Scientific highlights

While 5-methylcytosine (5mC) DNA methylation is a well-known player in genome stability and transcriptional regulation, the role of 5-hydroxymethylcytosine (5hmC), particularly prevalent in neurons, remains largely unknown. Here, we used long-read Oxford Nanopore Technology (ONT) to profile whole-genome, native 5mC and 5hmC levels in sorted neuronal nuclei samples from human post-mortem brain tissue. Genomic annotation revealed high 5hmC levels in actively transcribed genes and enhancers, which were enriched in neuron-related pathways, with functional variety when stratifying across chromatin states. Furthermore, 5hmC and 5mC modifications were enriched at transcription factor binding sites, affecting specific downstream regulatory networks. Altogether, our study demonstrates the potential of 5hmC and 5mC DNA modifications to provide biological insight into the regulatory landscape and function of human neurons.

The cancer-intrinsic network controlling the response to immune checkpoint blockade therapy remains elusive. Using a published single-cell RNA sequencing (scRNA-seq) dataset from patients with breast cancer, we found that pretherapy mRNA expression of NEDD8, which encodes the regulatory protein essential for neddylation, in cancer cells was negatively associated with T-cell expansion upon pembrolizumab treatment. Genetic ablation of protein neddylation in murine tumors induced a curative response to PD-1 or PD-L1 blockade in mice. Combined analysis of neddylation-deficient murine tumors using scRNA-seq and high-resolution spatial transcriptomics revealed distinct spatial immune co-localization and immune cell signatures. In a high-content phenotypic screen of known compounds, protein neddylation was required for the induction of phenotypic changes by lipid inhibitors and ferroptosis inducers in human breast cancer cells. In accordance, these compounds enhanced the efficacy of PD-1 blockade and protected mice from tumor rechallenge. Altogether, we delineate the unknown functions of protein neddylation on antitumor immunity and provide therapeutic options that can enhance immunotherapy.

Element Biosciences' avidity sequencing has emerged as a competing technology to Illumina's short-read sequencing platform. Prior benchmarks of avidity sequencing have not included the latest Illumina NovaSeq X/X Plus instruments with XLEAP chemistry. Here, we have run polymerase chain reaction-free whole-genome sequencing on four human tumor cell lines using both Illumina NovaSeq X Plus and Element AVITI instruments. AVITI showed low duplication rates and reported higher base qualities; the latter contributed to improved mapping confidence and fewer spurious variant candidates. Both platforms were found to be highly comparable when benchmarking variant calling, with AVITI only providing a minor improvement on INDELs at lower coverages. Stratifying by genomic context revealed further differences, where AVITI genome coverage and variant calls were superior in high-GC regions while being inferior in GC homopolymers. Error-rate analysis highlighted further differences between the platforms; in particular, AVITI in some instances displayed an increased error rate on read 2 related to short fragments. AVITI error rate was also found to be more stable downstream of repetitive regions, except for GC homopolymers. We further found that AVITI sequencing was sensitive to G-quadruplex motifs. Overall, despite these identified differences, both platforms performed highly comparable for variant analysis.

Precision medicine is increasingly applied in the cancer clinic, adapting treatment to genomic alterations of the tumor. However, whether alterations disrupt the function of a protein can depend on if both alleles of a gene are altered. While massively parallel sequencing technologies can identify sequence aberrations, they are limited in resolving the corresponding haplotype information. In this proof-of-concept case study, we applied the linked-read droplet barcode sequencing (DBS) technology to resolve the haplotype complexity of colorectal cancer genomes on paired tumor and normal samples. Several cancer-related genes carried multiple mutations in either one or both haplotypes. Additionally, a number of haplotype-resolved large structural variants and copy number alterations were detected and phased with short somatic variants. Nearly all characterized oncogenic pathways harbored some of the identified short somatic variants. The study demonstrates that linked-read DBS technology can characterize complex genetic variations in a haplotype context and may provide essential information for personalized approaches.

