ABSTRACT Approximately 75% of breast cancer cases are estrogen receptor (ER)-positive, with endocrine therapies forming the foundation of treatment. However, therapeutic resistance remains a major clinical challenge, necessitating the identification of new molecular targets. Myosin VI (MVI), a motor protein increasingly linked to cancer, directly interacts with the ER and plays a key role in the spatial regulation of RNA Polymerase II. Notably, expression levels of MVI and ER are positively correlated across breast cancer tissues. Here, we present a multidisciplinary investigation combining advanced imaging, genomic profiling, and phenotypic characterisation to elucidate the interplay between MVI and the ER. We demonstrate that MVI nuclear localisation is dynamically regulated by estrogen signalling and ER activity. Conversely, ER nuclear localisation requires active MVI, suggesting a reciprocal regulatory mechanism. Furthermore, MVI influences subnuclear architecture, modulating ER transcriptional activity and downstream gene expression programmes that govern cell proliferation and migration. Importantly, pharmacological inhibition of MVI enhances the effect of hormone therapy, resulting in greater disruption of ER function than monotherapy alone. Moreover, MVI inhibition also suppresses the activity of hormone-resistant ER mutants, highlighting its potential to overcome therapy resistance. Our findings establish MVI as a critical regulator of ER nuclear dynamics and gene expression, supporting its candidacy as a novel therapeutic target in ER-positive breast cancer.

Plasmodium falciparum , the most virulent human malaria parasite, is transmitted by Anopheles mosquitoes whose community composition varies geographically. Population genomic analyses reveal highly differentiated P. falciparum loci with roles in mosquito infection, which may be driven by adaptation to regional vectors. To test this, we generated transgenic parasites in which reference alleles were replaced with geographically alternative variants at candidate transmission-stage genes. Transmissibility was compared across four mosquito species representing distinct geographic ranges ( An. gambiae , An. stephensi , An. minimus , and An. albimanus ). Two of five tested polymorphisms increased oocyst and sporozoite burdens in sympatric parasite–vector combinations. Both substitutions occurred in ookinete micronemal proteins, CTRP and WARP, within von Willebrand factor A domains, suggesting that regional allelic variation modulates Plasmodium –vector compatibility by altering midgut adhesion interactions.

Abstract Rubella virus (RuV) causes rubella and, when acquired during pregnancy, congenital rubella syndrome (CRS). Despite its clinical significance, the cellular entry mechanism of RuV remains poorly characterized owing to limited knowledge of host receptors, hampering pathogenesis studies and therapeutic development. Here, we identify Nectin-4 as a functional RuV entry receptor through proximity biotin labeling-based proteomics coupled with CRISPR/Cas9 validation. Notably, Nectin-4 is shared with measles virus, revealing convergent evolution in viral entry despite the different viral families. Nectin-4 directly interacts with the RuV envelope glycoproteins through its ectodomain, facilitating receptor-mediated endocytosis following initial calcium-dependent viral attachment. In polarized respiratory epithelial cells, Nectin-4 mediates basolateral viral entry and subsequent apical release of progeny virions, enabling efficient respiratory transmission. Using human placental organoid models, we demonstrate that syncytiotrophoblasts serve as primary targets for transplacental RuV infection, with viral entry significantly inhibited by Nectin-4 blockade. These findings establish Nectin-4 as critical for both horizontal respiratory transmission and vertical maternal–fetal transmission of RuV. Our results provide fundamental insights into RuV pathogenesis and identify Nectin-4 as a promising therapeutic target for CRS. Importantly, the discovery of shared receptor utilization between RuV and measles virus opens new avenues for integrated elimination strategies targeting both pathogens.

Abstract Resistance to azole antifungals in Aspergillus fumigatus, a WHO Critical Priority fungal pathogen, is rising worldwide and threatens the efficacy of frontline therapies for invasive aspergillosis. This resistance has emerged largely through environmental selection driven by the extensive use of azole fungicides in agriculture and horticulture, yet its evolutionary origins remain obscure. Here, we analysed whole-genome sequences from 1,220 isolates collected over a century from 34 countries, revealing five distinct but interfertile genetic clusters spanning the global population. Azole resistance alleles were highly concentrated within particular clusters and were associated with mutator genotypes carrying defects in DNA repair, known to accelerate the accumulation of adaptive mutations. Two clusters exhibited resistance to multiple fungicide classes, including azoles, benzimidazoles, strobilurins and succinate dehydrogenase inhibitors, indicating the emergence of stacked multidrug resistance with no measurable fitness cost in vitro or in murine models. Molecular dating situates the expansion of resistant lineages in the latter half of the twentieth century, coinciding with the introduction and intensification of modern fungicide use. Phylogeographic reconstruction points to Western Europe—particularly intensive horticultural systems and the international bulb trade—as a major source of globally disseminated azole-resistant A. fumigatus, underscoring the urgent need for coordinated One Health surveillance and intervention.

