DNA-damaging antibiotics like ciprofloxacin (CIP) induce extensive double-strand breaks in Escherichia coli, triggering both the SOS response and rapid DNA supercompaction. To uncover genes involved in the latter process beyond the previously identified key orchestrators encoded by recN and recA, we developed a novel machine learning-assisted high-throughput screening workflow and applied it to nearly 4000 E. coli strains, including the Keio collection's single-gene deletion strains and additional in-house strains. Conservative validation identified 15 hit strains with impaired DNA supercompaction. While defects in recombinational repair genes were associated with the most severe impairments, our investigation also revealed genes not previously associated with DNA compaction or repair that had milder and more heterogeneous effects on supercompaction, including yaiW, which encodes a membrane-associated protein. Notably, several non-DNA-repair gene deletions affected RecN colocalization with the nucleoid, recN expression, SOS response activity, or survival after CIP exposure, supporting indirect or modulatory roles. Altogether, this work confirms RecN and RecA as primary drivers of DNA supercompaction and demonstrates that high-content imaging combined with machine learning-assisted analysis provides a scalable approach to explore bacterial DNA organization phenotypes and DNA damage responses.

Identifying functional mutation blocks (FMBs) that contribute to genome-wide transcriptional regulation remains a challenge. BayesPI-BAR version 3 (bpb3) is a Python-based tool for predicting FMBs by integrating DNA sequence data with gene expression data. Its application is demonstrated through four examples in two tasks: ranking transcription factors (TFs) most affected by 67 known single-nucleotide polymorphisms (SNPs) in regulatory regions and integrating the genomic distribution of SNPs with differential gene expression to identify FMBs that disrupt TF-DNA binding and gene expression in lymphoma.For complete details on the use and execution of this protocol, please refer to Yang et al.1

A substantial proportion of Common Variable Immunodeficiency (CVID) patients has autoimmune and inflammatory complications (CVIDCOMP), associated with T‑cell and monocyte pathology, but the underlying molecular mechanisms remain unclear. We aimed to identify novel immunopathogenic pathways in this inflammation‑prone CVID phenotype. We performed RNA‑seq and mass‑spectrometry–based proteomics on isolated CD3⁺ T cells and CD14⁺ monocytes from CVID patients and healthy controls (HC). Differentially expressed genes (DEGs), proteins (DEPs) and pathways (Hallmark GSEA) were assessed. Plasma IL‑1β and CXCL10 were measured by multiplex assay, IL‑18 and CXCL9 by EIA. CVIDCOMP patients (RNAseq: n = 5, proteomics: n = 13) showed broader transcriptional and proteomic changes in CD14⁺ monocytes than in CD3⁺ T cells. In CD14⁺ monocytes, RNAseq revealed enrichment of “TNFα/NF-κB” signalling and “Interferon γ response”, while proteomics showed enrichment of “Interferon α response”. Conversely, proteomic analyses of cells from CVID without inflammatory/autoimmune complications (n = 5–9) showed only minor differences compared with HC (n = 10). In CVIDCOMP, GBP1, GBP4, GBP5, STAT1 and LGALS3BP were upregulated at both mRNA and protein levels, and several of these interferon‑/inflammasome‑related genes were upregulated in an independent CVID cohort from a public dataset. Plasma levels of IL‑1β, IL‑18, CXCL9 and CXCL10, reflecting inflammasome and interferon activity, were increased in two additional CVID cohorts (CVID = 104/60, HC = 22/30), with the highest concentrations in CVIDCOMP. This integrated analysis supports an inflammatory signature in CVIDCOMP monocytes, involving interferon‑ and inflammasome‑related pathways. The findings highlight CD14⁺ monocytes and their downstream mediators as potential targets for future mechanistic and therapeutic studies.

