCGA thyroid cancer cohort (PTC, FTC, MTC, ATC) were preprocessed. scRNA-seq data were integrated (Seurat, DoubletFinder, Harmony) for single-cell resolution. Unsupervised clustering identified molecular subtypes and DEGs (Wilcoxon rank-sum, false discovery rate). Machine learning (ML) models predicted outcomes (10-fold cross-validation, AUC-ROC). Clinical integration (Cox models, Kaplan-Meier) and validation (GEO, CRISPR, immunohistochemistry) confirmed signatures. Reproducible pipelines (GitHub) ensured consistency. Results : Transcriptomic datasets from TCGA thyroid cancer cohort (500 samples) were preprocessed (Q30 > 90%, alignment > 85%, DESeq2, ComBat). scRNA-seq integration (25,000 cells) identified 12 cell types, with ATC showing immunosuppressive myeloid cells (p < 0.001). Unsupervised clustering revealed four molecular subtypes and 1,250 DEGs (BRAF, RET, TP53, PTEN). ML models (random forest, SVM) achieved high accuracy (AUC-ROC: 0.92, 0.89), identifying a 50-gene signature. Clinical integration linked high-risk subtypes to poor survival (HR: 2.5, p < 0.001). Validation (GEO, CRISPR, IHC) confirmed signature robustness (AUC-ROC: 0.89–0.93). Reproducible pipelines were shared via GitHub. Conclusion : This study identified robust transcriptomic signatures and subtype-specific ecosystems in thyroid cancer, validated through computational clustering, ML, and functional assays. Thus, this study advances in precision oncology by linking molecular profiles to clinical outcomes, supported by reproducible pipelines and high-performance computing.

Compensatory endocytosis is crucial for the presynapse to maintain a functional pool of fusion-competent vesicles. Slow Clathrin-mediated endocytosis has been widely regarded as the primary mechanism of retrieval but this has been challenged by competing Clathrin-independent endocytic models, most notably sub-second ultra-fast endocytosis, reported to be predominant at physiological temperature. Here, we sought to resolve the salience of the respective endocytic modes by using a purely presynaptic preparation, the Xenapse, amenable to total internal reflection fluorescence microscopy (TIRFM). While the role of Clathrin is in dispute, Dynamin is widely acknowledged to figure as the scission protein at the invaginated vesicle neck. Hence, we labelled the endogenous Dynamin I with EGFP by CRISPR-Cas9 techniques and visualized single synaptic Dynamin-mediated scission events at very high temporal resolution. This revealed only a single slow mode of Dynamin-dependent retrieval with a half time of ∼ 9 seconds at physiological temperature. Cross-correlational analysis with fluorescently labelled Clathrin confirmed these Dynamin events to be Clathrin-dependent. We thereby affirm Clathrin-mediated endocytosis as the primary mode of compensatory retrieval.

Glioblastoma (GBM) poses a formidable challenge to patients for several reasons. Given its grim prognosis, understanding the various mechanisms GBM tumors utilize to resist therapy is essential to improve patient outcomes. Using PubMed, this focused review identifies and characterizes five critical elements of GBM tumors that contribute to their resistance to treatment: DNA repair enzymes, temozolomide (TMZ) and radiation mechanisms, anti-apoptosis mechanisms, GBM tumor heterogeneity and its effects on the cell cycle. This review explores various challenges associated with GBM tumors, such as their resistance against standard treatments such as TMZ and radiation therapy (RT). We explore the importance of epigenetic reprogramming, genetic mutations critical for cell proliferation and tumor suppression, and the role of mismatch repair (MMR) processes that influence RT and immune response interplay as contributors to GBM resistance. In addition, this review highlights vital DNA repair enzymes such as O6-methylguanine-DNA methyltransferase (MGMT) and Alkylpurine-DNA N-Glycosylase (APNG), which repair DNA damage introduced by alkylating agents such as TMZ. The involvement of the NuRD complex, particularly CHD4, in regulating access to DNA repair enzymes. Recent advancements in understanding the transcriptional regulation of MGMT through NF-κB activity are examined. Further, we explore novel approaches, including using anticancer neural stem cells and targeting hexokinase 2 (HK2) with antifungal drugs. Examining critical elements of the GBM cell cycle, such as the role of CDK's, cyclin(s) and proliferation markers such as ki67, can also give us a foundation for identifying possible target proteins and kinases for cancer drugs. While targeting DNA repair enzymes, proteins, and regulatory elements shows promise in enhancing GBM treatment efficacy, we acknowledge the challenges, including potential side effects and the risk of secondary cancers. Future research should focus on leveraging personalized medicine approaches and emerging biotechnologies, such as CRISPR gene editing, to develop targeted therapies that can overcome resistance mechanisms of GBM and improve patient outcomes.

