T lymphocyte activation is a crucial process in the regulation of innate and adaptive immune responses. The ion channel-kinase TRPM7 has previously been implicated in cellular Mg 2+ homeostasis, proliferation, and immune cell modulation. Here, we show that pharmacological and genetic silencing of TRPM7 leads to diminished human CD4 T-cell activation and proliferation following TCR mediated stimulation. In both primary human CD4 T cells and CRISPR/Cas-9 engineered Jurkat T cells, loss of TRPM7 led to altered Mg 2+ homeostasis, Ca 2+ signaling, reduced NFAT translocation, decreased IL-2 secretion and ultimately diminished proliferation and differentiation. While the activation of primary human CD4 T cells was dependent on TRPM7, polarization of naïve CD4 T cells into regulatory T cells (T reg ) was not. Taken together, these results highlight TRPM7 as a key protein of cellular Mg 2+ homeostasis and CD4 T-cell activation. Its role in lymphocyte activation suggests therapeutic potential for TRPM7 in numerous T-cell mediated diseases. Summary TRPM7 is crucial to maintaining cellular Mg 2+ homeostasis and regulates human CD4 T-cell activation by modulating early Ca 2+ signaling events in response to TCR-mediated stimulation subsequently, influencing T-cell differentiation in a Mg 2+ dependent manner.

Group B Streptococcus (GBS; Streptococcus agalactiae ) is an important pathobiont capable of colonizing various host environments, contributing to severe perinatal infections. Surface proteins play critical roles in GBS-host interactions, yet comprehensive studies of these proteins’ functions have been limited by genetic manipulation challenges. This study leveraged a CRISPR interference (CRISPRi) library to target genes encoding surface-trafficked proteins in GBS, identifying their roles in modulating macrophage cytokine responses. Bioinformatic analysis of 654 GBS genomes revealed 66 conserved surface protein genes. Using a GBS strain expressing chromosomally integrated dCas9, we generated and validated CRISPRi strains targeting these genes. THP-1 macrophage-like cells were exposed to ethanol-killed GBS variants, and pro-inflammatory cytokines TNF-α and IL-1β were measured. Notably, knockdown of the sip gene, encoding the Surface Immunogenic Protein (Sip), significantly increased IL-1β secretion, implicating Sip in caspase-1-dependent regulation. Further, Δ sip mutants demonstrated impaired biofilm formation, reduced adherence to human fetal membranes, and diminished uterine persistence in a mouse colonization model. These findings suggest Sip modulates GBS- host interactions critical for pathogenesis, underscoring its potential as a therapeutic target or vaccine component.

The chemokine receptor CXCR4 is overexpressed in many cancers and contributes to pathogenesis, disease progression, and resistance to therapies. CXCR4 is known to form oligomers, but the potential functional relevance in malignancies remain elusive. Using a newly established nanobody-based BRET method, we demonstrate that oligomerization of endogenous CXCR4 on lymphoid cancer cell lines correlates with enhanced expression levels. Specific disruption of CXCR4 oligomers reduced basal cell migration and pro-survival signaling via changes in the phosphoproteome, indicating the existence of basal CXCR4-oligomer-mediated signaling. Oligomer disruption also inhibited growth of primary CLL 3D spheroids and sensitized primary malignant cells to clinically used Bcl-2 inhibitor venetoclax. Given its limited efficacy in some patients and the ability to develop resistance, sensitizing malignant B-cells to venetoclax is of clinical relevance. Taken together, we established a new, non-canonical and critical role for CXCR4 oligomers in lymphoid neoplasms and demonstrated that selective targeting thereof has clinical potential. Significance statement Class A GPCRs, including the chemokine receptor CXCR4, can form oligomers, but their functional relevance remains poorly understood. This study provides evidence for the role of basal CXCR4 oligomers in lymphoid neoplasms, where they drive pro-survival signaling, migration, and tumor growth. We use a novel nanobody-based BRET method to demonstrate that endogenous CXCR4 constitutively oligomerizes in lymphoid cancer cells, correlating with receptor expression levels. Pharmacological disruption of these oligomers reduces tumor- associated signaling, impairs spheroid growth, and sensitizes patient-derived malignant cells to the apoptosis-inducing drug Venetoclax. Since CXCR4 is frequently overexpressed and potentially clustered in various malignancies, this work offers broader implications for enhancing treatment efficacy, overcoming drug resistance, and potentially reducing side effects across multiple cancer types.