IntroductionCommon variable immunodeficiency (CVID) is frequently complicated by duodenal inflammation, but the underlying molecular mechanisms remain poorly understood. While epigenetic alterations have been described in CVID, the epitranscriptome is largely unexplored. We therefore investigated whether RNA N6-methyladenosine (m6A) modifications in duodenal tissue are altered in CVID and whether such changes are associated with the local microbiota or m6A-related enzymes.Methodsm6A modification levels were analysed in snap-frozen duodenal biopsies from CVID patients with intraepithelial lymphocytosis and inflammation (CVID_IEL; n = 5), CVID patients with normal duodenal histology (CVID_N; n = 5) and controls with normal biopsies (n = 5) using m6A-RNA immunoprecipitation followed by microarray profiling and gene set enrichment analysis. Duodenal bacterial microbiota from the same anatomical region were characterised by 16S ribosomal RNA gene sequencing, and selected m6A-regulating enzymes were quantified in biopsies by targeted proteomics.ResultsIn total, 4,134 differentially methylated transcripts were identified, and unsupervised principal component analyses revealed partially overlapping, but clearly divergent m6A signatures for CVID_IEL, CVID_N and controls, with a gradient along the first principal component. Pathway analysis showed relative hypermethylation of mitochondria- and ribosome-related gene sets in both CVID subgroups versus controls, and hypomethylation of pathways linked to ubiquitination, proteasomal degradation, glycosylation and post-transcriptional gene silencing in CVID_IEL versus CVID_N. Sparse canonical correlation models demonstrated significant associations between specific duodenal bacterial genera and m6A-modified transcripts in CVID, but not in controls, whereas expression levels of the examined m6A-regulating enzymes did not differ between groups.DiscussionThese findings suggest that duodenal inflammation in CVID may be associated with a distinct m6A epitranscriptomic signature that is linked to specific features of the mucosal microbiota, providing preliminary, hypothesis-generating evidence for a potential interaction between microbiota, epitranscriptomic regulation and local immune dysregulation in CVID.

Processed meat products are widely consumed worldwide, especially because they are affordable and easy to prepare. Their production often involves the use of water-binding additives such as phosphates, nitrites, and sodium chloride. Although these substances improve taste, texture, and shelf life, their potential effects on human health remain unclear. This review aims to summarize current knowledge about the possible biological and clinical effects of these additives. Injection (brining) technology is commonly used to increase product weight and improve quality. However, it may also lead to regular consumption of these additives. Phosphates may influence mineral balance and could be linked to changes in vitamin D metabolism and vascular health. Nitrites can participate in the formation of N-nitroso compounds, which have been shown to have harmful effects in experimental studies. High sodium intake is associated with increased blood pressure and cardiovascular risk. Some studies also suggest that these additives may affect the gut microbiota and promote low-grade inflammation. Certain groups, including people with chronic diseases, may be more sensitive to these effects. Cultivated meat has been proposed as a potential alternative that may reduce reliance on such additives; however, it often still requires similar ingredients to achieve desired texture and stability, and its potential health benefits remain uncertain. Current regulations in the United States and the European Union primarily focus on individual additives and do not fully account for total daily intake from different foods. In addition, food labels often do not provide detailed quantitative information. Further research is needed to better understand long-term effects and combined exposure.

DNA sliding clamps are central coordinators of genome replication and maintenance, yet the full binding network ("interactome") of the bacterial β-clamp remains incompletely defined. Here, we report a novel interaction between Escherichia coli β-clamp and the helicase-nuclease RecBCD complex. Using bacterial two-hybrid assays and co-immunoprecipitation, supported by fluorescence microscopy, we show that RecB associates with β-clamp. Nuclear magnetic resonance spectroscopy maps the interaction to the canonical ligand pocket of β-clamp and identifies a clamp-binding motif in RecB (residues 1018-1023, QVEMEF), whose mutation abolishes binding. Functional assays indicate that this interaction occurs upon conformational switching of RecBCD at a Chi site, and disruption of the motif reduces survival after DNA damage. We also find indications of a second binding site in the helicase domain of RecB. These findings expand the β-clamp interactome and suggest a previously unappreciated role for β-clamp in DNA double-strand break repair, with potential implications for antibacterial strategies.