Summary: This essay examines CRISPR-Cas technology, highlighting its potential and limitations in gene editing. While CRISPR enables precise DNA modifications for treating genetic diseases, its in vivo application faces major hurdles: low editing efficiency, delivery challenges, and off-target effects. Homology-directed repair (HDR) is inefficient, delivery methods are complex, and unintended mutations pose risks. A quantum-like genetic computation hypothesis suggests that CRISPR functions within a non-linear, probabilistic framework, challenging conventional methodologies. Ethical concerns include safety, consent, and legal regulation. The essay argues for a new quantum-informed research approach, integrating holistic, non-invasive methods like holistic medicine and nutrition to balance genetic interventions with natural biological processes.

A bstract Background Medulloblastoma is the most common malignant brain tumor of childhood. The highest-risk tumors are driven by recurrent Myc amplifications (Myc-MB) and experience poorer outcomes despite intensive multimodal therapy. The Myc transcription factor defines core regulatory circuitry for these tumors and acts to broadly amplify downstream pro-survival transcriptional programs. Therapeutic targeting of Myc directly has proven elusive, but inhibiting transcriptional cofactors may present an indirect means of drugging the oncogenic transcriptional circuitry sustaining Myc-MB. Methods Independent CRISPR-Cas9 screens were pooled to identify conserved dependencies in Myc-MB. We performed chromatin conformation capture (Hi-C) from primary patient Myc-MB samples to map enhancer-promoter interactions. We then treated in vitro and xenograft models with CDK9/7 inhibitors to evaluate effect on Myc-driven programs and tumor growth. Results Eight CRISPR-Cas9 screens performed across three independent labs identify CDK9 as a conserved dependency in Myc-MB. Myc-MB cells are susceptible to CDK9 inhibition, which is synergistic with concurrent inhibition of CDK7. Inhibition of transcriptional CDKs disrupts enhancer-promoter activity in Myc-MB and downregulates Myc-driven transcriptional programs, exerting potent anti-tumor effect. Conclusions Our findings identify CDK9 inhibition as a translationally promising strategy for the treatment of Myc-MB. K ey P oints CDK9 is an intrinsic dependency in Myc-driven medulloblastoma Dual CDK9/7 inhibition disrupts Myc-driven transcriptional circuitry CDK9 inhibitors should be developed as pharmaceutical agents for Myc-MB I mportance of the S tudy Medulloblastoma is the most common malignant brain tumor of childhood, and outcomes for high-risk subgroups remain unsatisfactory despite intensive multimodal therapy. In this study, we pool multiple independent CRISPR-Cas9 screens to identify transcriptional cofactors such as CDK9 as conserved dependencies in Myc-MB. Using Hi-C from primary patient samples, we map Myc enhancer-promoter interactions and show that they can be disrupted using inhibition of transcriptional CDKs. CDK9 inhibitor treatment depletes Myc-driven transcriptional programs, leading to potent anti-tumor effect in vitro and prolongation of xenograft survival in vivo . With a large number of CDK9 inhibitory compounds now in clinical development, this study highlights the opportunity for clinical translation of these for children diagnosed with Myc-MB.

