Publications - Publikationer

Roles of dimeric intermediates in RNA-catalyzed rolling circle synthesis

Fri, 06 Jun 2025 13:21:30 +0200

The RNA world hypothesis is supported by the discovery of RNA polymerase ribozymes that can perform RNA-catalyzed RNA replication processes on different RNA templates. Recently, RNA-catalyzed rolling circle synthesis (RCS) on small circular RNA (scRNA) templates has been demonstrated. However, the structural and dynamic properties of scRNA replication and its products and intermediates have not been explored. Here, we have used cryogenic electron microscopy (cryo-EM) to characterize products and intermediates relevant for RCS replication. We find that these form an unexpectedly diverse group of RNA nanostructures. The main structural motif observed is a fully hybridized dimeric complex composed of two scRNAs and their complement strands resolved to 5.3 Å. Cryo-EM also reveals higher-order dimer filaments and dimer assembly intermediates, suggesting an assembly mechanism for the observed complexes. We show that the dimer complexes are stable and inhibit RNA-catalyzed RCS but can be reactivated by addition of more scRNA templates. We propose dimer formation as a general property of RCS replication and speculate that dimers might have benefited a primordial RNA genetic system by providing a stable ''storage'' form for RNA replication products and by coordinated RNA replication on both scRNA template strands.

Protective role of Angiogenin in muscle regeneration in amyotrophic lateral sclerosis

Fabbrizio, P., Baindoor, S., Margotta, C., et al. — Tue, 01 Jul 2025 13:21:30 +0200

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease with no effective treatments, in part caused by variations in progression and the absence of biomarkers. Mice carrying the SOD1G93A transgene with different genetic backgrounds show variable disease rates, reflecting the diversity of patients. While extensive research has been done on the involvement of the central nervous system, the role of skeletal muscle remains underexplored. We examined the impact of angiogenin, including its RNase activity, in skeletal muscles of ALS mouse models and in biopsies from ALS patients. Elevated levels of angiogenin were found in slowly progressing mice but not in rapidly progressing mice, correlating with increased muscle regeneration and vascularisation. In patients, higher levels of angiogenin in skeletal muscles correlated with milder disease. Mechanistically, angiogenin promotes muscle regeneration and vascularisation through satellite cell-endothelial interactions during myogenesis and angiogenesis. Furthermore, specific angiogenin-derived tiRNAs were upregulated in slowly progressing mice, suggesting their role in mediating the effects of angiogenin. These findings highlight angiogenin and its tiRNAs as potential prognostic markers and therapeutic targets for ALS, offering avenues for patient stratification and interventions to mitigate disease progression by promoting muscle regeneration.

The prognostic potential of RNA in stage II colon cancer

Korsgaard, U., Kristensen, M. P., García-Rodríguez, J. L., et al. — Fri, 06 Jun 2025 13:21:30 +0200

The management of stage II colon cancer poses an important clinical challenge due to difficulties in identifying patients at risk of recurrence. RNA expression profiles in solid tissues have demonstrated a strong potential as prognostic markers. The objective of this study was to develop an RNA-based risk score to risk-stratify patients with stage II colon cancer using a large, well-characterized, and screened patient cohort. This multicenter cohort study encompasses tissue samples collected from all surgically resected stage II colon cancer patients in the Region of Southern Denmark from 2014 to 2016 (n = 739). Gene expression analysis was performed on 122 RNAs, previously described as holding prognostic potential in colorectal cancer, using NanoString nCounter analyses. The resulting RNA expression profiles were used to develop a score predicting the risk of recurrence. The primary endpoint for the study was time to recurrence (TTR), with overall survival (OS) as a secondary endpoint. A dichotomized score, derived from the combined expression of four RNAs (ZNF697, SNORA2B, CTSC and OXLD1), effectively predicted TTR in stage II colon cancer patients. The score revealed a hazard ratio (HR) of 6.84 in univariate analysis (p < 0.001) and a HR of 5.00 in multivariate analysis (p < 0.001), surpassing the prognostic performance of known clinical risk factors. Additionally, the score was significantly associated with OS in Kaplan-Meier analysis (p = 0.024). In conclusion, this four-gene expression score demonstrates a strong association with TTR in stage II colon cancer patients, providing valuable prognostic insights that extend beyond conventional clinical risk markers.

ciRS-7 expression is epigenetically regulated in cancer cells across human adenocarcinomas

Paasch, T. P., Olesen, M. T. J., García-Rodríguez, J. L., et al. — Mon, 02 Jun 2025 13:21:30 +0200

Circular RNAs (circRNAs) constitute a large class of non-coding RNAs with gene regulatory capabilities, mainly through microRNA binding, a mechanism that has been linked to cancer development. The circRNA ciRS-7 (also known as CDR1as) is an interesting candidate as it harbors over 60 binding sites for miR-7, which is known to have tumor-suppressing properties. Here, we investigated the spatial expression patterns and epigenetic regulation of ciRS-7 across nine different adenocarcinomas originating from the colon, pancreas, ovary, endometrium, breast, stomach, bile duct, lung, and prostate. The study included primary patient samples and 18 different cell lines. ciRS-7 expression was analyzed using Reverse Transcription-quantitative PCR (RT-qPCR), single molecule in situ hybridization, and Nanostring nCounter, while epigenetic modifications were examined through bisulfite sequencing, Sensitive Melting Analysis after Real Time – Methylation Specific PCR (SMART-MSP), and chromatin immunoprecipitation. The functional relevance of epigenetic modifications was examined using DNA methyltransferase and histone deacetylase inhibitors. Across all adenocarcinomas, ciRS-7 was absent in the cancer cells in most of the primary tumor specimens, except for the breast tumors, while being expressed in the tumor microenvironment (TME). In line with this, ciRS-7 was not detected in most of the cell lines. Moreover, we demonstrated that DNA methylation and H3K9 acetylation, but not H3K27 methylation, are important epigenetic modifications that impact ciRS-7 expression. In conclusion, our data show that ciRS-7 is mainly expressed in the TME and is regulated through DNA methylation and histone acetylation across all major types of adenocarcinomas.

Helicobacter pylori CagA protein is a potent and broad-spectrum amyloid inhibitor

Jin, Z., Olsen, W. P., Mörman, C., et al. — Fri, 13 Jun 2025 13:21:30 +0200

Bacteria, the smallest and most abundant life forms on Earth, have been a source of insights that have had a considerable impact on human health. Helicobacter pylori has captured substantial attention due to its role in provoking an array of gastrointestinal ailments and other human diseases. Here, we report that H. pylori releases the protein CagA (cytotoxin-associated gene A) that strongly inhibits formation of both functional (bacterial biofilm) and pathogenic amyloid assemblies by targeting various stages during fibril formation. CagA's broad substrate specificity reveals a mechanism whereby H. pylori interferes with other bacteria and humans, offering approaches to combat bacterial infections and human protein misfolding diseases.

Natural Design of a Stabilized Cross-β Fold

Peña Díaz, S., Zhang, Z., Jiang, Y., et al. — Wed, 11 Jun 2025 13:21:30 +0200

Identification of α-Synuclein Aggregation Inhibitors via High-Throughput Screening

Peña-Díaz, S., Manglano-Artuñedo, Z., Pinheiro, F., Ventura, S. — Wed, 01 Jan 2025 13:21:30 +0100

Parkinson’s disease is the second most common neurodegenerative disorder worldwide and, despite daunting efforts, remains incurable. α-Synuclein (α-Syn) aggregation into intracellular inclusions is the main pathological hallmark of this disease and, accordingly, is an important therapeutic target. Due to the disordered nature of the protein, high-throughput screening has become the gold-standard approach for identifying α-Syn aggregation inhibitors. Here, we describe a comprehensive protocol for developing α-Syn aggregation modulators. This approach combines a straightforward and highly reproducible method for screening large chemical libraries with a suite of orthogonal validation techniques, cytotoxicity assays, and in vivo testing. This strategy, which has crystallized in the discovery of potent α-Syn aggregation inhibitors, can be readily adapted by other laboratories, thus contributing to finding disease-modifying drugs for Parkinson’s disease.

Three cryo-EM structures of CD109 reveal its mechanism of protease inhibition

Almeida, A. V., Jensen, K. T., Harwood, S. L., et al. — Tue, 24 Jun 2025 13:21:30 +0200

CD109 is a glycosylphosphatidylinositol-anchored protein. In addition to regulating transforming growth factor β (TGF-β) network signaling, CD109 is also a protease inhibitor. Protease cleavage of its bait region triggers a conformational change releasing the major fragment from the cell surface, exposing a reactive thioester that can conjugate proteases. To understand this protease inhibition mechanism, we determined cryoelectron microscopy structures of CD109 in native, protease-activated, and methylamine-activated conformations. Despite CD109’s low sequence similarity with the protease inhibitor A2ML1, CD109 adopts a similar protease-activated conformation, suggesting a shared mechanism of protease inhibition. Deglycosylation of CD109 does not affect chymotrypsin conjugation but enhances substrate access, suggesting that CD109 glycans contribute to protease inhibition. Our data provide a structural basis for understanding CD109’s protease-triggered membrane release, its protease inhibitory mechanism, and additional biological functions.

