Publications - Peer-reviewed articles

The Medicago truncatula LYR4 intracellular domain serves as a scaffold in immunity signaling independent of its phosphorylation activity

Simonsen, B., Rübsam, H., Kolte, M. V., et al. — Thu, 01 May 2025 21:33:43 +0200

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

Wed, 01 Jan 2025 21:33:43 +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 21:33:43 +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 21:33:43 +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 21:33:43 +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. — Thu, 08 May 2025 21:33:43 +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 21:33:43 +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 21:33:43 +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 21:33:43 +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 21:33:43 +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 21:33:43 +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. — Tue, 22 Apr 2025 21:33:43 +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 21:33:43 +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 21:33:43 +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 21:33:43 +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, 18 Apr 2025 21:33:43 +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 21:33:43 +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. — Tue, 01 Apr 2025 21:33:43 +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.

Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence

Stevnsner, T., Mortensen, U. H., Westergaard, O., Bonven, B. J. — Sun, 01 Jan 1989 21:33:43 +0100

The interaction between eukaryotic DNA topoisomerase I and a high affinity binding sequence was investigated. Quantitative footprint analysis demonstrated that the substrate preference results from strong specific binding of topoisomerase I to the sequence. The specificity was conferred by a tight noncovalent association between the enzyme and its target DNA, whereas the transient formation of a covalently bound enzyme·nicked DNA intermediate contributed insignificantly to the overall affinity. Topoisomerase I protected both strands over a 20-base pair region in which the cleavage site was centrally located. DNA modification interference analysis revealed a 16-base pair interference region on the scissile strand. Essential bases were confined to the 5' side of the cleavage site. The 6-base pair interference region observed on the complementary strand did not contain essential bases.

Inhibition of gene-specific repair of alkylation damage in cells deplated of poly(ADP-ribose) polymerase

Stevnsner, T., Ding, R., Smulson, M., Bohr, V. A. — Fri, 11 Nov 1994 21:33:43 +0100

The role of the enzyme poly(adenosine diphosphateribose) polymerase (PADPRP) in DNA repair at the level of the gene was investigated with human HeLa cells in which PADPRP antisense transcripts are inducible with dexamethasone. After such induction, the cellular content of PADPRP is reduced by 90%. DNA damage and its repair was studied in the essential dihydrofolate reductase (DHFR) gene after exposure of the cells to either ultraviolet (UV) irradiation or the alkylating agent nitrogen mustard. The expression of the antisense construct had no effect on gene-specific repair of UVinduced pyrimidine dimers. In contrast, induced antisense cells were deficient in the gene-specific repair of nitrogen mustard-induced leslons. Dexamethasone itself did not inhibit gene-specific repair in control cells. Thus, PADPRP appears to participate in the gene-specific repair of alkylation damage, but not in the repair of UV-induced pyrimidine dimers. Clonal survival assays revealed that cells depleted of PADPRP showed an increased susceptibility to nitrogen mustard, supporting the notion that repair of essential genes is critical for cellular survival.

Repair of individual DNA strands in the hamster dihydrofolate reductase gene after treatment with ultraviolet light, alkylating agents, and cisplatin