SUMMARY Drugs targeting nucleoli and generating nucleolar stress (NS) such as RNA Polymerase I inhibitors have shown anticancer properties with some progressing to clinical trials. However, the mechanisms that modulate the sensitivity to NS remain poorly understood, which has limited the design of clinical trials on patients most likely responding to these therapies. To get a panoramic view of the genetic determinants that shape the response to NS in cancer cells, we conducted genome-wide CRISPR screens in cells treated with 2 independent RNA Polymerase I inhibitors: Actinomycin D (ActD) and BMH-21. As expected, mutations known to regulate the response to NS such as P53 or RB1 increased the resistance to both drugs. On the other hand, the toxicity of RNA Pol I inhibitors was increased in the context of mutations that enhance PI3K/mTOR signaling. Surprisingly, we found mutations that sensitized to ActD but increased the resistance to BMH-21, revealing that, while both drugs are used as RNA Pol I inhibitors, the must have additional unknown targets. Together our study provides a global landscape of the mechanisms that modulate the sensitivity to NS-inducing agents and illustrate the need for an in-depth analysis of the mechanism of toxicity of drugs, particularly when these are advancing to clinical use.

Crossover formation during meiosis is a tightly controlled process in which genetic information is exchanged between homologous chromosomes to increase the diversity of the progeny. In this process, an excess of double-strand breaks is introduced, but only a limited subset is ultimately processed into crossovers. Imbalances in the distribution of crossovers can lead to errors in chromosome segregation, with devastating consequences on the health of the progeny. However, the selection of which breaks are designated to become crossovers is still poorly understood, as both its timing and the ultimate molecular mechanisms are under debate. Here, we used 3D dual-color single-molecule localization microscopy and real-time confocal imaging, combined with advanced image analysis, to investigate the timing and mechanism of crossover designation in C. elegans . We show that meiotic crossover patterning is not established by a single decision point but depends on a dynamic, multi-layered regulation process. An initial, early selection process restricts potential crossovers to a small subset of double-strand break sites that already exhibit basic patterning features, including assurance and interference. A second, later step fine-tunes this pattern to ultimately ensure genome integrity and promote accurate chromosome segregation. Real-time imaging reveals that although the full process takes more than seven hours, key molecular events occur within minutes, high-lighting how rapid local dynamic changes can give rise to an overall slow but extremely robust crossover regulation program.

Out of context dsDNA is detected by cGAS, which produces the cyclic dinucleotide 2′3′-cGAMP to activate STING and trigger downstream responses including transcriptional reprogramming, cell death and autophagy. STING agonists, among them non-hydrolysable cyclic dinucleotide analogues, are in active development to enhance immune activation in cancer treatment and vaccine adjuvants. Detailed knowledge of STING’s nucleotide preferences is critical for the development of effective therapeutics, yet the full spectrum of cyclic dinucleotides capable of activating STING has not been comprehensively defined. Especially considering the recent diversity of cyclic nucleotides identified across bacteria and invertebrates, the pool of potential STING agonists to be tested has expanded considerably. Here, we systematically dissect STING nucleotide preferences and distinguish STING-dependent from STING-independent nucleotide signaling pathways by treating THP-1 monocytes and pancreatic cancer cells with a nucleotide library. We identify a set of 27 cyclic dinucleotides that induce STING activation to different extents. Our data indicate that STING’s bias toward 2′3′-linked cyclic dinucleotides underlies its ability to also be activated by nucleotides containing purine–pyrimidine hybrids. In addition, we show that STING can be activated by diverse non-hydrolysable c-di-AMP and cGAMP isomers, thereby expanding the opportunities for designing STING agonists for therapeutic applications.

