Thomas Fröhlich

Aims/hypothesis: Offspring of mothers with diabetes are at increased risk of developing type 2 diabetes, and this predisposition is established during fetal life. The aim of this study was to define the functional, morphological and molecular alterations in neonatal pancreatic islets associated with maternal hyperglycaemia, with a particular focus on beta cells, an area that remains poorly understood. Given its similarity to humans in endocrine pancreas development, the pig was used as a translational animal model. Methods: Using genetically diabetic and beta cell reporter pig models, we performed functional assessments of glucose-stimulated insulin secretion in vivo and in situ using fresh pancreatic tissue slices from neonatal piglets born to hyperglycaemic mothers (PHGs) or normoglycaemic mothers (PNGs). Morphological and molecular profiling, including proteomics and single-cell RNA-seq, were performed to elucidate molecular changes reflecting the developmental impact of maternal diabetes on offspring metabolic health, with a particular focus on neonatal beta cells. Results: PHGs were born hyperglycaemic, hyperinsulinaemic and insulin resistant, exhibiting markedly delayed glucose and insulin response curves following oral glucose administration, with more pronounced effects in male piglets. Elevated basal insulin secretion and an almost blunted glucose responsiveness of pancreatic tissue slices were associated with increased insulin and somatostatin content, expansion of beta (+35%) and delta (+47%) cell populations, and organisation into larger islet clusters. Neonatal PHG islets exhibited a molecular profile consistent with intrinsic beta cell dysfunction that partially overlapped with type 2 diabetes (e.g. RBP4 upregulation), together with alterations in glucose sensing (near absence of GLUT2), nutrient metabolism resembling congenital hyperinsulinism ( HADH downregulation), and cytosolic Ca ² ⁺ homeostasis (near absence of the Ca ² ⁺ buffer calbindin D28K). In addition to an ATF4-dominated stress signature, a shift towards a distinct PHG beta cell subcluster characterised by low expression of ribosomal genes was observed, with an adaptive early-response-gene-mediated transcriptional reactivity. In contrast to the Gαs–adenylyl cyclase–cAMP–PKA signalling axis that dominated in PNG beta cells, neonatal PHG beta cells displayed distinct alterations in Gαq–PLC–PKCα signalling components, the mTORC2 target PKCα, and CREB1, suggesting a restructured signalling architecture. Conclusions/interpretation: Our findings reveal altered islet modelling with a unique beta cell dysfunction profile that partially overlaps with, but differs from, that of type 2 diabetes. A shift towards a beta cell subcluster exhibiting an adaptive early-response-gene-mediated transcriptional reactivity and an ATF4-associated transcriptional stress signature provides insight into molecular alterations associated with fetal exposure to maternal diabetes and may contribute to the increased risk of metabolic disease in offspring.

In ruminants, embryonic interferon tau (IFNT) is widely considered the indispensable signal for maternal recognition of pregnancy, driving endometrial gene expression to prevent luteolysis. However, this canonical paradigm has never been directly tested using genomic loss-of-function models. Here we show that IFNT is completely dispensable for pregnancy recognition, luteal maintenance, and full-term gestation in cattle. Using CRISPR-Cas9, we generate bovine fibroblasts lacking all functional copies of the multigene IFNT locus for somatic cell nuclear transfer. IFNT -null embryos develop into blastocysts in vitro at normal rates and exhibit typical elongation at day 18 post-transfer without inducing endometrial interferon-stimulated genes. Crucially, these IFNT -null conceptuses successfully prevent luteal regression, establish normal placentation, and progress through full-term gestations, culminating in the birth of healthy female calves. These findings provide the first direct genetic evidence that embryonic IFNT is dispensable for pregnancy recognition and establishment in cattle, fundamentally revising the long-standing model of ruminant reproduction and uncovering alternative embryo-maternal signaling mechanisms.