Synthetic biology offers the possibility of synthetic genomes with customised gene content and modular organisation. In eukaryotes, building whole custom genomes is still many years away, but work in Saccharomyces cerevisiae yeast is closing-in on the first synthetic eukaryotic genome with genome-wide design changes. A key design change throughout the synthetic yeast genome is the introduction of LoxPsym site sequences. These enable inducible genomic rearrangements in vivo via expression of Cre recombinase via SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxPsym-mediated Evolution). When paired with selection, SCRaMbLE can quickly generate strains with phenotype improvements by diversifying gene arrangement and content in LoxPsym-containing regions. Here, we demonstrate how iterative cycles of SCRaMbLE can be used to reorganise synthetic genome modules and synthetic chromosomes for improved functional performance under selection. To achieve this, we developed SCOUT ( S CRaMbLE C ontinuous O utput and U niversal T racker), a reporter system that allows SCRaMbLEd cells to be sorted into a high diversity pool. When coupled with long-read sequencing, SCOUT enables high-throughput mapping of genotype abundance and correlation of gene content and arrangement with growth-related phenotypes. Iterative SCRaMbLE was applied here to yeast strains with a full synthetic chromosome, and to strains with synthetic genome modules encoding the gene set for histidine biosynthesis. Five synthetic designs for HIS modules were constructed and tested, and we investigated how SCRaMbLE reorganised the poorest performing design to give improved growth under selection. The results of iterative SCRaMbLE serve as a quick route to identify genome module designs with optimised function in a selected condition and offer a powerful tool to generate datasets that can inform the design of modular genomes in the future.

CRISPR-Cas systems are transformative tools for gene editing which can be tuned or controlled by anti-CRISPRs (Acrs) - phage derived inhibitors that regulate CRISPR-Cas activity. However, Acrs that are capable of inhibiting biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful, rapid, and generalisable approach that leverages de novo protein design to develop new-to-nature proteins for controlling CRISPR-Cas activity. Using CRISPR-Cas13 as a representative example, we demonstrate that AI-designed anti-CRISPRs (AIcrs) are capable of highly potent and specific inhibition of CRISPR-Cas13 proteins. We present a comprehensive workflow for design validation and demonstrate AIcrs functionality in controlling CRISPR-Cas13 activity in bacteria. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR-Cas tools in diverse applications across research, medicine, agriculture, and microbiology.

The Gram-negative pathogen, Acinetobacter baumannii , poses a serious threat to human health due to its role in nosocomial infections that are resistant to treatment with current antibiotics. Despite this, our understanding of fundamental A. baumannii biology remains limited, as many essential genes have not been experimentally characterized. These essential genes are critical for bacterial survival and, thus, represent promising targets for drug discovery. Here, we systematically probe the function of essential genes by screening a CRISPR interference knockdown library against a diverse panel of chemical inhibitors, including antibiotics. We find that most essential genes show chemical-gene interactions, allowing insights into both inhibitor and gene function. For instance, knockdown of lipooligosaccharide (LOS) transport genes increased sensitivity to a broad range of chemicals. Cells with defective LOS transport showed cell envelope hyper-permeability that was dependent on continued LOS synthesis. Using phenotypes across our chemical-gene interaction dataset, we constructed an essential gene network linking poorly understood genes to well-characterized genes in cell division and other processes. Finally, our phenotype-structure analysis identified structurally related antibiotics with distinct cellular impacts and suggested potential targets for underexplored inhibitors. This study advances our understanding of essential gene and inhibitor function, providing a valuable resource for mechanistic studies, therapeutic strategies, and future key targets for antibiotic development.