Lactate, a well-known metabolite and signalling molecule, holds therapeutic potential for neurodegenerative diseases. Here, we investigated the effects of chronic lactate treatment on cognition and molecular biomarkers in the 5XFAD mouse model of Alzheimer’s disease (AD) and in wild-type (WT) controls using behavioural testing alongside proteomic and transcriptomic analyses. Mice received lactate or vehicle injections 4 days per week for 11 weeks, with behavioural testing before and after the treatment period. Lactate improved working memory in late-treated AD mice, without eliciting anxiety-like behaviour. At the molecular level, lactate reduced Il1b expression, and in a sex-dependent manner, normalised NEFL, and enhanced synaptic integrity proteins (OPCML, PPFIA2, STXBP3, SYT1, VGLUT2, VSNL1) in AD mice, while also augmenting mitochondrial regulators (ATP5G2, GRPEL1, SLC25A23) across genotypes. Notably, lactate upregulated low-abundance ionotropic glutamate receptor mRNAs (Grik3, Grin2c, Grid2ip) in female AD mice, indicating enhanced glutamatergic signalling. In WT mice, lactate increased expression of neurotrophic factors (Bdnf, Igf1, Vegfa), anti-inflammatory cytokines (Il4 and Il13), and the neuronal lactate transporter Mct2, suggesting promoted neuronal resilience. Together, these findings indicate that lactate treatment can mitigate cognitive decline and enhance molecular pathways of resilience in AD, warranting larger, age-stratified studies to validate its therapeutic potential and elucidate underlying mechanisms.

A cost-effective, viral nucleic acid (NA) isolation kit based on NAxtra magnetic nanoparticles was developed at the Norwegian University of Science and Technology in response to the shortage of commercial kits for isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA during the coronavirus disease 2019 (COVID-19) pandemic. This method showed comparable sensitivity to available kits at significantly reduced cost, making its application for other biological sources an intriguing prospect. Thus, based on this low-cost nucleic acid extraction technology, we developed a simple, low- and high-throughput, efficient method for isolation of high-integrity total NA, DNA and RNA from mammalian cell lines (monolayer) and organoids (3D-cultures). The extracted NA are compatible with downstream applications including (RT-)qPCR and next-generation sequencing. When automated, NA isolation can be performed in 14 min for up to 96 samples, yielding similar quantities to available kits.

Abstract Background CLN3 Batten disease is a severe pediatric neurodegenerative disorder caused by mutations in the CLN3 gene, most commonly a 1 kb deletion encompassing exons 7 and 8. CLN3 deficiency is associated with lysosomal dysfunction, impaired cellular clearance and disrupted metabolism. While neurons are particularly vulnerable in CLN3 Batten disease and have been the primary focus of research, glial cells are increasingly recognized as active contributors to disease pathology. Among them, astrocytes—the most abundant glial cell type in the brain—play critical roles in maintaining neuronal health and homeostasis. However, astrocytes remain understudied in CLN3 patient-derived models. Methods We present the first iPSC-derived astrocyte model from a skin biopsy of a CLN3 patient carrying the common 1 kb deletion. Cellular and molecular features of iPSC and astrocytes derived from both healthy controls and the CLN3 patient were characterized via qPCR, immunocytochemistry and targeted mass spectrometry. In addition, comprehensive omics-based profiling, through transcriptomic and label-free quantitative proteomics, was performed to uncover novel molecular mechanisms and generate hypotheses that can guide future mechanistic and functional studies. Results Transcriptomic and proteomic analyses during astrocyte differentiation revealed an upregulation of mitochondrial respiratory chain complexes I and IV—contrasting with the downregulation typically observed in CLN3-deficient neurons. We also identified a metabolic shift favoring the elongation of very-long-chain saturated fatty acids, accompanied by reduced lipid synthesis and enhanced fatty acid oxidation. These metabolic alterations were paralleled by an upregulation of proteins involved in oxidative stress responses, likely reflecting a compensatory adaptation to mitochondrial and lipid metabolic dysregulation. Furthermore, we observed significant changes in chromatin organization during astrocyte differentiation in CLN3 cells, suggesting epigenetic remodeling as a contributing factor to disease pathology. Conclusion Our findings prompt the hypothesis that mitochondrial dysfunction may precede lysosomal defects in CLN3-deficient astrocytes. Restoring mitochondrial health could improve brain metabolism, inflammation control, neurotransmitter regulation, and neuronal survival, highlighting mitochondria as promising therapeutic targets in CLN3 Batten disease.

