This table shows the baseline characteristics of participants included in the analysis and those excluded because of missing INFLA-score data. Demographic, lifestyle, clinical, and biochemical variables are compared between the two groups to assess potential differences related to INFLA-score availability.

Background The rice ( Oryza sativa ) root microbiome plays a critical role in plant adaptation to salinity stress, and its targeted manipulation offers a promising strategy to enhance crop resilience. In this study, a multi-generation enrichment approach under saline conditions was used to restructure the rice root endosphere and enrich for salt stress–resistant bacterial taxa. Results Successive reinoculations led to the consistent enrichment of key plant growth–promoting genera detected by 16S rRNA amplicon sequencing analysis, including Rhizobium, Paenibacillus, Pseudomonas, Pantoea , and Kosakonia , indicating adaptation to the root environment under salt stress while maintaining overall microbial diversity. Based on these enriched communities seen in silico , halotolerant bacterial strains were then isolated, characterized, and phenotypically tested in vitro. Strains exhibiting in vitro plant growth promoting and complementary functional traits were selected and combined into synthetic consortia. An eight-strains consortium (C1) significantly improved plant growth under both non-stressed and saline conditions without disrupting the native microbiome, in addition, a reduced three-strains consortium (C5) retained similar beneficial effects. In contrast, individual strain inoculations were less effective and often impaired plant performance. Importantly, colonization assays, monitored through Oxford Nanopore sequencing as a targeted high-resolution approach, revealed that several strains unable to establish efficiently as single inoculants were able to persist when delivered within a consortium. This indicates that consortium assembly does not simply combine individually beneficial strains but can create a supportive microbial context that promotes colonization, persistence, and plant growth promotion. Conclusions This study demonstrates that salinity-driven microbiome enrichment, combined with rational consortium design, can generate effective multi-strain inoculants that outperform single strains in promoting rice growth and salt stress tolerance, highlighting synergistic interactions and strain compatibility as key determinants of successful root establishment and bioinoculant performance.

Paget's disease of bone (PDB) is a late-onset skeletal disorder characterized by excessive osteoclast-mediated bone remodelling and disorganized bone deposition. The P937R mutation in the ZNF687 gene causes a severe form of PDB complicated by giant cell tumour transformation. Although ZNF687 has been implicated in osteoclastogenesis, whether it regulates upstream haematopoietic progenitor dynamics and bone marrow myeloid output remains unclear. Using a constitutive Zfp687 knock-out mouse model, we showed that Zfp687 loss causes postnatal growth restriction, reduced bone marrow cellularity, impaired osteoclast differentiation in vitro and in vivo, and increased trabecular bone mass during adulthood. Flow cytometry revealed a marked reduction in osteoclast progenitors and macrophages in Zfp687-deficient bone marrow, whereas the pagetic P937R mutation promoted the expansion of the same myeloid populations in the Zfp687P937R knock-in mouse model. Single-cell RNA sequencing of bone marrow-derived c-Kit+ haematopoietic progenitors further demonstrated that Zfp687 loss selectively disrupted the myeloid progenitor compartment. This analysis identified 22 transcriptionally distinct populations and revealed a significant depletion of the early cycling granulocyte-monocyte progenitor cluster, without evidence of a global block in myeloid differentiation. Mechanistically, Zfp687 deficiency impaired the Brd4-c-Myc-NFATc1 axis in osteoclastogenic precursors and reduced Csf1 expression in bone marrow stromal and osteoblastic cells, linking intrinsic transcriptional competence to niche-derived M-CSF support. In pagetic patient iPSCs-derived haematopoietic progenitors, the P937R mutation enhanced clonogenic haematopoietic output, accelerated colony formation, and promoted the expansion of primitive/multipotent colony-forming progenitors, leading to hypercellular myeloid colonies. Together, our findings establish ZNF687 as a regulator of haematopoietic progenitor dynamics that couples bone marrow myeloid output to osteoclastogenesis, providing a progenitor-level mechanism for severe ZNF687-related PDB.