Effect of prolonged voluntary wheel running on oxidative stress and defence mechanisms in cortex and hippocampus of healthy female rats

Myrup Holst, C., Esperon-Abril, I., Bryske Juhl, F., et al. — Wed, 01 Jan 2025 13:21:30 +0100

Physical exercise promotes brain health and cognitive function possibly through mechanisms that include strengthened resistance to oxidative stress. However, limited research has explored the cumulative effects of regular voluntary exercise on both oxidative stress and defence mechanisms in hippocampus and cortex, two regions essential for cognitive function. Especially, adaptations in the young, healthy brain are insufficiently understood. This study investigates the impact of 8 weeks of voluntary wheel running on oxidative damage and counteracting defence mechanisms in the cortex and hippocampus of young, healthy female rats. To this end, we assessed oxidative damage to proteins and DNA, antioxidant defence, and DNA repair mechanisms, focusing on the base excision repair pathway. Our findings show that 8 weeks of voluntary exercise does not significantly modify oxidative damage or antioxidant defences in either cortical or hippocampal brain regions. Instead, the voluntary wheel running intervention led to a reduction in the levels of DNA polymerase β and mitochondrial apurinic/apyrimidinic endonuclease 1, key enzymes involved in base excision repair. Moreover, mitochondrial DNA copy number increased in the cortex, but decreased in the hippocampus, suggesting distinct regional adaptations. Collectively, these results indicate that the healthy young brain maintains redox homeostasis despite reduced DNA repair capacity. By analysing a comprehensive array of biomarkers in two brain regions, this study addresses gaps in our current knowledge on prolonged training and brain health and provides valuable insights into how regular exercise produces region-specific and shared responses in the healthy state.

Application of microRNA In Situ Hybridization on Long-term Stored Human Formalin-fixed Paraffin-embedded Brain Samples from Psychiatric Patients

Søkilde, R., Kaadt, E., Kristensen, L. S., et al. — Wed, 01 Jan 2025 13:21:30 +0100

Here, we report that long-term stored human brain samples, formalin-fixed paraffin-embedded (FFPE) from The Brain Collection, University of Southern Denmark, Denmark (https://www.sdu.dk/en/forskning/bridge/the-brain-collection), can be used for in situ hybridization (ISH) analysis of selected microRNAs (miRNAs). The Human Brain Collection consists of brains from 9479 subjects who died at a Danish State Mental Hospital in the period of 1945–1982. In the present study we included tissue specimens from prefrontal cortex (PFC) and hippocampus (HIP) from 163 patients diagnosed with schizophrenia, bipolar disorder, or major depressive disorder. Initially, the Nanostring nCounter platform was used to identify miRNA candidates for ISH analysis using the miRNAscope technology. Based on the Nanostring nCounter quantifications with bulk tissue, we identified and selected 10 miRNAs from PFC (miR-9-5p, miR-29b-3p, miR-30c-5p, miR-124-3p, miR-125b-5p, miR-138-5p, miR-181a-5p, miR-224-5p, miR-302d-3p, and miR-432-5p) and 6 miRNAs from HIP (let-7a-5p, miR-7-5p, miR-124-3p, miR-127-3p, miR-145-5p and miR-149-5p). miRNAscope ISH analysis was then performed with the respective probes on 30 PFC and 30 HIP samples, respectively. In the PFC six miRNAs (miR-9-5p, miR-29b-3p, miR-124-3p, miR-125b-5p, miR-138-5p, and miR-181a-5p) were detected and four (miR-145-5p, let-7a-5p, miR-124-3p and miR-7-5p) in the HIP samples. In both brain regions miR-124-3p was the most abundantly expressed. We conclude, that the combination of the Nanostring nCounter technology and the miRNAscope analysis is a valid approach to study spatial expression of specific miRNAs in these up to 76 years old FFPE blocks. This opens a new avenue of possibilities for studying the underlying epigenetic mechanisms in mental disorders.

Polygenic risk score prediction accuracy convergence

Henches, L., Kim, J., Yang, Z., et al. — Tue, 01 Jul 2025 13:21:30 +0200

Polygenic risk scores (PRSs) models trained from genome-wide association study (GWAS) results are set to play a pivotal role in biomedical research addressing multifactorial human diseases. The prospect of using these risk scores in clinical care and public health is generating both enthusiasm and controversy, with varying opinions among experts about their strengths and limitations. The performance of existing polygenic scores is still limited but is expected to improve with increasing GWAS sample sizes and the development of new, more powerful methods. Theoretically, the variance explained by PRS can be as high as the total additive genetic variance, but it is unclear how much of that variance has already been captured by PRS. Here, we conducted a retrospective analysis to assess progress in PRS prediction accuracy since the publication of the first large-scale GWASs, using data from six common human diseases with sufficient GWAS information. We show that although PRS accuracy has grown rapidly over the years, the pace of improvement from recent GWAS has decreased substantially, suggesting that merely increasing GWAS sample sizes may lead to only modest improvements in risk discrimination. We next investigated the factors influencing the maximum achievable prediction using whole-genome sequencing data from 125,000 UK Biobank participants and state-of-the-art modeling of polygenic outcomes. Our analyses suggest that increasing the variant coverage of PRS, using either more imputed variants or sequencing data, is a key component for future improvements in prediction accuracy.

FaceAge, a deep learning system to estimate biological age from face photographs to improve prognostication

Bontempi, D., Zalay, O., Bitterman, D. S., et al. — Wed, 01 Jan 2025 13:21:30 +0100

Background: As humans age at different rates, physical appearance can yield insights into biological age and physiological health more reliably than chronological age. In medicine, however, appearance is incorporated into medical judgements in a subjective and non-standardised way. In this study, we aimed to develop and validate FaceAge, a deep learning system to estimate biological age from easily obtainable and low-cost face photographs. Methods: FaceAge was trained on data from 58 851 presumed healthy individuals aged 60 years or older: 56 304 individuals from the IMDb–Wiki dataset (training) and 2547 from the UTKFace dataset (initial validation). Clinical utility was evaluated on data from 6196 patients with cancer diagnoses from two institutions in the Netherlands and the USA: the MAASTRO, Harvard Thoracic, and Harvard Palliative cohorts FaceAge estimates in these cancer cohorts were compared with a non-cancerous reference cohort of 535 individuals. To assess the prognostic relevance of FaceAge, we performed Kaplan–Meier survival analysis and Cox modelling, adjusting for several clinical covariates. We also assessed the performance of FaceAge in patients with metastatic cancer receiving palliative treatment at the end of life by incorporating FaceAge into clinical prediction models. To evaluate whether FaceAge has the potential to be a biomarker for molecular ageing, we performed a gene-based analysis to assess its association with senescence genes. Findings: FaceAge showed significant independent prognostic performance in various cancer types and stages. Looking older was correlated with worse overall survival (after adjusting for covariates per-decade hazard ratio [HR] 1·151, p=0·013 in a pan-cancer cohort of n=4906; 1·148, p=0·011 in a thoracic cohort of n=573; and 1·117, p=0·021 in a palliative cohort of n=717). We found that, on average, patients with cancer looked older than their chronological age (mean increase of 4·79 years with respect to non-cancerous reference cohort, p<0·0001). We found that FaceAge can improve physicians’ survival predictions in patients with incurable cancer receiving palliative treatments (from area under the curve 0·74 [95% CI 0·70–0·78] to 0·8 [0·76–0·83]; p<0·0001), highlighting the clinical use of the algorithm to support end-of-life decision making. FaceAge was also significantly associated with molecular mechanisms of senescence through gene analysis, whereas age was not. Interpretation: Our results suggest that a deep learning model can estimate biological age from face photographs and thereby enhance survival prediction in patients with cancer. Further research, including validation in larger cohorts, is needed to verify these findings in patients with cancer and to establish whether the findings extend to patients with other diseases. Subject to further testing and validation, approaches such as FaceAge could be used to translate a patient's visual appearance into objective, quantitative, and clinically valuable measures. Funding: US National Institutes of Health and EU European Research Council.

Differences in complement activation of serum-resistant and serum-sensitive Klebsiella pneumoniae isolates

Thomsen, M. E., Bennike, T. B., Christiansen, G., et al. — Tue, 01 Jul 2025 13:21:30 +0200

The gram-negative bacteria Klebsiella pneumoniae are genetically heterogeneous and a common cause of sepsis and bacteremia in humans. The complement system is the first line of defence against bacteria when they invade the body. We previously investigated K. pneumoniae isolates from sepsis patients. We found that complement factor (C) 3 is deposited on all isolates independent of serum sensitivity, but the membrane attack complex (MAC) was only formed on the serum-sensitive isolates. To investigate the mechanism for serum resistance, we incubated one serum-sensitive and one serum-resistant isolate in human serum and identified bound complement factors by mass spectrometry. The serum-sensitive isolate had all expected complement factors bound, including C4, while the serum-resistant isolate had only C3 bound. The serum resistance was caused by a fast cleavage of C3b to iC3b. Thereby, the C5 convertase, and thus MAC, cannot be formed. To confirm the role of C4 in serum sensitivity, C4 was inhibited by the nanobody hC4Nb8, resulting in the survival of the serum-sensitive isolate. This suggests that C4 is indispensable for MAC formation through the classical and lectin pathways. In contrast, when activated selectively, the alternative pathway primarily leads to the generation of iC3b, thereby enabling serum resistance by bypassing MAC assembly.