Acute myeloid leukemia (AML) is a heterogeneous malignancy with limited curative treatment options. Transposable elements (TEs) are now recognized as key regulators of genome function, with aberrant activation implicated in cancer. However, their tumor-type-specific roles remain poorly characterized. Using single-cell Perturb-seq, we systematically screened for chromatin-associated regulators in primary AML patient cells to uncover dependencies required for leukemia cell viability. Our screen identified IRF2BP2 as an AML-selective dependency, functioning as a repressor of TE expression. Loss of IRF2BP2 induced differentiation, apoptosis, and impaired leukemic cell fitness, phenotypes linked to transcriptional activation of TEs, particularly evolutionarily young human endogenous retrovirus K (HERV-K). Mechanistically, IRF2BP2 cooperates with TRIM28 and DNMT1 to epigenetically silence TE expression. CRISPR-mediated activation of HERV-K/LTR5_Hs recapitulated the phenotypic effects of IRF2BP2 loss, while targeted re-silencing of HERV-K/LTR5_Hs partially rescued the effects, establishing a causal link between TE regulation and AML maintenance. Our findings highlight tumor-suppressive functions of TEs in leukemia and reveal IRF2BP2 as a key regulator of TE silencing in AML. Targeting the epigenetic machinery governing TE repression may represent a promising therapeutic avenue for differentiation-inducing and immunomodulatory strategies in AML.

Membrane trafficking governs the transport of molecules to both intracellular and extracellular locations, thereby maintaining cell homeostasis. During cancer progression, alterations in membrane trafficking are frequently observed. However, the mechanisms underlying the dysregulation of membrane trafficking in cancer progression remain largely unresolved. Recent evidence has demonstrated that epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma (LUAD) employs a membrane trafficking program to coordinate cancer cell invasion and immunosuppression in the tumor microenvironment (TME). To further dissect the pro-tumorigenic membrane trafficking program, here we conducted a CRISPR interference (CRISPRi) in vivo screen for membrane trafficking regulators in a syngeneic mouse model with a complete immune system. This screen identified REEP2, an endoplasmic reticulum (ER) shaping protein, as a novel regulator of the EMT-dependent membrane trafficking program, which is associated with a poor prognosis in LUAD patients and is required for LUAD metastasis in a syngeneic orthotopic LUAD mouse model. Mechanistically, the EMT activator ZEB1 upregulates REEP2 expression through miR-183- and miR-193a-mediated regulation that promotes the transportation of secretory cargoes from the ER exit site (ERES) to the Golgi, thereby augmenting the secretion of pro-tumorigenic factors. The REEP2-driven secretion promotes cancer cell proliferation, migration, and the infiltration of myeloid-derived suppressor cells (MDSCs) in the TME. These findings identify REEP2 as a critical mediator of the EMT-driven pro-metastatic membrane trafficking program, revealing a specific vulnerability in mesenchymal LUAD.

Background The pathogenic G 4 C 2 repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies focused on delineating the underlying perturbed mechanisms resulting from this genetic mutation are often confounded by the heterogeneity present in current disease models, such as patient-derived iPSC lines, with estimations of up to 50% of the variation in iPSC cell phenotypes resulting from inter-individual differences. Isogenic models, in which the pathogenic mutation is introduced into a defined genetic background, offer a powerful approach to isolating mutation-specific effects and enable high-resolution comparison across distinct ALS/FTD-associated mutations. Such models are essential for uncovering convergent disease mechanisms and improving reproducibility in ALS/FTD research. Methods A two-step scarless CRISPR/Cas9 genome editing strategy was used to generate isogenic human iPSC lines carrying a de novo knock-in of a disease-length G 4 C 2 repeat expansion in the C9ORF72 locus. The resulting lines underwent thorough quality control and were differentiated into lower motor neurons and assessed for the presence of key ALS/FTD pathologies, including changes to C9ORF72 mRNA and protein expression, RNA foci and dipeptide repeat proteins. Results Two C9ORF72 knock-in iPSC lines were generated with 631 and 600 G 4 C 2 repeats, alongside an isogenic genome editing control line. The C9ORF72 G 4 C 2 repeat expansion knock-in iPSC lines exhibit both loss-of-function and gain-of-function pathological features characteristic of ALS/FTD. Compared to the parental wild-type KOLF2.1J line and isogenic (wild-type) CRISPR control line, these exhibit a significant reduction in C9ORF72 mRNA and protein levels, the presence of RNA foci accumulation, and a marked increase in poly(GA) and poly(GP) dipeptide repeat protein levels in iPSCs and motor neurons. Conclusions This is one of the first reports of a successful knock-in of the pathogenic C9ORF72 G 4 C 2 repeat expansion into a human iPSC line, establishing a genetically defined and physiologically relevant model of ALS/FTD. These isogenic lines recapitulate both key loss- and gain-of-function disease pathologies, providing a crucial complement to existing patient-derived iPSC banks. By eliminating confounding genetic background variability, these cell lines will enable more precise interrogation of C9ORF72 -linked pathomechanisms and offer a robust platform for comparative studies across the ALS and FTD spectrum, mechanistic investigations, and future therapeutic targeting with enhanced translational relevance.

