Scientific highlights

Transcriptomic and genomic analyses of bladder cancer (BC) reveal a highly diverse disease stratified into molecular subtypes with distinct molecular features and biological behaviors. Intratumor heterogeneity (ITH) and plasticity can significantly impact diagnosis and patient management, yet their extent in BC remains highly debated. Here, we investigated whether the three main bladder cancer subtypes maintain or alter their identity in response to changes in the microenvironment and during metastatic colonization. Seven patient-derived xenograft (PDX) models representing the major BC subtypes were propagated into three distinct tissue microenvironments: subcutaneous, mammary fat pad and under the kidney capsule. Metastatic lesions were generated via systemic injection of tumor cells. Tumor samples were analysed using RNA- and exome sequencing, SNP-arrays and histopathology to assess subtype fidelity, genomic evolution, and clonal dynamics. A comprehensive, longitudinal multiomics analysis showed that tumors consistently maintain their molecular subtype, as well as their transcriptomic and genomic profiles, across different environments. No evidence of emerging ITH or subtype transitions was observed, regardless of the microenvironment. The transcriptomic adaptations observed in metastases and different implantation sites are limited and are associated primarily with hypoxia, epithelial-mesenchymal transition (EMT), and invasion. Our results suggest that invasive bladder cancers have a strong intrinsic tumor identity that is not easily reprogrammed by the microenvironment.

Through biosilicification, organisms incorporate dissolved silica (dSi) and deposit it as biogenic silica (bSi), driving the silicon (Si) cycle in aquatic systems. While Si accumulation in marine picocyanobacteria has been recently observed, its mechanisms and ecological implications remain unclear. This study investigates biosilicification in marine and brackish picocyanobacteria of the Synechococcus clade and two model freshwater coccoid cyanobacteria. Brackish strains showed significantly higher Si quotas when supplemented with external dSi (100 µM) compared to controls (up to 60.0 ± 7.3 amol Si.cell-1 versus 9.2 to 16.3 ± 2.9 amol Si.cell-1). Conversely, freshwater strains displayed no significant differences in Si quotas between dSi-enriched treatments and controls, emphasizing that not all phytoplanktons without an obligate Si requirement accumulate this element. The Si-accumulating marine and brackish picocyanobacteria clustered within the Synechococcus clade, whereas their freshwater counterparts formed a distinct sister group, suggesting a link between phylogeny and silicification. Rapid culture growth caused increased pH and led to dSi precipitation, influencing apparent dSi uptake; this was mitigated by pH control through bubbling. This phenomenon has significant implications for natural systems affected by phytoplankton blooms. In such environments, pH-induced silicon precipitation may reduce dSi availability impacting Si-dependent populations like diatoms. Our findings suggest brackish picocyanobacteria could significantly influence the Si cycle through at least two mechanisms: cellular Si accumulation and biologically induced changes in dSi concentrations.IMPORTANCEThis work provides the first evidence of biogenic silica accumulation in brackish picocyanobacteria and uncovers a link between phylogeny and biosilicification patterns. Our findings demonstrate that picocyanobacterial growth induces pH-dependent silica precipitation, which could lead to overestimations of cellular Si quotas by up to 85%. This process may drive substantial silica precipitation in highly productive freshwater and coastal marine systems, with potential effects on silica cycling and the population dynamics of Si-dependent phytoplankton. The extent of biosilicification in modern picocyanobacteria offers insights into the rock record, shedding light on the evolutionary and ecological dynamics that influence sedimentary processes and the preservation of biosilicification signatures in geological formations. Overall, this research adds to the significant impact that microorganisms lacking an obligate silica requirement may have on silica dynamics.