Oncolytic virus (OV) therapy is a promising treatment for various tumors. However, in pancreatic ductal adenocarcinoma (PDAC), the high abundance of cancer-associated fibroblasts (CAFs) can limit OV therapy efficacy by impairing viral spread and anti-tumor immunity. We have previously shown that oncolytic reovirus infection of CAFs depends on expression of the reovirus entry receptor Junctional Adhesion Molecule A (JAM-A), which is not or lowly expressed in most PDAC CAFs. We propose that increasing JAM-A expression on CAFs will boost viral spread in a tumor. However, there are currently no known regulators of JAM-A expression. Therefore, we performed a genome-wide CRISPR/Cas9 knock-out screen to identify regulators of JAM-A expression. Ablation of the top negative regulator, Zinc Finger E-Box binding Homeobox 1 ( ZEB1 ), in pancreatic fibroblasts led to strong JAM-A upregulation. We show that ZEB1 directly regulates JAM-A expression by binding to the E-box regions located within the JAM-A promotor. Importantly, ZEB1 ablation increased the sensitivity of fibroblasts to reovirus infection and subsequent cell death. Our work provides a novel overview of genes regulating JAM-A expression and provides a rational approach of combining ZEB1 inhibition with reovirus therapy to target both CAFs and tumor cells in stroma-rich tumors such as PDAC.

Over the last few centuries, advancements in plant breeding have revolutionized agriculture, driving significant increases in global food production. Polyploidy, the increase in chromosome copies, can positively affect plant performance and is assumed to have played a critical role in the domestication of crop plants. Polyploidy is thought to be primarily caused by sperm that, due to meiotic aberrations, deliver unreduced chromosome sets. We have recently identified an alternative pathway to polyploidization by demonstrating that polyspermy, the fertilization of an egg cell by more than one sperm, occurs in planta and results in viable triploid plants. Capitalizing on a novel high-throughput polyspermy detection tool, we have shown that polyspermy involving two pollen donors can generate plants with three parents, one mother and two fathers. This 3PaTec technology not only speeds up breeding processes through an instant combination of beneficial traits from three parents; it also allows selective polyploidization of the egg cell, thereby bypassing the central cell-derived embryo-nourishing endosperm, a major hybridization barrier. Here, we further explore the genetic and developmental factors influencing polyspermy and show that the frequency of polyspermy and triparental plant formation varies among ecotypes and depends on pollen availability, suggesting that polyspermy is an adaptive trait. Additionally, we extend the application of 3PaTec to crops by successfully generating triparental sugar beet in-field using a wind pollination strategy. Our findings highlight the potential of 3PaTec for major crop plants. This innovative breeding technology does not rely on genetic engineering, requiring minimal technical expertise and infrastructure. As a result, it is highly accessible to a wide range of users, contributing to the democratization of plant breeding by empowering individuals from all backgrounds to collaborate and contribute to developing resilient and sustainable crops.

Immunocompetent and experimentally accessible alveolar systems to study human respiratory diseases are lacking. Here, we developed a single donor human induced pluripotent stem cell (iPSC)-derived Lung-on-Chip (iLoC) containing Type II and I alveolar epithelial cells, vascular endothelial cells, and macrophages in a microfluidic device that mimic lung 3D mechanical stretching and air-liquid interface. Imaging and scRNA-seq analysis revealed that the iLoC recapitulated cellular profiles present in the human distal lung. Infection of the iLoC with the human pathogen Mycobacterium tuberculosis (Mtb) showed that both macrophages and epithelial cells were infected and showed limited bacterial replication. Stochastically, large macrophage clusters containing necrotic core-like structure and Mtb replication were observed. A genetically engineered autophagy deficient iLoC revealed that after Mtb infection, macrophage necrosis was higher upon ATG14 deficiency without bacterial replication. Altogether, we report an autologous, genetically tractable human alveolar model to study lung diseases and therapies.

6-Thioguanine (6-TG), an FDA-approved antimetabolite drug, is widely used in the treatment of leukemia. Its cellular effects require metabolic activation and are regulated through interactions with various proteins such as NUDT15, which catalyzes the hydrolysis of the active 6-TG metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP. Recent genome-wide CRISPR loss-of-function studies have identified another NUDIX hydrolase, NUDT5, as a crucial mediator of 6-TG toxicity. Here, we present the development and characterization of potent and selective NUDT5 degraders, guided by a cell-based assay screening strategy. These degraders, in conjunction with orthogonal CRISPR knock-out and reconstitution experiments, reveal a novel and unexpected, non-enzymatic role for NUDT5 in modulating the cellular response to 6-TG. Depletion of NUDT5 protein is antagonistic to NUDT15 inhibition, suggesting a distinct mode-of-action with potential implications for patient therapy.