Polypogon australis (Brong.), a native Chilean grass (Poaceae), is a facultative metallophyte capable of colonizing copper mine tailing dams and adapting to saline and acidic substrates. These traits make it a promising candidate for phytoremediation of metal-contaminated soils. However, the lack of in vitro propagation, callus induction, and somatic embryogenesis protocols limits its use in large-scale applications and genetic improvement. This work aims to establish a reproducible in vitro regeneration system for P. australis . Mesocotyl explants were cultured on Murashige and Skoog (MS) medium supplemented with 2.5 mg L −1 Dicamba. Callus induction was achieved in 38.9% of explants, and 45.5% of embryogenic calli regenerated into plantlets producing leaves and radicles without requiring exogenous organogenesis-inducing hormones. The regenerated plants continued to develop further on MS + sucrose medium, confirming the totipotent capacity of mesocotyl-derived calli. The developed protocol provides a foundation for large-scale propagation and genetic transformation of P. australis . By overcoming propagation bottlenecks, this methodology strengthens the potential of this native metallophyte as a model for phytoremediation and future CRISPR-based biotechnological approaches to enhance copper tolerance and accumulation.

ABSTRACT Heterochromatin protein 1 (HP1) is a conserved chromatin-associated factor implicated in the establishment and maintenance of H3K9me-marked heterochromatin, potentially through phase separation–mediated condensation. Whether HP1 in molecular terms works primarily via dimerization or liquid–liquid phase separation (LLPS) is unresolved. Using the C. elegans HP1 orthologue HPL-2 and a combined in vitro–in vivo approach, we systematically dissected the molecular determinants of HPL-2 function in heterochromatin condensation. Through specific mutants, we demonstrate that HPL-2 dimerization, but not LLPS, is essential for condensing H3K9me chromatin arrays in vitro and for maintaining H3K9me heterochromatin foci in C. elegans embryos. We further show that HPL-2 dimerization is sufficient to mediate segregation of H3K9me from unmodified chromatin arrays in vitro , generating biphasic condensates reminiscent of cellular heterochromatin domains. Surprisingly, HPL-2 mutants defective in condensation cause only minor transcriptional changes at canonical heterochromatin loci, implying that HP1-dependent heterochromatin foci and gene silencing are not tightly coupled in vivo . Nonetheless, these mutant C. elegans exhibit profound physiological and developmental defects. Our findings establish dimerization as the principal molecular mechanism of HP1-driven H3K9me-chromatin condensation, elucidate the auxiliary role of LLPS, and reveal the uncoupling between HP1-dependent heterochromatin and transcriptional regulation. GRAPHICAL ABSTRACT

Hepatoblastoma (HB), the most common pediatric liver malignancy, is associated with high cure rates although patients with advanced or recurrent disease have less favorable outcomes. Because patients are invariably <4 years of age, chemotherapies can cause significant long-term morbidities. Immortalized HB cell lines could be of great utility for drug screening, for the identification of novel therapeutic susceptibilities, and for studies requiring highly regulated and/or rapidly changing in vitro environments. However, HB research is hampered by a paucity of established cell lines that could be used for such purposes, with only two human cell lines being readily available and neither of which is representative of the most common HB molecular subtypes. Recently, immortalized cell lines have been derived from murine HBs that are driven by several of the most common oncogenes associated with human tumors. These comprise five distinct groups associated with the deregulation of each of the possible combinations of oncogenic forms of the bcatenin, YAP and NRF2 transcription factors or the over-expression of MYC. All five groups share many of the attributes and molecular signatures of actual human HBs. In addition, they have been used for purposes as diverse as identifying novel molecular targets through the use of CRISPR-based screens and the demonstration that some HB cells can trans-differentiate into endothelial cells that support more robust tumor growth. The experience gained from these models and advances in the propagation of human hepatocytes in mice suggests that it will soon be possible to generate bespoke human HBs and immortalized cell lines.

