Publications - Publikationer

Retrospective and multifactorial single-cell profiling reveals sequential chromatin reorganization during X inactivation

Kefalopoulou, S., Rullens, P. M.J., de Luca, K. L., et al. — Tue, 01 Jul 2025 07:16:26 +0200

The regulation of gene expression is governed at multiple levels of chromatin organization. However, how gene regulation is co-ordinated remains relatively unexplored. Here we develop Dam&ChIC, a method that enables retrospective and multifactorial chromatin profiling in single cells. Dam&ChIC employs chromatin labelling in living cells with ^m6A to acquire a past chromatin state, coupled with an antibody-mediated readout to capture the present chromatin state. Analyses of diverse factor combinations highlight its versatility and superior resolution. By tracking lamina-associated domain inheritance over the cell cycle, we showcase that Dam&ChIC provides retrospective single-cell chromatin data. When applied in random X chromosome inactivation, Dam&ChIC disentangles the temporal order of chromatin remodelling events. Upon mitotic exit and following Xist expression, the inactive X chromosome undergoes extensive genome–lamina detachment, preceding spreading of Polycomb. We anticipate that Dam&ChIC will be instrumental in unravelling the interconnectivity and order of gene-regulatory events underlying cell-state changes during development.

DNA methylation cooperates with genomic alterations during non-small cell lung cancer evolution

Gimeno-Valiente, F., Castignani, C., Larose Cadieux, E., et al. — Mon, 01 Sep 2025 07:16:26 +0200

Aberrant DNA methylation has been described in nearly all human cancers, yet its interplay with genomic alterations during tumor evolution is poorly understood. To explore this, we performed reduced representation bisulfite sequencing on 217 tumor and matched normal regions from 59 patients with non-small cell lung cancer from the TRACERx study to deconvolve tumor methylation. We developed two metrics for integrative evolutionary analysis with DNA and RNA sequencing data. Intratumoral methylation distance quantifies intratumor DNA methylation heterogeneity. M_R/M_N classifies genes based on the rate of hypermethylation at regulatory (M_R) versus nonregulatory (M_N) CpGs to identify driver genes exhibiting recurrent functional hypermethylation. We identified DNA methylation-linked dosage compensation of essential genes co-amplified with neighboring oncogenes. We propose two complementary mechanisms that converge for copy number alteration-affected chromatin to undergo the epigenetic equivalent of an allosteric activity transition. Hypermethylated driver genes under positive selection may open avenues for therapeutic stratification of patients.

Pathological Folding of α-Synuclein on Polystyrene Nanoplastic Revealed by Sum Frequency Scattering and 2D Infrared Spectroscopy

Mishra, A., Golbek, T. W., Thomassen, A. B., et al. — Thu, 06 Nov 2025 07:16:26 +0100

The impact of micro- and nanoplastics (MNPs) on human health is a growing field of research. Reports that MNPs can breach the blood-brain barrier and accumulate inside the brain have raised concerns over their possible involvement in the development of neurogenerative diseases. The aggregation of the abundant neuronal protein α-synuclein (α-syn) is pertinent to almost 50 neurological diseases including Parkinson's disease (PD). The role of nanoplastics in the formation of toxic aggregates is unclear and has been shown to depend strongly on the type of plastics. Here we report the molecular structure and orientation of human α-syn adsorbed on polystyrene NPs using interface-specific sum frequency scattering (SFS) and structure-sensitive two-dimensional infrared (2D IR) spectroscopy. The SFS experimental data were compared with the calculated spectra of several thousands of α-syn conformations generated from molecular dynamics simulations. The SFS results reveal that α-syn folds on polystyrene nanoplastics, adopting a partly helical structure with the N-terminus and nonamyloid component regions directly bound on the polystyrene nanosurface, while the C terminus protrudes away from the polystyrene interface. 2D IR results suggest that the entire α-syn corona comprises of partly aggregated α-syn structures, built of an ordered core enclosed with flexible dynamic regions. The data shed light on the mechanism by which α-syn folds and forms aggregates at the plastic particle surfaces, a link that has been missing in understanding the role of nanoplastic in the pathogenesis of PD and related neurodegenerative diseases.

Open problems in ageing science

Talay, A., Belikov, A. V., To, P. K. P., et al. — Sat, 08 Nov 2025 07:16:26 +0100

The field of ageing science has gone through remarkable progress in recent decades, yet many fundamental questions remain unanswered or unexplored. Here we present a curated list of 100 open problems in ageing and longevity science. These questions were collected through community engagement and further analysed using Natural Language Processing to assess their prevalence in the literature and to identify both well-established and emerging research gaps. The final list is categorised into different topics, including molecular and cellular mechanisms of ageing, comparative biology and the use of model organisms, biomarkers and the development of therapeutic interventions. Both long-standing questions and more recent and specific questions are featured. Our comprehensive compilation is available to the biogerontology community on our website ( www.longevityknowledge.app ). Overall, this work highlights current key research questions in ageing biology and offers a roadmap for fostering future progress in biogerontology.

Catalytic mechanism and differential alarmone regulation of a conserved stringent nucleosidase

Bærentsen, R. L., Kronborg, K., Brodersen, D. E., Zhang, Y. E. — Wed, 05 Nov 2025 07:16:26 +0100

Insights into bacterial metabolic adaptation during stress is crucial for understanding early mechanisms of antibiotic resistance. In the Gram-negative bacterium Escherichia coli, the universal stringent response produces the alarmones (p)ppGpp that target many cellular proteins. The cellular nucleosidase PpnN is regulated by (p)ppGpp and was shown to balance bacterial fitness and persistence during fluoroquinolone exposure. pppGpp and ppGpp both activate PpnN, but differentially regulate its cooperativity via an unknown mechanism; furthermore, the catalytic mechanism of PpnN has remained unclear. Here, we provide mechanistic insights into the interaction of PpnN with a substrate analogue, reaction products, and alarmone molecules, which allows us to understand the catalytic mechanism of this family of nucleosidases and the differential modes of regulation by ppGpp and pppGpp, respectively. Comparison to the homologous plant cytokinin-producing LOG proteins reveals that PpnN utilizes an evolutionarily conserved purine hydrolysis mechanism, which in bacteria is regulated by alarmones during stress.

BRCA2 deficiency and replication stress drive APOBEC3-Mediated genomic instability

Situ, K., Duan, H., Godin, S. K., et al. — Mon, 01 Dec 2025 07:16:26 +0100

BRCA2 plays a critical role in stabilizing stalled replication forks, yet critical gaps remain in understanding how BRCA2 deficiency triggers fork collapse and drives genomic instability. Here, we identify cytidine deaminase APOBEC3B as a key driver of this process. Using a unique uracil-in-DNA probe, we show that BRCA2 loss promotes APOBEC3B-mediated uracil accumulation in single-stranded DNA (U-ssDNA) at stalled forks. These lesions when processed by UNG2 and APE1, trigger fork collapse and release ssDNA fragments into the cytoplasm, activating NF-κB signaling. This in turn upregulates APOBEC3B expression, establishing a self-reinforcing loop that amplifies cytidine deamination at stalled forks and exacerbates genomic instability. Depletion of APOBEC3B, UNG2, or APE1 rescues these defects. Notably, BRCA1-deficient cells do not accumulate U-ssDNA or induce APOBEC3B under replication stress, highlighting a BRCA2-specific vulnerability. Clinically, low APE1 expression correlates with poor survival in patients with BRCA2-mutant tumors, with high APOBEC3 levels further worsening outcomes. Together, our findings establish that replication stress, whether intrinsic or therapy induced, triggers APOBEC3B overexpression and potentially activates an APOBEC3B-driven mutagenic loop in BRCA2-deficient cells. These results position APOBEC3B, UNG2 and APE1 as critical regulators of BRCA2-mutant tumor evolution and therapy resistance.

Contrasting Risk Profiles for Suicide Attempt and Suicide Using Danish Registers and Genetic Data

Ge, F., Wang, Y., Agerbo, E., et al. — Tue, 21 Oct 2025 07:16:26 +0200

IMPORTANCE: Not all individuals who die by suicide have a history of nonfatal suicide attempt (SA); however, little is known about the extent to which the genetic and environmental etiologies of SA and suicide are shared or distinct.

OBJECTIVE: To examine shared and distinct risk factors for SA and suicide, focusing on clinically diagnosed health conditions and genetic factors.

DESIGN, SETTING, AND PARTICIPANTS: For health conditions, a nested case-control study was performed using data from the Danish registers. For genetic factors, a case-control analysis framework was used, with individual genotypes retrieved from the iPSYCH2015 dataset, which was nested within the entire Danish population. Individuals older than 10 years were included to minimize the risk of misclassification for SA and suicide. Data were analyzed from January 2024 to April 2025.

EXPOSURES: Twenty-eight health conditions and 35 polygenic scores (PGSs) for complex traits.