Genome-wide analyses identify 30 loci associated with obsessive–compulsive disorder

Strom, N. I., Gerring, Z. F., Galimberti, M., et al. — Wed, 01 Jan 2025 13:21:30 +0100

Obsessive–compulsive disorder (OCD) affects ~1% of children and adults and is partly caused by genetic factors. We conducted a genome-wide association study (GWAS) meta-analysis combining 53,660 OCD cases and 2,044,417 controls and identified 30 independent genome-wide significant loci. Gene-based approaches identified 249 potential effector genes for OCD, with 25 of these classified as the most likely causal candidates, including WDR6, DALRD3 and CTNND1 and multiple genes in the major histocompatibility complex (MHC) region. We estimated that ~11,500 genetic variants explained 90% of OCD genetic heritability. OCD genetic risk was associated with excitatory neurons in the hippocampus and the cortex, along with D₁ and D₂ type dopamine receptor-containing medium spiny neurons. OCD genetic risk was shared with 65 of 112 additional phenotypes, including all the psychiatric disorders we examined. In particular, OCD shared genetic risk with anxiety, depression, anorexia nervosa and Tourette syndrome and was negatively associated with inflammatory bowel diseases, educational attainment and body mass index.

The role of the extracellular matrix in TGFBI-related corneal dystrophy development

Poulsen, E. T., Nielsen, N. S., Enghild, J. J. — Thu, 20 Feb 2025 13:21:30 +0100

The transparent nature of the cornea makes it unique among tissues in the human body. The distinct composition of the corneal proteome appears to be a key factor in protein aggregation caused by mutations in the gene encoding transforming growth factor beta-induced protein (TGFBIp). This chapter reviews the current understanding of the biophysical and biochemical properties of corneal TGFBIp carrying one of the more than 70 known disease-causing mutations. Notably, TGFBIp exhibits a dual aggregation propensity, leading to either amorphous or amyloid insoluble protein aggregates in the cornea upon mutation. Emerging research utilizing protein characterization techniques such as mass spectrometry and X-ray crystallography suggests that the proteolytic machinery in the cornea plays a central role in driving TGFBIp aggregation.

Genetic Architecture and Risk of Childhood Maltreatment Across 5 Psychiatric Diagnoses

Nielsen, T. T., Bali, P., Grove, J., et al. — Fri, 09 May 2025 13:21:30 +0200

IMPORTANCE: Childhood maltreatment (CM) is associated with psychiatric disorders. The underlying mechanisms are complex and involve genetics.

OBJECTIVE: To investigate the polygenic architecture of CM-exposed individuals across psychiatric conditions and if genetics modulates absolute CM risk in the presence of high-impact risk factors such as parental psychiatric diagnoses.

DESIGN, SETTING, AND PARTICIPANTS: The population-based case-cohort iPSYCH was used to analyze 13 polygenic scores (PGS) in CM-exposed individuals across 5 psychiatric International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) diagnoses benchmarked against controls. Individuals were stratified into PGS quantiles, and absolute CM risk was calculated using Cox regression. Sex-specific analyses were also performed. Data were analyzed from June 2022 to December 2024.

EXPOSURES: PGS of phenotypes of psychiatric disorders, CM, educational attainment, and substance use.

MAIN OUTCOMES AND MEASURES: PGSs were generated using summary statistics from genome-wide association studies of phenotypes representing psychiatric disorders, CM, educational attainment, and substance use and tested for their association with CM across psychiatric disorders.

RESULTS: This study included 102 856 individuals (mean [SD] age, 22.6 [7.1] years; 54 918 male [53.4%]) 8 to 35 years old. A total of 2179 CM-exposed individuals were analyzed across individuals with attention-deficit/hyperactivity disorder (ADHD; n = 22 674), autism (n = 18 941), schizophrenia (n = 6103), bipolar disorder (n = 3061), depression (n = 28 896), and controls (n = 34 689). PGSs for ADHD and educational attainment were associated with CM across all psychiatric diagnoses. The absolute CM risk was increased in the highest PGS groups, eg, for ADHD, the absolute CM risk was 5.6% in the highest ADHD-PGS quartile whereas it was only 3.3% in the lowest ADHD-PGS quartile (hazard rate ratio quantile 4 vs quantile 1 = 1.81; 95% CI, 1.47-2.22). CM risk was more than twice as high for children with parents with a psychiatric diagnosis (5.7%) than for children with parents without a psychiatric diagnosis (2.5%), but even in the presence of this risk factor, individuals could still be stratified into risk groups based on their genetics. No genetic differences between CM-exposed males and females were observed, but there were striking sex differences in absolute CM risk, which reached 5.6% for females in the highest ADHD-PGS quartile and 2.0% for males.

CONCLUSIONS AND RELEVANCE: Results of this case-control study suggest that individuals with high ADHD-PRS and/or low educational attainment-PRS had an associated elevated risk of CM. Extra attention should be given to individuals at high risk for CM across all 5 psychiatric diagnoses, ie, females with a high ADHD-PGS and/or a parent diagnosed with a psychiatric disorder.

Convergent evidence linking neonatal vitamin D status and risk of neurodevelopmental disorders

Horsdal, H. T., Albiñana, C., Zhu, Z., et al. — Sun, 01 Jun 2025 13:21:30 +0200

BACKGROUND: There is growing evidence linking neonatal vitamin D deficiency to an increased risk of schizophrenia, ADHD, and autism spectrum disorder (ASD). The aim of this study was to examine the association between two vitamin D biomarkers (25 hydroxyvitamin D [25(OH)D] and vitamin D-binding protein [DBP], and their related genetic correlates) and the risk of six mental disorders.

METHODS: We used a population-based, case-cohort sample of all individuals born in Denmark between 1981 and 2005. Using Danish health registers with follow-up to Dec 31, 2012, we identified individuals diagnosed with major depressive disorder, bipolar disorder, schizophrenia, ADHD, ASD, and anorexia nervosa based on ICD-10 criteria. Additionally, a random subcohort from the general population was selected. Based on neonatal dried blood spots, we measured concentrations of 25(OH)D and DBP. Our primary analyses were based on hazard ratios (HR) with 95% CI and absolute risks for the six mental disorders according to measured concentrations of 25(OH)D and DBP. As secondary analyses, we examined the association between genetic predictors of 25(OH)D and DBP, and the six mental disorders, and Mendelian randomisation analyses based on published summary statistics for 25(OH)D, DBP, and the six mental disorders. People with lived experience contributed to the development of the guiding hypothesis.

FINDINGS: We used the total population from the iPSYCH2012 design (n=88 764), which included individuals who developed the six mental disorders, major depressive disorder (n=24 240), bipolar disorder (n=1928), schizophrenia (n=3540), ADHD (n=18 726), ASD (n=16 146), anorexia nervosa (n=3643), and the randomly sampled subcohort (n=30 000). Among those who met a range of inclusion criteria (eg, measured 25[OH]D, DBP or genotype, and predominantly European ancestry), we measured 25(OH)D or DBP in 71 793 individuals (38 118 [53·1%] male and 33 675 [46·9%] female); 65 952 had 25(OH)D and 66 797 the DBP measurements. Significant inverse relationships were found between 25(OH)D and schizophrenia (HR 0·82, 95% CI 0·78-0·86), ASD (HR 0·93, 95% CI 0·90-0·96), and ADHD (HR 0·89, 95% CI 0·86-0·92). A significant inverse relationship was found between DBP and schizophrenia (HR 0·84, 95% CI 0·80-0·88). Based on polygenic risk scores, higher concentrations of 25(OH)D (adjusted for DBP) were significantly associated with a reduced risk of both ASD and schizophrenia. Analyses based on Mendelian randomisation provided support for a causal association between both lower 25(OH)D and DBP concentrations and an increased risk of ADHD.

INTERPRETATION: Convergent evidence finds that neonatal vitamin D status is associated with an altered risk of mental disorders. Our study supports the hypothesis that optimising neonatal vitamin D status might reduce the incidence of a range of neurodevelopmental disorders.

FUNDING: The Danish National Research Foundation.

