Oral lichen planus (OLP) is a chronic inflammatory oral disease associated with immune dysregulation and malignant transformation risk. Vitamin D and probiotics may modulate immune and microbial pathways involved in OLP. In this study, we evaluated their effects on clinical outcomes and multi-omics profiles in 25 adult OLP patients (median age: 68 years). Vitamin D-deficient patients received 2000 IU/day vitamin D3, and all participants received a probiotic blend (Limosilactobacillus reuteri LRE11, Lacticaseibacillus rhamnosus LR04, and Lacticaseibacillus casei LC04) for 16 weeks. Clinical assessments and analyses of saliva, serum, oral swabs, and stool samples were performed before and after treatment. Clinical outcomes improved significantly, with reductions in lesion number (p < 0.001), lesion size (p = 0.004), and bleeding lesions (p = 0.014); 76% of patients were classified as in remission. Vitamin D levels increased significantly among deficient patients receiving correction (p < 0.01). Salivary and fecal metabolomics showed significant remodeling of amino acid and carbohydrate pathways, including decreases in branched-chain amino acids in saliva and modulation of nicotinamide- and amino acid-related metabolites in feces. Microbiome α-diversity remained stable, whereas β-diversity shifted significantly across oral and fecal sites, with enrichment of Lacticaseibacillus and other context-dependent commensals. Multi-omics integration identified three latent factors linking salivary cytokines, microbial taxa, metabolites, and systemic lipid profiles, suggesting coordinated mucosal-metabolic-immune remodeling across the oral-gut axis.

Olive pomace (OP), the solid residue left from olive oil production, is rich in bio-functional compounds, particularly phenolics with well-documented health-promoting properties. However, OP remains largely underutilised, leading to the waste of a valuable food-processing by-product. Its exploitation is further hindered by the inherent chemical instability of phenolic compounds. To address these limitations, we developed a novel nano-encapsulated hydroalcoholic OP extract embedded within a chitosan matrix cross-linked with tripolyphosphate (OPFE-lyo-NPs), a formulation designed to protect bioactive compounds and enhance their targeted delivery to the gut. Using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), we investigated the impact of OPFE-lyo-NPs and a placebo (lyo-NPs) on gut bacterial communities and their metabolic activity before (T0), after 3 and 10 days of treatment (T3, T10), and 7 days washout (W7). Both formulations increased total short-chain fatty acid (SCFA) production, with OPFE-lyo-NPs inducing higher butyrate levels than lyo-NPs in both proximal (21.68 vs 13.32 mM) and distal colon (24.50 vs 18.81 mM) at T10. Phenolics-loaded nanoparticles promoted a potentially beneficial modulation of the gut microbiota, defined by an increased relative abundance of health-associated taxa such as Lactobacillus and Faecalibacterium, and a shift toward a more butyrogenic profile. While lyo-NPs also enriched several health-associated taxa, OPFE-lyo-NPs uniquely promoted polyphenol-responsive genera with known health benefits and suppressed potentially detrimental genera, including Blautia and Microbacterium. These results suggest that the chitosan-based nanoparticles effectively deliver OP phenolics to the colon, where they exert targeted modulatory effects on the gut ecosystem, supporting potential benefits for host health.

Background: The accurate enumeration and identification of probiotic strains are essential for product quality. The plate count (PC) gold standard enumerates viable, culturable cells but does not by itself resolve individual strains within multi-strain consortia, and molecular methods (qPCR, ddPCR) are costly and face recognised challenges in quantifying relative strain abundance. Fourier transform infrared (FTIR) spectroscopy is a promising phenotypic alternative. Methods: We developed an FTIR-based artificial neural network classifier to identify and quantify a four-strain probiotic blend comprising Lactobacillus acidophilus LA02, Lacticaseibacillus rhamnosus LR04, Limosilactobacillus fermentum LF08, and Bifidobacterium animalis subsp. lactis BS01 compared against selective plate counting and species-specific PCR. Results: The classifier correctly identified all 36 test colonies (100%; 95% Clopper–Pearson CI: 90.3–100%); descriptive cluster analysis indicated spectral distinctiveness (silhouette = 0.908; Davies–Bouldin = 0.127; cophenetic correlation = 0.955). Enumeration agreement with selective plate counting was assessed descriptively (Pearson r = 0.78, 95% CI [−0.72, 1.00], n = 4 strain means; mean difference −0.028 log10 CFU/mL; all strain-level differences < 0.1 log10 CFU/mL). PCR confirmed all FTIR classifications. Conclusions: This proof-of-concept study demonstrates the feasibility of coupling cultivation with spectroscopic identification in a hybrid PC + FTIR workflow for multi-strain probiotic quality control. Because the findings derive from a single blend preparation analysed in technical replicates, they characterise this dataset and require confirmation on independently prepared batches before the approach can be regarded as a validated method.