MAIN OUTCOMES AND MEASURES: The primary outcomes were SA, suicide, and cumulative SA burden. Associations between health conditions and the risk of SA and suicide were assessed using conditional logistic regression. PGSs for complex traits were calculated using LDpred2-auto, and their associations with SA and suicide were evaluated via logistic regression. To assess whether effect sizes differed significantly between SA and suicide, bayesian model-based classification and Cochran Q test were applied.

RESULTS: A total of 81 713 cases of SA (50 512 [61.8%] female; mean [SD] age, 32.3 [14.9] years), with 408 490 age-matched controls (252 525 [61.8%] female; mean [SD] age, 32.3 [14.9] years), and 9362 cases of suicide (2360 [25.2%] female; mean [SD] age, 45.1 [14.6] years), along with 46 749 matched controls (11 796 [25.2%] female; mean [SD] age, 45.1 [14.6] years) who were alive at the date of the case's death, were included in the health conditions analysis. The PGS analysis included 8221 cases of SA (5944 [72.3%] female; mean [SD] age, 19.7 [4.4] years) and 225 cases of suicide (80 [35.6%] female; mean [SD] age, 24.6 [5.0] years). Chronic diseases (eg, dyslipidemia or hearing problems) showed stronger associations with SA, while severe conditions (eg, cancer) were more strongly associated with suicide. Suicide was influenced only by PGSs for mental disorders, whereas SA was associated with both psychiatric and broader health-related genetic risk factors. Notably, dose-response associations were observed for most health conditions and PGSs in relation to cumulative SA burden.

CONCLUSIONS AND RELEVANCE: A broad range of health conditions and genetic factors were associated with increased risk of both outcomes; however, their shared and distinct risk factors suggest that SA and death by suicide are not solely differentiated by liability severity.

Maternal body mass index in early pregnancy and autism in offspring

Morin, M., Yin, W., MacLean, H., et al. — Fri, 07 Nov 2025 07:16:26 +0100

BACKGROUND: Elevated maternal pre-pregnancy body mass index (BMI) has been suggested to increase risk of offspring autism spectrum disorder (ASD) but evidence is mixed across heterogeneous studies and robust estimates spanning the full BMI range are lacking. This study examined the association between maternal BMI and offspring ASD in a harmonized, two-nation study and across the full BMI range.

METHODS: We included all singleton children born in Denmark 2004-2018 and Sweden 1998-2019 to parents of Nordic origin (n = 2,072,445), with follow-up from age 2 until 31 December 2021, or 2022, respectively. Maternal BMI recorded at the first antenatal visit was obtained from the Swedish and Danish Medical Birth Registers and was analyzed as a continuous variable and in World Health Organization-defined categories of underweight (BMI < 18.5), normal weight (18.5-24.9), overweight (25-29.9), obese class I (30-34.9), and obese class II-III (≥ 35). The relative risk of ASD was estimated as hazard ratios (HR) from Cox regression models, adjusted for birth year and parental age, educational level, income, and psychiatric history at time of childbirth, using data from national health and population registers. Both country-specific and pooled analyses were conducted. Subgroup and sensitivity analyses, including a sibling comparison, were performed to address the specificity and robustness of findings.

RESULTS: A total of 58,416 (2.8%) children were diagnosed with ASD during follow-up. The risk of ASD exhibited a J-shaped association with BMI, which gradually increased for mothers with both lower and higher BMI compared to BMI 22 (mid-normal range) (HR = 1.16 [95% CI 1.06-1.27] for BMI 15, and HR = 1.50 [95% CI 1.46-1.53] for BMI 30 in the fully adjusted model). Adjustment for familial factors in a sibling comparison attenuated associations.

CONCLUSIONS: Both high and low maternal BMI are associated with an increased risk of ASD in the offspring. Familial factors, including genetic and environmental components consistent between siblings, may explain part of the association.

Atmospheric Biogenic Ice-Nucleating Particles Link to Microbial Communities in the Arctic Marine Environment in Western Greenland

Castenschiold, C. D.F., Mignani, C., Christiansen, S., et al. — Tue, 28 Oct 2025 07:16:26 +0100

Biogenic ice-nucleating particles (INPs) can significantly impact mixed-phase clouds by enhancing precipitation and reducing albedo. As Arctic sea ice diminishes, the exposure of open ocean may increase aerosolization rates of marine bioaerosols and INPs. We investigated INP concentrations and microbial communities in ambient marine air, sea bulk water (SBW), and sea surface microlayer (SML) along a transect from the Davis Strait to Baffin Bay. INP concentrations in SBW increased with latitude, regardless of the extent of terrestrial freshwater input. We further identified correlations between INP levels and abundances of specific microbial taxa, including Formosa, Lewinella, Micromonas, and Dino-Group-I-Clade-5, suggesting potential ice nucleation activity of these taxa. Air samples exhibited distinct microbiomes compared to seawater, indicating terrestrial contributions, but at the highest observed wind speeds (7–8 m/s), substantial contributions of the seawater microbiome were detected in the air. Elevated atmospheric INP concentrations at higher latitudes correlated with seawater INP levels, which was supported by laboratory sea spray experiments showing that INPs in SBW influenced aerosol INP levels. Our findings highlight the Arctic Ocean as a significant source of biogenic atmospheric INPs and enhance our understanding of marine microbes as contributors to biogenic INPs. By identification of potential ice nucleation active microbial taxa and examination of aerosolization processes, this study provides a framework for future research on Arctic marine-derived INPs and their atmospheric impact.

Nanoscale Precise Stamping of Biomolecule Patterns Using DNA Origami

Teodori, L., Shahrokhtash, A., Sørensen, E. A., et al. — Tue, 28 Oct 2025 07:16:26 +0100

Understanding the importance of ligand patterning in biological processes requires precise control over molecular positioning and spacing. While DNA origami structures offer nanoscale precision in biomolecule arrangement, their biological applications are limited by challenges related to their structural stability, scalability, and surface area. Here, we present a straightforward and rapid DNA origami stamping technique for transferring nanoscale oligonucleotide patterns onto surfaces, visualized using DNA-PAINT super-resolution microscopy to quantitatively assess the stamping efficiency and precision across different stamp types. Unlike traditional top-down methods that require specialized equipment, our technique provides an accessible, self-assembled platform for surface patterning, with versatility across various substrates via modifiable pattern-transfer oligonucleotides. We demonstrate reliable, efficient, and precise pattern transfer at single-molecule resolution, opening opportunities to study distance-dependent biological processes, including receptor activation, multivalent binding, and enzymatic cascades across broader spatial scales and different detection techniques. The use of the passivated surface limits nonspecific interactions with unpatterned areas and enables control over the interaction between the biological target and the patterned biomolecules. Our method advances surface patterning by combining DNA nanotechnology with single-molecule imaging techniques, expanding access to cost-effective analytical approaches and potentially enabling multiplexed detection and live measurements.

Changes in polygenic burden for psychiatric disorders across two decades of birth cohorts

Lousdal, M. L., LaBianca, S., Agerbo, E., et al. — Mon, 01 Sep 2025 07:16:26 +0200

During recent decades, the incidence of several psychiatric disorders has increased, but no previous study has investigated whether the polygenic burden based on common variants for psychiatric disorders in diagnosed individuals has changed over time. Here we aimed to explore changes in polygenic scores for schizophrenia, depression, autism and attention deficit hyperactivity disorder (ADHD) in the general population and in case populations according to birth cohorts. The iPSYCH2015 is a Danish population-based case–cohort study, including individuals born between 1981 and 2008, who were followed for a psychiatric diagnosis between 1994 and 2015. We included 41,132 individuals from the random subcohort and 60,293 individuals diagnosed with schizophrenia spectrum disorders, depression, autism or ADHD. We estimated changes in polygenic scores across birth years based on linear regression. The average polygenic score was stable in the random subcohort but decreased across birth years in case populations, most predominantly for schizophrenia (per 10 years: −0.13 s.d., 95% confidence interval (CI) −0.18 to −0.07) but also for depression (−0.06 s.d., 95% CI −0.10 to −0.03) and autism (−0.08 s.d., 95% CI −0.13 to −0.04) and to a limited degree for ADHD (−0.03 s.d., 95% CI −0.08 to 0.02). Moreover, we estimated how the hazard ratio for being diagnosed given a 1 s.d. increase in polygenic score changed according to birth year, which decreased for schizophrenia but remained stable for the other disorders. Finally, we estimated the number of additional cases per 1 s.d. increase in polygenic score according to birth year, which decreased for both schizophrenia and depression, whereas autism and ADHD showed increases. In conclusion, the polygenic burden for psychiatric disorders changed across two decades among diagnosed individuals in Denmark. For schizophrenia, the polygenic score itself and its predictive ability decreased over time, whereas depression, autism and ADHD showed diverse changes.