A cellular system to study responses to a collision between the transcription complex and a protein-bound nick in the DNA template

Herring, P., Roedgaard, M., Holst, C. M., et al. — Wed, 01 Jan 2025 13:21:30 +0100

We present a transcription-coupled Flp-nick system enabling a stable protein-bound nick mimicking a topoisomerase I–DNA cleavage complex. The nick is introduced at a single site within a controllable LacZ gene inserted into the Saccharomyces cerevisiae genome. This system allows unique single-site studies of a frequently occurring damage within a transcription unit in vivo. As proof of principle, we demonstrate RNA polymerase II accumulation at the damage site when MG132 inhibits the proteasome. Similarly, accumulation occurs when polymerase ubiquitination is abolished by deletion of the ubiquitinase ELC1 gene. This indicates that a topoisomerase I–DNA mimicking cleavage complex per se induces RNA polymerase II ubiquitination and degradation. These findings advance understanding of cellular responses to topoisomerase I-targeting drugs used in cancer chemotherapy.

Genomes of critically endangered saola are shaped by population structure and purging

Garcia-Erill, G., Liu, S., Le, M. D., et al. — Thu, 12 Jun 2025 13:21:30 +0200

The saola is one of the most elusive large mammals, standing at the brink of extinction. We constructed a reference genome and resequenced 26 saola individuals, confirming the saola as a basal member of the Bovini. Despite its small geographic range, we found that the saola is partitioned into two populations with high genetic differentiation (F_ST = 0.49). We estimate that these populations diverged and started declining 5,000–20,000 years ago, possibly due to climate changes and exacerbated by increasing human activities. The saola has long tracts without genomic diversity; however, most of these tracts are not shared by the two populations. Saolas carry a high genetic load, yet their gradual decline resulted in the purging of the most deleterious genetic variation. Finally, we find that combining the two populations, e.g., in an eventual captive breeding program, would mitigate the genetic load and increase the odds of species survival.

Low T cell diversity associates with poor outcome in bladder cancer

Kjær, A., Kristjánsdóttir, N., Juul, R. I., et al. — Tue, 20 May 2025 13:21:30 +0200

T cells are crucial effector cells in the endogenous defense against cancer, yet the clinical impact of their quantity, diversity, and dynamics remains underexplored. Here, we investigate the clinical relevance of the T cell receptor (TCR) repertoire in patients with bladder cancer. In advanced-stage disease, low pre-treatment peripheral TCR diversity is associated with worse overall survival (p = 0.024), particularly when coupled with low circulating T cell fractions (p = 0.00049). These low-diversity repertoires are dominated by hyper-expanded clones that persist throughout treatment. Further longitudinal analysis reveals reductions in TCR diversity after treatment, indicating adverse effects on the immune system. In early-stage disease, immunotherapy increases TCR diversity in patients with good outcomes. Furthermore, single-cell sequencing identifies most hyper-expanded clones as cytotoxic T cells, while non-expanded clones are predominantly naive T cells. Overall, this highlights TCR diversity as a promising biomarker, offering opportunities for tailored oncological treatments to enhance clinical outcomes.

Faba bean genetics and crop growth models – progress to date and opportunities for integration

Wed, 01 Jan 2025 13:21:30 +0100

Background: Faba bean (Vicia faba L.) is a globally adapted protein crop with a high yield potential and efficient nitrogen fixation. A decade ago, it was an orphan crop with limited genetic and genomic resources and little knowledge was available on the genetic basis of agronomic traits. Over the past few years, rapid progress has been made in genetic mapping and genomic prediction. A major remaining question is how to improve the understanding of associations between genes and environmental factors, including water and nutrient uptake and availability. Scope: We review recent progress in faba bean research, including the development of reference genome sequences and genotyping approaches, which has facilitated high-resolution genetic mapping. We anchor QTL from different studies to the same reference genome to provide a current overview of faba bean traits and associated QTL, highlighting robust signals supported by multiple lines of evidence. We then consider the state of the art in faba bean crop growth models (CGMs) and how they could inform future crop improvement in combination with genetic models. Conclusion: Genetic studies based on high-resolution genotype information and multi-location field trials are now providing a basis for genetic dissection of faba bean genotype by environment interactions and for predicting the performance of specific genotypes in unseen environments. Integration of process-based CGMs with genetic modelling could represent an important next step by capturing genotype-specific growth dynamics but await field trial data suited for supporting development of improved faba bean CGMs.

Industrial Scale Production and Characterization of a Whey Fraction Enriched in Extracellular Vesicle Material

Midtgaard, S. R., Hansen, M. S., Drachmann, N., et al. — Thu, 01 May 2025 13:21:30 +0200

Human milk serves the sole nutritional role for the developing infant. During lactation, nano-sized extracellular vesicles (EVs) in milk containing a multitude of biologically active components are transferred from mother to offspring. Infant formula (IF) based on cow milk-derived ingredients has been reported to contain reduced levels of EVs as compared to human milk. There is therefore an unmet need to produce large-scale volumes of milk EVs to improve IF composition. Here, we report a scalable industrial production protocol for a bovine whey-derived ingredient that is highly enriched in EV material using a large-scale sequential ceramic membrane filtration setup. Furthermore, we demonstrate a robust and generally applicable analytical approach to determine the relative contributions of EVs and milk fat globule membrane (MFGM) using molar ratios of the membrane-bound proteins butyrophilin (BTN) and CD9 as surrogate markers for MFGM and EVs, respectively. Taken together, our findings provide a basis for comparing bovine milk-containing foods and aid in developing specialized ingredients that can minimize the compositional difference between infant formula and human milk.

Importance of the leaf respiratory quotient

Bruhn, D., Fan, Y., Griffin, K. L., et al. — Tue, 01 Apr 2025 13:21:30 +0200

Rates of leaf respiratory CO₂-release (R_CO2) are important for terrestrial biosphere models that estimate carbon exchange between plants and the atmosphere. Hitherto, models of R_CO2 have primarily been based on considerations of respiratory energy demand (particularly ATP) for maintenance and growth purposes. Respiratory ATP synthesis is closely tied to the rate of respiratory O₂-uptake (R_O2), with relative engagement of the alternative oxidase influencing the ATP:O ratio. However, the extent to which respiratory ATP synthesis is coupled to leaf R_CO2 depends on the respiratory quotient (RQ, mol CO₂ efflux per unit mol O₂ uptake), with models predicting leaf R_CO2 assuming that the RQ is at unity. Here, we show systematic inter-specific, temporal and temperature-dependent variation in leaf RQ, with values of RQ ranging from 0.51 to 2.2, challenging model assumptions on the RQ. We discuss possible mechanisms underlying the variation in leaf RQ, potential ways forward in terms of new measurement protocols, and perspectives for modelled R_CO2. Our analyses highlight a range of outstanding research questions that need to be answered before we can mechanistically model leaf R_CO2 at various scales.

Comparative analysis of STP6 and STP10 unravels molecular selectivity in sugar transport proteins

Andersen, C. G., Bavnhøj, L., Brag, S., et al. — Tue, 29 Apr 2025 13:21:30 +0200

The distribution of sugars is crucial for plant energy, signaling, and defense mechanisms. Sugar Transport Proteins (STPs) are Sugar Porters (SPs) that mediate proton-driven cellular uptake of glucose. Some STPs also transport fructose, while others remain highly selective for only glucose. What determines this selectivity, allowing STPs to distinguish between compounds with highly similar chemical composition, remains unknown. Here, we present the structure of Arabidopsis thaliana STP6 in an inward-occluded conformational state with glucose bound and demonstrate its role as both a glucose and fructose transporter. We perform a comparative analysis of STP6 with the glucose-selective STP10 using in vivo and in vitro systems, demonstrating how different experimental setups strongly influence kinetic transport properties. We analyze the properties of the monosaccharide binding site and show that the position of a single methyl group in the binding site is sufficient to shuffle glucose and fructose specificity, providing detailed insights into the fine-tuned dynamics of affinity-induced specificity for sugar uptake. Altogether, these findings enhance our understanding of sugar selectivity in STPs and more broadly SP proteins.

SorCS2 Is Important for Astrocytic Function in Neurovascular Signaling

Staehr, C. ., Login, H., Melnikova, E., et al. — Sun, 01 Jun 2025 13:21:30 +0200

Introduction
The receptor SorCS2 is involved in the trafficking of membrane receptors and transporters. It has been implicated in brain disorders and has previously been reported to be indispensable for ionotropic glutamatergic neurotransmission in the hippocampus.

Aim
We aimed to study the role of SorCS2 in the control of astrocyte‐neuron communication, critical for neurovascular coupling.

Methods
Brain slices from P8 and 2‐month‐old wild‐type and SorCS2 knockout (Sorcs2 −/− ) mice were immunostained for SorCS2, GFAP, AQP4, IB4, and CD31. Neurovascular coupling was assessed in vivo using laser speckle contrast imaging and ex vivo in live brain slices loaded with calcium‐sensitive dye. Bulk and cell surface fraction proteomics was analyzed on freshly isolated and cultured astrocytes, respectively, and validated with Western blot and qPCR.