Studying how microenvironmental cues influence metabolic reprogramming can uncover mechanisms driving tumor progression. Using an in vitro model with proliferative stimuli of the in vivo lymph node niche (LN)-including interleukin-21 (IL-21)-we examined metabolic rewiring in chronic lymphocytic leukemia (CLL) cells. We found that the metabolic intermediates of upper glycolysis and its branching pathways are key in fulfilling metabolic demands of proliferating CLL cells. Among branching pathways, the pentose phosphate pathway (PPP) was the most transcriptionally upregulated in proliferating CLL cells. Increased expression of PPP genes was detected ex vivo at the bulk and single-cell level in the LN-resident and -emigrating CLL cells, with more consistency across enzymes of the nonoxidative PPP branch. Expression of the latter correlated with shorter failure-free survival in CLL patients. At the cellular level, metabolomics and 13C-glucose tracing confirmed high activity of the non-oxidative PPP in proliferating CLL cells. IL-21 regulated the expression of PPP enzymes, with STAT3 serving as the primary downstream effector. CRISPR/Cas9-mediated silencing of PPP enzymes revealed that, in vitro, proliferating CLL cells from most patients were not dependent on these enzymes. In contrast, silencing transketolase (TKT)-the rate-limiting enzyme of the non-oxidative PPP-abolished tumor engraftment in vivo, demonstrating that CLL cells rely on this pathway within the tumor microenvironment. These findings uncover a CLL-specific metabolic reprogramming wherein IL-21-STAT3 drives PPP activity and identify the nonoxidative PPP as a critical in vivo vulnerability of leukemic cells in the murine CLL model.

Mutations in TRIM32 cause limb-girdle muscular dystrophy recessive 8 (LGMDR8), a neuromuscular disorder primarily affecting the proximal muscles of hips and shoulders. However, the precise pathogenic mechanism remains unclear. In this study, we used Trim32 knock-out C2C12 murine myoblasts to investigate the impact of full Trim32 loss along the myogenesis process. We found that Trim32 deficiency alters global transcriptomics already in the early phases of the differentiation process leading to impaired myogenic signaling, ultimately resulting in delayed and abnormal myotube formation. Following this up, we discovered that lack of Trim32 disrupts the transition from proliferation to differentiation by limiting the necessary downregulation of the proto-oncogene c-Myc, thus delaying and altering the immediate early onset of differentiation. Interestingly, unlike previous reports that emphasized protein-level regulation, our data reveal that, at this precise stage of differentiation, Trim32 regulates the stability of c-Myc at mRNA level. Attenuating c-Myc expression level is able to partially recover the myogenesis defects observed in the absence of Trim32, suggesting that the Trim32-c-Myc axis may represent an essential hub, although likely not the exclusive mechanism, in muscle regeneration within LGMDR8 pathogenesis.

Abstract Parkinson’s disease (PD) exhibits substantial genetic heterogeneity, yet how combinations of rare variants converge on disease-relevant cellular mechanisms remains unclear. Here, we generated human induced pluripotent stem cell-derived dopaminergic neurons from PD patients carrying rare variants in recently implicated genes and performed integrated electrophysiological, proteomic, lipidomic, and genetic analyses. Patient-derived neurons showed reduced membrane capacitance and altered action potential firing, indicating impaired intrinsic excitability and synaptic dysfunction, with marked variability across genetic backgrounds. Multi-omics profiling revealed dysregulation of mitochondrial function, glycolysis, and oxidative phosphorylation, accompanied by extensive lipid remodeling, including increased fatty acids, acylcarnitines, and sphingolipids, and reduced gangliosides. These alterations were more pronounced in neurons harboring specific variant combinations in KIF21B, SLC6A3, HMOX2, TMEM175, and AIMP2. Integrative analyses uncovered coordinated protein–lipid changes linking mitochondrial dysfunction and membrane homeostasis. Notably, Calpastatin and CXCR4 were consistently dysregulated across PD neurons. Genetic association analyses in independent cohorts identified PD-associated variants in genes encoding dysregulated proteins, supporting the functional relevance of these pathways. Overall, our results define convergent and variant-specific mechanisms underlying PD and highlight candidate biomarkers and therapeutic targets.