BACKGROUND/OBJECTIVES:Exercise training has been associated with metabolic improvements in bariatric patients beyond weight and fat loss, potentially involving modulation of the gut microbiota. We investigated whether exercise-related microbial changes are associated with metabolic adaptations in women undergoing Roux-en-Y gastric bypass (RYGB) by combining a randomized controlled exercise intervention trial in women post-surgery with a human-to-mouse fecal microbiota transplantation (FMT) experiment. SUBJECTS/METHODS:Thirty-two women were randomized to RYGB (n = 16) or RYGB plus a 6-month exercise training program initiated three months post-surgery (RYGB + ET; n = 16), while a lean control group (LEAN; n = 16) was evaluated at baseline. Blood and fecal samples were collected before surgery, and at 3 (POST3) and 9 (POST9) months following surgery for biochemical, inflammatory, and microbiota analyses. RESULTS:Both surgical groups showed comparable improvements in body composition and inflammation; however, RYGB + ET was associated with greater improvements in HDL, triglycerides, fasting glucose, and fasting insulin. Exercise was also associated with increased gut microbiota α-diversity and shifts in microbial composition, including enrichment of genera previously linked to short-chain fatty acid (SCFA) metabolism and host metabolic health. To explore the potential contribution of these microbial communities, fecal microbiota collected at POST9 were transplanted into 36 high-fat diet-fed female mice, generating recipient groups rRYGB, rRYGB+ET, and rLEAN. Mice receiving RYGB + ET microbiota displayed similar inflammatory status and glucose tolerance, but lower fasting insulin and HOMA-IR, along with partial preservation of intestinal morphology, compared with mice receiving RYGB microbiota. CONCLUSIONS:These findings suggest that exercise following bariatric surgery is associated with distinct gut microbial configurations and metabolic improvements, and that exercise-conditioned microbiota may contribute to aspects of host metabolic regulation after surgery.

Cystic fibrosis (CF) is characterized by chronic airway inflammation, yet clinical observations have revealed more favorable COVID-19 outcomes than originally predicted. Several studies demonstrated a significant decrease of SARS-CoV-2 replication in CF-mutated bronchial cells suggesting that CFTR dysfunction may interfere with viral replication, though the underlying mechanisms remain unclear. To elucidate these mechanisms we performed transcriptomic profiling of SARS-CoV-2-infected bronchial epithelial cells with wild-type (WT) or mutated CFTR, using both immortalized and primary airway models. RNA-seq was performed on WT and CF cellular models before and at 24, 48, and 72-hours post-infection. The differentially expressed genes (DEGs) were defined as genes with a log2 fold change>1 between groups (p<0.05) and significant DEGs were subjected to Gene Ontology and KEGG enrichment analysis (p<0.05). Our results reveal that CFTR deficiency impairs SARS-CoV-2 replication not by altering receptor availability (e.g., ACE2, TMPRSS2), but through widespread intracellular remodeling defects. CF cells failed to activate key antiviral and inflammatory responses, including interferon signaling, AP-1 transcriptional complex, and IL-6-mediated pathways. Furthermore, they exhibited defective unfolded protein response, altered calcium signaling, and disrupted ER-mitochondrial communication. Crucially, pH dysregulation and impaired expression of V-ATPase subunits and autophagy-related genes hindered vesicle acidification, double-membrane vesicle formation, and viral assembly. These intrinsic alterations also blunted virus-induced senescence programs. Collectively, our findings indicate that CF cellular environment is intrinsically unfavorable to SARS-CoV-2, limiting its replication and propagation. This study provides a mechanistic basis for the reduced viral burden observed in CF and highlights intracellular pH regulation and organelle homeostasis as potential therapeutic targets against SARS-CoV-2 infection.