Chronic kidney disease (CKD) affects 10% of the human population, with only a small fraction genetically defined. CKD is also common in dogs and has been diagnosed in nearly all breeds, but its genetic basis remains unclear. Here, we performed a Bayesian mixed model genome-wide association analysis for canine CKD in a boxer population of 117 canine cases and 137 controls, and identified 21 genetic regions associated with the disease. At the top markers from each CKD region, the cases carried an average of 20.2 risk alleles, significantly higher than controls (15.6 risk alleles). An ANOVA test showed that the 21 CKD regions together explained 57% of CKD phenotypic variation in the population. Based on whole genome sequencing data of 20 boxers, we identified 5,206 variants in LD with the top 50 BayesR markers. Following comparative analysis with human regulatory data, 17 putative regulatory variants were identified and tested with electrophoretic mobility shift assays. In total four variants, three intronic variants from the MAGI2 and GALNT18 genes, and one variant in an intergenic region on chr28, showed alternative binding ability for the risk and protective alleles in kidney cell lines. Many genes from the 21 CKD regions, RELN, MAGI2, FGFR2 and others, have been implicated in human kidney development or disease. The results from this study provide new information that may enlighten the etiology of CKD in both dogs and humans.

As the only endemic neotropical parrot to have recently lived in the northern hemisphere, the Carolina parakeet (Conuropsis carolinensis) was an iconic North American bird. The last surviving specimen died in the Cincinnati Zoo in 1918 [1]. The cause of its extinction remains contentious: besides excessive mortality associated to habitat destruction and active hunting, their survival could have been negatively affected by its range having become increasingly patchy [2] or by the exposure to poultry pathogens [3, 4]. In addition, the Carolina parakeet showed a predilection for cockleburs, an herbaceous plant that contains a powerful toxin, carboxyatractyloside, or CAT [5], which did not seem to affect them but made the birds notoriously toxic to most predators [3]. To explore the demographic history of this bird, we generated the complete genomic sequence of a preserved specimen held in a private collection in Espinelves (Girona, Spain), as well as of a close extant relative, Aratinga solstitialis. We identified two non-synonymous genetic changes in two highly conserved proteins known to interact with CAT that could underlie a specific dietary adaptation to this toxin. Our genomic analyses did not reveal evidence of a dramatic past demographic decline in the Carolina parakeet; also, its genome did not exhibit the long runs of homozygosity that are signals of recent inbreeding and are typically found in endangered species. As such, our results suggest its extinction was an abrupt process and thus likely solely attributable to human causes.

High-throughput sequencing was applied to investigate the mutation/methylation patterns on 1q and gene expression profiles in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) with/without (w/wo) dup(1q). Sequencing of the breakpoint regions and all exons on 1q in seven dup(1q)-positive cases revealed non-synonymous somatic single nucleotide variants (SNVs) in BLZF1, FMN2, KCNT2, LCE1C, NES, and PARP1. Deep sequencing of these in a validation cohort w (n = 17)/wo (n = 94) dup(1q) revealed similar SNV frequencies in the two groups (47% vs. 35%; P = 0.42). Only 0.6% of the 36,259 CpGs on 1q were differentially methylated between cases w (n = 14)/wo (n = 13) dup(1q). RNA sequencing of high hyperdiploid (HeH) and t(1;19)(q23;p13)-positive cases w (n = 14)/wo (n = 52) dup(1q) identified 252 and 424 differentially expressed genes, respectively; only seven overlapped. Of the overexpressed genes in the HeH and t(1;19) groups, 23 and 31%, respectively, mapped to 1q; 60-80% of these encode nucleic acid/protein binding factors or proteins with catalytic activity. We conclude that the pathogenetically important consequence of dup(1q) in BCP ALL is a gene-dosage effect, with the deregulated genes differing between genetic subtypes, but involving similar molecular functions, biological processes, and protein classes.