May, A., Nairn, R. S., Okumoto, D. S., et al. — Mon, 25 Jan 1993 21:33:43 +0100

We have analyzed gene-specific and strand-specific DNA damage and repair in the dihydrofolate reductase gene in hamster cells. Cells were UV-irradiated or treated with two types of chemotherapeutics, alkylating agents or cisplatin. UV-induced pyrimidine dimers were detected using a previoulsy published technique in which the T4 endonuclease V enzyme is used to create nicks at the lesion sites. 6-4 photoproducts were detected in a similar assay using ABC excinuclease after prior reversal of the pyrimidine dimers with photolyase. Adducts formed by the alkylating agents nitrogen mustard and dimethyl sulfate were quantitated by generating strand breaks at abasic sites after neutral depurination. Cisplatin-induced intrastrand adducts were detected with ABC excinuclease, and cisplatin interstrand cross-links were detected using a denaturation-reannealing reaction before electrophoresis. In accord with previous reports by other investigators, we find distinct strand specificity of the repair of pyrimidine dimers after UV; the transcribed strand was much more efficiently repaired than the nontranscribed strand. In contrast, there was little or no strand bias in the repair of the 6-4 photoproducts. For alkylating agents, a slight bias toward repair in the transcribed strand was found after treatment with nitrogen mustard, but there appeared to be no bias in the repair after treatment with dimethyl sulfate. Cisplatin interstrand cross-links are repaired with equal efficiency from the two strands, but the more common cisplatin-induced lesion, the intrastrand adduct, is preferentially repaired from the transcribed strand. In conclusion, there is strand bias in the repair of pyrimidine dimers and cisplatin intrastrand adducts, but the strand specificity of repair may not be a general feature for all DNA lesions, as we found little or no strand bias in the repair of other lesions studied.

DNA repair in a UV resistant chinese hamster ovary cell line

Petersen, L. N., Stevnsner, T., Bohr, V. A. — Fri, 01 Dec 1995 21:33:43 +0100

We have established an ultraviolet (UV) resistant Chinese hamster ovary (CHO) cell line B11^UVres by repetitive UV exposure of the CHO cell line B11. We have characterized the resistant cell line with respect to growth, sensitivity to various DNA damaging agents, and the repair of UV induced DNA lesions. When examining sensitivity to UV in clonogenic survival studies, we find that the ID50 is increased 2.3-fold in the resistant cell line B11^UVres compared to the parental cell line B11. Although the doubling time of the resistant cell line is greater than that of the parental cell line, there is no difference in the rate of replication after UV irradiation. When measuring repair of UV induced DNA lesions in the overall genome we find no significant difference between the two cell lines. However, at early times after UV, there is a significant increase in the rate of repair of cyclobutane pyramidine dimers (CPDs) in the transcribed strand of the dihydrofolate reductase (DHFR) gene in the B11^UVres cells compared to the B11 cells. There is a small increase of steady state transcription of the DHFR gene in the UV resistant cells, but hardly enough to account for the repair increase. The UV resistant cell line B11^UVres is not cross-resistant to the cross-linking agents mitomycin C or cisplatin, but shows increased sensitivity to these compounds.

Caffeine inhibits gene-specific repair of UV-induced DNA damage in hamster cells and in human xeroderma pigmentosum group c cells

Link, C. J., Evans, M. K., Cook, J. A., Muldoon, R., Stevnsner, T., Bohr, V. A. — Mon, 01 May 1995 21:33:43 +0200

We have studied the effect of caffeine on gene- and strandspecific DNA repair after exposure of Chinese hamster ovary cells and human xeroderma pigmentosum complementation group C (XPC) cells to ultraviolet irradiation (UV). In hamster cells, caffeine inhibited the repair of cyclobutane dimers (CPDs) in the dihydrofolate reductase (DHFR) gene by up to 66% after 8 h of repair incubation. This effect was dose-dependent, with more inhibition at 10 than at 1.5 mM caffeine. The inhibition was due to decreased repair in the transcribed strand of the hamster DHFR gene. This decrease in repair of CPDs in the DHFR gene correlated with an enhancement of UV-induced cell killing by caffeine. DNA repair was also measured in the overall genome by repair-replication analysis. In hamster cells, caffeine caused a modest enhancement of repair. caffeine did not produce a significant effect on cell cycle progression up to 8 h after UV irradiation, but it caused a distinct block in early S phase during the 24 h post-irradiation period. In XPC cells, 10 mM caffeine inhibited the removal of CPDs from the transcribed strand of the DHFR gene by 92% The removal of all Photoproducts from the overall genome was inhibited by 26% in these cells. Since the residual repair in XPC cells is thought to occur in active genomic regions, we propose that caffeine preferentially inhibits gene-specific repair.

Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

Jones, J. C., Stevnsner, T., Mattern, M. R., Bohr, V. A. — Sun, 01 Sep 1991 21:33:43 +0200

We have studied the effect of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reducstase gene. Replication was measured in the overall genome. We tested inhibitors of DNA polymease α and δ (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topoisomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, we tested the effect of the potential topoisomerase inhibitors affected the overall genome repair; β-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair.