The intensity of environmental stressors increases due to global change. Under natural conditions, insect pollinators are experiencing multiple stressors simultaneously which may exacerbate the negative effects of individual stressors. Using a fully crossed factorial design, we investigated the effects of environmentally relevant ozone levels, heat, and LDPE microplastics (MP) on Bombus terrestris health. Changes in the fat body proteome suggest that ozone induces an oxidative stress response, heat induces changes in the metabolism, and MP induces tissue damage responses and detoxification reactions. We observed MP to have the strongest effect on mortality among the single stressors. Only in combination with MP, ozone, heat, and both combined significantly increased the mortality. For the combination of all three stressors, the effect strength exceeded expectations, indicating synergistic effects. We propose a reduced heat resistance in MP-exposed bumblebees as one possible underlying mechanism. Our study suggests that the progressive environmental accumulation of MP, rising temperatures and the associated increase in ozone levels could pose a serious health risk to pollinators in the future.

We investigated the role of Netrin-1 (NTN1) in human naïve pluripotency using complementary loss- and gain-of-function approaches. In primate embryos and human embryonic stem cells (hESCs), Netrin-1 expression is associated with the naïve pluripotent state. Disruption of NTN1 had no detectable effect on hESCs maintained on murine embryonic fibroblasts. However, under sub-optimal culture conditions, NTN1-knockout cells exhibited compromised naïve pluripotency, which was rescued with feeder cells overexpressing Netrin-1. Netrin-1 overexpression in hESCs accelerated acquisition of the naïve state and markedly increased resistance to differentiation. These effects were accompanied by extensive epigenetic remodeling, including H3K27ac and H2K27me3. Proteomic and phospho-proteomic analyses further revealed rapid Netrin-1-dependent alterations in pathways controlling cell adhesion, signaling, and chromatin regulation. Together, these findings extend the role of Netrin-1 beyond its established functions and identify it as a coordinator of extracellular cues, intracellular signaling, and nuclear regulatory mechanisms that support human naïve pluripotency.

Microplastics (MP) are widespread in aquatic ecosystems and pose a threat to freshwater biodiversity. While numerous studies examine physiological effects on aquatic organisms, less is known about how MP alter chemically mediated interactions that regulate predator–prey dynamics. Predator-induced defenses in Daphnia depend on detecting kairomones and represent an important form of adaptive phenotypic plasticity. Whether MP interfere with these responses, and through which mechanisms, remains unclear. Here, we show that polystyrene MP impair predator-induced defenses across Daphnia species by disrupting predator-cue-mediated plasticity at the behavioral, morphological, and molecular levels. In D. longicephala , chronic exposure to PS fragments weakened Notonecta -induced morphological defenses, whereas additive-containing PS fragments nearly suppressed defense formation and reduced body size. Consistent with these phenotypic effects, proteomic analyses revealed alterations in pathways related to molting and chitin metabolism, linking MP exposure to impaired defense formation. In D. magna , PS particles attenuated fish kairomone-induced diel vertical migration, with stronger effects for larger particles, consistent with reduced effective availability or perception of predator cues. Natural limestone particles caused only minor effects, indicating particle-specific rather than general particle-driven responses. Our findings demonstrate that MP can disrupt adaptive predator–prey interactions with potential cascading consequences for freshwater food webs.

Pollution of aquatic environments poses an increasingly severe threat to ecosystems worldwide, and understanding its molecular consequences for aquatic organisms requires extensive research and the development of advanced analytical tools. Phosphoproteomics can be particularly valuable for this purpose, as shifts in phosphorylation states can serve as early molecular indicators of toxic exposure. The cladoceran Daphnia is a keystone species in aquatic ecosystems, linking lower and higher trophic levels, and is therefore widely used as a model organism in ecotoxicology to study biological consequences of pollution. Here, we present a simple and effective strategy to analyse the phosphoproteome of Daphnia magna , a commonly used Daphnia species in ecotoxicology. Following TiO ₂ -based phosphopeptide enrichment and LC-MS/MS analysis, we identified a comprehensive dataset of 3,532 phosphorylation sites across 1,329 phosphoproteins. These proteins were especially involved in signaling pathways and cellular structure and the vast majority have not yet been demonstrated in other Daphnia species. In conclusion, our results demonstrate that a straightforward phosphoproteomic LC-MS/MS workflow in D. magna can serve as a powerful tool for investigating adverse molecular effects caused by anthropogenic pollution, such as microplastics or pharmaceuticals. Statement of significance: The dataset presented here demonstrates the feasibility of a simple yet effective strategy to perform phosphoprotemics in Daphnia magna , and it will be particularly valuable for future ecotoxicoproteomics research using this model organism.