The germline-restricted chromosome (GRC) of passerines is a remarkable tissue-specific chromosome that accumulated paralogs of genes from the regular "A chromosomes" over millions of years, often amplified into dozens of gene copies. In addition to its repetitive content, typically uniparental inheritance, and lack of recombination, the GRC resembles non-recombining sex chromosomes and some B chromosomes, for all of which assembly and single-nucleotide polymorphisms (SNPs) calling are difficult. Here, we first show that much of the Australian zebra finch macro-GRC can be assembled using accurate long reads. We then describe a paralog-aware Snakemake pipeline, ParaVar, to map short reads from the GRC to retrieve GRC regions suitable for haplotype-based analysis. ParaVar reliably calls hundreds of SNPs across the GRC, thereby providing an estimate of nucleotide diversity on the highly repetitive zebra finch macro-GRC. Our results show significantly lower nucleotide diversity (20- to 50-fold lower) on the GRC compared to the mitogenome and autosomes, and a strong phylogenetic discordance between the GRC and the mitochondrial genome. Beyond the contribution of background selection, our results suggest that a single GRC haplotype recently spread through the populations while jumping across matrilines via occasional paternal inheritance. We anticipate that our paralog-aware pipeline will be useful for SNP calling and population genetics analyses of repetitive GRCs, sex chromosomes, and B chromosomes.

Blood proteins have provided essential insights into how humans responded to the recent pandemic. To expand our understanding beyond patients seeking medical care, we conducted a citizen-centric survey with 2,000 random residents (age: 18-69 years) from Sweden's two largest cities in 2021. With self-sampled dried blood spots (DBS) and health information from 437 (22%) volunteers, we performed multi-analyte COVID-19 serology, measured autoantibodies (AAbs) against 22 interferons, and quantified 502 circulating low-abundant immune-related blood proteins. Antibody assays confirmed self-reported infections (26%) and vaccinations (40%), showed timing-dependent discrepancies in the immune response, and revealed anti-type I interferon AAbs co-occurring frequently alongside natural infections. Proteomics data added plausible mechanistic insights into cell-mediated processes: data-driven analyses revealed 24% of participants presented deviating immune phenotypes linked to infections, immunity, respiratory effects, and age. Multi-molecular DBS analysis of random layperson samples captured the broader spectrum of immune system states, adding relevant insights for clinical and public health investigations.

Basophils are implicated in various diseases including allergies, but a comprehensive single-cell characterization of human basophils has yet to be performed. Here, we aimed to generate a single-cell omics-based reference resource of circulating human basophils, integrating transcriptomic and large-scale immunoprofiling data. We also sought to investigate basophil heterogeneity at the molecular level. Circulating basophils were analyzed using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). Both short- and long-read single-cell RNA-sequencing platforms were used to capture the transcriptomic data. CITE-seq enabled accurate identification and profiling of side scatterlow lineage- CCR3+ FcεRI+ basophils. Short-read single-cell RNA-sequencing data revealed two previously unresolved basophil populations, defined by 66 differentially expressed genes and reproducibly identified across donors. Despite the transcriptional differences, the populations displayed similar immunophenotypes based on more than 100 investigated cell surface markers. Long-read single-cell RNA-sequencing analysis confirmed the existence of the two populations and provided further insights into their gene expression profiles. We present a multimodal single-cell resource that defines two novel transcriptionally distinct basophil populations. This resource, accessible through a user-friendly web interface, constitutes a cellular and molecular reference map for future studies of basophils in health and disease.

The reshuffling of genomic variation from multiple origins is an important contributor to phenotypic diversification, yet insights into the evolutionary trajectories of this combinatorial process and their interplay with genetic architecture remain scarce. We show that convergent plumage color evolution in wheatears involves a monogenic architecture with modular variation introgressed at the agouti signaling protein (ASIP) locus. Introgression of a new transposable element insertion and linked protein-coding variation underpin a transspecific throat color polymorphism, which stable isotopes suggest is associated with alternative foraging niches. Cointrogression of linked regulatory ASIP variation resulted in mantle color convergence in one species, whereas convergent color evolution at the genus level required new variation. Our results demonstrate evolutionary trajectories from introgressed variation realized within the constraints of a monogenic architecture.

Grasshoppers represent true outliers in genome sizes, both within insects and within animals in general. Their genomes are large and generally variable in sizes and feature a high abundance of repetitive DNA sequences. This has hampered the assembly of grasshopper genomes to chromosome level. Here we present a chromosome-level reference genome for the club-legged grasshopper (Gomphocerus sibiricus, Acrididae: Gomphocerinae) using PacBio HiFi long-read and Hi-C sequencing technologies. In male haploid cells, the species has a chromosome set of n = 9 with an X0 sex-determination system, characterized by an absence of a Y chromosome. Our assembly spans 9.57 Gb in total, with 8.87 Gb organized into nine chromosomes-eight autosomes and the X chromosome. The final assembly has a scaffold N50 value of 1.58 Gb, covers 96.7% single copy Insecta orthologs, and contains 42,665 predicted protein-coding genes and 43,385 mRNA transcripts. We compiled a curated, non-redundant, species-specific repeat library and used it to annotate repetitive DNA, covering 81.69% of the genome, mostly DNA transposons, LINE and LTR retrotransposons. The genome of the club-legged grasshopper shows high degree of synteny with the locusts Schistocerca gregaria and Locusta migratoria, and the analysis strongly indicates three autosome-autosome centric fusions in Gomphocerinae. The genome offers a valuable resource for grasshopper genomics and for exploring the genetic basis of a transspecies color polymorphism.

