Lactobacillaceae are Gram-positive, and lactic acid-positive (LAB) bacteria frequently serve as probiotics. We first systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered an altered carrying capacity with strain specificity during planktonic growth, calling for adding a buffering system during the formulation of probiotics. In addition, heterogeneous response to fermentable sugars was manifested in microbial aggregation (measured by image-stream flow cytometry), colony development, and attachment to mucin. Of all probiotic strains, L. rhamnosus GG (LGG), a prevalent probiotic specie, manifested an enhanced survival of self-imposed acid stress, consistent with enhanced cell wall modulation observed by transmitting electron microscopy and proteomic analysis. A comprehensive proteomic and metabolomic study revealed that the formation of biofilms and aggregation capacity is a specific response to glucose and independent of self-imposed acid stress. In contrast, the increased competitiveness and aggression of LGG and other LAB strains towards enteric pathogens were a synergistic outcome of a change in organic acid production, glucose-dependent bacteriocin production, and fermentation-specific volatile production. Our improved resolution into the cellular circuits (metabolome, proteome, and volatilome) of probiotic strains and their interactions can lead to developing novel therapeutic approaches to combat GI tract infections.

In order to characterize the glial response during retinal remodeling, a laser model was used to compare the degenerative changes in the mouse with the regenerative character of the response in zebrafish. These data were validated with human retinal samples. C57BL/6J mice and AB zebrafish underwent laser photocoagulation with a 532 nm diode laser in the outer nuclear layer (mouse: 300 μm; ZF: 50 μm). At different time points post injury induction, the kinetics of retinal changes were assessed by H&E. The gliotic response was observed with confocal microscopy for Müller cell markers (GS, CRALBP) in combination with gliotic markers (vimentin, nestin, S100β, GFAP) in the late stage of wound repair. In parallel, human donor retina section with hard drusen formation were used to investigate gliotic response. Focal laser treatment elevated the expression of glia markers in the area of the damage. This was associated with increased expression of S100β, GFAP, vimentin and nestin in mouse and human. In zebrafish, we could detect S100β at the first time point but no GFAP nor nestin positivity was found. However, in zebrafish no double positive GFAP/GS was found on days 10 and 17 as were no S100β/GS double positive cells on day 12. In all models, macroglia have the ability to undergo the same gliotic response, but zebrafish do not show expression of all detected gliotic markers. The data demonstrate upregulation of S100β in mice eyes that are comparable to human retinal tissue with early onset of retinal degeneration (drusen). No distinct staining of S100β could be found in zebrafish retinas. An interplay between astrocytes and Müller cells might also be involved in this process. The results offer new insight into the gliotic mechanism in retinal degeneration / regeneration.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate target mRNA expression, and altered expression of miRNAs is associated with liver pathological conditions. Recent studies in animal models have shown neutrophil/myeloid-specific microRNA-223 (miR-223) as a key regulator in the development of various liver diseases including fibrosis, where hepatic stellate cells (HSCs) are the key player in pathogenesis. However, the precise roles of miR-223 in human HSCs and its therapeutic potential to control fibrosis remain largely unexplored. Using primary human HSCs, we demonstrated that miR-223 suppressed the fibrogenic program and cellular proliferation while promoting features of quiescent HSCs including lipid re-accumulation and retinol storage. Furthermore, induction of miR-223 in HSCs decreased cellular motility and contraction. Mechanistically, miR-223 negatively regulated expression of smooth muscle α-actin (α-SMA) and thus reduced cytoskeletal activity, which is known to promote amplification of fibrogenic signals. Restoration of α-SMA in miR-223-overexpressing HSCs alleviated the antifibrotic effects of miR-223. Finally, to explore the therapeutic potential of miR-233 in liver fibrosis, we generated co-cultured organoids of HSCs with Huh7 hepatoma cells and challenged them with acetaminophen (APAP) or palmitic acid (PA) to induce hepatotoxicity. We showed that ectopic expression of miR-223 in HSCs attenuated fibrogenesis in the two human organoid models of liver injury, suggesting its potential application in antifibrotic therapy.

Prostate cancer (PC) is one of the most widespread malignancies among males worldwide. The androgen receptor (AR) drives its development and progression and still represents the main target of PC therapy. Second-generation antiandrogens have, indeed, improved the patient’s management. Nonetheless, hormone resistance and tumour progression frequently develop. While the majority of drugs currently used in PC target the AR functions in epithelial PC cells, the role of the receptor in PC-associated fibroblasts (CAFs) and PC progression remains still pending and few therapeutics affecting the stromal AR functions have been so far developed.

By combining several approaches, we have shown that AR associates with Filamin A (FLNa), thus promoting migration and invasion of androgen-challenged CAFs from PC patient’s specimens at different Gleason’s scores. By using 2 and 3D cultures, we have demonstrated that CAFs move towards epithelial PC cells and promote the increase in PC organoid size. The stapled peptide Rh-2025u disrupts the androgen-triggered AR/FLNa complex assembly and impairs these responses in monolayer cells as well as 3D models. Furthermore, it reduces the overall tumour area in androgen-treated 3D co-culture. Mechanistically, our findings posit that AR/FLNa complex recruits β1 integrin and the membrane type-matrix metalloproteinase 1 upon androgen challenging of CAFs. Activation of a protease cascade leading to extracellular matrix (ECM) remodelling then follows. Rh-2025u peptide interferes in the assembly of this multimolecular complex and impairs ECM remodelling. As such, CAFs cannot longer navigate through ECM.

In summary, we propose the Rh-2025u peptide as a new drug, which alone or in combination with other emerging therapies may allow a more rational treatment of PC. Pharmacological blockade of AR functions in CAFs is indeed neglected and the approach we propose would improve the treatment’s outcome in PC patients.

The pathology of late-onset Alzheimer disease (LOAD) is still poorly understood, but it is multifactorial and closely related to changes with age. We developed a cellular platform for LOAD collecting skin fibroblasts or blood cells from LOAD patients and non-demented control individuals that are used in the induced pluripotent stem cell (iPSC) paradigm to produce brain cells for determining LOAD pathogenic processes in context of age, disease, genetic background, cell development, and cell type. This model has provided evidence for an innate inefficient cellular energy management in LOAD that is associated with alterations of the cellular transcriptomes and lipid compositions, and interconnected cause-and-effect linkages, such as impaired insulin/IGF-1 signaling, bioenergetic substrate deficiencies, diminished glucose metabolism, disruption of the autophagic flux, and others. In addition, testing of compounds revealed some restoration of the altered bioenergetic and metabolic processes in LOAD cells. Altogether, our studies have identified an inherent LOAD-associated cellular metabolic phenotype as a potential risk factor to develop neurodegenerative disease with age. We propose that our cellular model allows for patient-oriented examination of numerous mechanisms and interactions in LOAD pathogenesis, as a basis for a personalized medicine approach to predict altered aging and risk for development of dementia, and to test or implement (customized) therapeutic or disease-preventive intervention strategies.