Epigenomes of mammalian oocytes and embryos undergo major transitions essential for successful development. Here, we provide genome-wide maps of histone variant H2A.Z during twelve stages of mouse oogenesis and preimplantation embryo development and relate it to histone marks and genomic features. This revealed that major waves of H2A.Z incorporation occur early in growing oocytes, forming distinct patterns of maternal, embryonic, and persistent H2A.Z enrichment. Late maternal enrichment is inherited by the zygote and precedes reduced formation of lamina associated domains and early replication in the maternal genome of 2-cell embryos. Persistent H2A.Z enrichment is strongly associated with CpG islands and H3K4me3 near transcription start sites of active genes, but thousands of maternal and embryonic H2A.Z incorporation sites exist elsewhere, frequently at transposable elements. The persisting H2A.Z enrichments across related developmental stages enable preservation of epigenetic information despite major concurrent changes in H3K4me3, H3K27me3, and DNA methylation. Altogether, this advances our understanding of how histone variants contribute to epigenetic reprogramming during mammalian oogenesis and early development.

A novel, cost-effective nucleic acid (NA) isolation method for purifying total NA, DNA, or RNA from both two- and three-dimensional cell cultures has been developed at the Norwegian University of Science and Technology utilizing NAxtra magnetic nanoparticles. This method achieves comparable yields to existing isolation kits while offering significant improvements in cost and processing speed. However, the original protocol was not optimized for small-cell numbers or single-cell applications. Given the growing interest in single-cell and rare-cell population studies, there is a critical need for more sensitive isolation techniques. In this study, we have enhanced the sensitivity of the NAxtra-based isolation method to facilitate mid- to high-throughput purification from as few as 10,000 cells down to single cells. Automated processing using KingFisher systems enables the rapid handling of 96 samples within 12-18 min. Our findings indicate that this method not only matches but can exceed the performance of existing alternatives in (RT)-qPCR detection while being significantly more economical and efficient. Additionally, it enables the extraction of high-quality RNA suitable for transcriptomics analyses from limited cell quantities, including single cells. This advancement holds substantial promise for improving the accessibility and efficiency of NA research, particularly in studies involving scarce cellular materials.

Mammalian oocytes and embryos have distinct epigenomes that undergo major transitions essential for transmitting life to the next generation. Here, we provide the first genome-wide maps of the histone variant H2A.Z during six different stages of mouse oogenesis. Utilizing picogram- scale chromatin immunoprecipitation and sequencing (picoChIP-seq), we reveal major waves of H2A.Z incorporation occurring early in growing oocytes. We identify distinct patterns of H2A.Z signal at oocyte-specific, embryo-specific, and constant H2A.Z loci. Late oocyte-specific loci precede reduced formation of lamina associated domains and early replication timing in the maternal compared to the paternal genome of 2-cell embryos. While constant H2A.Z is strongly associated with CpG islands (CGIs) and H3K4me3 near transcription start sites (TSS) of active genes, tens of thousands of oocyte- and embryo-specific H2A.Z incorporation sites exist independently of CGIs and TSSs. These TSS-distal H2A.Z sites are frequently enriched at transposable elements (TEs), and an intriguing inverse relationship exists between H2A.Z and H3K4me3 in low CpG environments, such as MTA and MTB retrotransposons (RTs). The existence of changes in H2A.Z distribution that persist across related developmental stages enable preservation of epigenetic information despite major concurrent changes in H3K4me3, H3K27me3, and DNA methylation. Altogether, this reveals new layers of regulation advancing our understanding of how histone variants contribute to the epigenetic landscape during mammalian oogenesis and preimplantation embryo development. ### Competing Interest Statement The authors have declared no competing interest. Danish National Research Foundation, https://ror.org/00znyv691, DNRF115 Novo Nordisk Foundation, https://ror.org/04txyc737, NNF22OC0080710 Norwegian Centres of Excellence scheme, 332713