How species adapt to diverse environmental conditions is essential for understanding evolution and the maintenance of biodiversity. The European cisco (Coregonus albula) is a salmonid that occurs in both fresh and brackish water, and this together with the presence of sympatric spring- and autumn-spawning lacustrine populations provides an opportunity for studying the genetics of adaptation in relation to salinity and timing of reproduction. Here, we present a high-quality reference genome of the European cisco based on PacBio HiFi long read sequencing and HiC-directed scaffolding. We generated low-coverage whole-genome sequencing data from 336 individuals across 12 population samples to explore population structure and genetics of ecological adaptation. We found a major subdivision between two groups of populations most likely reflecting colonisation from different glacial refugia. Within the two major groups, we detected further genetic differentiation between spring- and autumn-spawning populations and between populations from freshwater lakes, rivers and brackish water (Bothnian Bay). A genome-wide screen for genetic differentiation among populations identified a set of outlier SNPs strongly correlated with spawning timing and salinity. Several of the genes associated with spawning time, including BHLHE40, TIMELESS and CPT1A, have previously been shown to have a role in circadian rhythm biology. As many as 17 loci were associated with genetic differentiation between populations reproducing in fresh and brackish water. This study provides insights into the genomic basis of ecological adaptation in European cisco with implications for sustainable fishery management.

Chronic lymphocytic leukemia (CLL) comprises immunogenetically defined stereotyped subsets of patients with distinct B-cell receptor (BcR) immunoglobulin features and clinical trajectories, yet the molecular pathways underlying subset-specific differences remain incompletely characterized. To resolve disease-relevant heterogeneity obscured in bulk analyses, we performed integrated single-cell transcriptomic and immunogenetic profiling of 48 557 malignant and bystander immune cells from 13 treatment-naïve primary patient samples representing poor-prognostic subsets #1 and #2 and the indolent subset #4. Despite interpatient variability, leukemic cells exhibited pronounced subset-specific transcriptional features, with enrichment of hypoxia-related genes in subset #1, oxidative phosphorylation (OXPHOS), MYC/E2F targets, and mechanistic target of rapamycin complex 1 (mTORC1) signaling in subset #2, and negative enrichment of hypoxia, apoptosis, and reactive oxygen species-related pathways in subset #4. Notably, compared with indolent subset #4, aggressive subsets #1 and #2 harbored increased proportions of metabolically active and recently emigrated/proliferative leukemic cells, characterized by a CXCR4dim CD5bright transcriptional phenotype and concerted enrichment of MYC target genes, mTORC1 signaling, OXPHOS, and BcR signaling. Immunogenetic analyses revealed dominant malignant clones with evidence of intraclonal immunogenetic diversification in 11 of 13 cases. T cells were dominated by effector-memory phenotypes exhibiting progressive differentiation toward terminal cytotoxic states, accompanied by exhaustion-associated programs, and expanded T-cell clones largely restricted to terminal states. Ligand-receptor analysis further indicated costimulatory and inhibitory signaling between leukemic and immune cells. Together, this study provides an integrated view of the transcriptional and immunogenetic landscape of 3 major stereotyped CLL subsets, linking BcR immunoglobulin configuration with proliferative capacity and microenvironmental cross talk, thereby shaping clonal behavior.

Atypical B-cells (ABCs) are observed across infections, yet their ontogeny and differentiation remain unclear. Using high-dimensional flow cytometry, single-cell transcriptomics, V(D)J sequencing, and functional assays in samples from individuals with malaria and healthy donors, we identify markers of cellular origin while tracking ABC differentiation. Early ABCs express CD11c and arise predominantly from memory B-cells. Two intermediate states originate from germinal center-derived and marginal zone-like B-cells. Pseudotime and lineage analyses reveal trajectories marked by progressive loss of CD24 and CD27, which is recapitulated in vitro, defining bona fide differentiation programs. Differentiation culminates in late ABCs lacking CD24, CD27, and CD21, while expressing CD11c, T-bet, and NKG7 at high levels, exhibiting partial overlap with double-negative-2 B-cells. Early and intermediate stages show greater plasma-cell differentiation potential and cytokine production capacity, whereas later stages acquire transcriptomic signatures of antigen presentation and association with microbial particles, defining stage-dependent functions that reconcile conflicting ABC roles. Comparable ABC populations occur in healthy blood and tonsils, with tissue-associated class switching and CD86 expression consistent with recent T-cell interaction. This framework consolidates ABC definitions along a single differentiation axis and clarifies their cellular origins.