Repair of mitochondrial DNA after various types of DNA damage in chinese hamster ovary cells

Sun, 01 Nov 1992 21:33:43 +0100

Using methodology recently developed to assess gene-specific DNA repair, we have demonstrated that it is possible not only to study mitochondrial DNA repair, but also directly to compare mitochondrial and nuclear DNA repair in the same biological sample. Complex enzymatic mechanisms recognize and repair nuclear DNA damage, but it has long been thought that there was no DNA repair in mitochondria. Therefore, in an attempt to delineate more clearly which DNA repair mechanisms, if any, are functioning in mitochondria, we have investigated the repair of several specific DNA lesions in mitochondrial DNA. They include cyclobutane dimers, cisplatin intrastrand adducts, cisplatin interstrand crosslinks and alkali-labile sites. We find that pyrimidine dimers and complex alkylation damage are not repaired in mitochondrial DNA, and that there is minimal repair of cisplatin intrastrand crosslinks. In contrast, there is efficient repair of cisplatin interstrand crosslinks as evidenced by ∼ 70% of the lesions being removed by 24 h. Additionally, there is efficient repair of N-methylpurines following exposure to methylnitrosourea with ∼ 70% of the lesions being removed by 24 h. The results of these studies reveal that repair capacity of mitochondrial DNA damage depends upon the type of lesion produced by the damaging agent. We speculate that a process similar to the base excision mechanism for nuclear DNA exists for mitochondrial DNA but that there is no nucleotide excision repair mechanism to remove more bulky lesions in this organelle.

Studies on the role of topoisomerases in general, gene- and strand-specific DNA repair

Stevnsner, T., Bohr, V. A. — Wed, 01 Sep 1993 21:33:43 +0200

Using specific inhibitors we have assessed the role of topoisomerases I and II in DNA repair of the overall genome and in both strands of an essential gene, the dihydrofolate reductase (DHFR) gene in chinese hamster ovary (CHO) cells. In these studies we have: (1) used inhibitors of topoisomerases during the repair incubation and (2) studied the DNA repair in cells with altered levels of topoisomerase activity. When cells were allowed to repair after UV irradiation, the gene-specific DNA repair was not affected by either topoisomerase I or topoisomerase II inhibitors alone. However, when topoisomerase I and topoisomerase II inhibitors were added simultaneously the gene- and strand-specific DNA repair were markedly inhibited. In contrast, the overall genome DNA repair was only marginally affected. This suggests that topoisomerases are involved in gene-specific DNA repair and that one type may substitute for the other in the repair process. That concept is further supported by our findings using a mutant cell line with a decreased level of topoisomerase I: gene-specific DNA repair can be inhibited by a topoisomerase II inhibitor alone. By analyzing the steady-state expression of the DHFR gene we find that inhibition of repair in the DHFR gene is not ascribed to an obvious change in the messenger level. Furthermore, using agents other than UV, we observe that the inhibitors have no effect on gene-specific repair of DNA damage introduced by the chemotherapeutic agents cisplatin and nitrogen mustard.

Repair of ribosomal RNA genes in hamster cells after UV irradiation, or treatment with cisplatin or alkylating agents

Stevnsner, T., May, A., Petersen, L. N., Larminat, F., Pirsel, M., Bohr, V. A. — Sun, 01 Aug 1993 21:33:43 +0200