Neonatal hypoxic-ischemic (H-I) brain injury, a leading cause of neurodevelopmental disabilities, severely affects the metabolically active and neurogenic hippocampus. To investigate its acute effects and identify drug targets for early therapeutic windows, we applied single-nucleus RNA sequencing on postnatal day 8 (P8) mouse hippocampi under sham, hypoxic, and hypoxic-ischemic conditions. We constructed a comprehensive hippocampal cell atlas and developed a machine-learning classifier for precise cell type identification. Our analysis reveals early vulnerabilities in mature neurons and notable resilience in immature DG, GABAergic, and Cajal-Retzius cells following H-I. Gene regulatory network analysis identified key transcription factors associated with neuronal vulnerability, along with upregulated ribosome biogenesis and dysregulated calcium homeostasis pathways. We observed rapid activation of astrocytes and microglia, with Runx1 identified as a potential key transcription factor associated with early microglia immune responses. Endothelial cells displayed complex transcriptional changes and predicted intercellular signaling patterns that may influence vascular repair and recovery. Our study advances the understanding of immediate cellular and transcriptional responses to neonatal H-I injury, providing new insights into hippocampal cell heterogeneity and pathophysiology. The integrated hippocampal atlas, post-H-I atlas, and machine learning classifier are available at https://hippo-seq.org .

High-conflict parental divorce can result in parental alienation (PA), leading to chronic psychological stress in children that adversely affects their mental and physical health. This stress may manifest as anxiety, depression, eating disorders, immune dysfunction, and cardiovascular, neurological, endocrine, or gastrointestinal issues. The inconsistent legal recognition of PA highlights the need for objective diagnostic tools to inform judicial decisions. We propose a panel of molecular biomarkers, derived from well-established indicators of chronic stress, to address the limited data specific to PA. This panel targets hypothalamic-pituitary-adrenal (HPA) axis activation, neurotransmitter dysregulation, inflammation, oxidative stress, epigenetic modifications, and gut microbiota dysbiosis. Interdisciplinary collaboration among researchers, general practitioners, and legal professionals is crucial to validate and implement this panel, ultimately enhancing child welfare through early intervention.

Adaptive thermogenesis, or beiging, involves the conversion of energy-storing white adipocytes into energy-dissipating beige adipocytes, enabling physiological adaptation to environmental stressors such as hypothermia. While white and beige adipocytes share transcriptional similarity, this identity switch still requires transcriptional and epigenetic reprogramming. However, the molecular mechanisms governing this transition remain incompletely understood. Here, we identify SUMOylation as a critical repressor of adipocyte beiging. Using the small-molecule SUMOylation inhibitor TAK-981, we demonstrate that transient inhibition of SUMOylation primes human adipocytes to express beiging genes including UCP1 and inducing mitochondrial uncoupling in a rosiglitazone-dependent manner. Furthermore, SUMOylation suppresses both the de novo differentiation of human adipose stem cells into beige adipocytes and the transdifferentiation of mature white adipocytes into beige cells. Mechanistically, TAK-981 modulates the cAMP-PKA-p38 signaling axis, ultimately affecting downstream transcriptional programs under the control of PPARG and PPARA enhancers. Our findings establish SUMOylation as a fundamental barrier to white-to-beige adipocyte reprogramming and suggest that the pharmacological combination of PPARG agonists with TAK-981 can promote metabolically beneficial adipose tissue remodeling in clinical settings. ### Competing Interest Statement The authors have declared no competing interest.