Extrachromosomal arrays are unique chromosome-like structures created from DNA injected into the C. elegans germline. Arrays are easy to create and allow for high expression of multiple transgenes. They are, however, unstable unless integrated into a chromosome. Current methods for integration, such as X-rays and CRISPR, damage DNA and are low-efficiency. Here, we demonstrate that the viral integrase PhiC31, which mediates a non-mutagenic recombination between short attB and attP sequences, can be used for extremely efficient and targeted integration of arrays. In this method, a transgene, a selectable marker, and attP sites are injected into the gonad of a strain that (1) has an attB site in its genome, and (2) expresses PhiC31 in its germline. F1 extrachromosomal arrays are cloned, grown for multiple generations with selection, and then screened for homozygous array integrations. The procedure is simple, requires less time than screening for extrachromosomal arrays, and arrays can be screened for transgene function after stable integration. Arrays that transmit are integrated by PhiC31 with 50-95% efficiency, allowing for the isolation of many unique integrants from a single injection. Arrays can also be integrated at fluorescent landing pads and arbitrary sites in the genome. Using nanopore sequencing, we show that three new integrated arrays are between 1.6 and 18 megabases in length, assemble with large repeats, and can contain hundreds of copies of injected transgenes. We have built a collection of strains and plasmids to enable array integration at multiple sites in the genome using various selections. PhiC1-mediated Integration of Arrays of Transgenes (PhiAT) will allow C. elegans researchers to shift from using unstable extrachromosomal arrays to directly integrating arrays.

Abstract Syphilis, caused by Treponema pallidum , is a sexually transmitted infection that has re-emerged globally over the past decade, posing significant public health challenges. Conventional diagnostic methods are limited by lengthy processing times, operational complexity, and moderate sensitivity, highlighting the urgent need for rapid, sensitive, and user-friendly detection strategies. In this study, we developed a visual detection platform for T. pallidum DNA by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. The assay can be completed within one hour, with results directly interpreted via fluorescence readout. It demonstrated a detection limit as low as 11.34 copies/µL and high specificity, accurately distinguishing T. pallidum without cross-reactivity with common blood-borne pathogens, including HIV, HBV, HCV, and DENV. Validation with clinical samples showed complete concordance with standard diagnostic outcomes. To enhance suitability for point-of-care applications, the RPA-CRISPR/Cas12a system was further adapted to a lateral flow assay (LFA) format, achieving a detection sensitivity of 5.56×10² copies/µL while minimizing reliance on specialized instrumentation. Overall, this platform provides a rapid, sensitive, and robust approach for point-of-care syphilis diagnosis and offers a reference framework for detecting other pathogenic organisms.

Activation and proliferation of parietal epithelial cells (PECs), located along the inner rim of Bowman’s capsule, drives disease progression in subtypes of glomerulonephritis and focal segmental glomerulosclerosis. In examining the mechanisms contributing to PEC activation two established mouse models were utilized in this study, nephrotoxic serum nephritis (transient model) and podocyte-specific Klf4 knockout (progressive model). A role for transcription factor FRA2 ( Fosl2 ) was uncovered through single nuclear multiomic approaches relating to the regulation of PEC transcriptional/chromatin dynamics. Co-immunoprecipitation followed by mass spectrometry assessed the FRA2 protein interactome in cultured PECs, revealing a potential role for FRA2 in alternative splicing. Fosl2 expression was then blunted through CRISPR-Cas9 gene editing in cultured PECs, revealing reduced proliferative capacity and the downregulation of myofibroblast markers. In-vivo genetic lineage tracing of PECs after nephrotoxic serum revealed PEC-to-myofibroblast trans-differentiation events. Finally, immunostaining of human kidney biopsies with varied subtypes of glomerulonephritis confirmed Fosl2 expression in activated PECs within crescentic lesions, with single cell deconvolution strategies assigning PEC-skewed proportion ratios to bulk RNA-seq patient data from the NEPTUNE consortium. These results suggest that FRA2 ( Fosl2 ) directs a conserved molecular program of PEC-specific responses in subtypes of glomerulonephritis and focal segmental glomerulosclerosis.