Metschnikowia pulcherrima includes strains of applied agro-food interest, particularly due to the antimicrobial activity against plant pathogens, contribution to the aroma of fermented beverages, and preliminary evidence related to probiotic activity. This biotechnological relevance sheds new light of interest on the biology of this yeast. To better understand and expand its biotechnological potential and applicability, the genomes of M. pulcherrima NRRL Y-7111 T, NRRL Y-48695, CBS 10357, and NRRL Y-48712 were sequenced, and de-novo assembled. Between 10,671 and 14,548 genes were predicted and the cooperative genomic analyses were integrated with experimental assessments relating to traits relevant for biotechnological application and safety. In silico and in vitro safety assessment revealed intermediate sensitivity for itraconazole; furthermore, variants of the genes related to pulcherrimin production and transport were found in all the genomes. Moreover, an arsenal of carbohydrate-active enzymes (CAZymes) was unravelled, and their predicted localization was investigated. This study expands the body of knowledge on M. pulcherrima, including traits relevant for defining its safety as a bioresource, which is a pivotal aspect for its possible inclusion in the European Food Safety Authority (EFSA) Qualified Presumption of Safety (QPS) list and its application in REgulated food/feed PROducts (REPRO) both in the European Union aligned European countries. • A pipeline for genomic characterisation and safety assessment of unconventional yeasts, using M. pulcherrima as a model species was developed. • M. pulcherrima strains can be considered safe and safety data can be used to develop a body of knowledge to include M. pulcherrima in EFSA QPS list. • Analysis of the predicted localization of CAZymes allowed the detection of compounds as potential biological control agents.

Genome-scale metabolic models (GSMMs) can mechanistically explain phenotypic differences among closely related bacterial strains. However, high-throughput multi-strain reconstructions of GSMMs are still challenging: reference-based methods inherit curated information while missing new contents; alternatively (universe-based), reference-free methods could cover strain-specific reactions, but they disregard curated information. Ideally, references should be curated pan-GSMMs for species (or genus), but their reconstruction is extremely demanding, making them still rare in the literature. Here, Gempipe is presented, a computational tool streamlining the multi-strain reconstruction and analysis of GSMMs, going through the production of a pan-GSMM. Its reconstruction method is hybrid; as an optional reference, GSMM is automatically expanded with extra reactions taken from a reference-free reconstruction. Gempipe also downloads, filters, and annotates genomes; performs in-depth gene recovery; annotates models' contents; and predicts strain-specific capabilities. The companion programming interface includes functions ranging from the (pan-)GSMMs' curation to the multi-strain analysis. Gempipe was validated using multi-strain data sets, showing improved accuracy when compared with state-of-the-art tools. Moreover, metabolic diversities within Limosilactobacillus reuteri were explored, grouping strains into metabolically coherent clusters and systematically predicting health-related metabolites' biosynthesis.IMPORTANCEAvailable genome-scale metabolic model (GSMM) reconstruction tools present major limitations in the context of multi-strain modeling. Gempipe surpasses these limitations by implementing a novel, hybrid reconstruction strategy. Not only does it produce more accurate strain-specific GSMMs, but it also produces pan-GSMMs when the only available reference is a manually curated model for a single strain, which is currently the most common case. With the vast availability of genome sequences, the high-throughput, multi-strain GSMM reconstruction and analysis approach provided by Gempipe will facilitate large-scale studies of exploration and bioprospecting of strain-level bacterial metabolic diversity, moving a step forward in strains' screening and rational selection.