Population-representative inference for primary and secondary outcomes in extended case-cohort designs

Wimberley, T., Plana-Ripoll, O., Pedersen, C. B., et al. — Mon, 01 Dec 2025 07:16:26 +0100

Background: The case-cohort design is useful for obtaining population-representative inference with smaller samples, but the performance when dealing with secondary outcomes and extended follow-up remains unclear. We compare the case-cohort design with the full-cohort design across various outcomes, including additional case-groups and extended follow-up, illustrated using the iPSYCH study. Methods: From Danish nationwide registers, we identified the full-population cohort of all individuals born 1981–2008 (n = 1,657,449) and their clinical diagnoses until 2021. The iPSYCH case-cohort sample (n = 141,265) includes specific case-groups with specific psychiatric disorders diagnosed 1994–2015 (n = 93,608) and a random-population subcohort (n = 50,615). We applied inverse probability weights to estimate person-years at risk, age-specific incidence rates, absolute risks, and incidence rate ratios in various scenarios for primary outcomes (affective disorder, bipolar disorder, schizophrenia, autism, and attention-deficit hyperactivity disorder) and secondary outcomes (epilepsy, anxiety, migraine, asthma, diabetes, injury, traumatic brain injury, substance use disorder, and death). Results: Weighted estimates based on the iPSYCH sample aligned with those in the full cohort for both primary and secondary outcomes. For example, weighted absolute risks by age 40 were analogous to full-cohort estimates for both affective disorder (7.9% [7.5–8.3] versus 8.0% [7.9–8.1]) and epilepsy (2.3% [2.1–2.5] versus 2.3% [2.2–2.3]). Similarly, weighted incidence rates and incidence rate ratios mimicked full-cohort estimates. Conclusion: The extended case-cohort design yields valid estimates of age-specific incidence rates, absolute risks, person-years at risk, and incidence rate ratios for primary and secondary outcomes, even with multiple case-groups and extended follow-up.

Rhodamine6G and Hœchst33342 narrow BmrA conformational spectrum for a more efficient use of ATP

Gobet, A., Moissonnier, L., Zarkadas, E., et al. — Mon, 01 Dec 2025 07:16:26 +0100

Multidrug ABC transporters harness the energy of ATP binding and hydrolysis to translocate substrates out of the cell and detoxify them. While this involves a well-accepted alternating access mechanism, molecular details of this interplay are still elusive. Rhodamine6G binding on a catalytic inactive mutant of the homodimeric multidrug ABC transporter BmrA triggers a cooperative binding of ATP on the two identical nucleotide-binding-sites, otherwise michaelian. Here, we investigate this asymmetric behavior via a structural-enzymology approach, solving cryoEM structures of BmrA at defined ATP ratios, highlighting the plasticity of BmrA as it undergoes the transition from inward to outward facing conformations. Analysis of continuous heterogeneity within cryoEM data and structural dynamics, reveals that Rhodamine6G narrows the conformational spectrum explored by the nucleotide-binding domains. We observe the same behavior for the other drug Hœchst33342. Following on these findings, the effect of drug-binding showed an ATPase stimulation and a maximal transport activity of the wild-type protein at the concentration-range where the cooperative transition occurs. Altogether, these findings provide a description of the influence of drug binding on the ATP-binding sites through a change in conformational dynamics.

Novel and unscrutinized immune entities of the zebrafish gut

Wed, 01 Jan 2025 07:16:26 +0100

The zebrafish model offers a unique opportunity to study gut immunity due to its diverse applicability within several fields of research, combined with an evolutionarily conserved immune system, transparency, and genetic tractability. This review highlights recent advances in understudied immune cell types in the zebrafish gut, emphasizing their potential to illuminate immune processes of the vertebrate immune system. The biological function of the gut is highly conserved in zebrafish, which makes them a relevant model to study intestinal immune cells with advanced molecular and imaging techniques that enable in vivo visualization of immune mechanisms and cell trajectories. Rodent and pig models have successfully contributed to our understanding of many aspects of the immune system, while zebrafish have so far been underestimated in their potential role in furthering our knowledge in this field. We suggest how future study directions can help elucidate the complex nature of gut immunity and highlight similarities between mammalian and zebrafish immune systems. Provided that immune cell functions are conserved, zebrafish can offer great opportunities for translational studies and have an important impact in improving human health.

Transforming Computational Power into Environmental Solutions

Wed, 01 Jan 2025 07:16:26 +0100

The extensive accumulation of persistent synthetic polymers has made plastics a rapidly expanding global concern. In this context, enzymatic degradation offers a viable and environmentally friendly alternative to the conventional plastic recycling methods. Here, we discuss how multi-microsecond Molecular Dynamics (MD) simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) methods have allowed us to shed light on the conformational dynamics and catalytic mechanisms underlying the activity of the polyurethane-degrading enzymes UMG-SP2 and UMG-SP3. Our findings emphasize the role of these computational approaches for the identification of the rate-limiting step, stationary states' geometries, and residue-specific electrostatic effects, enabling subsequent mutagenesis studies to enhance the efficiency of these urethanases. The access to EuroHPC's computing resources was essential to this research, as it allowed the extensive sampling of enzyme:substrate dynamics over large simulation timescales and the QM/MM-level resolution of the free-energy profile associated with the cleavage of the substrate's urethane bond. This work demonstrates the relevant role of HPC-driven research in advancing mechanistic insights and highlights how the synergy between large-scale computational resources and enzymatic knowledge can enhance our understanding of plastic biodegradation.

The genetic diversity of Indonesian cattle has been shaped by multiple introductions and adaptive introgression

Wang, X., Nursyifa, C., Aninta, S. G., et al. — Mon, 01 Dec 2025 07:16:26 +0100

Genetic diversity is a crucial resource in livestock, determining their traits and ability to respond to selection. Indonesian cattle are unique due to their history of admixture involving both zebu (Bos indicus) and banteng (B. javanicus), and may therefore contain novel cattle genetic resources. We generated whole genome sequences from 126 Indonesian cattle, 51 domesticated banteng and three captive banteng. We show that Indonesian cattle have very high genetic diversity, especially the Madura breed due to introgression from banteng and possibly other Bos species, contributing up to 36.6% of the Madura’s genome. We find that Indonesian zebu ancestry can be traced to at least three distinct ancestral populations, two of which were introduced more than 1345 years ago from mainland Southeast or eastern Asia. Peaks and valleys in banteng ancestry across the genome in admixed breeds suggest that both negative and positive selection act on introgressed haplotypes. Despite adaptive introgression being mainly breed-specific, we found evidence that some phenotypes, such as coat color, have experienced convergent adaptive introgression. Overall, our results provide insights into the historical movement of cattle in Asia, and showcase the potential for genetic improvement of cattle by identifying ~3.5 million novel SNPs introgressed into Indonesian cattle.

SyFi

Selten, G., Gómez-Repollés, A., Lamouche, F., Radutoiu, S., de Jonge, R. — Wed, 01 Jan 2025 07:16:26 +0100

The plant root microbiome is a complex community shaped by interactions among bacteria, the plant host and the environment. Synthetic community (SynCom) experiments help disentangle these interactions by inoculating host plants with a representative set of culturable microbial isolates from the natural root microbiome. Studying these simplified communities provides valuable insights into microbiome assembly and function. However, as SynComs become increasingly complex to better represent natural communities, bioinformatics challenges arise. Specifically, accurately identifying and quantifying SynCom members based on, for example, 16S rRNA amplicon sequencing becomes more difficult due to the high similarity of the target amplicon, limiting downstream interpretations. Here, we present SynCom Fingerprinting (SyFi), a bioinformatics workflow designed to improve the resolution and accuracy of SynCom member identification. SyFi consists of three modules: the first module constructs a genomic fingerprint for each SynCom member based on its genome sequence and, when available, raw genomic reads, accounting for both copy number and sequence variation in the target gene. The second module extracts the target region from this genomic fingerprint to create a secondary fingerprint linked to the relevant amplicon sequence. The third module uses these fingerprints as a reference to perform pseudoalignment-based quantification of SynCom member abundance from amplicon sequencing reads. We demonstrate that SyFi outperforms standard amplicon analysis by leverag-ing natural intragenomic variation, enabling more precise differentiation of closely related SynCom members. As a result, SyFi enhances the reliability of microbiome experiments using complex SynComs, which more accurately reflect natural commu-nities. This improved resolution is essential for advancing our understanding of the root microbiome and its impact on plant health and productivity in agricultural and ecological settings. SyFi is available at https://github.com/adriangeerre/SyFi.

Structural basis for the synergistic assembly of the snRNA export complex

Dubiez, E., Garland, W., Finderup Brask, M., et al. — Fri, 01 Aug 2025 07:16:26 +0200

The nuclear cap-binding complex (CBC) and its partner Arsenite-Resistance Protein 2 (ARS2) regulate the fate of RNA polymerase II transcripts via mutually exclusive interactions with RNA effectors. One such effector is PHAX, which mediates the nuclear export of U-rich small nuclear RNAs (snRNAs). Here we present the cryo-electron microscopy structure of the human snRNA export complex comprising phosphorylated PHAX, CBC, CRM1–RanGTP and capped RNA. The central region of PHAX bridges CBC to the export factor CRM1–RanGTP, while also reinforcing cap dinucleotide binding. Additionally, PHAX interacts with a distant region of CRM1, facilitating contacts of the essential phosphorylated region of PHAX with the prominent basic surface of RanGTP. CBC engagement within the snRNA export complex is incompatible with its binding to other RNA effectors such as ALYREF or NCBP3. We demonstrate that snRNA export complex formation requires synergistic binding of all its components, which in turn displaces ARS2 from CBC and commits the complex for export.

