When organisms encounter pathogens, they rapidly activate complex defense programs to ensure survival. While these immune responses are vital, they often also incur trade-offs, such as reduced growth and development and must therefore be tightly controlled. In this study, we reveal that the steroid hormones brassinosteroids (BRs) contribute to this control in Arabidopsis thaliana by repressing immunity-related genes. We provide evidence that the BR-regulated basic helix-loop-helix (bHLH) transcription factor CESTA (CES), along with its homologs BR ENHANCED EXPRESSION (BEE)1-3, mediate DNA methylation changes at transposable element (TE)-rich loci containing nucleotide-binding leucine-rich-repeat (NLR)-type receptor genes, including SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1 (SNC1). These CES-induced methylation changes correlate with altered splicing of SNC1 pre-mRNA, a process that requires the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). In support, we show that CES associates with components of the chromatin remodeling and splicing machinery. Together, our findings reveal a previously unrecognized BR-induced mechanism that modulates the epigenetic and post transcriptional regulation of immune genes, enabling plants to prioritize growth over defense.

Xanthan gum, a natural heteropolysaccharide produced by Xanthomonas species, has many biotechnological applications across industries due to its unique rheological properties. Expanding its utility requires specific enzymes capable of targeted xanthan modification or degradation. In this study, a novel bacterial strain, isolated from a spoiled xanthan sample and identified as Paenibacillus taichungensis I5, was shown to degrade xanthan using a plate screening assay with Congo red. Activity tests of crude enzyme in culture supernatant demonstrated the secretion of xanthan-degrading enzymes. Genome and proteome analyses suggest a chromosomal xanthan utilization locus encoding a suite of enzymes, including a xanthanase (Pt_XanGH9), two xanthan lyases (Pt_XanPL8a and Pt_XanPL8b), two unsaturated glucuronidases, two α-mannosidases, as well as transport and regulator proteins. Functional characterization through recombinant protein expression and enzyme assays confirmed the functions of Pt_XanGH9, Pt_XanPL8a and Pt_XanPL8b on native xanthan and xanthan-derived oligosaccharides. The polysaccharide degradation products released by these enzymes were identified via LC–MS analysis and suggested two xanthan lyases with divergent cleavage preferences. In contrast to Pt_XanPL8a, Pt_XanPL8b is synthesized with an N-terminal signal peptide, yet both lyases were detected in cell-free supernatant during growth on xanthan. Based on the composition of the xanthan utilization gene cluster and preliminary enzyme characteristics, a working model for xanthan utilization by P. taichungensis I5 is proposed. Reaching a better understanding of bacterial xanthan degrading pathways and the enzymes involved may help to develop modified xanthan derivatives and xanthan degrading enzymes that align with the specific demands of various industrial process. • The genome of P. taichungensis I5 encodes a xanthan utilization locus. • P. taichungensis I5 employs a twin lyase-dependent xanthan utilization system. • The two xanthan lyases differ in cellular localization and in cleavage specificity.

Some filamentous plant‑pathogenic fungi have comparably large genome sizes within the fungal kingdom due to the proliferation of transposable elements (TEs). Blumeria hordei ( Bh ), the causal agent of the powdery mildew disease on barley, is a filamentous obligate biotrophic fungus. Compared to other ascomycetes, it contains a low number of genes but a high genomic TE content of approximately 75%. Yet, a comprehensive understanding of the contribution of TEs to the RNA and protein landscape of Bh is lacking. Here, we use Bh as a model to study transcripts and proteins derived from genes and individual TEs. Therefore , we created two high‑quality genome assemblies of the German Bh isolate TUM1 and the Australian Bh isolate AUS1. We applied deep proteomics with mass spectrometry, long‑read and short‑read sequencing on both DNA and RNA. Based on these multi‑omic resources, we completed nearly gapless genome assemblies, new gene and TE annotations, and effector predictions. Using long-read RNA sequencing, we detected extensive co-transcription of TEs and genes as TE-gene chimeric transcripts. We identified previously unpredicted splice variants or genes, partially supported by proteomics. The intergenic and TE genomic space of Bh TUM1 gives rise to thousands of transcripts and several novel TE-derived proteins that lack from previous TE protein predictions. Together, this supports an existing potential for expression of novel transcripts and proteins from highly abundant TEs in the Bh genome.