Short-read Illumina sequencing of hypervariable regions of the 16S rRNA gene and long-read Oxford Nanopore Technologies (ONT) sequencing of the full-length 16S gene are increasingly used to profile microbial communities. However, differences in sequencing chemistry, read length, and taxonomic assignment methods raise concerns about the comparability of microbiome profiles and their impact on biological interpretation. Faecal samples from two groups of healthy dogs, 8 young (32 < age < 59 months) and 8 old (age > 109 months), were analysed. Taxonomic relative abundances obtained from Illumina and ONT sequencing were compared after nomenclature harmonisation. Agreement between workflows was assessed using Bland–Altman analysis on log2-transformed relative abundances. Although the mean bias at the genus level was small (0.229), limits of agreement indicated poor interchangeability between workflows. When restricted to taxa detected by both workflows, variability decreased but a larger negative bias emerged, suggesting abundance-dependent discrepancies. The effects of sequencing workflow on biologically relevant signals related to host age were evaluated by computing alpha and beta diversity metrics independently within each workflow. Alpha diversity differed between workflows, with ONT yielding higher Shannon diversity and richness values than Illumina. Beta diversity analyses indicated significant (p < 0.05) age-related shifts in community composition only in ONT, with differences in variance explained and effect sizes. Workflow comparisons revealed taxon-specific differences at phylum, family, and genus levels, affecting both moderately and highly abundant taxa. These findings indicate that sequencing workflow choice impacts microbiome profiling and downstream interpretation, underscoring the need for care in cross-workflow comparisons. Illumina and Nanopore show limited interchangeability in microbiome profiling. Workflow choice significantly alters relative abundance estimates across taxa. Bias increases at finer taxonomic resolution. Detection differences drive cross-workflow discrepancies. Sequencing workflow impacts alpha and beta diversity metrics.

Supplementary Figure S1 shows a flowchart detailing how the analytic study population was derived from the source population, including application of inclusion and exclusion criteria and the number of participants remaining at each stage.

Supplementary Table S4 shows the vause-specific and Fine–Gray competing-risk hazard ratios and 95% confidence intervals for cancer mortality, cancer hospitalization, and fatal and non-fatal colorectal cancer (INFLA-score Q5 versus Q1-Q4).

Background: Celiac disease (CD) is a gluten-dependent autoimmune disorder whose pathogenesis is only partially explained by HLA-DQ2/DQ8 predisposition and adaptive immunity. Although HLA-DQ2/DQ8 alleles are carried by 30–40% of the population, only 1–2% develop CD, implicating additional non-genetic factors. Intestinal epithelial cells (IECs) are among the first cells exposed to dietary gluten, yet their epigenetic contribution across the clinical spectrum of CD remains undefined. The aims were to characterise CD-specific DNA methylation and transcriptional alterations in highly purified IECs and to assess their persistence after a gluten-free diet (GFD). Methods: We profiled genome-wide DNA methylation (Illumina EPIC array) and bulk RNA sequencing in IECs from duodenal biopsies of 181 children — untreated classical and potential CD, GFD-treated CD, and controls with other gastrointestinal disorders. Findings were validated by immunohistochemistry in an independent adult cohort and in patient-derived intestinal epithelial organoids. Findings: Differentially methylated regions were shared across all CD groups versus controls and were enriched in antigen processing and presentation pathways; this signature persisted in GFD-treated patients, indicating independence from active inflammation. Transcriptomic analysis in classical CD confirmed upregulation of these pathways, and immunohistochemistry showed increased TAP1, CIITA and MHC-II protein in IECs at diagnosis and after GFD. Organoids retained CD-specific methylation changes in vitro. A model combining 12 differentially methylated regions and HLA haplotype classified classical CD patients and controls with 95% accuracy. Interpretation: The intestinal epithelium displays stable, CD-specific epigenetic alterations associated with enhanced antigen processing and presentation pathway, suggesting an active epithelial contribution to CD pathogenesis.FundingThis work was supported by Italian Society for Celiac Disease with Project Investigator Grant N.003_2020 and by the Italian Ministry of Health (Rome, Italy) through the contribution given to the Institute for Maternal and Child Health IRCCS Burlo Garofolo (Trieste, Italy) with grant RC21/20.

Supplementary Table S3 shows the cause-specific and Fine–Gray competing-risk hazard ratios and 95% confidence intervals for cancer mortality, cancer hospitalization, and fatal and non-fatal colorectal cancer according to fifths of low-grade inflammation (INFLA-score).