CRISPR-mediated gene activation (CRISPRa) is among the most efficient and reliable strategies for mimicking sustained activation of endogenous promoters and their corresponding genes at physiological levels. By leveraging guide-RNA (gRNA) library design, CRISPRa screens can be applied on a whole-genome scale and are compatible with both arrayed and pooled formats, depending on assay requirements. Compared with conventional arrayed CRISPRa libraries that use single or dual gRNAs and often require multiple gRNA candidates per target, a recently developed CRISPRa library (termed T. gonfio) incorporates four tandem gRNAs per lentivector per target, thereby reducing library complexity and representing the smallest arrayed genome-wide CRISPRa library. To streamline genome-wide arrayed CRISPRa screening, this study developed a high-throughput automated workflow using the Biomek i7 Hybrid liquid-handling platform, integrated with multiple peripheral instruments. The workflow comprises three pipelines: lentiviral library transduction, cell library passaging, and assay processing. These pipelines together establish and maintain the transduced cell library for extended screening times. This enables assay processing at desired extended time points and improves the likelihood of identifying phenotypes that require longer time to develop, making the workflow suitable even for rapidly proliferating cell models. In a pilot arrayed screen using a T. gonfio mini-library targeting kinases and phosphatases, activation of the EPHA2 receptor promoter induced a growth reduction phenotype in the HEK293 cell model. This phenotype was recapitulated in a parallel pooled CRISPRa screen using the same mini-library and further validated in a co-culture assay.

Missense variants in O-GlcNAc transferase (OGT) result in OGT congenital disorder of glycosylation (OGT-CDG), an intellectual disability syndrome associated with O-GlcNAc dyshomeostasis and a range of neurodevelopmental defects. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for removing protein O-GlcNAcylation, has been explored as a target for modulating brain O-GlcNAc homeostasis in neurodegenerative diseases and may also be a target for OGT-CDG. Here, we describe an OGT-CDG mouse line that exhibits microcephaly, motor deficits, and brain O-GlcNAc dyshomeostasis, closely mirroring patient symptoms. We genetically explored OGA as a target for OGT-CDG by crossing these mice with a line carrying catalytically inactive OGA. Encouragingly, this partially restored O-GlcNAc homeostasis in brain and blood, although it did not result in significant phenotypic rescue. These findings suggest that OGA inhibition can modulate enzymatic imbalance in OGT-CDG mice, and that blood can be used to monitor the effects of interventions targeting O-GlcNAc dyshomeostasis.

Cotton (Gossypium hirsutum) is globally cultivated for its high-quality fiber; yet, its seed, rich in oil and protein, offers untapped potential to support various applications, including food, feed, and industry. With cottonseed oil gaining renewed attention as a valuable co-product, efforts to enhance oil content must contend with longstanding breeding priorities focused on lint yield and fiber quality. A central challenge lies in the complex and often antagonistic genetic relationships between oil accumulation and key agronomic traits. Notably, negative correlations between seed oil content and fiber yield, as well as the pleiotropic nature of several regulatory genes and Quantitative Trait Loci (QTLs), present significant barriers to dual-trait improvement. This review synthesizes current knowledge on the genetic and molecular interplay between cottonseed oil content and other agronomic traits. We examine the architecture of oil-related QTLs and pleiotropic loci, co-expression patterns of shared transcriptional regulators, and metabolic trade-offs influencing carbon allocation between seed and fiber. Recent advances in genomics, transcriptomics, and systems biology are explored as tools to disentangle these trait interactions. We highlight strategies such as multi-trait genomic selection, CRISPR-based uncoupling of antagonistic loci, and the use of wild and exotic germplasm to overcome linkage drag. By providing an integrative overview of the constraints and opportunities at the intersection of oil and agronomic trait improvement, this review lays the groundwork for the development of dual-purpose cotton ideotypes. We propose a conceptual framework for breeding programs to simultaneously enhance fiber yield and oil productivity in a sustainable and climate-resilient manner.