Immune marker staining, collagen features, and αSMA-associated stromal analyses in KC/AKC and KPC/AKPC tumors.

BACKGROUND:Long-read sequencing and multi-omic analytical frameworks are increasingly being adopted in rare disease diagnostics. However, clinical workflows comprehensively integrating these methodologies remain uncommon. OBJECTIVE:This study aimed to assess the potential and limitations of integrating long-read genomic, transcriptomic, and proteomic analyses to characterize complex structural variants. METHODS:Two unrelated patients presenting with dystonia and comorbid neurological features underwent nanopore-based long-read DNA sequencing. In patient 1, complementary transcriptomic and proteomic analyses were performed. RESULTS:The workflow enabled the identification and characterization of two pathogenic complex structural variants: a homozygous AluY-mediated inversion disrupting PANK2, underlying neurodegeneration with brain iron accumulation (patient 1), and a heterozygous de novo 16p13.3 duplication-triplication event associated with an atypical dystonia-parkinsonism phenotype (patient 2). CONCLUSIONS:Our findings underscore the diagnostic potential of integrated long-read and multi-omic approaches for complex structural variant characterization, while illustrating persistent limitations of automated pipelines and highlighting unpredictable relationships between genomic, transcriptomic, and proteomic findings. © 2026 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Bacterial extracellular vesicles (EVs) are known to mediate intercellular communication, virulence, and immune modulation. Here we show that bacteria can utilise EVs also as recyclable nutrient reservoirs. Using Bacillus cereus as a model organism, we demonstrate that EVs exhibit distinct dynamics depending on growth conditions: EVs produced in complex nutrient-rich media undergo time-dependent degradation, while those produced in defined nutrient-limited conditions remain stable and accumulate. We observe similar EV degradation patterns in Staphylococcus aureus. Time-resolved multi-omics profiling reveals that EVs containing the lipid sphingomyelin undergo progressive degradation. Using pharmacological inhibition, knockout mutants, and enzymatic complementation, we show that this process is driven by secreted sphingomyelinase (SMase). This enzyme contributes to degradation of sphingomyelin-containing EVs, thereby releasing their biomolecular cargo which can be used as a nutrient source. Growth assays confirm that SMase-mediated EV degradation provides a growth advantage when nutrients become depleted, thus establishing EVs as dynamic nutrient reservoirs.

Abstract The tumor microenvironment (TME) actively contributes to pancreatic ductal adenocarcinoma (PDAC) pathogenesis through dynamic bidirectional tumor–stroma interactions. In this study, we demonstrated that ATM-deficient tumor epithelium reprograms the TME in a genotype-specific manner to enhance cancer aggressiveness. In genetically engineered mouse models, pancreatic stellate cell and cancer-associated fibroblast (CAF) coculture systems, single-nucleus multiomics, and human PDAC models, tumoral loss of ATM serine/threonine kinase drove CAFs toward αSMA+ myofibroblastic (myCAF) differentiation, independently of p53 status. The myCAFs, in turn, promoted cancer aggressiveness and chemoresistance. Mechanistically, ATM deficiency increased reactive oxygen species and contractility signaling, enhancing TGFβ1 secretion. Pharmacologic TGFβ inhibition reversed myCAF differentiation, sensitized tumors to chemotherapy, and impaired tumor progression in both murine and human ATM-null models. These findings reveal that ATM-deficient tumors shape a cancer-promoting niche via TGFβ signaling and identify dual targeting of intrinsic and extrinsic vulnerabilities as a promising precision oncology strategy. Significance: TGF-β-driven myofibroblastic stromal differentiation in ATM-deficient pancreatic cancer generates a genotype-specific tumor microenvironment, providing a targetable axis and highlighting the need to integrate epithelial genotype and stromal context in pancreatic cancer therapy.