Glioblastoma (GBM) is the most common and lethal form of primary brain tumor, characterized by poor prognosis and limited treatment options, with a median survival of only 12 to 15 months post-diagnosis. Despite standard therapeutic protocols involving temozolomide (TMZ) and radiotherapy, effective treatment is challenging, underscoring the need to explore alternative therapeutic strategies. One promising approach is the inhibition of DNA damage response (DDR) pathways. RNaseH2, a key enzyme involved in ribonucleotide excision repair (RER), plays a critical role in maintaining genomic stability by removing ribonucleotides misincorporated into DNA. Thus, inhibition of RNaseH2 may enhance tumor mutational burden (TMB) and potentially transform immunogenically “cold” tumors into immunogenically “hot” tumors. In this study, a high-throughput screening (HTS) assay resulted in the identification of 52 potential RNaseH2 inhibitors from a library of 71227 compounds. We selected six of these inhibitors for further investigation, evaluating their effects both alone and in combination with TMZ in commercially available U87 MG wild-type and IDH1 mutant (IDH1 WT and MUT) glioma cells and patient-derived cells established from glioma organoids (GBO-PDC). This study highlights the therapeutic potential of RNaseH2 inhibition in combination with TMZ for GBM therapy, validated in patient-derived model, offering a promising avenue for treating this highly aggressive and yet uncurable cancer.

The respiratory tract is colonized with low-density microbial communities, which have been shown to impact human respiratory health through microbiota–host interactions. However, a lack of fast and cost-effective nucleic acid extraction method for low-microbial biomass samples hinders investigation of respiratory microbiota. Here, we performed a pilot study to assess the suitability of the NAxtra nucleic acid extraction protocol for profiling bacterial microbiota in respiratory samples. A small number of nasopharyngeal aspirate (n = 8), nasal swab (n = 8), and saliva samples (n = 8) were collected, nucleic acids were isolated using the NAxtra protocol, and 16 S rRNA gene sequencing was performed to characterize bacterial microbiota, which were compared to the same sample types from previous studies using other protocols. The bacterial composition in nasal and saliva samples were consistent with previous reports. Saliva microbiota was significantly richer than nasal microbiota and varied less among individual samples than nasal microbiota. Bacterial composition in nasal samples was distinct from nasopharyngeal aspirates, but closer to saliva samples. A sequencing depth of 50,000 reads/sample was sufficient for microbiota profiling in low biomass respiratory samples. Our pilot study indicates the potential of the NAxtra protocol for bacterial microbiota characterization of low-microbial biomass samples and supports a more comprehensive study to fully evaluate the value of the NAxtra protocol in microbiota research and clinical diagnostics of respiratory pathogens.

Adult neurogenesis in the hippocampus, involving the generation and integration of new neurons, is essential for behavioral pattern separation, which supports accurate memory recall and cognitive plasticity. Here, we explore the role of the DNA repair protein NEIL3 in adult hippocampal neurogenesis and behavioral pattern separation. NEIL3 is required for efficient proliferation and neuronal differentiation of neonatal NSPCs and adult-born NPCs in the hippocampus following a behavioral pattern separation task. NEIL3-depleted mice exhibited a reduced preference for the novel object location, indicating a deficit in pattern separation. NEIL3-deficient adult-born neurons exhibited a significant reduction in mature-like membrane properties, indicating impaired functional maturation. Interestingly, these impairments were not associated with the decreased genomic integrity but with the altered transcriptional regulation of the Wnt signaling pathway. Given the importance of adult neurogenesis in cognitive function, targeting NEIL3 could offer therapeutic potential for addressing age-related hippocampal dysfunction and cognitive decline.

The SMUG1 DNA glycosylase is the primary enzyme responsible for excising 5-hydroxymethyluracil (5hmU) from genomic DNA. SMUG1 activity is high in the brain, suggesting its crucial role in controlling 5hmU levels in this organ. 5hmU has been proposed as an epigenetic mark, but it is still unclear whether, and in which contexts, it may have regulatory functions. Here we show that accumulation of 5hmU in Smug1-deficient mice leads to widespread gene expression alterations across multiple brain regions. Chromatin immunoprecipitation revealed that SMUG1 is recruited to AT-rich promoters of olfactory receptor genes, where it regulates 5hmU levels. The persistence of 5hmU in the olfactory receptor promoters leads to deficits in sensory perception of smell and influences reward-related behaviour. Thus, we identify a specific function for SMUG1 in promoting transcription of olfactory receptor genes.