Adrian Schwarzenberg

The probiotic potential of Bacillus velezensis is increasingly recognized in companion animal nutrition; however, the strain-specific mechanisms underlying host responses remain poorly understood. Here, we investigated metabolite-driven effects of three B. velezensis strains (LSSA01, 15AP4, and 2084) using cell-free supernatants (CFS) to decouple host responses from bacterial colonization. All findings are derived from in vitro models using cell-free supernatants (CFS), enabling assessment of metabolite-mediated effects under controlled conditions. Canine proximal epithelial (MCA-B1) cells and macrophage-like (DH82) cells were used to assess epithelial barrier regulation, cellular stress and apoptosis pathways, immune signaling, and innate effector function. LSSA01 CFS significantly enhanced transcription of epithelial junction and resilience markers, including claudin-1 (6.2-fold, p < 0.0001), ZO-1 (1.4-fold, p < 0.01), and BCL-2 (1.4-fold, p < 0.01), accompanied by an increased BCL-2/BAX ratio (p < 0.05), suggesting strengthened barrier function and enhanced expression of markers associated with cellular resilience. In contrast, 15AP4 and 2084 primarily modulated protein-level junctional organization and stress-response pathways and significantly enhanced macrophage phagocytic activity under inflammatory challenge (170–286% of control, p < 0.01–0.0001). Untargeted metabolomics revealed distinct strain-specific signatures, including marked enrichment of nucleotides such as adenosine monophosphate (31–39-fold vs. control, p < 0.0001), alongside vitamins and bioactive peptides. Together, these findings indicate that B. velezensis strains exhibit complementary, non-redundant metabolite-mediated activities on canine epithelial and immune cells, highlighting the importance of strain-resolved evaluation for mechanistic understanding and supporting future investigation, while acknowledging that in vivo validation will be important to confirm relevance for canine health applications.

Good canine gastrointestinal health depends on the suppression of enteric pathogens and maintenance of epithelial barrier integrity. Limosilactobacillus reuteri ATCC PTA 6127 (Lr6127) is a dog-derived probiotic, but evidence supporting its functional properties remains limited. Here, we evaluated the antimicrobial and epithelial-supportive effects of Lr6127 using a canine epithelial cell model. Cell-free supernatant (CFS) from Lr6127 significantly inhibited the growth of canine-relevant pathogens, including Enterotoxigenic Escherichia coli (52.0 ± 1.3%), Clostridium perfringens (54.0 ± 2.7%), and Salmonella enterica subsp. enterica serovar Typhimurium (48.6 ± 1.2%), compared with the medium control (p < 0.0001). Pathogen inhibition increased in a dose-dependent manner with increasing CFS concentration. Untargeted metabolomic analysis revealed enrichment of multiple antimicrobial-associated metabolites, indicating a multi-component profile consistent with pathogen suppression, with genomic analysis supporting the aromatic amino acid-derived metabolite findings. In addition, viable Lr6127 significantly reduced the epithelial adhesion of all the tested pathogens (p < 0.01). Beyond direct antimicrobial effects, Lr6127 CFS promoted epithelial wound healing at later time points, accompanied by the coordinated modulation of proteins associated with cytoskeletal remodeling and barrier repair. Collectively, these findings support the idea that Lr6127 is associated with antimicrobial and epithelial-related effects, highlighting its potential to contribute to epithelial function under controlled in vitro conditions.

Coordinated responses of intestinal epithelial and immune cells are essential for maintaining barrier integrity and immune homeostasis in dogs, yet our mechanistic understanding of probiotic-derived metabolites remains limited due to reliance on non-canine experimental models, highlighting the need for studies in canine-derived systems. Here, we investigated the effects of metabolites derived from Limosilactobacillus reuteri strain ATCC PTA6127 (Lr6127), delivered as a cell-free supernatant (CFS), on canine epithelial MCA-B1 cells and macrophage-like DH82 cells subjected to lipopolysaccharide (LPS)-induced inflammatory stress. Lr6127 CFS significantly reduced epithelial permeability, decreasing FITC–dextran leakage to 94.9 ± 1.9% (normalized relative to LPS-treated control, which was set as 100%) (p < 0.001), despite no detectable transcriptional changes in tight junction, adherens junction, or mucin genes. Barrier effects were instead associated with changes in markers of cellular stress responses, with heme oxygenase expression decreasing from 0.9 ± 0.1 to 0.7 ± 0.1 (p < 0.05). In DH82 immune cells, Lr6127-derived metabolites altered LPS-induced stress- and inflammation-related gene expression patterns; enhanced anti-apoptotic responses, as reflected by the increased BCL2 expression (1.4 ± 0.1 vs. 1.0 ± 0.0; p < 0.01) and elevated BCL2/BAX ratios (p < 0.01); and reduced expression of pro-inflammatory mediators including IL-6 and CCL2 (p < 0.05–0.001). Proteomic analysis corroborated that Lr6127-derived metabolites reduced the abundance of inflammatory and STAT-associated signaling proteins under LPS challenge, while indicating context-dependent changes in immune-related protein profiles under resting condition. Collectively, these results suggest that Lr6127-derived metabolites improved epithelial barrier function, which was accompanied by coordinated changes in cellular stress-related and inflammatory pathways, highlighting their potential to positively influence host responses.