Corpus luteum (CL) is an endocrine structure that undergoes substantial changes over short intervals during the bovine reproductive (estrous) cycle. These changes are regulated by a vast array of signaling molecules, mainly proteins, that govern reproduction. We collected a comprehensive set of CLs from non-pregnant (days 1-2, 3-4, 5-7, 8-12, 13-17, and >18) and pregnant (months 1-2, 3-4, 5-6, and >7) cows, enabling a detailed characterization of temporal changes in CL biology. While previous studies have focused on a limited number of proteins, we employed a broader approach to exploring additional proteins potentially involved in CL regulation across different stages. Using high-resolution mass spectrometry (HR-LC-MS/MS), we identified approximately 3,783 individual proteins. These proteins may be useful for research in species where access to well-characterized CL tissue is limited. Our findings indicate diverse changes in protein expression across groups, highlighting proteomic patterns that may reflect key processes of bovine reproduction, particularly during the transition toward regression, a critical period that influences whether the CL is maintained to support pregnancy or undergoes regression.

Clinical characteristics and mutation profiles of the patient-derived organoids used in this study.

Analyses of TGFβ ligand/receptor expression, reporter activity, inferred signaling interactions, and PSC differentiation in ATM-deficient tumor models.

Abstract Sepsis accounts for nearly 20% of global mortality, with antibiotic resistance worsening clinical outcomes. Rapid antibiotic administration and accurate pathogen identification remain crucial. It is well now known that extracellular vesicles (EVs) from human cells and bacterial membrane vesicles (bMVs) play a central role in the interaction between host and pathogen and represent promising biomarkers for early infections. This study investigated how antibiotic exposure alters EV responses in Staphylococcus aureus (SA)spiked blood and compared these findings with EV proteome profiles from bacteremia patients. In an in vitro model, whole blood from healthy donors was spiked with SA at a multiplicity of infection (MOI) of 0.001, treated with clinically relevant concentrations of piperacillin–tazobactam, vancomycin, or moxifloxacin, and plasma was subsequently isolated for EV analysis. EVs were isolated using the Miltenyi Pan EV Kit and analyzed by bead-based flow cytometry and high-resolution LC–MS/MS. In parallel, serum EVs from healthy controls ( n = 6) and bacteremia patients ( n = 12; 6 blood culture–positive and 6 culture-negative) were analyzed using the same workflow. Flow cytometry revealed increased levels of CMO⁺ CD45⁺ PanEV⁺ SA⁺ vesicles in SA-spiked samples, particularly following low-dose piperacillin–tazobactam and high-dose vancomycin treatment, despite minimal changes in vesicle size and total particle counts. Proteomic analysis of plasma EVs showed significant alterations in protein composition, including increased abundance of the SA-derived ribosomal protein rplU and host defense–associated proteins. Functional enrichment highlighted pathways related to neutrophil degranulation, vesicle-mediated transport, and antibacterial responses. In patient samples, serum EVs were enriched in acute-phase and immune-related proteins, including SERPINA1, SERPINA3, CRP, and SAA2, along with canonical EV markers such as CD81 and syntenin-1, irrespective of blood culture status. Antibiotic exposure and SA infection are associated with measurable changes in the human EV proteome, characterized by enrichment of immune and host defense–related proteins despite stable vesicle numbers. Similar EV-associated protein patterns were observed in both blood culture–positive and –negative patient samples, reflecting shared features of the systemic host response to infection and highlighting the potential of EV profiling to capture infection-associated biological signals.