SOX transcription factors have important roles during astrocyte and oligodendrocyte development, but how glial genes are specified and activated in a sub-lineage-specific fashion remains unknown. Here, we define glial-specific gene expression in the developing spinal cord using single-cell RNA-sequencing. Moreover, by ChIP-seq analyses we show that these glial gene sets are extensively preselected already in multipotent neural precursor cells through prebinding by SOX3. In the subsequent lineage-restricted glial precursor cells, astrocyte genes become additionally targeted by SOX9 at DNA regions strongly enriched for Nfi binding motifs. Oligodendrocyte genes instead are prebound by SOX9 only, at sites which during oligodendrocyte maturation are targeted by SOX10. Interestingly, reporter gene assays and functional studies in the spinal cord reveal that SOX3 binding represses the synergistic activation of astrocyte genes by SOX9 and NFIA, whereas oligodendrocyte genes are activated in a combinatorial manner by SOX9 and SOX10. These genome-wide studies demonstrate how sequentially expressed SOX proteins act on lineage-specific regulatory DNA elements to coordinate glial gene expression both in a temporal and in a sub-lineage-specific fashion.

DNA-assisted proteomics technologies enable ultra-sensitive measurements in multiplex format using DNA-barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio-conjugation protocols. Here, we introduce a magnetic bead-assisted DNA-barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand-fold. The success of DNA-barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno-PCR assays. Specific DNA-barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read-out on a massively parallel sequencing platform in a procedure denoted Immuno-Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications.

The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important pathogenic DNA mutation signature in genomic disorders and cancer genomes, its architecture remains unresolved. Here, we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the DNA of 24 patients identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted structural variant (SV) haplotypes. Using a combination of short-read genome sequencing (GS), long-read GS, optical genome mapping, and single-cell DNA template strand sequencing (strand-seq), the haplotype structure was resolved in 18 samples. The point of template switching in 4 samples was shown to be a segment of ∼2.2-5.5 kb of 100% nucleotide similarity within inverted repeat pairs. These data provide experimental evidence that inverted low-copy repeats act as recombinant substrates. This type of CGR can result in multiple conformers generating diverse SV haplotypes in susceptible dosage-sensitive loci.

Persistent taxane-induced peripheral neuropathy (TIPN) is highly prevalent among early-stage breast cancer survivors (ESBCS) and has detrimental effect on quality of life. We leveraged logistic regression models to develop and validate polygenic prediction models to estimate the risk of persistent PN symptoms in a training cohort and validation cohort taking clinical risk factors into account. Based on 337 whole-exome sequenced ESBCS two of five prediction models for individual PN symptoms obtained AUC results above 60% when validated. Using the model for numbness in feet (35 SNVs) in the test cohort, 73% survivors were correctly predicted. For tingling in feet (55 SNVs) 70% were correctly predicted. Both models included SNVs from the ADAMTS20, APT6V0A2, CCDC88C, CYP2C8, EPHA5, NR1H3, PSKH2/APTV0D2, and SCN10A genes. For cramps in feet, difficulty climbing stairs and difficulty opening a jar the validation was unsuccessful. Polygenic prediction models including clinical risk factors can estimate the risk of persistent taxane-induced numbness in feet and tingling in feet in ESBCS.

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.

Organisms inhabiting highly seasonal environments must cope with a wide range of environmentally induced challenges. Many seasonal challenges require extensive physiological modification to survive. In winter, to survive extreme cold and limited resources, insects commonly enter diapause, which is an endogenously derived dormant state associated with minimized cellular processes and low energetic expenditure. Due to the high degree of complexity involved in diapause, substantial cellular regulation is required, of which our understanding primarily derives from the transcriptome via messenger RNA expression dynamics. Here we aim to advance our understanding of diapause by investigating microRNA (miRNA) expression in diapausing and direct developing pupae of the butterfly Pieris napi. We identified coordinated patterns of miRNA expression throughout diapause in both head and abdomen tissues of pupae, and via miRNA target identification, found several expression patterns to be enriched for relevant diapause-related physiological processes. We also identified two candidate miRNAs, miR-14-5p and miR-2a-3p, that are likely involved in diapause progression through their activity in the ecdysone pathway, a critical regulator of diapause termination. miR-14-5p targets phantom, a gene in the ecdysone synthesis pathway, and is upregulated early in diapause. miR-2a-3p has been found to be expressed in response to ecdysone, and is upregulated during diapause termination. Together, the expression patterns of these two miRNAs match our current understanding of the timing of hormonal regulation of diapause in P. napi and provide interesting candidates to further explore the mechanistic role of microRNAs in diapause regulation.