Effective canine gastrointestinal health depends on suppression of enteric pathogens and maintenance of epithelial barrier integrity. Limosilactobacillus reuteri ATCC PTA 6127 (Lr6127) is a dog‑derived probiotic, but evidence supporting its protective capacity remains limited. Here, we evaluated the antimicrobial and epithelial‑supportive effects of Lr6127 using a canine epithelial cell model. Cell‑free supernatant (CFS) from Lr6127 significantly inhibited the growth of canine‑relevant pathogens, including Escherichia coli (52.0 ± 1.3%), Clostridium perfringens (54.0 ± 2.7%), and Salmonella typhimurium (48.6 ± 1.2%), compared with the medium control (P < 0.0001). Pathogen inhibition increased in a dose‑dependent manner with increasing CFS concentrations. Untargeted metabolomic analysis revealed enrichment of multiple antimicrobial‑associated metabolites, indicating multi‑component pathogen suppression, with genomic analysis supporting the aromatic amino acid–derived metabolite findings. In addition, viable Lr6127 significantly reduced epithelial adhesion of all tested pathogens (P < 0.01). Beyond direct antimicrobial effects, Lr6127 CFS promoted epithelial wound healing at later time points, accompanied by coordinated modulation of proteins associated with cytoskeletal remodeling and barrier repair. Collectively, these findings suggest that Lr6127 is associated with pathogen suppression, reduced adhesion, and epithelial wound repair mechanisms.

Coordinated responses of intestinal epithelial and immune cells are essential for maintaining barrier integrity and immune homeostasis in dogs, yet mechanistic understanding of probiotic‑derived metabolites remains limited due to reliance on non‑canine experimental models. Here, we investigated metabolites derived from Limosilactobacillus reuteri strain ATCC PTA6127 (Lr6127), delivered as cell-free supernatants (CFS), using canine epithelial MCA-B1 cells and macrophage-like DH82 cells subjected to lipopolysaccharide (LPS)–induced inflammatory stress. Lr6127 CFS significantly reduced epithelial permeability, decreasing FITC‑dextran leakage to 94.9 ± 1.9% relative to LPS-treated controls normalized to 100% (P < 0.001), despite no detectable transcriptional changes in tight junction, adherent junction, or mucin genes. Barrier effects were instead associated with changes in markers of cellular stress re-sponses, with heme oxygenase expression reduced from 0.9 ± 0.1 to 0.7 ± 0.1 (P < 0.05). In DH82 immune cells, Lr6127-derived metabolites suppressed LPS-induced stress and inflammatory signaling, enhanced anti-apoptotic responses as reflected by increased BCL2 expression (1.4 ± 0.1 vs. 1.0 ± 0.0; P < 0.01) and elevated BCL2/BAX ratios (P < 0.01), and reduced pro-inflammatory mediators including IL-6 and CCL2 (P < 0.05–0.001). Proteomic analysis corroborated reduced abundance of inflammatory and STAT-associated signaling proteins under LPS challenge while promoting immune readiness under resting conditions. Collectively, these results suggest that Lr6127‑derived metabolites may support epithelial barrier integrity and immune re-balancing potentially through modulation of cellular stress and inflammatory path-ways.

Bacillus velezensis-based probiotics are increasingly recognized for their potential to enhance intestinal health in companion animals, yet their mechanisms of action in canine epithelial systems remain incompletely defined. This study aimed to evaluate whether a live Bacillus velezensis probiotic consortia (BC) modulates epithelial barrier integrity, immune signaling, apoptosis-renewal pathways, and metabolic activity in canine-relevant intestinal and macrophage cell models. MCA-B1 proximal gastrointestinal epithelial cells and DH82 macrophage-like cells were exposed to BC cultures, followed by quantification of tight-junction expression, permeability (FITC-Dextran), cytokine responses, phagocytic activity, apoptosis-related markers, and metabolomic profiles. BC treatment significantly strengthened the epithelial barrier, inducing a marked upregulation of Claudin 1 (CLDN1) (11.3 fold), CLDN4 (2.4 fold), Occludin (OCLN, 1.7 fold), and increasing key proteins including ZO-2 and cingulin while reducing LPS-induced FITC-Dextran permeability to 94.5%. BC concurrently modulated innate immune signaling, increasing MyD88 (33.2%), IL-8 (14.6 fold), IL-18 (2.6 fold), and IFNB1 protein levels, while enhancing anti-inflammatory regulation, including a robust rise in DH82-derived IL-10. Apoptosis-renewal markers shifted toward physiological turnover, with increased BCL2 (1.9 fold) and reduced BAK1. Metabolomic profiling of BC activity revealed elevated AMP, abundant Peptide Transporter 1 (PEPT1)-transportable peptides, increased γ-glutamyl metabolites, and lower Glutathione disulfide (GSSG), consistent with AMPK-linked tight-junction assembly and glutathione-supported redox buffering. Together, these data indicate that Bacillus velezensis-derived metabolites positively influence barrier-related, immunological, and metabolic responses in a canine proximal intestinal epithelial system and modulate functional responses in macrophage-like cells. These in vitro findings contribute to the mechanistic understanding of host cellular responses to Bacillus-associated metabolites.