We have measured the DNA damage formation and repair in the ribosomal and the dihydrofolate reductase (DHFR) genes after treatment of hamster cells with different types of DNA damaging agents. In mammalian cells, the ribosomal DNA (rDNA) is transcribed by RNA polymerase I, whereas the DHFR is transcribed by RNA polymerase II. Cells were treated with agents that induce different types of lesions, and that are known to be repaired via different pathways. We used UV (254 nm) irradiation, treatment with cisplatin and treatment with the alkylating agents nitrogen mustard (HN2) and methyl methanesulphonate (MMS). UV induced pyrimidine dimers were detected with the enzyme T4 endonuclease V, which creates nicks at the dimer sites; the breaks are then resolved and identified by denaturing electrophoresis and Southern blot. Intrastrand adducts formed by the alkylating agents HN2 and MMS were quantitated by generating strand breaks at abasic sites after neutral depurination. Interstrand crosslinks (ICL) formed by HN2 and cisplatin were detected by a denaturation-reannealing reaction before neutral agarose gel-electrophoresis. We find that the repair of the pyrimidine dimers is significantly less efficient in the RNA polymerase I transcribed rDNA genes than in RNA polymerase II transcribed DHFR gene at 8 and 24 h after irradiation. ICL and intrastrand adducts induced by HN2 are also removed more slowly from the rDNA than from the DHFR gene. In contrast, MMS induced intrastrand adducts and cisplatin induced ICL are repaired equally efficiently in the RNA polymerase I and RNA polymerase II transcribed genes. We conclude that for some types of DNA damage, there is less repair in the ribosomal genes than in the DHFR; but for other DNA lesions there is no difference. The difference in repair efficiency between the rDNA and the DHFR genes may reflect the different RNA polymerases involved in their transcription. It may, however, alternatively, reflect the different nuclear localization of these genes.

Increased gene specific repair of cisplatin induced interstrand crosslinks in cisplatin resistant cell lines, and studies on carrier ligand specificity

Petersen, L. N., Mamenta, E. L., Stevnsner, T., Chaney, S. G., Bohr, V. A. — Mon, 01 Jan 1996 21:33:43 +0100

Development of resistance to cisplatin in previously treatment-responsive malignancies is a major obstacle to successful treatment. Enhanced DNA repair as well as enhanced replicative bypass of DNA adducts have been suggested to play a role in the development of resistance to cisplatin. However, the relative contribution of these mechanisms is unknown. Second generation platinum compounds containing the 1,2-diaminocyclohexane (dach) carrier ligand have been of particular interest in the studies of resistance mechanisms since they have been effective in treatment of cells resistant to cisplatin. We have investigated the formation and repair of interstrand crosslinks (ICL) in the mouse leukemia cell line L1210/0 and its carrier ligand specific resistant derivatives L1210/DDP and L1210/DACH after treatment with ethylenediamine (en)-Pt and diaminocyclohexane (dach)-Pt compounds. ICL in the overall genome were examined using a modification of the alkaline elution assay. A Southern blot technique was employed for the study of ICL in specific regions of the genome. In the overall genome we found decreased formation of ICL with either -en or -dach carrier ligands in the two resistant cell lines without carrier ligand specificity. Some carrier ligand specificity of ICL formation was observed in the dihydrofolate reductase (DHFR) gene, but it did not correlate with the carrier ligand specificity of resistance. At the level of the overall genome there was no difference in repair of ICL between the sensitive and the two resistant cell lines. When measured in the DHFR gene, however, there was enhanced repair of ICL in the two resistant cell lines compared with the sensitive cell line. The enhanced repair at the level of the gene did not display any carrier ligand specificity.

Distamycin inhibition of topoisomerase I-DNA interaction

Wed, 25 Apr 1990 21:33:43 +0200

Inhibition of eukaryotic DNA topoisomerase I by the minor groove binding ligand, distamycin A, was investigated. Low concentrations of the ligand selectively prevented catalytic action at a high affinity topoisomerase I binding sequence. A restriction enzyme protection assay indicated that the catalytic cycle was blocked at the binding step. Distamycin binding sites on DNA were localized by hydroxyl radical footprinting. A strongly preferred site mapped to a homopolymeric (dA)·(dT)-tract partially included in the essential topoisomerase I binding region. Mutational elimination of the stable helix curvature associated with this ligand binding site demonstrated that (i) the intrinsic bend was inessential for efficient binding of topoisomerase I, and (ii) distamycin inhibition did not occur by deformation of a stable bend. Alternative modes of inhibition are discussed.