Results
SorCS2 was strongly expressed in astrocytes, primarily in their endfeet, of P8 mice; however, it was sparsely represented in 2‐month‐old mice. Sorcs2 −/− mice demonstrated reduced neurovascular coupling associated with a reduced astrocytic calcium response to neuronal excitation. No differences in vascularization or endothelium‐dependent relaxation ex vivo between the 2‐month‐old groups were observed. Proteomics suggested changes in glutamatergic signaling and suppressed calcium signaling in Sorcs2 −/− brains from both P8 and 2‐month‐old mice. The increased abundance of glutamate metabotropic receptor 3 in Sorcs2 −/− astrocytes was validated by PCR and Western blot. In cultured Sorcs2 −/− astrocytes, AQP4 abundance was increased in the bulk lysate but reduced in the cell surface fraction, suggesting impaired trafficking.

Conclusion
The results suggest that SorCS2 expression is important for the development of neurovascular coupling, at least in part by modulating glutamatergic and calcium signaling in astrocytes.

Immunological drivers and potential novel drug targets for major psychiatric, neurodevelopmental, and neurodegenerative conditions

Dardani, C., Robinson, J. W., Jones, H. J., et al. — Tue, 01 Apr 2025 13:21:30 +0200

Immune dysfunction is implicated in the aetiology of psychiatric, neurodevelopmental, and neurodegenerative conditions, but the issue of causality remains unclear impeding attempts to develop new interventions. Using genomic data on protein and gene expression across blood and brain, we assessed evidence of a potential causal role for 736 immune response-related biomarkers on 7 neuropsychiatric conditions by applying Mendelian randomization (MR) and genetic colocalisation analyses. A systematic three-tier approach, grouping biomarkers based on increasingly stringent criteria, was used to appraise evidence of causality (passing MR sensitivity analyses, colocalisation, False Discovery Rate and Bonferroni thresholds). We provide evidence for a potential causal role of 29 biomarkers for 7 conditions. The identified biomarkers suggest a role of both brain specific and systemic immune response in the aetiology of schizophrenia, Alzheimer’s disease, depression, and bipolar disorder. Of the identified biomarkers, 20 are therapeutically tractable, including ACE, TNFRSF17, SERPING1, AGER and CD40, with drugs currently approved or in advanced clinical trials. Based on the largest available selection of plasma immune-response related biomarkers, our study provides insight into possible influential biomarkers for the aetiology of neuropsychiatric conditions. These genetically prioritised biomarkers now require examination to further evaluate causality, their role in the aetiological mechanisms underlying the conditions, and therapeutic potential.

Analysis of Processing, Post-Maturation, and By-Products of shRNA in Gene and Cell Therapy Applications

Hong, Z., Tesic, N., Bofill-De Ros, X. — Tue, 01 Apr 2025 13:21:30 +0200

Short hairpin RNAs (shRNAs) are potent tools for gene silencing, offering therapeutic potential for gene and cell therapy applications. However, their efficacy and safety depend on precise processing by the RNA interference machinery and the generation of minimal by-products. In this protocol, we describe how to systematically analyze the processing of therapeutic small RNAs by DROSHA and DICER1 and their incorporation into functional AGO complexes. Using standard small RNA sequencing and tailored bioinformatic analysis (QuagmiR), we evaluate the different steps of shRNA maturation that influence processing efficiency and specificity. We provide guidelines for troubleshooting common design pitfalls and off-target effects in transcriptome-wide profiling to identify unintended mRNA targeting via the miRNA-like effect. We provide examples of the bioinformatic analysis that can be performed to characterize therapeutic shRNA. Finally, we provide guidelines for troubleshooting shRNA designs that result in suboptimal processing or undesired off-target effects. This protocol underscores the importance of rational shRNA design to enhance specificity and reduce biogenesis by-products that can lead to off-target effects, providing a framework for optimizing the use of small RNAs in gene and cell therapies.

Recurrent innovation of protein-protein interactions in the Drosophila piRNA pathway

Riedelbauch, S., Masser, S., Fasching, S., et al. — Thu, 24 Apr 2025 13:21:30 +0200

Despite being essential for fertility, genome-defense-pathway genes often evolve rapidly. However, little is known about the molecular basis of this adaptation. Here, we characterized the evolution of a protein interaction network within the PIWI-interacting small RNA (piRNA) genome-defense pathway in Drosophila at unprecedented scale and evolutionary resolution. We uncovered the pervasive rapid evolution of a protein interaction network anchored at the heterochromatin protein 1 (HP1) paralog Rhino. Through cross-species high-throughput yeast-two-hybrid screening, we identified three distinct evolutionary protein interaction trajectories across ~40 million years of Drosophila evolution. While several protein interactions are fully conserved, indicating functional conservation despite rapid amino acid-sequence change, other interactions are preserved through coevolution and were detected only between proteins within or from closely related species. We also identified species-restricted protein interactions, revealing insight into the mechanistic diversity and ongoing molecular innovation in Drosophila piRNA production. In sum, our analyses reveal principles of interaction evolution in an adaptively evolving protein–protein interaction network, and support intermolecular interaction innovation as a central molecular mechanism of evolutionary adaptation in protein-coding genes.

True length of diverse capped RNA sequencing (TLDR-seq)

Auxillos, J., Stigliani, A., Vaagensø, C. S., et al. — Fri, 11 Apr 2025 13:21:30 +0200

Analysis of transcript function is greatly aided by knowledge of the full-length RNA sequence. New long-read sequencing enabled by Oxford Nanopore and PacBio devices have the potential to provide full-length transcript information; however, standard methods still lack the ability to capture true RNA 5' ends and select for polyadenylated (pA+) transcripts only. Here, we present a method that, by utilizing cap trapping and 3'-end adapter ligation, sequences transcripts between their exact 5' and 3' ends regardless of polyadenylation status and without the need for ribosomal RNA depletion, with the ability to characterize polyadenylation length of RNAs, if any. The method shows high reproducibility, can faithfully detect 5' ends, 3' ends and splice junctions, and produces gene-expression estimates that are highly correlated to those of short-read sequencing techniques. We also demonstrate that the method can detect and sequence full-length nonadenylated (pA-) RNAs, including long noncoding RNAs, promoter upstream transcripts, and enhancer RNAs, and present cases where pA+ and pA- RNAs show preferences for different but closely located transcription start sites. Our method is therefore useful for the characterization of diverse capped RNA species and analysis of relationships between transcription initiation, termination, and RNA processing.

Stem cell culture conditions affect in vitro differentiation potential and mouse gastruloid formation

Blotenburg, M., Suurenbroek, L., Bax, D., et al. — Sat, 01 Mar 2025 13:21:30 +0100

Aggregating low numbers of mouse embryonic stem cells (mESCs) and inducing Wnt signalling generates ‘gastruloids’, self-organising complex structures that display an anteroposterior organisation of cell types derived from all three germ layers. Current gastruloid protocols display considerable heterogeneity between experiments in terms of morphology, elongation efficiency, and cell type composition. We therefore investigated whether altering the mESC pluripotency state would provide more consistent results. By growing three mESC lines from two different genetic backgrounds in different intervals of ESLIF and 2i medium the pluripotency state of cells was modulated, and mESC culture as well as the resulting gastruloids were analysed. Microscopic analysis showed a pre-culture-specific effect on gastruloid formation, in terms of aspect ratio and reproducibility. RNA-seq analysis of the mESC start population confirmed that short-term pulses of 2i and ESLIF modulate the pluripotency state, and result in different cellular states. Since multiple epigenetic regulators were detected among the top differentially expressed genes, we further analysed genome-wide DNA methylation and H3K27me3 distributions. We observed epigenetic differences between conditions, most dominantly in the promoter regions of developmental regulators. Lastly, when we investigated the cell type composition of gastruloids grown from these different pre-cultures, we observed that mESCs subjected to 2i-ESLIF preceding aggregation generated gastruloids more consistently, including more complex mesodermal contributions as compared to the ESLIF-only control. These results indicate that optimisation of the mESCs pluripotency state allows the modulation of cell differentiation during gastruloid formation.

Transport of phenoxyacetic acid herbicides by PIN-FORMED auxin transporters

Schulz, L., Ung, K. L., Zuzic, L., et al. — Thu, 01 May 2025 13:21:30 +0200

Auxins are a group of phytohormones that control plant growth and development. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane. We go on to characterize the molecular determinants of substrate specificity using a variety of different substrates as well as protein mutagenesis to probe the binding site. Finally, we present cryogenic electron microscopy structures of Arabidopsis thaliana PIN8 bound to either 2,4-dichlorophenoxyacetic acid or 4-chlorophenoxyacetic acid. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with the transport cycle. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results provide a foundation for future development of novel synthetic auxins and for precision breeding of herbicide-resistant crop plants.