Why species that in their core areas mainly reproduce sexually become enriched with clones in marginal populations ("geographic parthenogenesis") remains unclear. Earlier hypotheses have emphasized that selection might promote clonality because it protects locally adapted genotypes. On the other hand, it also hampers recombination and adaptation to changing conditions. The aim of the present study was to investigate the early stages of range expansion in a partially clonal species and what drives an increase in cloning during such expansion. We used genome-wide sequencing to investigate the origin and evolution of large clones formed in a macroalgal species (Fucus vesiculosus) during a recent expansion into the postglacial Baltic Sea. We found low but persistent clonality in core populations, while at range margins, large dominant clonal lineages had evolved repeatedly from different sexual populations. A range expansion model showed that even when asexual recruitment is less favourable than sexual recruitment in core populations, repeated bottlenecks at the expansion front can establish a genetically eroded clonal wave that spreads ahead of a sexual wave into the new area. Genetic variation decreases by drift following repeated bottlenecks at the expansion front. This results in the emerging clones having low expected heterozygosity, which corroborated our empirical observations. We conclude that Baker's Law (clones being favoured by uniparental reproductive assurance in new areas) can play an important role during range expansion in partially clonal species, resulting in a complex spatiotemporal mosaic of clonal and sexual lineages that might persist during thousands of generations.

We examined transposable element (TE) content of 248 placental mammal genome assemblies, the largest de novo TE curation effort in eukaryotes to date. We found that although mammals resemble one another in total TE content and diversity, they show substantial differences with regard to recent TE accumulation. This includes multiple recent expansion and quiescence events across the mammalian tree. Young TEs, particularly long interspersed elements, drive increases in genome size, whereas DNA transposons are associated with smaller genomes. Mammals tend to accumulate only a few types of TEs at any given time, with one TE type dominating. We also found association between dietary habit and the presence of DNA transposon invasions. These detailed annotations will serve as a benchmark for future comparative TE analyses among placental mammals.

Oomycetes cause several damaging diseases of plants and animals, and some species also act as biocontrol agents on insects, fungi, and other oomycetes. RNA silencing is increasingly being shown to play a role in the pathogenicity of Phytophthora species, either through trans-boundary movement of small RNAs (sRNAs) or through expression regulation of infection promoting effectors. To gain a wider understanding of RNA silencing in oomycete species with more diverse hosts, we mined genome assemblies for Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDRP) proteins from Phytophthora plurivora, Ph. cactorum, Ph. colocasiae, Pythium oligandrum, Py. periplocum, and Lagenidium giganteum. Moreover, we sequenced small RNAs from the mycelium stage in each of these species. Each of the species possessed a single DCL protein, but they differed in the number and sequence of AGOs and RDRPs. SRNAs of 21nt, 25nt, and 26nt were prevalent in all oomycetes analyzed, but the relative abundance and 5' base preference of these classes differed markedly between genera. Most sRNAs mapped to transposons and other repeats, signifying that the major role for RNA silencing in oomycetes is to limit the expansion of these elements. We also found that sRNAs may act to regulate the expression of duplicated genes. Other sRNAs mapped to several gene families, and this number was higher in Pythium spp., suggesting a role of RNA silencing in regulating gene expression. Genes for most effector classes were the source of sRNAs of variable size, but some gene families showed a preference for specific classes of sRNAs, such as 25/26 nt sRNAs targeting RxLR effector genes in Phytophthora species. Novel miRNA-like RNAs (milRNAs) were discovered in all species, and two were predicted to target transcripts for RxLR effectors in Ph. plurivora and Ph. cactorum, indicating a putative role in regulating infection. Moreover, milRNAs from the biocontrol Pythium species had matches in the predicted transcriptome of Phytophthora infestans and Botrytis cinerea, and L. giganteum milRNAs matched candidate genes in the mosquito Aedes aegypti. This suggests that trans-boundary RNA silencing may have a role in the biocontrol action of these oomycetes.