Barley cysteine endoprotease B (HvEPB) is an important enzyme in plant biology and food production. Pilot-scale fed-batch fermentation with Komagataella phaffii in minimal FM22 medium enabled controlled recombinant HvEPB (r-HvEPB) secretion. Peptidomics across four technological brewing matrices - raw barley, malt, wort and beer - revealed hydrophobic P2 preference (V, L, Y) and heterogeneous P1 selection (T, Q, G), with certain residues disfavoured (S, A, G at P2; P, I, L at P1). Cleavage motifs shifted depending on the underlying matrix, primarily reflecting differences in substrates properties and availability. Immunogenic gluten peptides, particularly from C- and B-hordeins, were efficiently cleaved. Enzyme-linked immunosorbent assay confirmed 85-89% gluten reduction across all four matrices under controlled in vitro incubation. r-HvEPB hydrolysed brewing-relevant hordeins, α/β-amylases, while LTP I and serpin Z4 were less cleaved. Weighted motif analysis and inhibition trials underscored HvEPB's pivotal role as a broad-specificity endoprotease in malt-based systems, with r-HvEPB showing significantly higher gluten hydrolysis than endogenous malt proteases.

PSC-induced chemotherapy resistance and in vitro and ex vivo assays assessing apoptosis, invasion, wound closure, and drug response after perturbation of tumor–CAF signaling.

Patients with suspected monogenic disorders often remain undiagnosed after exome sequencing. We report a family with two sisters affected by a complex spastic paraplegia. Initial exome sequencing had identified monoallelic pathogenic nonsense variants in AP4S1 and AP4B1, subunits of the adaptor protein complex 4 (AP-4), suggesting digenic inheritance. As digenic inheritance has not been established for AP-4-associated disorders, we applied a multiomics approach including genome sequencing, RNA sequencing and proteomics to clarify the genetic cause. By RNA sequencing a predicted synonymous variant (NM_006594.5:c.969G > A), compound heterozygous to the nonsense variant in AP4B1 and previously considered as benign, was re-prioritized as aberrant splicing was demonstrated. Proteomics showed reduced abundance of AP-4 components AP4B1 and AP4M1 and an upregulation of the cargo protein ATG9A, confirming AP-4 deficiency. Although the AP4S1 variant resulted in nonsense-mediated decay, the identification of biallelic causative variants in AP4B1 established the diagnosis of monogenic "Spastic paraplegia 47, autosomal recessive" while the initial hypothesis of digenic inheritance was refuted. This study illustrates the value of multiomics approaches in the diagnostic workflow of rare diseases and the potential for pathogenicity of synonymous variants.

Abstract The uptake of nitrogen-fixing bacteria into living plant cells and the intracellular accommodation of arbuscular mycorrhiza (AM) fungi requires the plasma membrane-localised Symbiosis Receptor-like Kinase (SymRK). AM is widespread across terrestrial vascular plant lineages, while the nitrogen-fixing root nodule symbiosis (RNS) is restricted to one clade within the eurosids. This distribution led to the concept that SymRK was adopted during evolution to mediate RNS. Comparative analyses revealed that SymRK orthologs from the eurosid clade support RNS while SymRK from the phylogenetically distant species Solanum lycopersicum (tomato) does not. To dissect the molecular basis for this different functionality, we carried out complementation analyses of the Lotus japonicus symrk-3 mutant which is unable to form AM or RNS. Domains swap chimera from the tomato and L. japonicus SymRK orthologs revealed that the intracellular domain of L. japonicus SymRK is necessary and for cortical infection thread (IT) and symbiosome development at 21 days post inoculation. Notably, this signalling specificity could be overcome by ectopic expression of tomato SymRK, pointing to altered protein dosage as a potential determinant of function. Consistent with this idea, SINA family E3 ubiquitin ligases interacted with and ubiquitinylated L. japonicus SymRK, but not tomato SymRK. In yeast two hybrid analysis, the interaction of SymRK with SINA2 and SINA4 depended on the C-terminal intrinsically disordered tail region of L. japonicus SymRK. We conclude that the SymRK intracellular domain evolved interaction capabilities with SINA E3 ligases which correlates with its ability to support RNS.

Fibrosis and CAF marker analyses, MRI imaging, survival, proliferation, apoptosis, and flow cytometry from orthotopic treatment experiments.