TRIM8 is an E3 ubiquitin ligase that functions as both a tumour suppressor and an oncoprotein. Earlier, we reported that TRIM8 interacts with key regulators of mitotic spindle assembly, and that TRIM8 knockdown results in mitotic delay and aneuploidy. In this study, we implemented a multi-omics strategy with differential transcriptomic (single-cell RNA sequencing or scRNA-seq), translatomic (polysome profiling with RNA-seq), and proteomic (LC-MS/MS) approaches to elucidate the involvement of TRIM8 in different levels (transcription, translation, post-translation) and stages (G0/G1, S, G2/M) of mitotic cell cycle regulation and progression. With the aid of differential transcriptomic (scRNA-seq) and proteomic (LC-MS/MS) approaches, we show that depletion of TRIM8 perturbs the canonical 'Cell Cycle Control of Chromosomal Replication' pathway and demonstrate that TRIM8 negatively regulates the expression of TOP2A, known to be essential for genomic integrity. We also show that TRIM8 downregulation induces substantial alterations in the translation activity of cells and results in the upregulation of polysome-bound MALAT1 lncRNA by means of significant changes in polysome profiling coupled with RNA-sequencing. Moreover, we unveil endogenous TRIM8 as a novel ciliary protein that co-localizes with CEP170, required for ciliary function, in the centrosomal region throughout all mitotic phases. Our work shows the dynamic role played by a TRIM family protein across various stages of mitosis for the first time, laying the foundation for exploring the therapeutic potential of TRIM8 in addressing cell cycle-related diseases, including cancer. ### Competing Interest Statement The authors have declared no competing interest.

Electrical stimulation (ES) is widely employed in both clinical therapies and research settings where it has shown promise in promoting tissue regeneration, wound healing, and inflammation control. Research has also highlighted ES as a regulator of DNA demethylation, which plays a critical role in nerve regeneration and cellular repair mechanisms. While the impact of ES on epigenetic processes is recognized, its broader effects on cellular functions, particularly in inflammation and wound healing, are less understood. We recently showed how ES impacts inflammatory states by modulating transcriptomic and metabolomic profiles in a 3Din vitromodel where human fibroblasts and keratinocytes are included in a collagen matrix, i.e., even in the absence of the nervous system. Here, we propose to deepen our exploration on the differential effects on DNA methylation, including an investigation of the correlation with age acceleration using a mitotic clock. These results confirm and caution on the differential effect of DC on inflamed and non-inflamed samples and suggest an involvement of direct current stimuli at 1 V (DC1) in the control of senescent processes associated with mitosis and inflammation; the mechanistic details of these will have to be clarified with additional experiments.

Colitis-associated cancer (CAC) arises from a complex interplay between host and environmental factors. In this report, we investigated the role of the gut microbiome using Winnie mice, an ulcerative colitis-like (UC-like) model with a missense mutation in the Muc2 gene. Upon rederivation from a conventional (CONV) to a specific pathogen-free (SPF) facility, Winnie mice developed severe colitis and, notably, spontaneous CAC that progressively worsened over time. In contrast, CONV Winnie mice showed only mild colitis but no tumorigenesis. By comparison, when re-derived into germ-free (GF) conditions, SPF Winnie mice were protected from colitis and colon tumors, indicating an essential role for the gut microbiome in the development of CAC in these mice. Using shotgun metagenomics, metabolomics, and lipidomics, we identified a distinct proinflammatory microbial and metabolic signature that potentially drives the transition from colitis to CAC. Using either SPF Winnie or WT (Bl/6) donors, fecal microbiota transplantation (FMT) into GF Winnie recipients demonstrated that, while colitis developed regardless of the donor, only FM from SPF Winnie donors resulted in CAC in recipient mice. Our studies present a relevant model of CAC, providing strong evidence that the microbiome plays a key role in its pathogenesis, thus challenging the concept of colon cancer as a strictly nontransmissible disease.

Endometrial cancers (ECs) are mainly adenocarcinomas arising from the uterine endometrium. In this work, we employed data-independent acquisition (DIA) mass spectrometry (MS)-based label-free quantification (LFQ-MS) proteomics to analyze the proteome of tissue washings collected from 25 control (CTRL) subjects, 25 patients with low-grade type 1 endometrial cancer (EC), and 24 patients with high-grade type 1 EC. Following quantification and statistical analysis, we identified 42 proteins able to discriminate CTRL from EC patients, and 151 proteins differentiating high-grade EC cases from low-grade EC cases. Notably, PRRC2A and SYDE2 effectively distinguished both EC patients from controls and advanced EC cases from low-grade EC cases. Validation by Western blot analysis in an independent cohort comprising 19 CTRL patients, 19 patients with low-grade EC, and 19 patients with high-grade EC confirmed the upregulation of PRRC2A and SYDE2. These proteins are implicated in the translocation of SLC2A4, the regulation of MECP2, and extracellular matrix (ECM) proteoglycan pathways, all of which are associated with tumor growth. Our results demonstrate that DIA-based proteomic analysis of tissue washings enables the identification of potential biomarkers for endometrial cancer (EC). Moreover, this study highlights tissue washings as a promising biological fluid for biomarker discovery in EC.