Intraflagellar transport

Yadav, S. K., Kidmose, R. T., Lorentzen, E. — Mon, 06 Oct 2025 07:16:26 +0200

A combination of AlphaFold predictions and functional validation has identified IFT88 as the direct adaptor linking kinesin-2 motors to IFT trains through a conserved TPR-ARM interface, solving a decades-old mystery.

MULoc-target

Jiang, Y., Wang, D., Zeng, S., et al. — Tue, 01 Jul 2025 07:16:26 +0200

Protein targeting, often guided by targeting peptides, is a critical biological process that directs proteins to their specific cellular destinations, ensuring proper cellular functionality and organization. Accurate classification and detection of targeting peptides are fundamental to understanding protein sorting mechanisms. This study introduces MULoc-Target, a novel deep-learning method designed to detect and classify targeting peptides in eukaryotic proteins. To support its development and evaluation, we curated a benchmark dataset comprising eight types of eukaryotic targeting peptides with manually curated annotations. Comprehensive evaluations on this dataset and external datasets from the literature demonstrate that MULoc-Target achieves state-of-the-art or competitive performance in detecting and classifying targeting peptides. Additionally, it enables the extraction of enriched motif patterns, offering valuable insights into their properties and the underlying targeting mechanisms. The identified motifs align closely with established biological features, further validating MULoc-Target's capabilities. A web server for MULoc-Target is integrated into our MULocDeep localization suite as a new toolkit, publicly accessible at https://mu-loc.org/MULoc-Target, and the inference code is available at https://github.com/yuexujiang/MULoc-Target.

Flippase-Mediated Hybrid Vesicle Division

Thu, 02 Oct 2025 07:16:26 +0200

The assembly of synthetic systems with the ability for protein-mediated division remains a challenge in bottom-up synthetic biology. Here, the reconstitution of an active Drs2p–Cdc50p lipid flippase in polymer lipid hybrid vesicles (HVs) made from phospholipids and 1 or 2.5 mol% amphiphilic block copolymers, with poly(carboxyethyl acrylate) or poly(6-O-methacryloyl-d-galactopyranose) as the hydrophilic extension and either cholesteryl methacrylate or butyl methacrylate or combinations thereof as the hydrophobic blocks is demonstrated. The reconstitution of Drs2p–Cdc50p in HVs flip 2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) lipids from the inner to the outer leaflet, leading to transmembrane asymmetry. Importantly, the chemical nature of the hydrophobic block in the amphiphilic block copolymers used to assemble the HVs is crucial to support changes in the spontaneous curvature of the bilayers due to translocation of DOPS lipids that results in HV constriction and division. Taken together, this effort is a step forward in imitating cell division in synthetic assemblies toward potentially bottom-up assembled self-replicating units.

Application of a mouse model humanized for cytochrome P450–mediated drug metabolism to predict drug-drug interactions between a peptide and small molecule drugs

Kapelyukh, Y., Gabel-Jensen, C., MacLeod, A. K., et al. — Wed, 01 Oct 2025 07:16:26 +0200

Conventional preclinical in vitro approaches inaccurately predicted clinical trial outcomes of drug-drug interactions involving the peptide NN1177, a glucagon and glucagon-like peptide 1 receptor coagonist. To further study the mechanisms behind this discrepancy, we have exploited a mouse model (8HUM) humanized for the major cytochrome P450 (P450) enzymes involved in drug disposition in humans. We show that NN1177 administration to 8HUM mice suppressed hepatic in vivo expression of CYP3A4 (82% compared to vehicle) and CYP1A2 (58% compared to vehicle). This was consistent with in vitro sandwich culture hepatocyte data reported previously. However, reduction in CYP3A4 and CYP1A2 levels in vivo appeared to resolve over time, despite daily NN1177 administration. These findings suggest an adaptive response to the metabolic effects of NN1177. In vivo pharmacokinetic studies in 8HUM closely matched the findings observed in the clinical trial, because there was no relevant increase in the exposure of the CYP3A4 and CYP1A2 probe drugs. Furthermore, no suppression effects were observed when the mice had been pretreated with the inducing agents, St. John’s wort or phenobarbital, respectively, suggesting that the mechanism of P450 reduction does not involve the transcription factors constitutive androgen receptor or pregnane X receptor. These data highlight the complexities associated with therapeutic peptide drug-drug interactions and the remaining challenges for accurate predictions of P450 suppression and potential clinical implications. The humanized 8HUM model provides a promising and informative preclinical tool that can add high value during drug development by providing further insights into the effects on P450 expression, together with the subsequent impact of coadministered probe drugs in an in vivo model. Significance Statement: The current work describes the application of a humanized cytochrome P450 mouse model that provides further insight into the potential mechanisms and outperforms conventional in vitro approaches for preclinical predictions of peptide drug-drug interaction risk. The results showed no significant effects on the Cooperstown 5 + 1 cocktail, in line with clinical findings, and thereby represent an exciting model to further explore future therapeutic peptide projects during drug development.

Impact of abiotic stress on miRNA profiles in tomato-derived extracellular vesicles and their biological activity

Huang, Z., Pych, E., Whitehead, B. J., et al. — Fri, 01 Aug 2025 07:16:26 +0200

Edible plant-derived extracellular vesicles (EVs), which are rich in bioactive macromolecules such as proteins and microRNAs (miRNAs), contribute to their health benefits. While abiotic stress induces molecular changes in plants, its impact on the secretion and composition of EVs remains poorly understood. This study isolated and characterized EVs from tomatoes under nutrient deficiency, salt stress, and drought stress, analyzing changes in protein and nucleic acid content. Digital droplet polymerase chain reaction (PCR) was used to investigate the impact on selected miRNA levels. Results showed that abiotic stress significantly increased EV secretion and protein content, with varied effects on nucleic acids and miRNAs. Specifically, salt stress decreased miR-9476 levels, weakening its inhibition of the metallothionein 1× (MT1X) gene, leading to increased zinc content in intestinal cells and reduced zinc transport. These findings suggest environmental factors can modulate the biomolecular composition and functionality of plant-derived EVs, guiding the enrichment of specific biomolecules.

Convergent evolution of H4K16ac-mediated dosage compensation in the ZW species Artemia franciscana

Zimmer, F., Fox, A. M., Pan, Q., et al. — Wed, 01 Oct 2025 07:16:26 +0200

Sex chromosomes impact chromatin organization and histone modification dynamics differently between males and females, particularly those involved in dosage compensation (DC). The evolutionary diversity, as well as the tissue- and age-dependent variations of DC mechanisms are incompletely understood. Here, we investigate the occurrence of histone H4 lysine 16 acetylation (H4K16ac), previously known for its role in sex chromosome DC in the male-heterogametic fruit fly Drosophila melanogaster and the green anole lizard Anolis carolinensis. By sampling multiple arthropods, we find the convergent evolution of H4K16ac for DC in a female-heterogametic (ZW) species, the crustacean Artemia franciscana. CUT&Tag analysis demonstrates that H4K16ac is confined to the non-recombining stratum of the Z chromosome in females. H4K16ac-mediated DC is established during embryogenesis. In aged individuals, we observe an overall decline in nuclear organization, disrupted H4K16ac territories and increased variability in local acetylation levels on the female Z chromosome. Our findings shed light on the evolutionary diversity of DC across species and raise the possibility of sex-specific histone acetylation contributing to male-female differences in lifespan.

Hydrolysis of the Urethane Bond Catalyzed by Pseudomonas sp. MIS38 Lipase

Teixeira, L. M.C., Paiva, P., Johansen, M. B., et al. — Wed, 01 Jan 2025 07:16:26 +0100

Plastic accumulation has become a major global concern due to the lack of efficient and environmentally friendly strategies to manage the end-of-life of these materials. Among the most used families of plastics is polyurethane (PU), which is valued for its versatility and low production cost. A promising strategy to address the end-of-life challenges of PU is employing efficient PU-degrading enzymes. Notably, an extracellular lipase from the I.3 family originating from Pseudomonas sp. MIS38 has shown significant promise in this regard. In this study, we investigated the enzyme’s capability to hydrolyze a PU fragment. Employing QM/MM computational methodologies, we studied the hydrolysis mechanism of MIS38 lipase and found it to follow the prototypical serine esterase mechanism involving acylation and deacylation stages. The rate-limiting step occurred between the formation of the acyl-enzyme intermediate and the formation of the second tetrahedral intermediate. The Gibbs activation barrier for this step was 19.67 kcal·mol^–1, confirming the lipase’s potential to biodegrade PU efficiently. More importantly, we observed that the enzyme preferentially cleaved the C–N bond instead of the C–O bond. This preference was due to the arrangement of the active site and the substrate, which made C–N the more favorable cleavable site. Furthermore, we find that the C–O group is not a suitable cleavable bond due to steric hindrances. This study suggests that the mechanism of urethane bond hydrolysis is more complex than currently assumed because bond cleavage is context-dependent and may differ depending on the enzyme and the substrate.