Gene drives are engineered alleles that bias their own inheritance in offspring, enabling the spread of specific traits throughout a population. Targeting female fertility genes in a gene drive can be an efficient strategy for population suppression. In this study, we investigated nine female fertility genes in Drosophila melanogaster using CRISPR-based homing gene drives. Employing a multiplexed gRNA approach to prevent formation of functional resistance alleles, we aimed to maintain high drive conversion efficiency with low fitness costs in female drive carriers. Drive efficiency was assessed in individual crosses and had varied performance across different target genes. Notably, drives targeting the octopamine β2 receptor ( oct ) and stall ( stl ) genes exhibited the highest drive conversion rates and were further tested in cages. A drive targeting stl successfully suppressed a cage population with a high release frequency, though suppression failed in another replicate cage with lower initial release frequency. Fitness costs in female drive carriers were observed in test cages, impacting the overall efficiency of population suppression. Further tests on the fertility of these lines using individual crosses indicated that some fitness costs were possibly due to the maternal deposition of Cas9 combined with new gRNA expression, which would only occur in progeny of drive males when testing split drives with separate Cas9 (when mimicking cages with complete drives) but not for complete drive systems. This could enable success in complete drives with higher maternal Cas9 deposition, even if cage experiments in split drives fail. Our findings underscore the potential and challenges of assessing gene drives for population control, providing valuable insights for optimizing and testing suppression gene drive designs.

Abstract Senescent cells drive tissue dysfunction through the senescence-associated secretory phenotype (SASP). We uncovered a central role for mitochondria in the epigenetic regulation of the SASP, where mitochondrial-derived metabolites, specifically citrate and acetyl-CoA, fuel histone acetylation at SASP gene loci, promoting their expression. We identified the mitochondrial citrate carrier (SLC25A1) and ATP-citrate lyase (ACLY) as critical for this process. Inhibiting these pathways selectively suppresses SASP without affecting cell cycle arrest, highlighting their potential as therapeutic targets for age-related inflammation. Notably, SLC25A1 inhibition reduces systemic inflammation and extends healthspan in aged mice, establishing mitochondrial metabolism as pivotal to the epigenetic control of aging.

ABSTRACT Mapping how pathogens interact with their host cells can reveal unexpected pathogen and host cell biology, paving the way for new treatments. Cryptosporidium is an intracellular parasite of intestinal epithelial cells, and a leading cause of diarrheal death and disease in infants worldwide. Despite this, very little is known about the cell biology of infection of this eukaryotic pathogen. Here, we designed and implemented a unique microscopy-based arrayed CRISPR-Cas9 screen to interrogate the effects of the loss of every protein-coding human gene on a Cryptosporidium infection. As the experimental readout is image-based, we extracted multiple phenotypic features of infection, including parasite growth, progression of the parasite to its sexual life stage, and recruitment of host actin to ‘pedestals’ beneath the parasite vacuole. Using this dataset, we discovered a tipping point in the host cholesterol biosynthesis pathway that controls Cryptosporidium infection. Parasite growth can either be inhibited or promoted by the intermediary metabolite squalene. A build-up of squalene in epithelial cells creates a reducing environment, with more reduced host glutathione available for uptake by the parasite. Because Cryptosporidium has lost the ability to synthesise glutathione, this uptake from the host cell is required for growth and progression through its life cycle. We demonstrate that this dependency can be leveraged for treatment with the abandoned drug lapaquistat, an inhibitor of host squalene synthase that has efficacy against Cryptosporidium in vitro and in vivo .

ABSTRACT Genetic assays are an invaluable tool for both fundamental biological research and translational applications. Variable Dose Analysis (VDA) is an RNAi-based method for cell-based genetic assays that offers several advantages over approaches such as CRISPR and other RNAi-based methods including improved data quality (signal-to-noise ratio) and the ability to study essential genes at sub-lethal knockdown efficiency. Here we report the development of three new variants of the VDA method called high-throughput VDA (htVDA), VDA-plus and pooled-VDA. htVDA requires 10-fold reduced reagent volumes and takes advantage of liquid handling automation to allow higher throughput screens to be performed while maintaining high data quality. VDA-plus is a modified version of VDA that further improves data quality by 4.5-fold compared to standard VDA to allow highly sensitive detection of weak phenotypes. Finally, Pooled VDA allows greatly increased throughput by analysing multiple gene knockdowns in a single population of cells. Together, these new methods enhance the toolbox available for genetic assays, which will prove valuable in both high-and low-throughput applications. In particular, the low noise and ability of VDA to study essential genes at sub-lethal knockdown levels will support identification of novel drug-targets, among which essential genes are often enriched. While these tools have been developed in Drosophila cells, the underlying principles are transferrable to any cell culture system.