How sexual selection affects the genome ultimately relies on the strength and type of selection, and the genetic architecture of the involved traits. While associating genotype with phenotype often utilizes standard trait morphology, trait representations in morphospace using geometric morphometric approaches receive less focus in this regard. Here, we identify genetic associations to a sexual ornament, the comb, in the chicken system (Gallus gallus). Our approach combined genome-wide genotype and gene expression data (>30k genes) with different aspects of comb morphology in an advanced intercross line (F8) generated by crossing a wild-type Red Junglefowl with a domestic breed of chicken (White Leghorn). In total, 10 quantitative trait loci were found associated to various aspects of comb shape and size, while 1,184 expression QTL were found associated to gene expression patterns, among which 98 had overlapping confidence intervals with those of quantitative trait loci. Our results highlight both known genomic regions confirming previous records of a large effect quantitative trait loci associated to comb size, and novel quantitative trait loci associated to comb shape. Genes were considered candidates affecting comb morphology if they were found within both confidence intervals of the underlying quantitative trait loci and eQTL. Overlaps between quantitative trait loci and genome-wide selective sweeps identified in a previous study revealed that only loci associated to comb size may be experiencing on-going selection under domestication.

The relative role of genetic adaptation and phenotypic plasticity is of fundamental importance in evolutionary ecology [M. J. West-Eberhard, Proc. Natl. Acad. Sci. U.S.A. 102 (suppl. 1), 6543-6549 (2005)]. European eels have a complex life cycle, including transitions between life stages across ecological conditions in the Sargasso Sea, where spawning occurs, and those in brackish and freshwater bodies from northern Europe to northern Africa. Whether continental eel populations consist of locally adapted and genetically distinct populations or comprise a single panmictic population has received conflicting support. Here we use whole-genome sequencing and show that European eels belong to one panmictic population. A complete lack of geographical genetic differentiation is demonstrated. We postulate that this is possible because the most critical life stages-spawning and embryonic development-take place under near-identical conditions in the Sargasso Sea. We further show that within-generation selection, which has recently been proposed as a mechanism for genetic adaptation in eels, can only marginally change allele frequencies between cohorts of eels from different geographic regions. Our results strongly indicate plasticity as the predominant mechanism for how eels respond to diverse environmental conditions during postlarval stages, ultimately solving a long-standing question for a classically enigmatic species.

Ancient DNA provides a powerful means to investigate the timing, rate and extent of population declines caused by extrinsic factors, such as past climate change and human activities. One species probably affected by both these factors is the arctic fox, which had a large distribution during the last glaciation that subsequently contracted at the start of the Holocene. More recently, the arctic fox population in Scandinavia went through a demographic bottleneck owing to human persecution. To investigate the consequences of these processes, we generated mitogenome sequences from a temporal dataset comprising Pleistocene, historical and modern arctic fox samples. We found no evidence that Pleistocene populations in mid-latitude Europe or Russia contributed to the present-day gene pool of the Scandinavian population, suggesting that postglacial climate warming led to local population extinctions. Furthermore, during the twentieth-century bottleneck in Scandinavia, at least half of the mitogenome haplotypes were lost, consistent with a 20-fold reduction in female effective population size. In conclusion, these results suggest that the arctic fox in mainland Western Europe has lost genetic diversity as a result of both past climate change and human persecution. Consequently, it might be particularly vulnerable to the future challenges posed by climate change. This article is part of a discussion meeting issue 'The past is a foreign country: how much can the fossil record actually inform conservation?'