Therapeutic AASS inhibition by AAV-miRNA rescues glutaric aciduria type I severe phenotype in mice

Segur-Bailach, E., Mateu-Bosch, A., Bofill-De Ros, X., et al. — Wed, 01 Oct 2025 07:16:26 +0200

Glutaric aciduria type I (GA1) is an inherited disorder caused by the enzymatic defect of glutaryl-coenzyme A dehydrogenase in the lysine degradation pathway, characterized by the accumulation of toxic metabolites in the central nervous system. We reasoned that substrate reduction therapy targeting the α-aminoadipic semialdehyde synthase (AASS), the first enzyme in the catabolism of lysine, could provide an attractive therapeutic alternative. We explored reducing the expression of AASS by an artificial microRNA with AASS target sequences embedded in a miR-16 backbone (miR_AASS). We analyzed several delivery routes and AAV serotypes and evaluated the therapeutic efficacy of a systemic neonatal delivery of AAV9_miR_AASS in the Gcdh^−/− mouse model of GA1. We detected dose-dependent miR-AASS expression and AASS inhibition in liver and striatum, the main tissues affected in GA1. Treatment with AAV9_miR_AASS in lysine overload-challenged mice reduced the accumulation of neurotoxic metabolites up to 6 months post-treatment in the striatum, prevented the neuropathological alterations, and improved mouse survival. Our results show that AAV9_miR_AASS supports AASS lowering as a potential gene therapy strategy for GA1.

Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment

Demir, F., Kovalenko, E., Lassé, M., et al. — Mon, 27 Oct 2025 07:16:26 +0100

Dysregulated proteolysis is central to autoimmune pathogenesis. The complement cascade, a major protease network, generates fragments that modulate immunity and tissue injury. We developed a scalable blood plasma N-terminomics workflow that markedly expands detection of proteolytic events in vitro and in vivo. Applied to 143 systemic lupus erythematosus (SLE) patients, Multi-Omics Factor Analysis (MOFA) linked N-terminal signatures to immunological and clinical heterogeneity. This revealed a previously unrecognized complement fragment, C3-LHF1, encompassing the C345C domain and rivaling, based on intensity detected by mass spectrometry, the abundance of canonical fragments like C3a and C3b. C3-LHF1 associated with renal function and remission in lupus nephritis, and exhibited dual functions: inhibiting classical and lectin complement pathways and acting as a partial IL6ST (gp130) agonist, independent of IL6Rα. In human kidney organoids, C3-LHF1 induced JAK/STAT3 signaling, amplified TNFα-driven CXCL10 secretion, and reduced podocyte marker expression, suggesting a role in tissue remodeling. These findings reveal unanticipated complexity in complement-mediated signaling and provide a comprehensive atlas of protein N-termini in human plasma, which enables discovery of novel immunoregulatory mechanisms and therapeutic targets in inflammatory disease.

Polygenic prediction of body mass index and obesity through the life course and across ancestries

Smit, R. A.J., Wade, K. H., Hui, Q., et al. — Mon, 01 Sep 2025 07:16:26 +0200

Polygenic scores (PGSs) for body mass index (BMI) may guide early prevention and targeted treatment of obesity. Using genetic data from up to 5.1 million people (4.6% African ancestry, 14.4% American ancestry, 8.4% East Asian ancestry, 71.1% European ancestry and 1.5% South Asian ancestry) from the GIANT consortium and 23andMe, Inc., we developed ancestry-specific and multi-ancestry PGSs. The multi-ancestry score explained 17.6% of BMI variation among UK Biobank participants of European ancestry. For other populations, this ranged from 16% in East Asian-Americans to 2.2% in rural Ugandans. In the ALSPAC study, children with higher PGSs showed accelerated BMI gain from age 2.5 years to adolescence, with earlier adiposity rebound. Adding the PGS to predictors available at birth nearly doubled explained variance for BMI from age 5 onward (for example, from 11% to 21% at age 8). Up to age 5, adding the PGS to early-life BMI improved prediction of BMI at age 18 (for example, from 22% to 35% at age 5). Higher PGSs were associated with greater adult weight gain. In intensive lifestyle intervention trials, individuals with higher PGSs lost modestly more weight in the first year (0.55 kg per s.d.) but were more likely to regain it. Overall, these data show that PGSs have the potential to improve obesity prediction, particularly when implemented early in life.

The oral fingerprint

Kofod Petersen, A., Villesen, P., Staun Larsen, L. — Wed, 01 Jan 2025 07:16:26 +0100

Intoduction: The palatal rugae have been suggested to be just as unique as the human fingerprint. Therefore, endeavors have been made to utilize this uniqueness for the identification of disaster victims. With the rise of digital 3D dental data, computational comparisons of palatal rugae have become possible. But a direct comparison of the full palatal scan by iterative closest point (ICP) has shown to be tedious and demands a knowledge of superimposition software. Methods: Here, we propose (1) an automatic extraction of the palatal rugae ridges from the 3D scans, followed by (2) ICP of the extracted ridges. Results: Pairwise comparisons of palates take less than a second, and in this study, it was possible to distinguish between palates from the same individual vs. palates from different individuals with a receiver operating characteristic area-under-the-curve of 0.994. Discussion: This shows that the extraction of the palatal rugae ridges is a potential efficient addition to the toolbox of a forensic odontologist for disaster victim identification.

Dysfunction of cortical GABAergic projection neurons as a major hallmark in a model of neuropsychiatric syndrome

Asenjo-Martinez, A., Dragicevic, K., Hou, W. H., et al. — Wed, 01 Oct 2025 07:16:26 +0200

Neuropsychiatric disorders have a strong genetic component and are linked to developmental risk factors, yet it is unclear why symptoms appear only later in life and which neuronal types contribute to brain dysfunction. We addressed these questions using a robust mouse model of a neuropsychiatric syndrome—the 15q13.3 microdeletion. Single-nucleus transcriptomics revealed the largest gene expression alterations in the somatostatin (Sst) Sst_Chodl subtype, the long-range γ-aminobutyric acid (GABAergic) projecting neurons. Despite the developmental onset of perturbations, impairments in Sst_Chodl neurons manifested only at late maturation. Calcium imaging and patch-clamp recordings unraveled impaired responsivity overall in deep-layer Sst neurons, with only the Sst_Chodl subtype exhibiting increased activity. Patch-seq analysis connected molecular changes to cellular dysfunction of Sst_Chodl neurons. Finally, microdeletion mice displayed sleep disturbances associated with impaired activity of deep-layer Sst neurons, which were rescued by chemogenetic inhibition of Sst_Chodl neurons. Our findings spotlight GABAergic projection neurons as potential vulnerable targets in neuropsychiatric disorders.

Bridging experiment and theory

Teixeira, L. M.C., Paiva, P., Ferreira, P., et al. — Wed, 01 Oct 2025 07:16:26 +0200

Understanding how dynamical behaviour and structural features influence protein function and stability is crucial. While extensive experimental data exist, studying real-time protein dynamics and enzyme catalysis remains challenging. Computational advances have been instrumental in overcoming experimental limitations, enabling molecular-level insights into biological macromolecules. The integration of experimental and computational approaches has proven to be very valuable in protein studies. Here, we demonstrate this synergy by investigating the conformational stability of the urethanase UMG-SP3, which exhibited a lower optimum temperature than expected and rapid loss of activity. Molecular dynamics simulations of the UMG-SP3-substrate complex at various temperatures revealed structural rearrangements outside the optimum temperature range (25–35 °C), leading to loss of the native protein fold and impaired substrate binding. Even at the optimum temperature for activity, the enzyme struggled to maintain a catalytically favourable orientation, aligning with experimental findings. Unfolding profiles were determined through differential scanning fluorimetry. Notably, the computational results provided a rationaly for the structural instability observed experimentally, emphasizing the strength of computational methods in elucidating protein behaviour at the atomic level. This study highlights the importance of combining experimental and computational approaches to deepen our understanding of protein stability and function.