The Proteinase PAPP-A has Deep Evolutionary Roots Outside of the IGF System

Kjeldsen, C. M.N., Oxvig, C. — Sat, 01 Mar 2025 13:21:30 +0100

The animal pappalysin metalloproteinases, PAPP-A and PAPP-A2, are highly specific regulatory enzymes of the insulin-like growth factor (IGF) system. Cleavage of their only known substrates, a subset of IGF binding proteins (IGFBPs), releases bioactive IGFI and IGFII, thus promoting IGF signaling. Stanniocalcin-1 and -2 (STC1 and STC2) are potent pappalysin inhibitors, completing the STC-PAPP-A-IGFBP-IGF axis. Utilizing homology searches and phylogenetic analyses, we examined the occurrence of pappalysins in the animal kingdom and their functional conservation. This revealed the extensive presence of pappalysins across metazoans, as well as the presence of 3 pappalysins: PAPP-A, PAPP-A2, and a third group of invertebrate pappalysins, which we name invertebrate PAPP-A (invPAPP-A). We show that PAPP-A and PAPP-A2 arose by duplication during early vertebrate evolution. Despite significant evolutionary distance, the domain architecture of the metazoan pappalysins is completely conserved, and several functional domains and motifs are highly conserved across all pappalysins. However, invPAPP-A exists outside the context of IGFBPs, suggesting that the animal pappalysins may have substrates beyond the IGFBPs for PAPP-A and PAPP-A2 that remain to be discovered. Since PAPP-A is an emerging drug target, it is important to understand potential involvement in regulatory systems other than the IGF system, which might be affected upon targeting of PAPP-A.

Genome-wide association study identifies candidate genes contributing to flowering time variation in Lotus japonicus in Japan

Wakabayashi, T., Andersen, S. U., Tanaka, S., et al. — Wed, 01 Jan 2025 13:21:30 +0100

Flowering time is an important factor in plant fitness and local adaptation. Genome-wide association (GWA) studies have allowed the identification of candidate genes in certain plant species for various traits, including flowering time. Lotus japonicus is widely found throughout the Japanese archipelago. To obtain flowering time data with more prominent difference as more suitable indicator of environmental adaptation, flowering time data were collected for 132 wild accessions originating from various points across this region under shorter day length conditions than in previous studies. The results showed latitudinal variations in flowering time, with southern accessions flowering earlier. Comparing data from four flowering times with varying conditions revealed greater differences under a shorter day length. It is likely that day length significantly affects flowering time in this species. GWA analyses were conducted on flowering time variation measured in this study and the ratios between flowering time under different conditions. Candidate genes different from previous study were detected, including orthologues of known flowering time genes in each analysis. Correlation tests between flowering time and strongly detected single-nucleotide polymorphisms (SNPs) in the GWA analysis suggested that approximately 60% of flowering time variation can be explained by the two main SNPs. This result suggests that the majority of the variation could be explained by a small number of genetic factors. Considering the strong association with flowering time variation, these candidates may be responsible for these differences and therefore can be related to local adaptation in this species.

Quaternary stabilization of a GH2 β-galactosidase from the psychrophile A. ikkensis, a flexible and unstable dimeric enzyme

Nowak, J. S., Kruuse, N., Rasmussen, H. Ø., et al. — Thu, 01 May 2025 13:21:30 +0200

Studies of cold-active enzymes may elucidate the basis for low-temperature activity and contribute to their wider application in energy-efficient processes. Here we investigate the cold-active GH2 β-galactosidase from the psychrophilic bacterium Alkalilactibacillus ikkensis (AiLac). AiLac has a specific activity twice as high as its closest structural homolog (the mesophilic Escherichia coli GH2 β-galactosidase) toward the lactose analog ONPG at room temperature and neutral pH, and shows biphasic behavior in Michaelis-Menten plots. AiLac is activated by Mg 2+ and Na + and is most effective at pH 7.0 and 30°C. However, early unfolding events are observed already at room temperature. Stability studies using intrinsic fluorescence, circular dichroism, and small-angle x-ray scattering (SAXS), combined with activity assays, showed AiLac to be highly sensitive to heat and urea and to be stabilized, but also inhibited, by loss of structural flexibility induced by the osmolyte trehalose. AlphaFold structure prediction combined with SAXS and flow-induced dispersion analysis support a reversible monomer-dimer model, suggesting structural adaptation to cold temperatures on a quaternary level. The low amount of dimeric buried surface area, high flexibility, and remarkably low chemical and thermal stability present an extreme example of cold adaptation promoted by high levels of solvent interactions. To investigate the relationship between evolution and oligomerization, we trained a generative deep learning model to successfully engineer functional variants that form stabilized dimers and tetramers by introducing high evolutionary fitness mutations at the interface, demonstrating an efficient way to explore the local sequence fitness landscape to modulate the equilibrium of oligomerization.

Parkinson's disease and gut microbiota metabolites

Mirab, F., Pirhaghi, M., Otzen, D. E., Saboury, A. A. — Fri, 01 Aug 2025 13:21:30 +0200

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the abnormal accumulation of alpha-synuclein (α-Syn). Recent research emphasizes the significant role of the gut microbiota, the diverse community of microbes living in the intestines, in modulating α-Syn pathology. This review explores the bi-directional communication along the microbiota-gut-brain axis, highlighting the paradoxical impact of two gut microbiota metabolites-functional bacterial amyloids (FuBA) and vitamins-on neurodegenerative diseases, particularly PD. FuBA contributes to PD pathogenesis by promoting α-Syn aggregation, while vitamins offer neuroprotection through their anti-amyloidogenic, antioxidant, and anti-inflammatory properties. Understanding these processes could lead to precision clinical approaches and novel strategies for managing and preventing PD.

Milk osteopontin has high iron-binding capacity and facilitates iron absorption in intestinal cells

Buhl, E. H., Christensen, B., Pedersen, F. H., S⊘rensen, E. S. — Wed, 01 Jan 2025 13:21:30 +0100

Insufficient absorption of iron and the consequent development of iron deficiency have serious health consequences. Hence, identification and development of iron delivery systems that can increase the bioavailability and uptake of dietary iron are important. Osteopontin (OPN) is an acidic and highly phosphorylated integrin-binding protein found in milk where it exists as a full-length protein and as N-terminally derived fragments. Milk OPN can be taken up by enterocytes and transported across the intestinal barrier into the circulation. Milk OPN has previously been shown to bind calcium and magnesium. This study investigates milk OPN as a carrier of iron and its potential to increase iron absorption in intestinal cells. Full-length OPN and N-terminal fragments of OPN were shown to bind ∼30 and ∼10 mol of iron, respectively, and the phosphorylated residues were crucial for iron binding. Osteopontin retained iron bound after simulated gastrointestinal digestion. Immunodetection of digested OPN and OPN-Fe complexes showed that the OPN-Fe complexes were more resistant to pepsin digestion than OPN without bound iron. The cellular uptake of iron was investigated by measuring intracellular ferritin formation and mRNA expression of divalent metal transporter 1 in Caco-2 cells. Osteopontin increased the uptake of iron even in the presence of phytic acid, a dietary inhibitor of iron absorption. These data indicate that OPN can function as an iron carrier for use in alternative strategies for delivering iron in a bioavailable form for intestinal uptake.

Toward Design Principles for Biomolecular Condensates for Metabolic Pathways

André, A. A.M., Rehnberg, N., Garg, A., Kjærgaard, M. — Thu, 01 May 2025 13:21:30 +0200

Biology uses membrane-less organelles or biomolecular condensates as dynamic reaction compartments that can form or dissolve to regulate biochemical pathways. This has led to a flurry of research aiming to design new synthetic organelles that function as reaction crucibles for enzymes and biomolecular cascades in biotechnology. The mechanisms by which a condensate can enhance multistep biochemical processes including mass action, tuning the chemical environment, scaffolding and metabolic channelling is reviewed. These mechanisms are not inherently beneficial for the rate of enzymatic processes but can also inhibit a reaction. Similarly, some aspects of condensates are likely intrinsically inhibitory including retardation of diffusion, where the net effect of a condensate will be a trade-off between inhibitory and stimulatory effects. It is discussed which generalizable conclusions can be drawn so far and how close it is to design principles for condensates for enzyme cascades in microbial cell factories including which reactions are likely to be enhanced by condensates and which type of condensate will be suited for which reaction.