A human topoisomerase I cleavage complex is recognized by an additional human topisomerase I molecule in vitro

Søe, K., Dianov, G., Nasheuer, H. P., Bohr, V. A., Grosse, F., Stevnsner, T. — Wed, 01 Aug 2001 21:33:43 +0200

Several recent studies have shown that human topoisomerase I (htopol) can recognize various DNA lesions and thereby form a covalent topoisomerase I-DNA complex, which is known to be detrimental to cells. We have investigated whether htopol recognizes another htopol that is covalently trapped on a DNA substrate. For this purpose we created an artificial DNA substrate containing a specific topoisomerase I binding sequence, where the enzyme was trapped in the covalently bound form. We demonstrate that, in vitro, free htopol stimulates the formation of an additional cleavage complex immediately upstream of the covalently bound topoisomerase I. The predominant distance between the two cleavage sites is 13 nt. In addition we find that these two enzymes may form direct protein-protein contacts and we propose that these may be mediated through the formation of a dimer by domain swapping involving the C-terminal and the core domains. Finally, we discuss the possibility that the double cleavage reaction may be the initial step for the removal of the recognized cleavage complex.

Base excision repair in nuclear and mitochondrial DNA

Mon, 01 Jan 2001 21:33:43 +0100

Base excision repair mechanisms have been analyzed in nuclear and mitochondrial DNA. We measured the size and position of the newly incorporated DNA repair patch in various DNA substrates containing single oxidative lesions. Repair of 8-oxoguanine and of thymine glycol is almost exclusively via the base excision repair (BER) pathway with little or no involvement of nucleotide excision repair (NER). The repair mode is generally via the single-nucleotide replacement pathway with little incorporation into longer patches. Extension of these studies suggests that DNA polymerase β plays a critical role not only in the short-patch repair process but also in the long-patch, PCNA-dependent pathway. Mitochondria are targets for heavy load of oxidative DNA damage. They have efficient BER repair capacity, but cannot repair most bulky lesions normally repaired by NER. In vitro experiments performed using rat and human mitochondrial extracts suggest that the repair incorporation during the removal of uracil in DNA occurs via the short-patch repair BEr pathway. Oxidative DNA damage accumulates with age in mitochondrial DNA, but this cannot be explained by an attenuation of DNA repair. In contrast, we observe that mitochondrial incision of 8-oxoG increases with age in rodents.

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 21:33:43 +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 21:33:43 +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 21:33:43 +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 21:33:43 +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 21:33:43 +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.

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 21:33:43 +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 21:33:43 +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 21:33:43 +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.

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 21:33:43 +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 21:33:43 +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 21:33:43 +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.

Genetic predictions of eye and hair colour in the Danish population

Cabrejas-Olalla, A., Jørgensen, F. G., Cheng, J. Y., et al. — Sun, 01 Jun 2025 21:33:43 +0200

Genetic predictions of eye and hair colour are prominent examples of forensic DNA phenotyping that can help resolve criminal cases. The advent of high-throughput genotyping technologies in forensic genetics opens up the possibility of applying polygenic risk scores in forensic settings. In this work, we compare the performance of HIrisPlex with PRSice-2 in predicting eye and hair colour to gain insights into the relative benefits of new approaches. Predictions were carried out on 584 Danish high school students for which genetic and self-reported phenotype data were available. Prediction of brown eye colour was very accurate (AUC = 0.98), followed by blue eye colour (AUC = 0.82), while it failed for intermediate eye colour (AUC = 0.57). As for hair colour, red and black were overall better predicted than blond and brown, and PRSice-2 performed better in all but the black hair colour. Despite the limitations of the study, HIrisPlex exhibited its usual high performance in the prediction of brown and blue eye colour, as well as red and black hair colour. However, PRSice-2 offered overall improvements in hair colour prediction over HIrisPlex suggesting that there is room for improvement in forensic DNA phenotyping by using polygenic risk scores.