ABSTRACT Background and Aims Gastrointestinal (GI) enterochromaffin (EC) cells are specialised sensors of luminal stimuli. They secrete most of the body’s serotonin (5-HT), and are critical for modulating GI motility, secretion, and sensation, while also signalling satiety and intestinal discomfort. The aim of this study was to investigate mechanisms underlying the regulation of human EC cells, and the relative importance of direct nutrient stimulation compared with neuronal and paracrine regulation. Methods Intestinal organoids from human duodenal biopsies were modified using CRISPR-Cas9 to specifically label EC cells with either the fluorescent protein Venus or the cAMP sensor Epac1-S. EC cells were purified by fluorescence-activated cell sorting for analysis by bulk RNA sequencing and liquid chromatography mass spectrometry peptidomics. The function of human EC cells was studied using single cell patch clamp, calcium and cAMP imaging and 5-HT ELISA assays. Results Human EC cells showed expression of receptors for nutrients (including GPR142 , GPBAR1, GPR119, FFAR2, OR51E1, OR51E2 ), gut hormones (including SSTR1,2&5 , NPY1R, GIPR ) and neurotransmitters ( ADRA2A , ADRB1 ). Functional assays revealed EC responses (calcium, cAMP and/or secretion) to a range of stimuli, including bacterial metabolites, aromatic amino acids and adrenergic agonists. Electrophysiological recordings showed that isovalerate increased action potential firing. Conclusions 5-HT release from EC cells controls many physiological functions and is currently being targeted to treat disorders of the gut-brain axis. Studying ECs from human organoids enables improved understanding of the molecular mechanisms underlying EC cell activation, which is fundamental for the development of new strategies to target 5-HT-related gut and metabolic disorders. Synopsis Human duodenal organoids expressing fluorescent proteins in enterochromaffin cells were used to study mechanisms underlying serotonin secretion. Different expression of key sensory receptors was identified by transcriptomic analysis, and validated by live cell second messenger imaging and secretion assays.

CRISPR-associated endoribonucleases (Cas RNases) cleave single-stranded RNA in a highly sequence-specific manner, by recognizing and binding to short RNA sequences known as direct repeats (DRs). Here we investigate the potential of exploiting Cas RNases for the regulation of target genes with one or more DRs introduced into the 3’ untranslated region, an approach we refer to as DREDGE ( d irect r epeat- e nabled d own-regulation of g ene e xpression). The DNase-dead version of Cas12a (dCas12a) was identified as the most efficient among 5 different Cas RNases tested and was subsequently evaluated in doxycycline-regulatable systems targeting either stably expressed fluorescent proteins or an endogenous gene. DREDGE performed superbly in stable cell lines, resulting in up to 90% downregulation with rapid onset, notably, in a fully reversible manner. Successful control of an endogenous gene with DREDGE was demonstrated in two formats, including one wherein both the DR and the transgene driving expression of dCas12a were introduced in one step by CRISPR-Cas. Our results establish DREDGE as an effective method for regulating gene expression in a targeted, highly selective, and fully reversible manner, with several advantages over existing technologies.

Stem cells are highly resistant to viral infection compared to their differentiated progeny, and this resistance is associated with stem cell-specific restriction factors and intrinsic interferon stimulated genes (ISGs). In HIV infection, proviral DNA has been detected in certain bone marrow hematopoietic stem cells, yet widespread stem cell infection in vivo is restricted. Intriguingly, exposing bone marrow stem cells to HIV in vitro led to viral replication selectively only in the CD34 - population, but not in the CD34 + cells. The mechanism dictating this CD34-based HIV restriction remained a mystery, especially since HIV has a capacity to antagonize restriction factors and ISGs. CD34 is a common marker of hematopoietic stem and progenitor cells. Here, we report the intrinsic antiviral properties of CD34. Expression of CD34 in HIV-1 producer cells results in the loss of progeny virion infectivity. Conversely, removal of CD34 using CRISPR/Cas9 knockout or stem cell differentiation cytokines promotes HIV-1 replication in stem cells. These results suggest that in addition to restriction factors and intrinsic ISGs, CD34 serves as a host innate protection preventing retrovirus replication in stem cells. Mechanistically, CD34 does not block viral entry, integration, and release. Instead, it becomes incorporated onto progeny virions, which inactivates virus infectivity. These findings offer new insights into innate immunity in stem cells, and highlight intriguing retrovirus-host interactions in evolution.