Développement de méthodes basées sur la spectrométrie de masse pour la détection de composés organophosphorés et d'explosifs: Au cours des années, l'utilisation de composés nocifs a augmenté de façon exponentielle, et c'est la raison pour laquelle développer des méthodes pour l'identification de composés dangereux, tels que les composés organophosphorés (OP) et les explosifs organiques nitroaromatiques, est indispensable. L'analyse des composés organophosphorés et des explosifs est un enjeu important dans la sécurité intérieure, la médecine légale et les sciences de l'environnement. Il est crucial de développer des méthodes analytiques fiables, sensibles et efficaces pour identifier précisément les composés organophosphorés et les explosifs. Dans ce contexte, le but de ce travail de recherche était de développer des méthodes basées sur la spectrométrie de masse pour l'identification précise et sans ambiguïté de ces composés. Un arbre d'identification a été développé pour l'élucidation structurale de composés organophosphorés. Cette approche a été évaluée à l'aide d'une matrice biologique. D'autre part, les explosifs nitroaromatiques ont été étudiés et plusieurs nouveaux résultats ont été trouvés et reportés. L'application de l'analyse directe en temps réel (DART) couplée à la spectrométrie de masse Orbitrap (DART-FTMS) est décrite ici pour le screening rapide et la caractérisation des échantillons de frottis sur coton obtenus à partir d'armes militaires.

Despite increasing interest in probiotics as antibiotic alternatives in swine production, few studies have directly compared the functional efficacy of different commercial probiotic formulations under controlled conditions. We conducted an in vitro study using porcine intestinal epithelial (IPEC-J2) and macrophage-like (3D4/21) cell models to compare the efficacy of three commercial probiotic consortia (C1: three strains of Bacillus velezensis; C2: B. licheniformis + B. subtilis; C3: Clostridium butyricum). Treatments were evaluated for their ability to inhibit pathogenic Escherichia coli, Clostridium perfringens, and Salmonella spp., enhance epithelial barrier integrity, and modulate immune responses. Experimental endpoints included pathogen inhibition assays, adhesion to IPEC-J2 cells, transepithelial electrical resistance (TEER), tight junction protein expression, and cytokine profiling via RT-qPCR and proteomics. Data were analyzed using the Kruskal–Wallis test with false discovery rate (FDR) control at 5%. C1 cell-free supernatant (CFS) strongly inhibited pathogen growth (84.8 ± 5.3% inhibition of ETEC F4⁺F18⁻ vs. medium control; p < 0.05), whereas C2 had no effect, and C3 inhibited only one isolate. The coculture of IPEC-J2 cells with C1 CFS increased the expression of TJ proteins ZO-1, MUC13, and MUC20 (+12.9–46.6% vs. control; p < 0.001) and anti-inflammatory TGF-β; reduced pro-inflammatory IL-6 in LPS-stimulated 3D4/21 cells. In comparison, C2 and C3 showed minimal impact on epithelial barrier integrity and immune modulation, as indicated by negligible changes in TEER values, tight junction protein expression (ZO-1, MUC13, MUC20), and cytokine profiles relative to the control. In conclusion, C1 demonstrated greater in vitro efficacy than C2 (B. licheniformis + B. subtilis) and C3 (Clostridium butyricum), including pathogen inhibition assays, epithelial adhesion, TEER measurements, and cytokine modulation, suggesting its potential as a leading candidate for functional probiotic applications.

Editorial: Metabolomics: a tool to understand plant protection against biotic and abiotic stresses

In temperate trees, optimal timing and quality of flowering directly depend on adequate winter dormancy progression, regulated by a combination of chilling and warm temperatures. Physiological, genetic and functional genomic studies have shown that hormones play a key role in bud dormancy establishment, maintenance and release. We combined physiological and transcriptional analyses, quantification of abscisic acid (ABA) and gibberellins (GAs), and modeling to further investigate how these signaling pathways are associated with dormancy progression in the flower buds of two sweet cherry cultivars. Our results demonstrated that GA-associated pathways have distinct functions and may be differentially related with dormancy. In addition, ABA levels rise at the onset of dormancy, associated with enhanced expression of ABA biosynthesis PavNCED genes, and decreased prior to dormancy release. Following the observations that ABA levels are correlated with dormancy depth, we identified PavUG71B6, a sweet cherry UDP-GLYCOSYLTRANSFERASE gene that up-regulates active catabolism of ABA to ABA glucosyl ester (ABA-GE) and may be associated with low ABA content in the early cultivar. Subsequently, we modeled ABA content and dormancy behavior in three cultivars based on the expression of a small set of genes regulating ABA levels. These results strongly suggest the central role of ABA pathway in the control of dormancy progression and open up new perspectives for the development of molecular-based phenological modeling.