TGFBI R124H mutant allele silencing in granular corneal dystrophy type 2 using topical siRNA delivery

McLain, A., Kowalczyk, A., Baran-Rachwalska, P., et al. — Sun, 01 Jun 2025 13:21:30 +0200

In recent years, success has been achieved in treating several eye conditions with oligonucleotide-based therapies. Herein, we outline the experimentation involved in progressing selection and development of a lead therapeutic siRNA for R124H mutation of TGFBI gene which causes Granular Corneal Dystrophy Type 2 (GCD2/Avellino CD). Firstly, a series of siRNA designs, generated by a gene walk across the R124H TGFBI mutation site, were tested and a lead siRNA identified. The lead siRNA was delivered into an immortalised human corneal epithelial cell line to assess on-target efficacy and off-target effects. The in vivo efficacy of the lead R124H TGFBI siRNA, complexed with Bio-Courier technology, silicon stabilized hybrid lipid nanoparticles (sshLNP), was assessed in a mouse model of GCD2 which expressed the human R124H TGFBI transgene. Following topical siRNA application for 5 consecutive days, expression of the R124H mutant TGFBI transgene was measured and shown to be reduced by 22.4 % (± 15.7 %, p < 0.05). We investigated gene expression in the mouse cornea and showed expression of murine Tgfbi was 20-fold lower than TGFBI in human cornea, and expression of the mutant TGFBI transgene was a further 3-fold lower. This estimated 60-fold lower mutant transgene expression may explain the low frequency of corneal deposits observed in this mouse model, limiting its usefulness to test whether siRNA silencing is capable of phenotypic improvement or regression of GCD2/Avellino corneal dystrophy. We assessed WT TGFBI silencing in human primary corneal epithelial cells (PCEC) derived from human corneal limbal biopsy material, which express TGFBI at a similar level to human corneal biopsy. We demonstrated that a single 100 nM siRNA treatment, delivered by the sshLNP to the primary human corneal epithelial cells, gave 26.6 % (± 6.6 %, p < 0.001) reduction in TGFBI mRNA and a 15.4 % (±10.5 %, p < 0.05 %) reduction in TGFBi protein after 48 h. In consideration of the mutant gene expression levels in existing models of GCD2 disease, an ex vivo model of mutation-expressing primary corneal epithelial cells generated from corneal limbal biopsies from GCD2 patients would be more suitable than existing transgenic mouse models for future pre-clinical work in the development of gene silencing therapies for corneal dystrophies.

The IGF System and Aging

Conover, C. A., Oxvig, C. — Tue, 01 Apr 2025 13:21:30 +0200

There is strong evidence that IGF signaling is involved in fundamental aspects of the aging process. However, the extracellular part of the IGF system is complex with various receptors, ligand effectors, high-affinity IGF-binding proteins, proteinases, and endogenous inhibitors that all, along with their biological context, must be considered. The IGF system components are evolutionarily conserved, underscoring the importance of understanding this system in physiology and pathophysiology. This review will briefly describe the different components of the IGF system and then discuss past and current literature regarding IGF and aging, with a focus on cellular senescence, model organisms of aging, centenarian genetics, and 3 age-related diseases - pulmonary fibrosis, Alzheimer disease, and macular degeneration - in appropriate murine models and in humans. Commonalities in mechanism suggest conditions where IGF system components may be disease drivers and potential targets in promoting healthy aging in humans.

Role of electrostatics in cold adaptation

Tue, 01 Jul 2025 13:21:30 +0200

Psychrophilic (cold-active) organisms have developed enzymes that facilitate sufficient metabolic activity at low temperatures to sustain life. This occurs through molecular adaptations that tend to increase protein flexibility at the expense of stability. However, psychrophiles also vary in their growth conditions. Eurypsychrophiles thrive over a wide temperature range and often prefer temperatures above 20 °C, while stenopsychrophiles grow optimally below 15 °C and are more narrowly adapted to cold temperatures. To elucidate differences between these two classes of enzymes, we here compare the stability and unfolding kinetics of two orthologues of the basal household enzyme triose phosphate isomerase, one from the stenopsychrophilic Antarctic permafrost bacterium Rhodonellum psychrophilum (sTPI) and the other from the eurypsychrophilic Greenland ikaite column bacterium Rhodococcus sp. JG-3 (eTPI). Remarkably, sTPI proved significantly more thermostable and resistant to chemical denaturation than its eurypsychrophilic counterpart, eTPI, in the absence of ionic components in solution, whereas inclusion of electrostatic screening agents in the form of sodium chloride or the charged denaturant guanidinium chloride largely cancelled out this difference. Thus, electrostatics play a prominent role in stabilizing the stenopsychrophilic sTPI, and a mandatory low-temperature growth environment does not preclude the development of considerable thermotolerance for individual enzymes. We were able to increase the thermostability of sTPI using an evolutionary machine learning model, which transferred several sTPI residues into the eTPI active site. While the stabilizing effect was modest, the combination of individual mutations was additive, underscoring the potential of combining multiple beneficial mutations to achieve enhanced enzyme properties.

Gfa1 (glutamine fructose-6-phosphate aminotransferase) is essential for Aspergillus fumigatus growth and virulence

Qin, Q., Wei, P., Usman, S., et al. — Mon, 01 Dec 2025 13:21:30 +0100

Background: Aspergillus fumigatus, the primary etiological agent of invasive aspergillosis, causes over 1.8 million deaths annually. Targeting cell wall biosynthetic pathways offers a promising antifungal strategy. Gfa1, a rate-limiting enzyme in UDP-GlcNAc synthesis, plays a pivotal role in the hexosamine biosynthetic pathway (HBP). Results: Deletion of gfa1 (Δgfa1) results in auxotrophy for glucosamine (GlcN) or N-acetylglucosamine (GlcNAc). Under full recovery (FR) conditions, where minimal medium is supplemented with 5 mM GlcN as the sole carbon source, the Δgfa1 mutant shows growth comparable to the wild-type (WT). However, when supplemented with 5 mM GlcN and 55 mM glucose, growth is partially repressed, likely due to carbon catabolite repression, a condition termed partial repression (PR). Under PR conditions, Δgfa1 exhibits compromised growth, reduced conidiation, defective germination, impaired cell wall integrity, and increased sensitivity to endoplasmic reticulum (ER) stress and high temperatures. Additionally, Δgfa1 demonstrates disruptions in protein homeostasis and iron metabolism. Transcriptomic analysis of the mutant under PR conditions reveals significant alterations in carbohydrate and amino acid metabolism, unfolded protein response (UPR) processes, and iron assimilation. Importantly, Gfa1 is essential for A. fumigatus virulence, as demonstrated in Caenorhabditis elegans and Galleria mellonella infection models. Conclusions: These findings underscore the critical role of Gfa1 in fungal pathogenicity and suggest its potential as a therapeutic target for combating A. fumigatus infections.

3D dental similarity quantification in forensic odontology identification

Kofod Petersen, A., Forgie, A., Villesen, P., Staun Larsen, L. — Thu, 01 May 2025 13:21:30 +0200

Forensic odontology identification largely depends on comparing dental work like fillings and crowns with the dental records of potential victims. This process can be challenging, especially regarding victims with minimal or no dental work. Alternatively, 3D tooth morphology can be used for identification by automated dental surface similarity scoring. However, high-resolution 3D intraoral photo scans contain hundreds of thousands of datapoints from each individual jaw, making database searches difficult and slow. Here, we reduce full 3D scans to keypoints, which are small points located in areas of high curvature on tooth surfaces. We use Difference of Curvature (DoC) for robust keypoint detection and evaluate different keypoint representation methods to distinguish between scans of the same individual and scans of different individuals, assigning them a similarity score.The results demonstrate that combining DoC with the Signature of Histograms of OrienTations (SHOT) representation method effectively separates matches from mismatches. This indicates the potential for automatic scoring of dental surface similarity. This can be valuable for forensic odontology identification, especially in cases where traditional methods are limited by the lack of dental work.

Endogenous viral elements constitute a complementary source of antigens for personalized cancer vaccines

Garde, C., Pavlidis, M. A., Garces, P., et al. — Mon, 01 Dec 2025 13:21:30 +0100

Personalized cancer vaccines (PCVs) largely leverage neoantigens arising from somatic mutations, limiting their application to patients with relatively high tumor mutational burden (TMB). This underscores the need for alternative antigens to design PCVs for low TMB cancers. To this end, we substantiate endogenous retroviral elements (EVEs) as tumor antigens through large-scale genomic analyses of healthy tissues and solid cancers. These analyses revealed that the breadth of EVE expression in tumors stratify checkpoint inhibitor-treated melanoma patients into groups with differential overall and progression-free survival. To enable the design of PCVs containing EVE-derived epitopes with therapeutic potential, we developed a computational pipeline, ObsERV. We show that EVE-derived peptides are presented as epitopes on tumors and can be predicted by ObsERV. Preclinical testing of ObsERV demonstrates induction of sustained poly-functional CD4+ and CD8+ T-cell responses as well as long-term tumor protection. As such, EVEs may facilitate and improve PCVs, especially for low-TMB patients.

Structure of the [Ca]E2P intermediate of Ca²⁺-ATPase 1 from Listeria monocytogenes

Basse Hansen, S., Flygaard, R. K., Kjaergaard, M., Nissen, P. — Mon, 07 Apr 2025 13:21:30 +0200

Active transport by P-type Ca²⁺-ATPases maintain internal calcium stores and a low cytosolic calcium concentration. Structural studies of mammalian sarco/endoplasmic reticulum Ca²⁺-ATPases (SERCA) have revealed several steps of the transport cycle, but a calcium-releasing intermediate has remained elusive. Single-molecule FRET studies of the bacterial Ca²⁺-ATPase LMCA1 revealed an intermediate of the transition between so-called [Ca]E1P and E2P states and suggested that calcium release from this intermediate was the essentially irreversible step of transport. Here, we present a 3.5 Å resolution cryo-EM structure for a four-glycine insertion mutant of LMCA1 in a lipid nanodisc obtained under conditions with calcium and ATP and adopting such an intermediate state, denoted [Ca]E2P. The cytosolic domains are positioned in the E2P-like conformation, while the calcium-binding transmembrane (TM) domain adopts a calcium-bound E1P-ADP-like conformation. Missing density for the E292 residue at the calcium site (the equivalent of SERCA1a E309) suggests flexibility and a site poised for calcium release and proton uptake. The structure suggests a mechanism where ADP release and re-organization of the cytoplasmic domains precede calcium release.