Diagnosis and Staging of Metabolic Dysfunction-Associated Steatotic Liver Disease Using Biomarker-Directed Aptamer Panels

Kjær, M. B., Jørgensen, A. G., Fjelstrup, S., et al. — Sat, 01 Feb 2025 21:33:43 +0100

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects one-third of adults globally. Despite efforts to develop non-invasive diagnostic tools, liver biopsy remains the gold standard for diagnosing metabolic dysfunction-associated steatohepatitis (MASH) and assessing fibrosis. This study investigated RNA aptamer panels, selected using APTASHAPE technology, for non-invasive MASLD diagnosis and fibrosis stratification. Aptamer panels were selected in a cohort of individuals with MASLD (development cohort, n = 77) and tested in separate cohorts: one with MASLD (test cohort, n = 57) and one assessed for bariatric surgery (bariatric cohort, n = 62). A panel distinguishing MASLD without steatohepatitis from MASH accurately stratified individuals in the development cohort (AUC = 0.83) but failed in the test and bariatric cohorts. It did, however, distinguish healthy controls from individuals with MASLD, achieving an AUC of 0.72 in the test cohort. A panel for fibrosis stratification differentiated F0 from F3–4 fibrosis in the development cohort (AUC = 0.68) but not in other cohorts. Mass spectrometry identified five plasma proteins as potential targets of the discriminative aptamers, with complement factor H suggested as a novel MASLD biomarker. In conclusion, APTASHAPE shows promise as a non-invasive tool for diagnosing and staging MASLD and identifying associated plasma biomarkers.

Stem cells in ageing and longevity: a new section in Biogerontology

Fraifeld, V., Rattan, S. — Tue, 01 Apr 2025 21:33:43 +0200

The genomic consequences and persistence of sociality in spiders

Sat, 01 Mar 2025 21:33:43 +0100

In cooperatively breeding social animals, a few individuals account for all reproduction. In some taxa, sociality is accompanied by a transition from outcrossing to inbreeding. In concert, these traits reduce effective population size, potentially rendering transitions to sociality “evolutionarily dead-ends.” We addressed this hypothesis in a comparative genomic study in spiders, in which sociality has evolved independently at least 23 times, but social branches are recent and short. We present genomic evidence for the evolutionary dead-end hypothesis in a spider genus with three independent transitions to sociality. We assembled and annotated high-quality, chromosome-level reference genomes from three pairs of closely related social and subsocial Stegodyphus species. We timed the divergence between the social and subsocial species pairs to be from 1.3 million to 1.8 million years. Social evolution in spiders involves a shift from outcrossing to inbreeding and from an equal to a female-biased sex ratio, causing severe reductions in effective population size and decreased efficacy of selection. We show that transitions to sociality only had full effect on purifying selection at 119, 260, and 279 kya, respectively, and follow similar convergent trajectories of progressive loss of diversity and shifts to an increasingly female-biased sex ratio. This almost deterministic genomic response to sociality may explain why social spider lineages do not persist. What causes species extinction is not clear, but either could be selfish meiotic drive eliminating the production of males or could be an inability to retain genome integrity in the face of extremely reduced efficacy of selection.

A roadmap toward the synthesis of life

Kriebisch, C. M.E., Bantysh, O., Baranda Pellejero, L., et al. — Thu, 13 Mar 2025 21:33:43 +0100

The synthesis of life from non-living matter has captivated and divided scientists for centuries. This bold goal aims at unraveling the fundamental principles of life and leveraging its unique features, such as its resilience, sustainability, and ability to evolve. Synthetic life represents more than an academic milestone—it has the potential to revolutionize biotechnology, medicine, and materials science. Although the fields of synthetic biology, systems chemistry, and biophysics have made great strides toward synthetic life, progress has been hindered by social, philosophical, and technical challenges, such as vague goals, misaligned interdisciplinary efforts, and incompletely addressing public and ethical concerns. Our perspective offers a roadmap toward the synthesis of life based on discussions during a 2-week workshop with scientists from around the globe.