Multimodal single-cell profiling provides a powerful approach for unravelling the gene regulatory mechanisms that drive development, by simultaneously capturing cell-type- specific transcriptional and chromatin states. However, its inherently destructive nature hampers the ability to trace regulatory dynamics between mother and daughter cells. Taking advantage of the invariant cell lineage of Caenorhabditis elegans, we constructed a lineage- resolved single-cell multimodal map of pre-gastrulation development, which allows the tracing of chromatin and gene expression changes across cell divisions and regulatory cascades. We characterise the early dynamics of genome regulation, revealing that zygotic genome activation occurs on an accessible chromatin landscape pre-patterned both maternally and zygotically, and we identify a redundant family of transcriptional regulators that drive a transient pre-gastrulation program. Our findings demonstrate the power of a lineage-resolved atlas for dissecting the genome regulatory events of development.

As members of the α-proteobacteria group, Caulobacter crescentus and its relatives are known for their asymmetric life cycle and comprehensive applications in gene delivery, agricultural biotechnology, and the production of high-value compounds. However, genetic manipulations of these bacteria are often time-consuming and labor-intensive due to the lack of efficient genome editing tools. Here, we report a practical CRISPR/ Sp Cas9M-reporting system that overcomes the limitations of Sp Cas9 expression, enabling efficient, markerless, and rapid genome editing in C. crescentus . As a demonstration, we successfully knocked out two genes encoding the scaffold proteins, achieving apparent editing efficiencies up to 80%. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized to enhance the editing efficiency or decrease the cell lethality. A nearly zero off-target ratio was observed after the curing of the editor plasmid in editing strains. Furthermore, we applied the CRISPR/ Sp Cas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti , establishing it as an efficient and reliable editing strategy. We anticipate that this system could be applied to other hard-to-edit organisms, accelerating both basic and applied research in α-proteobacteria.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by widespread inflammation and autoantibody production. Its development and progression involve genetic, epigenetic, and environmental factors. Although genome-wide association studies (GWAS) have repeatedly identified a susceptibility signal at 16p13, its fine-scale source and its functional and mechanistic role in SLE remain unclear. We used bioinformatics to prioritize likely functional variants and validated the top candidate through various experimental techniques, including CRISPR-based genome editing in B cells. To assess the functional impact of the proposed causal variant in CLEC16A , we compared autophagy levels between wild-type (WT) and knock-out (KO) cells. Systematic bioinformatics analysis identified the highly conserved non-coding intronic variant rs17673553, with the risk allele apparently affecting enhancer function and regulating several target genes, including CLEC16A itself. Luciferase reporter assays followed by ChIP-qPCR validated this enhancer activity, demonstrating that the risk allele increases the binding of enhancer histone marks (H3K27ac and H3K4me1), CTCF-binding factor, and key immune transcription factors (GATA3 and STAT3). Knock-down of GATA3 and STAT3 via siRNA led to a significant decrease in CLEC16A expression. These regulatory effects on the target gene were further confirmed using CRISPR-based genome editing and CRISPR-dCas9-based epigenetic activation/silencing. Functionally, WT cells exhibited higher levels of starvation-induced autophagy compared to KO cells, highlighting the role of CLEC16A and the rs17673553 locus in autophagy regulation. These findings suggest that the rs17673553 locus – particularly the risk allele – drives significant allele-specific chromatin modifications and binding of multiple transcription factors, thereby mechanistically regulating the expression of target autophagy-associated genes, including CLEC16A itself. This mechanism could potentially explain the association between rs17673553 and SLE, and underlie the signal at 16p13.