Parkinson's disease (PD) represents one of the most frequent neurodegenerative disorders for which genetic diagnosis is still challenging due to the high genetic heterogeneity associated with the disease and to the difficulty in interpreting test results. We have recently reported the identification of rare new gene variants in PD patients that support polygenic contribution to the disease. Here we report the identification of novel candidate PD genes and an exploratory protocol for predictive analysis of PD risk. The study includes the whole exome data of 22 PD families, 300 unrelated familiar PD, 504 unrelated sporadic PD and 664 healthy subjects. Family-based approach identified rare and disrupting variants in 44 candidate PD genes co-inherited by affected relatives. The analysis of the entire cohort discovered a significant excess of rare and deleterious variants in PD patients compared to controls in 7 genes out of the 44 identified in the families. Five of these, known as ANKK1, ANKRD50, GRK5, PACSIN1 and VPS8, were novel candidate PD genes, expressed in human dopaminergic neurons, and involved in signal transduction pathways and in endocytic recycling. In these genes, we identified both rare probably damaging variants, altering protein structure and dynamics, as well as frequent variants associated with PD risk. Moreover, we demonstrated that the co-inheritance of multiple rare variants (≥ 2) in a panel of 37 PD genes selected in this study, may predict disease risk in about 26 % of patients, both familial and sporadic cases, with high specificity (> 92 %; p ≤0.00001). Furthermore, patients carrying multiple rare variants showed higher risk of manifesting dyskinesia induced by levodopa treatment (p = 0.004), severe cognitive impairment (p = 0.009) and an earlier age at onset of the disease (p = 0.01). Despite the still exploratory nature of the study, these data provide novel insights into the genetic of PD and may be relevant for its prediction, diagnosis and treatment.

Feline Idiopathic Cystitis (FIC), is a chronic lower urinary tract condition in cats analogous to PBS/IC in women, which presents significant treatment challenges due to its idiopathic nature. Recent advancements in regenerative medicine highlight the potential of Adipose Tissue-Derived Stem Cells (ADSCs), particularly through their secretome, which includes mediators, bioactive molecules, and extracellular vesicles (EVs). Notably, exosomes, a subset of EVs, facilitate cell-to-cell communication and, when derived from ADSCs, exhibit anti-inflammatory properties and contribute to tissue regeneration. In this work, we aim to characterize the content of exosomes derived from feline ADSCs (fADSCs) to elucidate their mechanisms of action on recipient cells and assess their therapeutic potential for FIC. Exosomes were isolated from fADSCs and their microRNA (miRNA) content sequenced using Illumina technology. Our findings demonstrate that fADSC-derived exosomes harbor miRNAs that can induce regenerative processes, such as cell proliferation, immune modulation, angiogenesis, and anti-inflammatory responses. Key miRNAs identified include fca-miR-221, fca-let-7f-5p, fca-miR-337-5p, fca-miR-542-5p, fca-miR-24-3p, fca-miR-205, and fca-miR-23a, which promote proliferative, angiogenic, differentiation, and regenerative mechanisms. Additionally, miRNAs with anti-inflammatory effects, such as fca-miR-193a-5p and fca-miR-127-3p, and those positively regulating the immune system, including fca-let-7a-5p and fca-miR-chrC1_18846-5p, were identified. Of particular interest, fca-miR-219-5p (has-miR-6766-3p) has been reported to suppress liver fibrosis.These results underline the therapeutic potential of fADSC-derived exosomes in treating FIC and suggest innovative strategies for feline veterinary medicine.

Liver transplantation faces several biological challenges, including those related to ischemia/reperfusion injury, graft rejection or tolerance, and disease recurrence. These processes significantly impact posttransplant outcomes and highlight the need for a deeper understanding to improve patient care. The progress of omics sciences has been remarkable over the past decades, and omics techniques are widely used in clinical investigations. In this review, we have explored and briefly described investigations using omics technologies (epigenomics, transcriptomics, and proteomics) to better understand the processes affecting liver transplantation. Additionally, we have highlighted recent studies that use cutting-edge omics technologies, including single-cell RNA sequencing, spatial transcriptomics, and multiplex proteomics, often integrated into multiomics approaches, which enable a more detailed and holistic analysis of the produced data, paving the way for the discovery of precise biomarkers for liver transplant monitoring and the development of novel therapies to prevent allograft rejection.