Precise genome editing in Enterobacteriaceae is essential for studying gene function, pathogenesis, and antimicrobial resistance, yet many current systems face host-specific and efficiency limitations. We developed pGGTOX, a modular plasmid platform that enables efficient homologous recombination–mediated genome editing across diverse Enterobacteriaceae , including Escherichia coli , Klebsiella pneumoniae , Salmonella enterica , and Enterobacter intestinihominis . The system integrates a rhamnose-inducible toxin (MqsR) for stringent counterselection, a sfGFP reporter for visual tracking of recombination events, Golden Gate cloning for rapid assembly of homologous arms, an FRT-flanked resistance cassette for marker removal, and an oriT sequence for conjugative transfer. Together with the companion plasmid pCP20-oriT, pGGTOX supports precise, marker-free genomic modification. Using pGGTOX, we achieved targeted deletions of dapA in E. coli and mrkCD in carbapenem-resistant K. pneumoniae , both with 100% efficiency. The dapA mutant exhibited diaminopimelate auxotrophy, while mrkCD deletion markedly reduced biofilm formation, consistent with the loss of function associated with these genes. pGGTOX also enabled deletion of a 43.1-kb type IV secretion gene cluster ( tra ) from an IncN/FII plasmid in E. intestinihominis and insertion of a 10-kb CRISPR–Cas9 plasmid-curing module (pCasCure) into an S. enterica IncX1 plasmid. Deletion of the tra gene cluster resulted in a substantial reduction in plasmid conjugation efficiency. Conjugative transfer of the engineered IncX1-pCasCure plasmid into K. pneumoniae facilitated CRISPR-mediated curing of bla KPC , sensitizing carbapenem resistance to susceptibility. In summary, pGGTOX provides a versatile, efficient, and broadly applicable platform for genome engineering and CRISPR delivery in Enterobacteriaceae , expanding the toolkit for bacterial genetics and translational antimicrobial research. Importance Precise genetic manipulation in Enterobacteriaceae remains a major technical challenge, particularly for non-model or multidrug-resistant strains. We developed pGGTOX, a versatile and broadly applicable plasmid platform that enables efficient, marker-free genome editing through homologous recombination. By integrating stringent counterselection, visual screening, modular cloning, and conjugative transfer, pGGTOX simplifies construction and streamlines editing across multiple clinically relevant species. We demonstrate its utility in deleting chromosomal and plasmid-borne loci, inserting large genetic modules, and delivering CRISPR–Cas9 systems for targeted elimination of antibiotic resistance genes. This platform expands the molecular toolkit for functional genomics and provides a powerful new strategy for dissecting bacterial virulence, resistance, and plasmid biology.

Programmable nucleic-acid therapeutics, including lipid nanoparticle (LNP)–formulated mRNA vaccines, genome editors delivered as mRNA, and viral vectors, are transforming precision medicine but remain constrained by innate reactogenicity despite the introduction of chemical modifications into mRNA. Here, we show that mRNA/LNPs and viral vectors elicit a transient inflammatory burst that peaks ∼6 h after dosing in vivo. Co-administration or prophylaxis with hydroxychloroquine (HCQ), a clinically established 4-aminoquinoline, potently attenuated cytokine and chemokine induction across modalities, including strong responses to unmodified mRNA, without compromising therapeutic efficacy. Humoral and cellular immunity to SARS-CoV-2 Spike mRNA vaccines (BNT162b2 and non-modified mRNA/LNP) were preserved, as was live-virus neutralization. Transcriptomic profiling indicated selective dampening of TLR/cGAS–STING pathways with retention of type-I interferon elements compatible with effective vaccination. HCQ further mitigated AAV9-associated blood–brain barrier (BBB) disruption after stereotactic delivery to the mouse brain and reduced mRNA/LNP-associated hepatotoxicity and thrombocytopenia while maintaining therapeutic transgene expression and CRISPR base-editing in vivo. These findings identify HCQ as an anti-reactogenic adjunct that widens the safety window of nucleic-acid therapeutics without sacrificing performance.

Abstract High pathogenicity avian influenza virus (HPAIV) has significantly impacted upon avian and mammalian populations across the Antarctic region. All viruses detected have been genotype B3.2 with phylogenetic analyses indicating multiple independent incursions from continental South America to, and transmission between, sub-Antarctic islands. From a zoonotic perspective, several isolates contained markers of mammalian adaptation in PB2 with functional characterisation of mutants demonstrating efficient replication in primary human airway epithelial cell cultures, demonstrating that these PB2-mutations alone contributed to enhanced polymerase activity in human cell lines. No mammalian-adaptive mutations were detected in the haemagglutinin or neuraminidase genes, with viruses retaining avian receptor binding preferences. Antigenic characterisation demonstrated cross-reactivity with existing pre-pandemic candidate vaccine strains and all viruses remained susceptible to licensed frontline antiviral therapeutics. We demonstrate a complex evolving viral ecology in the sub-Antarctic region involving both avian and marine mammal hosts, with significant implications for regional wildlife populations and zoonotic risk.