Studying the spatiotemporal dynamics of cells in living organisms is a current frontier in bioimaging. Intravital Microscopy (IVM) provides direct, long-term observation of cell behavior in living animals, from tissue to sub-cellular resolution. Hence, IVM has become crucial for studying complex biological processes in motion and across scales, such as the immune response to pathogens and cancer. However, IVM data are typically kept in private repositories inaccessible to the scientific community, hampering large-scale analysis that aggregates data from multiple laboratories. To solve this issue, we introduce Immunemap, an atlas of immune cell motility based on an Open Data platform that provides access to over 58’000 single-cell tracks and 1’049’000 cell-centroid annotations from 360 videos in murine models. Leveraging Immunemap and unsupervised learning, we systematically analyzed cell trajectories, identifying four main patterns of cell migration in immune cells. Two patterns correspond to behaviors previously characterized: directed movement and arresting. However, we identified two other patterns, characterized by low directionality and twisted paths, often considered random migration. We show that the newly defined patterns can be subdivided into two distinct types: within small areas, suggesting a focused patrolling around one or a few cells, and over larger areas, indicative of a more extended tissue patrolling. Furthermore, we show that the percentage of cells displaying these motility patterns changes in response to immune stimuli. Altogether, Immunemap embraces the FAIR principles, promoting data reuse to extract novel insights from immune cell dynamics through an image-based systems biology approach.

ABSTRACT Metabarcoding is a valuable tool for characterising the communities that underpin the functioning of ecosystems. However, current methods often rely on PCR amplification for enrichment of marker genes. PCR can introduce significant biases that affect quantification and is typically restricted to one target loci at a time, limiting the diversity that can be captured in a single reaction. Here, we address these issues by using Cas9 to enrich marker genes for long-read nanopore sequencing directly from a DNA sample, removing the need for PCR. We show that this approach can effectively isolate a 4.5 kb region covering partial 18S and 28S rRNA genes and the ITS region in a mixed nematode community, and further adapt our approach for characterising a diverse microbial community. We demonstrate the ability for Cas9-based enrichment to support multiplexed targeting of several different DNA regions simultaneously, enabling optimal marker gene selection for different clades of interest within a sample. We also find a strong correlation between input DNA concentrations and output read proportions for mixed-species samples, demonstrating the ability for quantification of relative species abundance. This study lays a foundation for targeted long-read sequencing to more fully capture the diversity of organisms present in complex environments.

Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukemia (AML) RBPs are key regulators of tumor proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5’UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.

SUMMARY Neurons need to adjust synaptic output according to the targets. However, the target-specific synaptic structures within individual neurons in the central nervous system remains unresolved. Applying the CRISPR/Cas9-mediated split-GFP tagging, we visualized the endogenous active zone scaffold protein, Bruchpilot (Brp), in specific cells. This technology enabled the spatial characterization of presynaptic machineries only within the Kenyon cells (KCs) of the Drosophila mushroom bodies. We found the patterned accumulation of Brp among the compartments of axon terminals, where a KC synapses onto different postsynaptic neurons. Mechanistically, the localized octopaminergic modulation along γ KC terminals regulate this compartmental Brp heterogeneity via Octβ2R and cAMP signaling. We further found that acute food deprivation reorganizes this spatial pattern in an octopamine-dependent manner. Such coordinated regulation of local synaptic machineries thus explains how the mushroom bodies integrate changing physiological states. This subcellular information processing represents an elegant solution to expand computational capacity of the circuit.

Background: The ability to generate endogenous Cre recombinase drivers using CRISPR–Cas9 knock–in technology allows lineage tracing, cell type specific gene studies, and in vivo validation of inferred developmental trajectories from phenotypic and gene expression analyses. This report describes endogenous zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR–Cas9 precision targeted integration. Results: hand2–2A–cre and hand2–2A–creERT2 knock–ins crossed with ubiquitous loxP–based Switch reporters led to broad labeling in expected mesodermal and neural crest–derived lineages in branchial arches, cardiac, fin, liver, intestine, and mesothelial tissues, as well as enteric neurons. Novel patterns of hand2 lineage tracing appeared in venous blood vessels. CreERT2 induction at 24 hours reveals late emerging hand2 progenitors in the 24–48 hour embryo contribute to the venous and intestinal vasculature. Induction in 3 dpf larva restricts hand2 lineage labeling to mesoderm–derived components of the branchial arches, heart, liver and enteric neurons. Conclusions: hand2 progenitors from the lateral plate mesoderm and ectoderm contribute to numerous lineages in the developing embryo. Later emerging hand2 progenitors become restricted to a subset of lineages in the larva. The hand2 Cre and CreERT2 drivers establish critical new tools to investigate hand2 lineages in zebrafish embryogenesis and larval organogenesis.