BACKGROUND AND AIMS:We performed a randomized, double-blind, placebo-controlled, trial to investigate the changes in microbiome composition induced by Butyrate-Lsc-Microincapsulated (BLM) supplementation in IBD patients and its impact on disease activity. METHODS:140 IBD patients (n=60 Crohn's disease, CD and n=80 Ulcerative Colitis, UC) were randomized to oral administration of BLM, plus conventional therapy. Stool samples were assessed by 16S sequencing and fecal calprotectin (fCal) analysis. For the microbiota analysis, the Firmicutes/Bacteroidota (F/B) ratio was used. Clinical disease activity was assessed by using the Harvey-Bradshaw-Index (HBI) for CD and partial-Mayo-Score for UC, Quality-of-life (QoL) by using Inflammatory-Bowel-Disease-Questionnaire-32 (IBDQ) and adherence-dietary-recommendation was evaluated before and after supplementation RESULTS: microbiota analysis revealed two principal enterotypes, defined by the F/B ratio, in both CD and UC patients. BLM exerted a more pronounced effect on Enterotype 1 (low F/B ratio), resulting in greater clinical and biochemical improvements and potentially identifying a target population. After supplementation, clinical disease activity (p=0.013) and fCal (p=0.047) improved significantly in CD, while fCal showed a marginal reduction in UC (p=0.09). QoL increased significantly in both CD (p<0.001) and UC (p=0.003). CONCLUSIONS:Supplementation with BLM, by modulating the gut microbiota, significantly improved disease outcomes and QoL in patients with IBD. CLINICALTRIAL: GOV REGISTRATION:NCT04879914.

Immune mechanisms contribute to the neuropathology of Alzheimer's disease (AD) but the role of adaptive immune cells is unclear. Here we show that the brain CD8+ T cell compartment is dysregulated in AD patients and in the 3xTg-AD mouse model, accumulating activated CD103- tissue-resident memory T cells that produce large amounts of granzyme K (GrK). These CD103-CD8+ T cells originate from the circulation and migrate into the brain using LFA-1 integrin. Ablation of brain CD103-CD8+ T cells in 3xTg-AD mice ameliorates cognitive decline and reduces neuropathology. GrK induces neuronal dysfunction and tau hyperphosphorylation in human and mouse cells via protease-activated receptor-1 (PAR-1), which is expressed at higher levels in the AD brain, revealing a key immune-mediated neurotoxic axis. We conclude that communication between CD8+ T cells and the nervous system is altered in AD, paving the way for therapies targeting T cell-dependent neurotoxic inflammation.

Considering that global warming is changing berry ripening timing and progression, uncovering the molecular mechanisms and identifying key regulators governing berry ripening could provide important tools in maintaining high quality grapes and wine. NAC (NAM/ATAF/CUC) transcription factors represent an interesting family due to their key role in the developmental processes control, such as fruit-ripening-associated genes expression, and in the regulation of multiple stress responses. Between the 74 NAC family members, we selected 12 of them as putative regulators of berry ripening: NAC01, NAC03, NAC05, NAC11, NAC13, NAC17, NAC18, NAC26, NAC33, NAC37, NAC60 and NAC61. Genome wide analyses and functional assays permitted to reconstruct a hierarchical intra-family regulatory network in which most of the selected NACs resulted as transcriptional activators of other NACs. Moreover, to investigate the common regulative role of the selected NACs on the grapevine transcriptome, all the annotated V. vinifera genes were listed and the most represented genes between all the DAP-seq results were identified. Interestingly, at the top of the ranking we found many genes related to maturation and senescence such as an indole-3-acetic acid-amido synthetase, which could be involved in the establishment and maintenance of low IAA concentrations in ripening berries, a laccase, encoding for a phenylpropanoid pathway-related enzyme, the senescence-inducible chloroplast stay-green protein 1, triggering Chl degradation, and the UTP-glucose-1-phosphate uridylyltransferase, encoding for a carbohydrate-metabolism-related enzyme which is highly expressed in berries at veraison. All these results lay a foundation stone in understanding the genetic regulation of such a complex process as fruit ripening.