Structures and mechanism of the AUX/LAX transporters involved in auxin import

Ung, K. L., Schulz, L., Zuzic, L., et al. — Fri, 01 Aug 2025 07:16:26 +0200

Auxins are plant hormones that direct the growth and development of organisms on the basis of environmental cues. Indole-3-acetic acid (IAA) is the most abundant auxin in most plants. A variety of membrane transport proteins work together to distribute auxins. These include the AUX/LAX protein family that mediate auxin import from the apoplast to the cytosol. Here we use structural and biophysical approaches combined with molecular dynamics to study transport by Arabidopsis thaliana LAX3, which is essential for plant root formation. Transport assays document high-affinity transport of IAA, as well as competitive behaviour of the synthetic phenoxyacetic acid auxin herbicide 2,4-dichlorophenoxyacetic acid and the auxin transport inhibitors 1-naphthoxyacetic acid and 2-naphthoxyacetic acid. Four cryo-EM structures were solved with resolutions of 2.9–3.4 Å: an inward open apo structure, two inward semi-occluded structures in complex with IAA and 2,4-dichlorophenoxyacetic acid, and a fully occluded structure in complex with 2-naphthoxyacetic acid. Structurally, LAX3 consists of a bundle and a scaffold domain. The ligand-binding site is sandwiched between these domains with two histidines occupying positions analogous to the sodium-binding sites in distantly related sodium:neurotransmitter transporters. This architecture suggests that these histidines couple transport to the proton motive force. Molecular dynamics simulations are used to explore substrate binding and release, including their dependence on specific protonation states. This study advances our understanding of auxin recognition and transport by AUX/LAX, providing insights into a fundamental aspect of plant physiology and development.

Topical mutant allele-specific siRNA delivery for treatment of Meesmann epithelial corneal dystrophy and elucidation of disease biomarkers

McLain, A., Saffie-Siebert, S. R., Nielsen, N. S., et al. — Fri, 17 Oct 2025 07:16:26 +0200

Proven in vivo efficacy is a key requirement for novel gene therapies proceeding to clinical trial. No gene therapies for corneal dystrophies have been able to progress into patient-led studies due to a lack of suitable animal models expressing a disease phenotype comparable to that which develops in humans. Herein, we show an allele-specific siRNA targeting the L132P KRT12 mutation, causative of a severe form of Meesmann Epithelial Corneal Dystrophy (MECD), when complexed with silicon-stabilized hybrid lipid nanoparticles (sshLNP), and applied topically twice daily for seven days, results in restoration of corneal thickness in a humanized mouse model (Krt12 +/hL132P) for MECD. Furthermore, expression of disease biomarkers elevated in the MECD mouse model and verified in corneal epithelial cells of MECD family members, was reduced to that observed in unaffected wild-type (Krt12 +/+) mice. This represents the first successful use of topical mutant allele-specific siRNA in the treatment of an autosomal genetic corneal dystrophy. The successful silencing of disease biomarkers after topical siRNA treatment indicates expected clinical benefit. Furthermore, we demonstrated that siRNA-sshLNPs can be successfully formulated in sodium hyaluronate-containing eye-drops without impairing efficacy of the siRNA.

Actin Polymerizing Motors to Assist Cytoskeleton-like Networks Formation in Artificial Cells

Thu, 16 Oct 2025 07:16:26 +0200

Artificial cells are man-made systems that imitate specific functions of biological cells to study or harness cellular behavior. Biological cells can respond to external forces and signals by altering their shape, undergoing deformation, and generating the mechanical forces required for their movement. The cytoskeleton orchestrates this process through the coordinated action of actin filaments, intermediate filaments, and microtubules. Examples of artificial cells that sense and adapt to changes in their environment owing to cytoskeleton rearrangement have extensively been explored. These efforts focus on the use of biomolecules that stochastically self-assemble in the lumen of an artificial cell. Here, we employ actin polymerizing nanomotors to assist cytoskeleton formation inside artificial cells. Nano- and micromotors are a class of active colloids that can self-propel outperforming Brownian motion. Inspired by natures' way of leveraging biopolymerization reactions to sustain locomotion in microorganisms or in organelles within cells, we imitate the mechanism of motion of the food-born bacteria Listeria monocytogenes. Specifically, we coat polystyrene particles with an actin recruiting protein that allows for actin filament polymerization in a mammalian cell lysate environment. This polymerization results in up to a 3-fold increase in the propulsion of the motors compared to their Brownian motion. Lastly, we show that these motors can be encapsulated inside hybrid vesicle-based artificial cells made of amphiphilic block copolymers and phospholipids, forming actin filaments that assemble into a cytoskeleton-like network. Taken together, this effort highlights the synergistic integration of bottom-up synthetic biology and active matter, demonstrating how their convergence can advance the design of life-like systems.

Screening de novo designed protein binders in unpurified lysate using flow induced dispersion analysis

Pinheiro, F., Nowak, J. S., Zueva, E., et al. — Wed, 01 Oct 2025 07:16:26 +0200

Computational protein design can create binders against targets of interest, but identifying binders with sufficient affinity still requires biochemical screening of many designs. In this work, we test flow-induced dispersion analysis (FIDA) as a method for screening binders in a time and cost-effective manner. FIDA uses Taylor dispersion analysis to determine the hydrodynamic radius of fluorescently labeled biomolecules and their complexes. Here, we use FIDA to assess the binding of RFdiffusion-designed protein binders against the small helical peptide ALFA-tag and the guanylate kinase (GK) domain of PSD-95. Successful binders can be identified in a single measurement using heat-treated bacterial lysates, allowing rapid identification of binders with high affinity and thermostability. Subsequent titration experiments show micromolar affinities for ALFA-tag binders and nanomolar affinities for GK domain binders. The lack of immobilization, the minimal sample volume, and the compatibility with complex biological samples position FIDA as a valuable tool for the screening and characterization of computationally generated protein binders.

Dynamic visualization of extracellular matrix components in S. aureus colony biofilms reveals functional amyloids leading to the formation of cap-like structures

Zhang, T., Bär, J., Risberg, L., et al. — Mon, 01 Dec 2025 07:16:26 +0100

Staphylococcus aureus infections represent a clinical challenge due to their propensity to form biofilms and the increasing prevalence of antibiotic resistance. The ability of S. aureus to form biofilm affects clinical outcome, but techniques to study extracellular matrix (ECM) in S. aureus biofilms are lacking. Here, we present an agar-based method in which the optotracer EbbaBiolight 680 (Ebba680) is used to visualize ECM formation alongside evaluation of colony growth dynamics in agar colonies. As models for colony biofilms, we use drop inoculation for macrocolony formation or spread-plating for single-cell derived colonies. Kinetic fluorescence spectroscopy combined with time-lapse microscopy showed bright fluorescence signals, revealing different spatial-temporal appearance of ECM in macrocolonies versus single-cell derived colonies. In contrast, the microstructure was conserved between the two types of colonies. Detailed characterization of the biofilm microstructures by confocal microscopy revealed Ebba680 binding targets interspersed between cells as well as in a cap-like structure formed on the outer surface of the biofilm. Accessory gene regulator ( agr) controlled expression of Ebba680 binding target(s) and the binding of Ebba680 to synthetic fibrillated phenol soluble modulins (fPSMs) suggests these functional amyloids act as targets for Ebba680 in the biofilm ECM. By upgrading ColTapp, an application developed for colony radius quantification, to also analyze fluorescence images, concurrent analysis of Ebba680-stained ECM and colony growth was achieved. This provided a new dimension to the assessment of colony biofilms. Detailed phenotypic characterization of clinical isolates is critical for treatment decision making, and enhanced screening which includes ECM as presented here has potential to facilitate treatment decisions in problematic staphylococcal infections.

Multi-domain O-GlcNAcase structures reveal allosteric regulatory mechanisms

Hansen, S. B., Bartual, S. G., Yuan, H., et al. — Mon, 01 Dec 2025 07:16:26 +0100

Nucleocytoplasmic protein O-GlcNAcylation is a dynamic modification catalysed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAc hydrolase (OGA), whose activities are regulated through largely unknown O-GlcNAc-dependent feedback mechanisms. OGA is a homodimeric, multi-domain enzyme containing a catalytic core and a pseudo-histone acetyltransferase (pHAT) domain. While a catalytic structure has been reported, the structure and function of the pHAT domain remain elusive. Here, we report a crystal structure of the Trichoplax adhaerens pHAT domain and cryo-EM data of the multi-domain T. adhaerens and human OGAs, complemented by biophysical analyses. Here, we show that the eukaryotic OGA pHAT domain forms catalytically incompetent, symmetric homodimers, projecting a partially conserved putative peptide-binding site. In solution, OGA exist as flexible multi-domain dimers, but catalytic core-pHAT linker interactions restrict pHAT positional range. In human OGA, pHAT movements remodel the active site environment through conformational changes in a flexible arm region. These findings reveal allosteric mechanisms through which the pHAT domain contributes to O-GlcNAc homeostasis.