Gene editing has been revolutionised by the CRISPR-Cas9 technology. The versatility and ease-of-use of the technology far exceeds its predecessors, however, the selection of a high-quality guide RNA (gRNA) is critical to directing it to a target site. Selecting gRNA calls upon high-performance algorithms that evaluate nuclease activity at on-target and off-target sites. While there are a suite of programs available, many struggle to analyse the largest genomes, or their predictive accuracy is low. We have previously published a program, named Crackling that is amongst the fastest and most accurate tools available, however, it requires an end-user to have access to a traditional high-performance computing environment. Here, we present an adaptation of Crackling, named Crackling Cloud, that takes advantage of modern serverless cloud technologies that are widely available to anyone, and do not consume resources and incur costs when sitting idle, but can scale to use large volumes of resources when analyses require that. Crackling Cloud is provided as a templated solution using technologies of Amazon Web Services, and is available for free on GitHub under the terms of the BSD 3-clause licence: https://github.com/bmds-lab/Crackling-AWS

Abstract Immunotherapy has revolutionised cancer treatment, yet few patients respond clinically, necessitating alternative strategies that can benefit these patients. Novel immune-oncology targets can achieve this through bypassing resistance mechanisms to standard therapies. To address this, we introduce MIDAS, a multimodal graph neural network system for immune-oncology target discovery that leverages gene interactions, multi-omic patient profiles, immune cell biology, antigen processing, disease associations, and phenotypic consequences of genetic perturbations. MIDAS generalises to time-sliced data, outcompetes existing methods, including OpenTargets, and distinguishes approved from prospective targets. Moreover, MIDAS recovers immunotherapy response-associated genes in unseen trials, thus capturing tumour-immune dynamics within human tumours. Interpretability analyses reveal a reliance on autoimmunity, regulatory networks, and relevant biological pathways. Functionally perturbing the OSM-OSMR axis, a proposed target, in TRACERx melanoma patient-derived explants yielded reduced dysfunctional CD8 + T cells, which associate with immunotherapy response. Our results present a machine learning framework for analysing multimodal data for immune-oncology discovery.

Domain insertion engineering is a powerful approach to juxtapose otherwise separate biological functions, resulting in proteins with new-to-nature activities. A prominent example are switchable protein variants, created by receptor domain insertion into effector proteins. Identifying suitable, allosteric sites for domain insertion, however, typically requires extensive screening and optimization. We present ProDomino, a novel machine learning pipeline to rationalize domain recombination, trained on a semi-synthetic protein sequence dataset derived from naturally occurring intradomain insertion events. ProDomino robustly identifies domain insertion sites in proteins of biotechnological relevance, which we experimentally validated in E. coli and human cells. Finally, we employed light- and chemically regulated receptor domains as inserts and demonstrate the rapid, model-guided creation of potent, single-component opto- and chemogenetic protein switches. These include novel CRISPR-Cas9 and -Cas12a variants for inducible genome engineering in human cells. Our work enables one-shot domain insertion engineering and substantially accelerates the design of customized allosteric proteins.

SUMMARY The corpus callosum (CC) is the large axon bundle connecting the telencephalic hemispheres. The CC is formed exclusively in placental mammals, and the lack of comparable structures in other amniotes obscures the evolutionary origin of the CC. We here demonstrate that interhemispheric remodeling, a prior developmental step for CC formation, is highly conserved in non-mammalian amniotes, such as reptiles and birds. In these animal groups, the spatiotemporal dynamics of interhemispheric remodeling are tightly connected with distinct commissural formations. We observed a high degree of similarity between the mammalian CC and reptilian rostral pallial commissure, (RPC) and significant modifications in the avian pallial projection. Furthermore, we determined that Satb2 plays crucial roles in interhemispheric remodeling, which is associated with proper formation of both the CC and RPC in mice and geckoes, via the use of CRISPR-mediated gene-targeting. Our findings suggest that developmental mechanisms for midline remodeling were already present in the common ancestor of amniotes, which contributed to the evolution of eutherian-specific CC formation.