The serotonin system is the main therapeutic target for selective serotonin reuptake inhibitors (SSRIs) in treating depression, yet the mechanism of action of SSRIs remains incompletely understood. To investigate the molecular and transcriptional effects of SSRI administration on serotonin neurons, we performed spatial transcriptomics, a spatially resolved RNA-sequencing method in intact brain tissue. Mouse brain sections containing the dorsal raphe nucleus and adjacent midbrain structures were analyzed, revealing six distinct serotonergic subpopulations with unique molecular signatures and spatial distributions. Both acute and chronic fluoxetine treatment induced a large number of changes in gene expression in the dorsal raphe nucleus. Notably, Htr1a expression increased following acute treatment but decreased after chronic administration, supporting previous findings on serotonin transporter blockade effects on 5-HT1A autoreceptors. Gene enrichment and network analysis identified key pathways modulated by SSRI administration, including Ras, MAPK and cAMP signaling pathways as well as pathways involved in axonal guidance. Additionally, we observed treatment-dependent opposing transcriptional changes in neuropeptides, particularly Thyrotropin-releasing hormone (Trh) and Prodynorphin (Pdyn), with distinct spatial localization within the dorsal raphe nucleus. Collectively, our transcriptomic and in situ hybridization analyses reveal spatial and cell-type-specific heterogeneity in SSRI action within the dorsal raphe nucleus, providing new insights into the molecular basis of SSRI treatment effects.

Human genetic diversity generates an astonishing variety of facial shapes, and craniofacial anomalies rank among the most common birth defects. Identifying the cellular mechanisms that mediate the genome's influence on facial variation remains a challenge. Here we created a multimodal facial atlas across embryonic weeks 6-11, providing single-cell transcriptomics, chromatin accessibility and spatial transcriptomics, all at single-cell resolution. We characterized 56 cell states, mapping mesenchymal subtypes and their gene-enhancer cis-regulatory landscapes in space and time. Gene expression associations with facial traits were strongest in early mesenchymal progenitor cells, gradually becoming more region restricted. Autocorrelation analysis revealed patterning genes that define spatial neighborhoods of mesenchyme, potentially explaining trait specificity of their nearby variants. Enhancers of key pathology-related genes were found to be likely vehicles for generating facial variation in modern human populations. One such enhancer, linked to PAX1 expression, appears to be important for normal skeletal development in mice. Finally, facial effects inferred from the genome-wide association study, cell signaling interaction analysis and validation in mice revealed that peripheral nerves fine-tune maxilla shape during embryonic development. Together, these data offer new insights into the mechanisms underlying human phenotypic individuality and yield a multimodal atlas useful for studying craniofacial development and abnormalities.

Clinical immunity to malaria develops after repeated malaria episodes. In this process, the inflammatory response is modulated to respond less vigorously upon reinfection. Monocytes are a major source of pro-inflammatory mediators during blood-stage infection and are known to adapt to repeated pathogen exposure. Here, we investigated the impact of previous malaria exposure on monocytes during blood-stage malaria by comparing the response in previously exposed and primary infected individuals. We observed reduced levels of several proinflammatory chemokines in previously exposed individuals, linked to changes in monocytes. Similarly, BAFF levels were lower in these individuals and associated with modulation of monocyte and dendritic cells. This affected the BAFF-BAFF-R axis, crucial for B cell responses, correlating with increasing parasite-specific antibody levels. Collectively, we present insights into how previous malaria exposure shapes monocyte responses during acute malaria and how these in turn correlate with modulation of the B cell compartment and humoral immune response. .

Gene activation and repression is an integral part of embryonic development and tissue formation. Changes in chromatin organisation dictate accessibility to gene regulatory elements, controlling gene expression. Although several molecular regulators of lymphatic endothelial cell (LEC) development have been identified, the role of chromatin organisation in the acquisition of LEC identity remains unclear. In this study, we combine HiC and ATAC-sequencing to map 3D chromatin architecture and accessibility in LECs and blood endothelial cells (BECs). We identify cell type-specific topologically associating domains (TADs), and discovered TAD boundaries changes and differentially segregating enhancers in lymphatic-associated loci, such as prox1a and tbx1. Our multi-omic approach also defines the regulatory logic of nine LEC-enriched genes. In vivo validation of ATAC- and HiC-based enhancers confirms their activity in LECs. Leveraging these datasets, we reconstructed mafba tissue-specific regulatory networks identifying a genetic interaction with tfe3a in vivo limiting ectopic vessel formation. Overall, our work provides a powerful resource of multi-omic datasets that can be used to systematically determine the regulatory networks governing LEC identity and genes linked to lymphatic disease.