The growth orientation of the Marchantia polymorpha thallus – a system of dorsiventralized, indeterminate axes – is modulated by light. We show that red and blue light act antagonistically to control thallus growth tropisms, with red light signalling promoting epinasty and blue light signalling promoting hyponasty. We found that loss-of-function mutations in the blue light receptor Mp PHOT led to epinasty, while loss-of-function mutations in the red light receptor Mp PHY resulted in hyponasty. We hypothesize that these antagonistic activities of blue and red light signalling are balanced in white light, resulting in the development of flat thalli. Using time-resolved transcriptomics, we identified genes that were rapidly induced upon light exposure. Among these genes were all six members of the M. polymorpha BBX gene family. Mutants harbouring loss-of-function mutations in two of the six MpBBX transcription factors developed defective thallus tropisms. Mp bbx1 loss-of-function mutants formed hyponastic thalli, while Mp bbx5 loss-of-function mutants developed epinastic thalli. Double mutants Mpbbx1 Mp bbx5 grew flat, supporting the hypothesis that they function antagonistically. Together, these data indicate that phototropin-mediated blue light and phytochrome-mediated red light signalling antagonistically modulate thallus tropism, and that BBX transcription factors also act antagonistically to regulate thallus flatness.

Acute myeloid leukemia (AML) is characterized by differentiation arrest and uncontrolled proliferation. Differentiation therapy aims to treat AML by de-repressing latent myeloid maturation programs to induce cell cycle arrest and subsequent cell death. This approach is curative in the promyelocytic AML subtype, but has met with limited success in other subtypes. Genes such as LSD1 have emerged as intriguing non-APL AML differentiation therapy targets, but results as monoagents in clinical trials have been mixed. Here, we performed differentiation-specific CRISPR screens to identify targets whose inhibition synergizes with LSD1 inhibition to induce terminal differentiation of non-APL AML cells. Intriguingly, the MLL co-factor Menin scored as the top hit. Using cell lines, primary patient samples, and mouse AML models, we find that dual inhibition of LSD1 and Menin is a highly promising approach for differentiation therapy. Mechanistically, we determine that inhibition of Menin downregulates drivers of proliferation and stemness such as MEIS1, and inhibition of LSD1 induces inflammatory and interferon-related pro-myeloid differentiation expression programs. Surprisingly, we find that this combination is effective in selected AML models without mutations in MLL or NPM1, thus nominating dual inhibition of LSD1 and Menin as an attractive therapeutic approach for a mutationally diverse set of non-APL AMLs. Highlights Inhibition of LSD1 and Menin synergizes to induce differentiation of MLL-r and MLL-WT AMLs. Inhibition of Menin downregulates drivers of proliferation and stemness. Inhibition of LSD1 induces differentiation-associated inflammatory and interferon responses. LSD1 and Menin occupy different areas of the genome.

The wide variety of protocols and applications for DNA and RNA sequencing makes flexible tools for read processing an important step in sequence analysis. Beyond trimming and demultiplexing, custom read-level processing is commonly required for data exploration, QC and analysis. Existing tools are often task-specific and don’t generalise to new bioinformatic problems. Thus, there is a need for a tool flexible enough to handle the full variety of read processing tasks, and fast and scalable enough to retain high performance on growing sequencing datasets. We introduce matchbox , a read processor that enables fluent manipulation and analysis of FASTA/FASTQ/SAM/BAM files. With a lightweight scripting language designed around error-tolerant pattern-matching, users can write their own matchbox scripts to tackle a wide variety of bioinformatic problems, and incorporate them into existing pipelines and work-flows. We demonstrate matchbox ’s versatility in a number of contexts: demultiplexing long-read scRNA-seq data with 10X or SPLiT-seq barcodes; restranding RNA-seq reads; assessing CRISPR editing efficiency; and haplotyping macrosatellite repeat regions. matchbox achieves a computational performance comparable to existing tools, while addressing a broader range of bioinformatic needs, representing a new state-of-the-art in sequence processing. matchbox is implemented in Rust and available open-source at https://github.com/jakob-schuster/matchbox .