Abstract Background Ischemic stroke is a leading cause of disability and death worldwide, with limited treatment options, leaving many survivors with long-term neurological issues. Bone marrow mesenchymal stem cells (BMSCs) show promise in improving recovery, but few studies have examined their role during the recovery phase. This present study aims to explore whether and how BMSCs improve neurological function during the recovery period of ischemic stroke(IS). Methods Male Sprague-Dawley rats (weighing 280-300g) underwent transient middle cerebral artery occlusion(tMCAO). BMSCs and Bone marrow mesenchymal stem cell exosomes (BMSC-Exos) were isolated and characterized by flow cytometry, transmission electron microscopy, and western blotting assay. Neurological function was assessed through postural reflex, tactile, visual, proprioceptive placing tests, rotarod test, and Morris water maze. Angiogenesis and neurogenesis were observed by immunofluorescence staining (IF). Exosomal miRNA profiling was performed using a microRNA array. For the mechanism study, BMSCs + miR-195 CRISPR or BMSCs + miR-195 agomirs were administered intracerebroventricularly. Genes and protein expression levels were measured using qRT-qPCR, Western blotting, and IF staining. Results BMSCs enhance neurological function by promoting angiogenesis and neurogenesis during ischemic stroke recovery. MiR-195-5p, derived from BMSC exosomes, reduces Nogo-A induced by cerebral ischemia. Mechanistically, miR-195-5p stimulates vascular regeneration by inhibiting the Nogo-A/S1PR2 signaling pathway. Additionally, miR-195-5p inhibits the Nogo-A/NgR1 pathway, promoting neurogenesis. Conclusions BMSCs inhibit the Nogo-A/NgR1/S1PR2 signaling pathway via exosomal miR-195, promoting neurogenesis and angiogenesis during the ischemic stroke recovery phase, thereby reducing neurological deficits. These findings suggest that targeting Nogo-A with BMSCs during stroke recovery offers a promising therapeutic approach for survivors.

Cell Painting images offer valuable insights into a cell’s state and enable many biological applications, but publicly available arrayed datasets only include hundreds of genes perturbed. The JUMP (Joint Undertaking in Morphological Profiling) Cell Painting Consortium perturbed roughly 75% of the protein-coding genome in human U-2 OS cells, generating a rich resource of single-cell images and extracted features. These profiles capture the phenotypic impacts of perturbing 15,243 human genes, including overexpressing 12,609 genes (using open reading frames, ORFs) and knocking out 7,975 genes (using CRISPR-Cas9). We mitigated technical artifacts by rigorously evaluating data processing options and validated the dataset’s robustness and biological relevance. Analysis of phenotypic profiles revealed novel gene clusters and functional relationships, including those associated with mitochondrial function, cancer, and neural processes. The JUMP Cell Painting genetic dataset is a valuable resource for exploring gene relationships and uncovering novel functions.

Multiple histone H2A variants are known in eukaryotes. However, the functional relationship between H2A and its variants in plants remains largely obscure. Using CRISPR/Cas9 editing, we generated a mutant lacking four H2A isoforms in Arabidopsis and analyzed the functional and structural relationship between H2A and its variants H2A.Z and H2A.W. RNA-sequencing and phenotype analyses revealed mild changes in gene transcription and plant development in the mutants lacking H2A, H2A.Z, or H2A.W compared with the wild-type plants. Chromatin immunoprecipitation sequencing analysis showed that H2A is able to substitute for both H2A.Z and H2A.W across the genome, including in euchromatin and heterochromatin regions. However, H2A.Z replaced both H2A and H2A.W primarily within the euchromatin regions. By using DNA and histones derived from Arabidopsis, we constructed nucleosomes containing H2A, H2A.Z, or H2A.W and resolved their cryogenic electron microscopy structures at near-atomic resolution. Collectively, the results reveal the structural similarity and functional redundancy of H2A and H2A variants in Arabidopsis.