Mosquitoes transmit arboviruses that represent major global public health challenges. Increasing insecticide resistance and absence of effective antiviral therapies underscore the need for novel vector control strategies. Insect-specific viruses have emerged as candidates for biological control, however, the cellular mechanisms underlying their interactions with mosquito hosts remain poorly understood. Here, we examined the immune response of Aedes albopictus U4.4 cells to Kamiti River virus (KRV) infection, an insect-specific flavivirus. Cells were infected with KRV, and transcriptomic and small RNA profiles were analyzed at 24, 48 and 72 h post-infection. KRV infection induced production of virus-derived small interfering RNAs (vsiRNAs) and virus-derived PIWI-interacting RNA (vpiRNAs) from 24 to 72 h. The vsiRNAs predominantly mapped to the 3' untranslated region of the KRV genome, whereas vpiRNAs formed distinct hotspots in regions encoding the NS1, NS3, NS4A/B and NS5 proteins. Transcriptomic analysis revealed upregulation of genes associated with the humoral immune response, including defensin, cecropin, and glutathione S-transferase, and downregulation of Toll-like receptors and ecdysone-induced transcripts at later stages of infection. These gene expression patterns suggest an early activation followed by suppression of key immune signaling pathways. Collectively, the findings indicate that KRV leads to coordinated modulation of antiviral RNAi and host transcriptional responses, consistent with a balanced, commensal-like interaction in mosquito cells.

Vibrio vulnificus, a coastal marine bacterium and opportunistic pathogen, poses increasing health risks due to rising sea water temperatures. While it harbors diverse virulence factors, its disease mechanisms remain poorly understood. To address this, we sequenced 82 environmental isolates from the Baltic Sea and combined them with 325 published genomes (clinical and environmental) for comparative analysis. Phylogenetic reconstruction revealed four major lineages, with Baltic strains restricted to L2 and L4. Clinical and environmental strains were found across all lineages, suggesting phylogeny reflects environmental adaptation more than pathogenicity. Using our newly developed PhyloBOTL pipeline, we identified 128 orthologs enriched in clinical isolates, grouped into 36 co-localization clusters. These include both known and novel virulence candidates, such as chaperone-usher pili, type VI secretion effectors, and spermidine synthases. Some clusters showed convergent loss across clades, implying niche-specific evolution. We also designed PCR primers targeting these genes to aid in the surveillance of pathogenic V. vulnificus strains.IMPORTANCEVibrio vulnificus is a naturally occurring marine bacterium that can cause life-threatening infections, with incidence increasing as coastal waters warm due to climate change. Determining which environmental strains pose the greatest risk remains a major challenge for public health surveillance. By analyzing hundreds of genomes from globally distributed strains, including extensive sampling from the rapidly warming Baltic Sea, this study shows that pathogenic potential is not restricted to specific evolutionary lineages but is instead associated with distinct sets of genes enriched in clinical strains. To support pathogen detection, we developed PCR primers targeting a subset of these clinically associated genes. Together, these findings provide new insight into the molecular basis of V. vulnificus infections and offer practical tools for improved detection, monitoring, and risk assessment of harmful strains in coastal waters and seafood under ongoing climate change.

22q11.2 deletion syndrome (22q11DS) is associated with congenital anomalies and variable thymic hypoplasia with T cell lymphopenia and immune dysregulation. However, the spatial organization of human thymic lymphopoiesis and stromal mechanisms contributing to thymic dysfunction in 22q11DS remain incompletely defined. We applied spatial transcriptomic and spatial proteomic analyses on thymic samples from two 22q11DS patients and compared them with healthy controls. Across 22q11DS samples, we observed alterations in the corticomedullary organization and in the frequencies of fibroblasts, B cells, regulatory T cells, and mTEC subsets. These features coincided with a prominent remodeling of the mesenchymal compartment, including increased expression of extracellular matrix programs and collagens, and predicted disruption in mesenchymal-epithelial cell crosstalk. In the medulla, we observed alterations in interferon-associated gene programs within a colocalized niche comprising B cells, antigen-presenting cells, and mTEC subsets. Together, this provides an integrated spatial map of the 22q11DS thymus and nominates stromal remodeling as a candidate driver of impaired central tolerance induction in 22q11DS.