Polygenic and developmental profiles of autism differ by age at diagnosis

Zhang, X., Grove, J., Gu, Y., et al. — Wed, 01 Oct 2025 07:16:26 +0200

Although autism has historically been conceptualized as a condition that emerges in early childhood1,2, many autistic people are diagnosed later in life3-5. It is unknown whether earlier- and later-diagnosed autism have different developmental trajectories and genetic profiles. Using longitudinal data from four independent birth cohorts, we demonstrate that two different socioemotional and behavioural trajectories are associated with age at diagnosis. In independent cohorts of autistic individuals, common genetic variants account for approximately 11% of the variance in age at autism diagnosis, similar to the contribution of individual sociodemographic and clinical factors, which typically explain less than 15% of this variance. We further demonstrate that the polygenic architecture of autism can be broken down into two modestly genetically correlated (rg = 0.38, s.e. = 0.07) autism polygenic factors. One of these factors is associated with earlier autism diagnosis and lower social and communication abilities in early childhood, but is only moderately genetically correlated with attention deficit-hyperactivity disorder (ADHD) and mental-health conditions. Conversely, the second factor is associated with later autism diagnosis and increased socioemotional and behavioural difficulties in adolescence, and has moderate to high positive genetic correlations with ADHD and mental-health conditions. These findings indicate that earlier- and later-diagnosed autism have different developmental trajectories and genetic profiles. Our findings have important implications for how we conceptualize autism and provide a model to explain some of the diversity found in autism.

An OGT Missense Variant With Impaired Enzyme Activity in a Child With Severe Developmental Delay and Hepatoblastoma

D'Alessio, A. M., Yuan, H., Soria, L. R., et al. — Wed, 01 Jan 2025 07:16:26 +0100

O-GlcNAc transferase (OGT) and its antagonist O-GlcNAcase (OGA) regulate protein O-GlcNAcylation, a highly conserved post-translational modification involved in metabolic sensing. Pathogenic variants in the OGT gene cause an X-linked congenital disorder of glycosylation (OGT-CDG) presenting developmental delay, hypotonia, intellectual disability, and dysmorphic features. Here, we report on a child with developmental delay, hypotonia, and dysmorphic features who was found to carry a hemizygous novel OGT variant. This child also developed hepatoblastoma by the age of 17 months. OGT-CDG was diagnosed by exome sequencing that identified a de novo missense variant in the OGT gene. Functional validation by Western blot on patient-derived fibroblasts showed reduced O-GlcNAcylation and OGA expression, while significantly reduced enzyme activity in vitro confirmed the pathogenicity of the variant. To date, no patients with OGT-CDGs have been reported with hepatoblastoma or other malignancies. Although the occurrence of hepatoblastoma in the proband might be coincidental, the role of O-GlcNAcylation in cancer suggests that the deficiency of OGT activity might be associated with increased cancer risk.

Cancer-associated fibroblasts shape the formation of budding cancer cells at the invasive front of human colorectal cancer

García-Rodríguez, J. L., Korsgaard, U., Vissing, S. M., et al. — Mon, 01 Dec 2025 07:16:26 +0100

The formation of budding cancer cells at the invasive front of solid tumors is one of the first steps of metastasis. However, this process is still incompletely elucidated. Here, we used spatial molecular imaging to disentangle the complex interactions between cancer cells and the tumor microenvironment at the invasive front of colorectal tumors. Employing a 1000-plex gene panel, we defined all major cell types in tumors and adjacent normal tissue with accurate spatial information. Individual cancer cell clusters were located together, consistent with an expected mutation- and epigenetic-driven clonal evolution. However, cancer cell clusters encompassing budding cells exhibited a markedly different spatial distribution as they also contained cells that were scattered around the periphery of the main cancer cell masses. Moreover, these cells were frequently in contact with cancer-associated fibroblasts (CAFs) and underwent broad gene expression changes, mainly related to epithelial-mesenchymal transition (EMT), remodeling of the extracellular matrix (ECM), and migration. In addition, we defined an 11-gene signature (TYK2, IL2RG, KRT17, HLA-B, NPPC, WIF1, IL32, B2M, CCND1, CRIP1, ITGB1), which characterizes cancer cells en route to metastasis and is associated with inferior outcomes. Collectively, our findings suggest that CAFs induce pro-invasive gene expression changes involved in EMT, ECM remodeling, and migration.

Determinants of de novo mutations in extended pedigrees of 43 dog breeds

Zhang, S., Ma, J., Riera Belles, M., et al. — Mon, 01 Dec 2025 07:16:26 +0100

Background: Understanding the determinants of de novo mutation is critical for elucidating evolutionary processes and genetic disease susceptibility. But the interplay between life history, genomic architecture, and recombination remains poorly understood in non-model species. Domestic dogs, lacking the recombination regulator PRDM9 and subject to intense artificial selection, offer a unique system for dissecting factors that jointly influence mutation accumulation. Here, we leverage large-scale trio sequencing to unravel the determinants of de novo mutations across diverse dog breeds. Results: By analyzing 390 trios from 43 breeds, we estimate a germline mutation rate of 4.89 × 10⁻⁹ per base pair per generation. Parental age, especially paternal age, strongly influences mutation rates, with a 1.5-fold greater paternal age effect in dogs compared to humans. Larger breeds exhibit elevated early-life mutations, aligning with accelerated developmental trajectories. Strikingly, CpG Islands in dogs exhibit a 2.6-fold higher mutation rate than the genomic average, unlike humans where no such increase occurs. We also find a tenfold hypermutated dog and suggest a unique maternal mechanism of MLH1-mediated germline instability during gametogenesis. Conclusions: The unique mutational landscape in canids is determined by paternal age, body size, and CpG Islands recombination. Despite extensive breeding, germline mutation rates in dogs remain stable across breeds. The elevated mutation rate in CpG Islands due to recombination in the absence of PRDM9 underscores a distinct evolutionary mechanism in canids. These findings enhance our understanding of mutation dynamics, with implications for canine genetic diversity, disease susceptibility, and broader genomic studies in species lacking PRDM9.

STING signals to NF-κB from late endolysosomal compartments using IRF3 as an adaptor

Zhang, B., Pedersen, A., Reinert, L. S., et al. — Fri, 19 Sep 2025 07:16:26 +0200

NF-κB is central for activation of immune responses. Cytosolic DNA activates the cGAS-STING pathway to induce type I interferons (IFNs) and signaling through NF-κB, thus instigating host defenses and pathological inflammation. However, the mechanism underlying STING-induced NF-κB activation is unknown. Here we report that STING activates NF-κB in a delayed manner, following exit from the Golgi to endolysosomal compartments. Activation of NF-κB is dependent on the IFN-inducing transcription factor IRF3 but is independent of type I IFN signaling. This activation pattern is evolutionarily conserved in tetrapods. Mechanistically, the monomer IRF3 is recruited to STING pS358, with delayed kinetics relative to IRF3 recruitment to STING pS366, which promotes type I IFN responses. IRF3 engagement with STING pS358 induces trafficking to late endolysosomal compartments, supporting recruitment of TRAF6 and activation of NF-κB. We identify a TRAF6 binding motif in IRF3 that facilitates recruitment of TRAF6. This work defines a signaling surface on STING and a function for IRF3 as an adaptor in immune signaling. These findings indicate that STING signaling to NF-κB is enabled only within a short time window between exit from the Golgi and lysosomal degradation, possibly limiting inflammation under homeostatic and danger-sensing conditions.

A low frequency damaging SORCS2 variant identified in a family with ADHD compromises receptor stability and quenches activity

Kaas, M., Dinesen, S. B., Ahlgreen, O., et al. — Thu, 18 Sep 2025 07:16:26 +0200

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder affecting 5% of children and 2.5% of adults worldwide. ADHD is considered a polygenic disorder caused by a combination of both common and rare risk variants, each with low individual effect size. The Vps10p domain receptor SorCS2 is involved in neuronal development and synaptic plasticity by modulating brain-derived neurotrophic factor (BDNF) signaling. We here describe the identification and characterization of a heterozygous damaging variant in the SORCS2 gene found in two members of a family with persistent ADHD. The SORCS2 variant results in an arginine to tryptophan substitution in the 10CC region of the extracellular Vps10p domain, leading to aberrant posttranslational receptor processing, subcellular localization and ligand binding. Furthermore, the variant abrogates BDNF signaling in a dominant negative manner. Biochemical analysis of additional rare missense variants from ADHD cohorts suggested that SorCS2 structural stability and function is susceptible to such variation in the Vps10p domain. Our findings provide insights into how low frequency damaging variants in SORCS2 may contribute to the risk of ADHD.