Abstract Background Butyrate has shown anti-inflammatory effects and plays an important role in maintaining gut homeostasis, providing symptomatic relief when orally supplemented in patients suffering from various colonic diseases. Methods In this randomised, double-blind, placebo-controlled study 140 IBD patients in remission or with mild disease (n=60 Crohn's disease, CD and n=80 Ulcerative Colitis, UC) were randomized to oral administration of BLM/placebo for 90 days in addition to conventional therapy. As primary aim: changes in microbiome composition induced by BLM treatment. Fecal microbiota from stool samples was assessed by 16S sequencing. Clinical disease activity assessed by using Harvey Bradshaw Index (HBI) for CD and partial-Mayo Score (pMS) for UC, quality of life assessed by using Inflammatory Bowel Disease Questionnaire 32 (IBDQ-32) and adherence-dietary-raccomandation( WCRF) were evaluated before and after treatment. Results Demographics, clinical and dietary characteristics were similar between the two populations. The microbiota sequencing highlighted two different enterotypes in CD population: the first one was characterized by a low F/B ratio (with a genus prevalence of Fusobacteriota, Alistipes, and Blautia). The second enterotype was characterized by a higher F/B ratio (with a genus prevalence of Escherichia-Shigella and Lachnoclostridium)(Fig1). BLM had a more pronounced effect on enterotype-1 where the taxa associated with intestinal comfort, SCFA production, increased after treatment, while the taxa associated with CRC and intestinal inflammation, decreased. After treatment was observed: a)improved quality of life (IBDQ-32: CD: treat. 170/184 vs Pb. 188/197 p<0.001 vs p=0.021; UC: treat.185/195 vs Pb.188/188 p=0.003 vs p=0.4), b)lower disease activity in CD patients (the HBI score, showed a significant improvement for those treated with BLM as compared to placebo (McNemar’s p=0.013 vs p>0.9).c) reduced Calprotectin levels (Table1.) Finally, a significant separation was observed in samples from patients who have undergone minor surgery (ileo-colon), compared to non-surgery patients. The variable "surgery" seems to be associated with the enterotype-1. Conclusion Sodium butyrate supplementation significantly improved clinical outcomes and quality of life in patients with CD. Our data showed that the differential response to BLM treatment is based on enterotype, suggesting that Enterotype-1 patients may experience greater clinical benefits and improvements in QoL compared to Enterotype-2 patients in both UC and CD populations.

The study of species groups in which the presence of interspecific hybridization or introgression phenomena is known or suspected involves analysing shared bi-parentally inherited molecular markers. Current methods are based on different categories of markers among which the classical microsatellites or the more recent genome wide approaches for the analyses of thousands of SNPs or hundreds of microhaplotypes through high throughput sequencing. Our approach utilizes intron-targeted amplicon sequencing to characterise multi-locus intron polymorphisms (MIPs) and assess genetic diversity. These highly variable intron regions, combined with inter-specific transferable loci, serve as powerful multiple-SNP markers potentially suitable for various applications, from species and hybrid identification to population comparisons, without prior species knowledge. We developed the first panel of MIPs highly transferable across fish genomes, effectively distinguishing between species, even those closely related, and populations with different structures. MIPs offer versatile, hypervariable nuclear markers and promise to be especially useful when multiple nuclear loci must be genotyped across different species, such as for the monitoring of interspecific hybridization. Moreover, the relatively long sequences obtained ease the development of single-locus PCR-based diagnostic markers. This method, here demonstrated in teleost fishes, can be readily applied to other taxa, unlocking a new source of genetic variation.

Immune mechanisms contribute to the neuropathology of Alzheimer’s disease (AD) but the role of adaptive immune cells is unclear. Here we show that the brain CD8+ T cell compartment is dysregulated in AD patients and in a mouse model with the hallmarks of AD, accumulating activated CD103– tissue-resident memory T cells that produce large amounts of granzyme K (GrK). These CD103–CD8+ T cells originate from the circulation and migrate into the brain using LFA-1 integrin. Ablation of brain CD103–CD8+ T cells in AD mice ameliorated cognitive decline and reduced neuropathology. GrK induced neuronal dysfunction and tau hyperphosphorylation in human and mouse cells via protease-activated receptor-1 (PAR-1), which is expressed at higher levels in the AD brain, revealing a novel immune-mediated neurotoxic axis. We conclude that communication between CD8+ T cells and the nervous system is altered in AD, paving the way for therapies targeting T cell-dependent neurotoxic inflammation.