Integrative in silico and biochemical analyses demonstrate direct Arl3-mediated ODA16 release from the intraflagellar transport machinery

Wang, J., Kidmose, R. T., Boegholm, N., et al. — Sat, 01 Mar 2025 21:33:43 +0100

Outer dynein arms (ODAs) are essential for ciliary motility and are preassembled in the cytoplasm before trafficking into cilia by intraflagellar transport (IFT). ODA16 is a key adaptor protein that links ODAs to the IFT machinery via direct interaction with the IFT46 protein. However, the molecular mechanisms regulating the assembly, transport, and release of ODAs remain poorly understood. Here, we employ AlphaPulldown, an in silico screening method, to identify direct interactors of ODA16, including the dynein adaptor IDA3 and the small GTPase Arl3. We use structural modeling, biochemical, and biophysical assays on Chlamydomonas and human proteins to elucidate the interactions and regulatory mechanisms governing the IFT of ODAs. We identify a conserved N-terminal motif in Chlamydomonas IFT46 that mediates its binding to one side of the ODA16 structure. Biochemical dissection reveals that IDA3 and Arl3 bind to the same surface of ODA16 (the C-terminal β-propeller face), which is opposite to the IFT46 binding site, enabling them to dissociate ODA16 from IFT46, likely through an allosteric mechanism. Our findings provide mechanistic insights into the concerted actions of IFT and adaptor proteins in ODA transport and regulation.

The Composition of the Fecal and Mucosa-adherent Microbiota Varies Based on Age and Disease Activity in Ulcerative Colitis

Malham, M., Vestergaard, M. V., Bataillon, T., et al. — Sat, 01 Feb 2025 21:33:43 +0100

Background: Pediatric-onset ulcerative colitis (pUC) represents a more aggressive disease phenotype compared with adult-onset UC. We hypothesized that this difference can, in part, be explained by the composition of the microbiota. Methods: In a prospective, longitudinal study, we included pediatric (N=30) and adult (N=30) patients with newly or previously (>1 year) diagnosed UC. We analyzed the microbiota composition in the mucosa-adherent microbiota at baseline, using 16S rRNA gene sequencing, and the fecal microbiota at baseline and at 3-month intervals, using shotgun metagenomics. Results: For fecal samples, the bacterial composition differed between pUC and aUC in newly diagnosed patients (β-diversity, Bray Curtis: R²=0.08, P=.02). In colon biopsies, microbial diversity was higher in aUC compared with pUC (α-diversity, Shannon: estimated difference 0.54, P=.006). In the mucosa-adherent microbiota, Alistipes finegoldii was negatively associated with disease activity in pUC while being positively associated in aUC (estimate: -0.255 and 0.098, P=.003 and P=.02 in pUC and aUC, respectively). Finally, we showed reduced stability of the fecal microbiota in pediatric patients, evidenced by a different composition of the fecal microbiota in newly and previously diagnosed pUC, a pattern not found in adults. Conclusions: Our results indicate that pediatric UC patients have a more unstable fecal microbiota and a lower α diversity than adult patients and that the microbiota composition differs between aUC and pUC patients. These findings offer some explanation for the observed differences between pUC and aUC and indicate that individualized approaches are needed if microbiota modifications are to be used in the future treatment of UC.

Polygenic scores for autism are associated with reduced neurite density in adults and children from the general population

Gu, Y., Maria-Stauffer, E., Bedford, S. A., et al. — Mon, 24 Feb 2025 21:33:43 +0100

Genetic variants linked to autism are thought to change cognition and behaviour by altering the structure and function of the brain. Although a substantial body of literature has identified structural brain differences in autism, it is unknown whether autism-associated common genetic variants are linked to changes in cortical macro- and micro-structure. We investigated this using neuroimaging and genetic data from adults (UK Biobank, N = 31,748) and children (ABCD, N = 4928). Using polygenic scores and genetic correlations we observe a robust negative association between common variants for autism and a magnetic resonance imaging derived phenotype for neurite density (intracellular volume fraction) in the general population. This result is consistent across both children and adults, in both the cortex and in white matter tracts, and confirmed using polygenic scores and genetic correlations. There were no sex differences in this association. Mendelian randomisation analyses provide no evidence for a causal relationship between autism and intracellular volume fraction, although this should be revisited using better powered instruments. Overall, this study provides evidence for shared common variant genetics between autism and cortical neurite density.