High-dose vitamin D supplementation in pregnancy ameliorates obesity-induced increase in maternal IL-1β level without affecting obesity-induced increase in IL-6 and MCP

Sun, 01 Jun 2025 13:21:30 +0200

Background Maternal and placental inflammatory activity is carefully regulated during pregnancy and changes in inflammatory status are associated with pregnancy complications and health deficits in the offspring including adverse effects on neurodevelopment. Overweight/obesity is associated with chronic inflammation, thereby contributing to adverse effects. Disturbingly, overweight and obesity are highly prevalent among pregnant women worldwide. Vitamin D (vitD) possess immunomodulatory effects and is believed to support healthy pregnancy. Endocrinological societies recommend empiric vitD supplementation in pregnancy but there is no consensus on the minimal supplementation dose Methods An adjacent study to GRAVIT-D (no. NCT04291313, ClinicalTrial.gov), a double-blinded randomized trial investigating the clinical benefits of increasing vitD supplementation in pregnancy from 400IU to 3600IU/day from gestational week 11–16 onwards. In a subgroup, (n = 156), multiplex ELISA targeting third-semester serum levels of IL-1β, IL-6, IL-10, TNFα, MCP-1, and IL-17A was performed. Inflammation signals were correlated with the vitD dose given, subsequently analysing the effect of vitD in relation to the pre-pregnancy body mass index (BMI) within each treatment arm comparing the inflammatory response in WHO-defined BMI groups,  30 kg/m2. Main Results High pre-pregnancy BMI was associated with increased IL6 and MCP1 in both the 400IU and the 3600 IU exposed group. IL1β levels increased with BMI if using a 400IU/day supplement. High dose vitD supplementation ameliorated BMI effects on IL1β. Conclusion and Perspectives Increased vitD supplementation during pregnancy may ameliorate some overweight/obesity-induced inflammatory activity. Further studies are needed to determine the vitD need in pregnancies complicated by obesity and overweight.

Cold-Active Starch-Degrading Enzymes from a Cold and Alkaline Greenland Environment

Bendtsen, M. K., Nowak, J. S., Paiva, P., et al. — Fri, 14 Mar 2025 13:21:30 +0100

Cold-active enzymes hold promise for energy-efficient processes. Amylases are widely used in household and industrial applications, but only a few are cold-active. Here we describe three novel secreted amylases, Rho13, Ika2 and I3C6, all from bacteria growing in the cold and alkaline ikaite columns in Greenland. They all hydrolyzed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolyzed cyclodextrins, and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal active temperatures around 30-35 °C and significant enzymatic activity below 20 °C, but Rho13 and I3C6 had an alkaline optimal pH, while Ika2 was markedly acidophilic. They showed complex dependence on Ca 2+ concentration, with the activity of Rho13 and I3C6 following a bell-shaped curve and Ika2 being unaffected; however, removal of Ca 2+ reduced the stability of all three enzymes. Loss of structure occurred well above the temperature of optimal activity, showing the characteristic psychrophilic divorce between activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca 2+ binding site, while Rho13 and I3C6 both maintained one stably bound Ca 2+ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, based on a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation was also found in regions outside the active site and may provide easier substrate access and accommodation, as well as faster barrier transitions. Our work sheds further light on the many ways in which psychrophilic enzymes adapt to increased catalysis at lower temperatures.

TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

Lu, W. T., Zalmas, L. P., Bailey, C., et al. — Wed, 01 Jan 2025 13:21:30 +0100

Chromosomal instability (CIN) is common in solid tumours and fuels evolutionary adaptation and poor prognosis by increasing intratumour heterogeneity. Systematic characterization of driver events in the TRACERx non-small-cell lung cancer (NSCLC) cohort identified that genetic alterations in six genes, including FAT1, result in homologous recombination (HR) repair deficiencies and CIN. Using orthogonal genetic and experimental approaches, we demonstrate that FAT1 alterations are positively selected before genome doubling and associated with HR deficiency. FAT1 ablation causes persistent replication stress, an elevated mitotic failure rate, nuclear deformation and elevated structural CIN, including chromosome translocations and radial chromosomes. FAT1 loss contributes to whole-genome doubling (a form of numerical CIN) through the dysregulation of YAP1. Co-depletion of YAP1 partially rescues numerical CIN caused by FAT1 loss but does not relieve HR deficiencies, nor structural CIN. Importantly, overexpression of constitutively active YAP1^5SA is sufficient to induce numerical CIN. Taken together, we show that FAT1 loss in NSCLC attenuates HR and exacerbates CIN through two distinct downstream mechanisms, leading to increased tumour heterogeneity.

Distribution, activity, and storage of xanthine oxidase in bovine milk fractions

Gadegaard, I. S. E., Madsen, C. M., Johansen, P. M., Rasmussen, J. T. — Wed, 01 Jan 2025 13:21:30 +0100

Mammal enzyme xanthine dehydrogenase/oxidase exists in two interconvertible forms, with oxidase dominating in milk. Initially, the enzyme was affiliated to the milk fat globule membrane, but it is more widely distributed. Xanthine dehydrogenase/oxidase is involved in purine catabolism, but accumulated evidence reveals a much more complex and versatile role in milk, prompting the need for more accurate knowledge about its fractional presence. The content and activity of xanthine oxidase were estimated in a wide range of bovine milk fractions, with 220 mg L⁻¹ and 3.93 U g⁻¹ protein in whole milk, respectively. Besides buttermilk and fat globule membrane, high amounts of xanthine oxidase activity were found in extracellular vesicles and whey. Inclusion of detergent was shown to be important for release of all embedded xanthine oxidase activity. Finally, the conducted experiments disclosed effects of different storage conditions on the enzymatic integrity, showing that storage at −20 °C is not recommendable.

CRISPR/Cas9-mediated knockout of DYRK1B in triple-negative breast cancer cells

Rashidi, A., Füchtbauer, E. M., Vahabzadeh, Z., et al. — Wed, 01 Jan 2025 13:21:30 +0100

Breast cancer is the most common cancer among women worldwide, with the triple-negative subtype (TNBC) having a poor prognosis and limited treatment options. DYRK1B is a dual-specificity kinase that regulates the cell cycle and quiescence. While its role in several cancers has been characterized, its role in TNBC remains unknown. In this study, we used CRISPR/Cas9 to delete DYRK1B in MDA-MB-231 cells, a model of TNBC and investigated its effects on cell proliferation, apoptosis, invasion, migration, angiogenesis, and response to Paclitaxel. The DYRK1B knockout (KO) was confirmed by PCR, Real-time qPCR, and Sanger sequencing. KO cells showed a significant reduction in cell proliferation, colony formation, invasion, and migration. Additionally, there were alterations in mRNA expression levels of several genes related to the cell cycle, angiogenesis, and cell motility, such as CCND1, MCM2, PCNA, CDKN1B, HIF1A, VEGFA, and WASF3, compared to MDA-MB-231 wild type (WT) cells. Immunocytochemistry results assessing Ki67 expression, a marker of cell proliferation, indicated that DYRK1B knockout cells had significantly lower Ki67 expression than WT cells. Furthermore, KO cells exhibited increased apoptosis and sensitivity to contact inhibition. Additionally, the IC₅₀ for Paclitaxel was significantly decreased in KO cells. These results suggest that DYRK1B plays an important role in the survival and invasion of TNBC cells and might be a potential candidate as a new therapeutic target for this disease.

Comprehensive genomic characterization of early-stage bladder cancer

Prip, F., Lamy, P., Lindskrog, S. V., et al. — Wed, 01 Jan 2025 13:21:30 +0100

Understanding the molecular landscape of nonmuscle-invasive bladder cancer (NMIBC) is essential to improve risk assessment and treatment regimens. We performed a comprehensive genomic analysis of patients with NMIBC using whole-exome sequencing (n = 438), shallow whole-genome sequencing (n = 362) and total RNA sequencing (n = 414). A large genomic variation within NMIBC was observed and correlated with different molecular subtypes. Frequent loss of heterozygosity in FGFR3 and 17p (affecting TP53) was found in tumors with mutations in FGFR3 and TP53, respectively. Whole-genome doubling (WGD) was observed in 15% of the tumors and was associated with worse outcomes. Tumors with WGD were genomically unstable, with alterations in cell-cycle-related genes and an altered immune composition. Finally, integrative clustering of multi-omics data highlighted the important role of genomic instability and immune cell exhaustion in disease aggressiveness. These findings advance our understanding of genomic differences associated with disease aggressiveness in NMIBC and may ultimately improve patient stratification.