RNA is the central molecule that connects genetic makeup to the function of living cells and organisms. Since genes will also cease to be transcribed, RNA must decay so that the cellular RNA-pool can reflect the current transcriptional state. Undesired degradation is therefore a major challenge when RNA is isolated from living cells, tissues or organisms. Strongly protein-denaturing conditions, often involving toxic organic solvents, are usually employed to quickly inactivate RNases prior to separating nucleic acids from protein. We propose to combine high concentrations of urea, a compound used in fertilizers, with SDS as a non-volatile, low toxicity denaturant. The use of urea and SDS has previously been combined with organic solvent extraction, chaotropic salt-mediated column purification or selective precipitation of long RNA with LiCl. Here, we demonstrate that – akin to the classic alkaline lysis protocol for plasmid preparation – addition of potassium acetate forms insoluble potassium dodecyl sulfate, which can be removed along with denatured proteins and part of the genomic DNA by centrifugation. The RNA is then precipitated with isopropanol, recovering also small non-coding RNAs. We demonstrate that these somewhat crude RNA preparations are nonetheless sufficiently clean to be quantified via classic UV absorption measurements, stable over the time-course of a typical molecular biology reaction and do not harbor inhibitors of reverse transcriptase. While it seems unlikely that we are the first to conceive this simple strategy, we are not aware of any corresponding prior publication.

There is increasing evidence that microplastic (MP) particles cannot be viewed as a single, specific stressor. Instead, MPs have been shown to cause various, mainly sublethal effects that depend on their physical and chemical properties, including those related to size and shape. However, the molecular mechanisms explaining how MP shapes and sizes lead to different effects are not well understood. Therefore, we examined transcriptomic and proteomic changes in the freshwater mussel Dreissena bugensis after six days of exposure to two size classes of monodispersed PS spheres (6 μm; 20 μm) and fragments (d90: 5.7 μm; d90:18 μm) as well as fibers (Ø 3 μm, mean length 75.5 μm), at a concentration of 1000 particles/ml. Our findings suggest that for smaller MPs, the molecular responses were mainly driven by particle size, whereas particle shape played a larger role for larger MPs. Overall, our results show that MP-induced molecular responses are highly specific to shape and size, emphasizing that environmentally relevant mixtures of microplastic types may lead to complex biological effects. The involvement of MP particle size and shape-dependent pathways related to immune regulation, cellular maintenance, and reproduction suggests that even mainly sublethal effects could impact organismal fitness and, in the long term, populations and ecosystems.

Summary: The early steps of eukaryotic large ribosomal subunit assembly remain poorly understood due to the structural flexibility of pre-60S intermediates, whose rRNA is extensively modified by small nucleolar RNPs (snoRNPs). Some snoRNPs, however, lack any modification function and instead scaffold ribosome assembly through largely unknown mechanisms. Here, we show that the H/ACA snoRNP snR37 integrates both modifying and scaffolding roles. Biochemical and structural analyses reveal a canonical H/ACA core that pseudouridylates a conserved uridine in the A site of the peptidyl transferase center, the catalytic heart of the 60S subunit. Additional RNA helices recruit non-core proteins, the Upa1-Upa2 heterodimer and Rbp95, which mediate stable snR37 association with pre-60S complexes. These proteins cooperate with the Npa1 rRNA chaperone complex to link four rRNA domains, thereby structurally organizing early pre-60S intermediates and promoting proper formation of the PTC. This dual organization establishes a paradigm for snoRNPs combining rRNA modification and scaffolding functions.