LRP2 Expression in Melanoma Is Associated With a Transitory Cell State, Increased T Cell Infiltration, and Is Upregulated by IFNy Signaling

Rasmussen, M. Q., Bønnelykke-Behrndtz, M. L., Merrild, C., et al. — Mon, 01 Sep 2025 07:16:26 +0200

Low density lipoprotein receptor-related protein 2 (LRP2) is a 600 kilodalton multi-ligand endocytic membrane receptor ex-pressed in several cell types during fetal development, including neuroepithelial cells, and in select absorptive epithelial cellsin the adult. In epithelial cancers, LRP2 expression is associated with a differentiated tumor cell state and better prognosis. Inprevious work, we found that while LRP2 is not expressed in benign naevi, it is frequently acquired in melanoma. However, themolecular drivers of LRP2 expression in melanoma and characteristics of LRP2- expressing melanoma have yet to be described.Here, we show that LRP2 expression is related to a transitory melanoma cell state defined by co-expression of melanocyte lineageand neural crest transcriptional programs. Further, we reveal that melanoma LRP2 expression is increased in T cell-inflamedtumors and is directly upregulated through interferon-gamma signaling. Correlation of melanoma LRP2 expression with clin-icopathological variables demonstrates that LRP2 expression is associated with low Breslow thickness and low clinical stage inprimary melanomas. Taken together, the present study describes the characteristics of LRP2- expressing melanoma and revealsinterferon-gamma signaling as a novel strong positive regulator of LRP2 expression in melanoma.

Single-vesicle Tracking of α-Synuclein Oligomers Reveals Pore Formation by a Three-Stage Model

Bro̷chner, B. V., Zhang, X., Nielsen, J., Kjems, J., Otzen, D. E., Malle, M. G. — Tue, 16 Sep 2025 07:16:26 +0200

Neurodegenerative disorders, such as Parkinson's disease (PD) pose significant health challenges. A major hallmark of PD is the aggregation of α-synuclein into toxic oligomers (αSO) and fibrils. While many efforts focus on slowing disease progression, the molecular origins and mechanisms of αSO toxicity remain poorly understood, particularly regarding its proposed link to membrane disruption. To address this, we have developed a single-vesicle analysis platform for direct, and real-time measurements of αSO and membrane interaction. This platform allows us to demonstrate real-time translocation of dyes through αSO pores with single-particle resolution and use single-channel electrical recordings to analyze pore formation in planar lipid bilayers. Across methods, our data provide evidence for a three-stage model of αSO and membrane interactions, comprising initial membrane recruitment followed by partial pore insertion and subsequent full pore formation. Notably, while αSO recruitment was found to favor curved membranes, pore formation occurred more efficiently in less curved membranes, hence, recruitment is decoupled from a membrane charge-promoted reorientation and pore integration. Single αSO pore formations undergo multiple translocation steps making pore formation highly dynamic, cycling back and forth between partial insertion and full pore formation. The dynamic nature of pore formation can be modulated by lipid charge, lipid headgroup class, and ligand binding. Our findings suggest that increased dynamic pore formation could imply increased membrane toxicity. Evidence for the three-stage model is important for developing future targeting strategies to block αSO-mediated PD-related cellular dysfunction. We envision that the single-vesicle assay will enable screening of ligands modulating the pore formation.

Programmable RNA Nanostructures Enable Nanopore Detection of Cotranscriptionally Introduced RNA Modifications

Wed, 13 Aug 2025 07:16:26 +0200

Tracking RNA synthesis and metabolic histories requires cotranscriptional incorporation of modified nucleotides. However, identifying the incorporation of modified nucleotides into nascent RNA remains challenging, particularly for short RNAs. In this work, we developed a method utilizing solid-state nanopores and DNA:RNA nanostructures to detect modified nucleotide incorporation across different RNA length scales, from short to long RNAs transcribed in vitro. We identified the incorporation of biotin-modified uridine in short RNAs using a DNA nanostructure coupled with a nanopore readout. As a proof of concept for tracking RNA synthesis, we evaluated the incorporation of azide-modified uridine into long RNAs. To achieve quantitative labeling, we optimized conditions for click chemistry using cyclooctyne-DNA oligonucleotides. Subsequently, we successfully decorated long RNAs with azide-modified uridine and quantified the relative incorporation levels using nanopores. Our study establishes a robust platform for solid-state nanopore characterization of modified nucleotide-containing RNAs, advancing single-molecule analyses of RNA dynamics.

Polystyrene Nanoplastic Contaminants Denature Human Apolipoprotein A-1

Mishra, A., Golbek, T. W., Zubia-Aranburu, J., et al. — Wed, 03 Sep 2025 07:16:26 +0200

The subject of nanoplastics is of growing importance, as the use of plastics in our everyday lives has caused nanoplastics to be abundant in the air, water, and soil, bringing them in contact with humans and animals. Understanding how proteins bind and structure themselves at these nanoplastic interfaces is critical for determining the toxicity and health implications of nanoplastics. Proteomics has determined the most abundant protein in human protein corona formed around nanoplastics; however, the structure and orientation of these proteins is extremely challenging to determine. We use in situ sum frequency scattering vibrational spectroscopy and two-dimensional infrared spectroscopy to probe the structure of human liver protein apolipoprotein A-1 (ApoA-1) when adsorbed to polystyrene (PS) nanoparticles. The spectra indicate that ApoA-1 aggregates and forms fibrillar structures at the PS nanoplastic interface, leading to clustering of PS nanoplastics, which may pose a significant risk to human health.

Preventing CpG hypermethylation in oocytes safeguards mouse development

Kawamura, Y. K., Ozonov, E. A., Papasaikas, P., et al. — Fri, 29 Aug 2025 07:16:26 +0200

Except for regulatory CpG-island sequences, genomes of most mammalian cells are widely DNA-methylated. In oocytes, though, DNA methylation (DNAme) is largely confined to transcribed regions. The mechanisms restricting de novo DNAme in oocytes and their relevance thereof for zygotic genome activation and embryonic development are largely unknown. Here we show that KDM2A and KDM2B, two histone demethylases, prevent genome-wide accumulation of histone H3 lysine 36 di-methylation, thereby impeding DNMT3A-catalyzed DNAme. We demonstrate that aberrant DNAme at CpG islands inherited from Kdm2a/Kdm2b double-mutant oocytes represses gene transcription in two-cell embryos. Aberrant maternal DNAme impairs pre-implantation embryonic development, which is suppressed by Dnmt3a deficiency during oogenesis. Hence, KDM2A/KDM2B are essential for confining the oocyte methylome, thereby conferring competence for early embryonic development. Our research implies that the reprogramming capacity eminent to early embryos is insufficient for erasing aberrant DNAme from maternal chromatin, and that early development is susceptible to gene dosage haplo-insufficiency effects.

Selection of locally administered ocular tissue-targeting RNA aptamers using in vivo SELEX

Stenberg, K., Korhonen, S., Seemab, U., et al. — Mon, 01 Sep 2025 07:16:26 +0200

The number of patients affected by retinal diseases is increasing worldwide, and more effective ocular drug delivery strategies are needed. Intravitreal anti-VEGF therapy has reduced the rates of visual impairment and blindness; however, frequent injections cause a significant burden on patients, resulting in poor compliance and therapeutic outcome. Thus, a targeted approach with less invasive administration could extend the dosing intervals and offer a patient-friendly alternative to the current standard of intravitreal injection. Here, in vivo Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was conducted to select ocular tissue-targeting aptamers in rat using topical eye drops and subconjunctival injection. Data analysis of the sequenced aptamer pools revealed significant enrichment of Apt2 and Apt4 in ocular tissues. Their in vivo biodistribution was further evaluated using quantitative real-time PCR (qRT-PCR) at 0.5, 7, and 24 h after subconjunctival administration. Both aptamers accumulated preferentially in posterior ocular tissues, with Apt2 levels exceeding those of the control aptamer by a factor of two at 0.5 h and four at 24 h. This study demonstrates that in vivo SELEX is a viable tool to select ocular tissue-targeting aptamers from the ocular surface. This method could be utilized in the discovery of ocular targeting aptamers for therapeutic purposes and help uncover novel drug targets for various ocular disorders.

In silico prediction of variant effects

Sendrowski, J., Bataillon, T., Ramstein, G. P. — Fri, 01 Aug 2025 07:16:26 +0200

Key message: Sequence-based AI models show great potential for prediction of variant effects at high resolution, but their practical value in plant breeding remains to be confirmed through rigorous validation studies. Abstract: Plant breeding has traditionally relied on phenotyping to select individuals with desirable traits—a process that is both costly and time-consuming. Increasingly, breeding strategies are shifting toward precision breeding, where causal variants are directly targeted based on their effects. To predict the effects of causal variants, in silico methods are emerging as efficient alternatives or complements to mutagenesis screens. Here, we review state-of-the-art machine learning methods for predicting variant effects in plants across both coding and noncoding regions, contrasting supervised approaches in functional genomics with unsupervised methods in comparative genomics. We discuss challenges in validating predictions, and compare these methods with traditional association and comparative genomics techniques. We argue that modern sequence models extend traditional methods by generalizing across genomic contexts, fitting a unified model across loci rather than a separate model for each locus. In doing so, they address inherent limitations of traditional quantitative and evolutionary comparative genetics techniques. However, the accuracy and generalizability of sequence models heavily depend on the training data, highlighting the need for validation experiments. We point to successful applications of sequence models, especially with protein sequences, and identify areas for further improvement, especially in modeling regulatory sequences. While not yet mature for in silico-driven precision breeding, sequence models show strong potential to become an integral part of the breeder’s toolbox.