Abstract Limited resource availability in the gut promotes competitive interactions between bacteria, which drive adaptive within-host evolution ( 1–3 ). While adaptive evolution of bacterial communities has been increasingly studied in the recent years ( 4–7 ), its functional implications for host physiology remain unknown. Here, we show that within-host evolution of the human commensal Enterococcus faecalis boosts colonization resistance to enteric Salmonella enterica serovar Typhimurium ( S . Typhimurium) infection. During gut colonization, E. faecalis evolves the ability to metabolize fructoselysine, an abundant Amadori rearrangement product generated by thermal food processing. The depletion of this diet-derived nutrient prevents S . Typhimurium colonization by restricting an essential resource. This protective mechanism was conserved across independent mouse colonies and arises via diverse evolutionary trajectories, including nucleotide polymorphisms, gene amplifications, and a horizontal gene transfer event. Additionally, analysis of E. faecalis isolates from human infants revealed that adaptation to fructoselysine availability occurs in a diet-dependent manner. Isolates from infants fed with fructoselysine-rich formula were able to utilize fructoselysine, whereas those from infants fed with fructoselysine-poor breast milk were not. Conclusively, our results identify an inherent microbiome-driven self-healing mechanism, wherein bacterial evolution restores colonization resistance against enteric pathogens through evolved nutrient depletion. Understanding these evolutionary dynamics will inform microbiome-targeted approaches to prevent and treat infectious diseases by harnessing adaptive bacterial metabolism.

BACKGROUND:Luminal proteases have been implicated in epithelial barrier dysfunction and visceral hypersensitivity in irritable bowel syndrome (IBS), yet their impact on the enteric nervous system (ENS), the principal regulator of gastrointestinal function, remains unknown. OBJECTIVE:To investigate whether faecal mediators differentially activate enteric neurons across IBS subtypes and whether proteolytic and proteomic profiles explain neuronal phenotypes. DESIGN:The effects of faecal supernatants (FSN) from 21 IBS-D (diarrhoea-predominant), 9 IBS-C (constipation-predominant) and 18 healthy control (HC) patients recruited across centres in three countries on guinea pig distal colon submucous plexus neurons were assessed using a neuroimaging technique. Faecal proteolytic activities and proteomic profiles were analysed. RESULTS:IBS-D and IBS-C supernatants evoked significantly stronger neuronal activation than HC, demonstrating that FSN directly modulate ENS. In IBS-D, but not IBS-C, effects were mediated by serine and cysteine proteases and PAR-1. Proteome analysis revealed a significant difference in 47 proteins between IBS-D and HC, including several immunoglobulin components, underlying the role of microinflammation in IBS-D. A combination of amylases, trypsin-2 and an immunoglobulin protein demonstrated high diagnostic performance to distinguish IBS-D from HC. CONCLUSION:These findings uncover a previously unrecognised luminal-ENS axis in IBS and reveal fundamentally different pathological mechanisms between IBS-D and IBS-C. IBS-D is characterised by proteases and PAR-1-dependent neuronal activation and a distinct immune-enriched faecal proteome, whereas mediators in IBS-C act independently of these factors. These findings establish a functional link between faecal protease activity, ENS signalling and molecular biomarkers, highlighting new therapeutic and diagnostic avenues for subtype-specific management of IBS.

Abstract Escherichia coli couples the initiation of DNA replication with cell size by modulating the activity of the replication initiator protein DnaA. The activity of DnaA is regulated by both its interconversion between an active and inactive form and its titration on binding sites on the chromosome. Whereas its interconversion has been thoroughly studied, the extent to which DnaA titration can control replication initiation is poorly understood. Here, we describe the control of E. coli DNA replication via titration by modulating the expression of an ‘always - active’ DnaA variant in four growth conditions. While we obtained stable cell cycles during slow growth, faster growth associated with overlapping replication forks led to replicative instability and DNA damage. Overall, our results provide insights into the limits of titration-based systems in the control of genome replication and their potential role in the evolutionary trajectory of E. coli . Finally, this study provides design principles for a simplified, titration-only regulatory mechanism for DNA replication in synthetic cells.

ROS measurements, cytoskeletal marker expression, migration assays, and PSC differentiation analyses following genetic and pharmacologic perturbation of ROS and cytoskeletal pathways in ATM-deficient tumor models.