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.

Mitochondrial pathologies are clinically complex and show highly variable phenotypes among all inherited disorders, mainly due to their heteroplasmic nature. Mutations in mitochondrial DNA (mtDNA) and nuclear genome (gDNA) or both have been reported in mitochondrial diseases, suggesting common pathophysiological pathways. Nuclear gene defects identified in mitochondrial alterations are primarily responsible for mtDNA replication, transcription and translation, oxidative phosphorylation (OXPHOS), biogenesis of mtDNA, nucleoside transport, salvage or synthesis, maintenance of balanced mitochondrial deoxyribonucleoside triphosphates (dNTP) pool. The m.3243 A>G mtDNA mutation in the MT-TL1 gene coding for the tRNALeu (UUR) is one of the most common mitochondrial disease-causing mutations, with a carrier rate as high as 1:400. Recent studies suggest that patients with m.3243 A>G mutation exhibiting a huge clinical heterogeneity underpinning the necessity to investigate nuclear genome for a better understanding of complex mitochondrial disorders, such as mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), maternally inherited diabetes and deafness (MIDD) and myopathy. MIDD is a multi-system disorder characterized by diabetes, hearing impairment and maculopathy but can present several other clinical manifestations. This study aimed to sequence the whole mitochondrial genome and the whole exome of a clinically characterized MIDD family, negative to m.3243 A>G variant, and identify mutations in both nuclear and mitochondrial genome and their biological contribution to its heterogeneous phenotype. Obtained results permitted us to hypothesize that the mitochondrial abnormalities might be due to epigenetic deregulation of mitochondrial and nuclear-encoded genes that code for mitochondrial structure and functions. Thus, epigenetic modifications in the context of mitochondrial dysfunctions represent an emerging area of research, possibly useful to innovative mtDNA-related disease differential analyses.

Drug resistance still represents the main reason for therapy failure in melanoma patients. In this regard, cancer stem cells (CSCs) are thought to be responsible for treatment evasion and tumor relapse. Here, we used A375 and WM115 human melanoma cells to dissect the role of mitochondria in conferring the drug-resistant CSC phenotype. More specifically, we conducted flow cytometry analysis to fractionate this cell line into ABCG2+ (stem-like) and ABCG2- (non-stem) subpopulations: interestingly, ABCG2+ cells exhibited higher mitochondrial mass. Likewise, A375 and WM115 melanospheres, known to be enriched in ABCG2+ CSCs, showed enhanced mitochondrial content. In particular, an increase in mitochondrial biogenesis (PGC1-α protein levels), OXPHOS (complex I-V protein levels) and fusion (OPA1 and MFN2 protein levels) was found in spheroids with respect to the parental cell line, leading to a metabolic switch towards an oxidative phenotype characterized by higher oxygen consumption, ATP synthesis and ROS production. Notably, PGC1-α silencing led to the suppression of sphere formation and ABCG2 enrichment. Similarly, SR-18292 and XCT790, two PGC1-α inhibitors, were able to block melanosphere propagation and ABCG2+ cell proliferation. In summary, increased mitochondrial content is associated with a vemurafenib-resistant stem-like phenotype in melanoma, and therapeutically targeting the mitochondria-enriched CSC subpopulation might overcome drug resistance.

Cerebral cavernous malformation (CCM, OMIM #116860) is a vascular disorder of central nervous system capillaries. Affected vessels appear tangled and enlarged due to cell junction impairment, resulting in an increased blood-brain barrier (BBB) permeability, further worsened by deficiency of pericytes. The familial form of the disease arises following germline mutations at the three loci KRIT1/CCM1 (HGNC ID: 1573; 7q11.2-21), MGC4607/CCM2 (HGNC ID: 21708; 7p13) and PDCD10/CCM3 (HGNC ID: 8761; 3q26.1). However, a small percentage of patients affected by the inherited form of the disease harbors no mutations, suggesting the existence of a fourth CCM locus. In this context, by whole exome sequencing we identified the novel missense mutation c.2973C>T (p.Phe991Leu) in the PIEZO1 gene (HGNC ID: 28993; 16q24.3) and it was shown to segregate with the CCM phenotype, within the family. PIEZO1 encodes for a mechanosensitive Ca2+ ion channel that, in endothelial cells, contributes to maintaining BBB integrity. We found that PIEZO1 impairment results in increased apoptotic rate of endothelial cells, as well as in CCM gene expression perturbation. Moreover, by immunofluorescence, we showed that PIEZO1 impairment leads to endothelial cell morphology loss and to KRIT1 nuclear translocation. We used human cerebral microvascular endothelial cells (hCMECs) as cell model, Yoda1 as PIEZO1 agonist and gadolinium as PIEZO1 antagonist. Untreated cells were consider as control condition. According to our results, we think that PIEZO1 can be considered for further investigation in CCM pathogenesis.

The modified phenanthridine PJ34 blocks the post-translational modifications of specific proteins highly expressed in human malignant cells. This exclusively arrested mitosis in human malignant cells by inserting flaws in their mitotic spindle structure. Cancer cells were efficiently eradicated by Mitotic catastrophe cell death, while similarly treated healthy proliferating cells are spared and continue to proliferate as untreated cells. This cytotoxic effect was examined in a variety of human epithelial cancer cells in tissue culture and in xenografts. Three affected proteins were identified out of all tested proteins implicated in mitosis in epithelial malignant cells compared to healthy epithelial cells. Two kinesins, KifC1/HSET and Kif18A, and NuMA were identified. The identified kinesins are already examined for their potential cancer therapy. Blocking the post-translational modifications of NuMA exclusively prevented the protein binding capacity of NuMA in cancer cells. This prevented its clustering in the spindle poles, which stabilizes the spindles and enables the alignment of chromosomes in the spindle mid-zone. Unaligned chromosomes and dispersed NuMA and centrosomes were detected in distorted spindles of human cancer cells treated with PJ34. Mitosis was arrested in the anaphase and this lead to cell death via cytochrome-c release activating caspase cascades. Thus, the cytotoxic activity of PJ34 unveiled a new mechanism causing self-eradication of human cancer cells, including cancer cells that are not responsive to current therapies.

Cohen-Armon, Drug Discovery Today, 27; 1205-1209, 2022

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to the shortage of donor organs. Developing renewable sources of human liver tissue is therefore attractive. Pluripotent stem cell-derived liver tissue represents a potential alternative to cadaver derived hepatocytes and whole organ transplant. At present, two dimensional differentiation procedures deliver tissue lacking certain functions and phenotypic instability. Efforts to overcome these limiting factors have led to the building of three-dimensional (3D) cellular aggregates. Although enabling for the field, their widespread application and adoption is limited due to the reliance on variable biological components. Our studies focus on developing 3D liver tissue under defined conditions. We demonstrate that 3D derived tissue can be generated at scale and implanted underneath the skin of mice. Excitingly, implanted human tissue provides support to mice with metabolic liver disease. This includes immunocompetent recipients. In addition to their clinical application, in vitro generated 3D tissues have important roles to play in developing safe and efficacious medicines to treat human diseases. We demonstrate that stem cell derived 3D liver tissue exhibits liver cell phenotype for over one year in culture, providing an attractive resource for long-term disease modelling and screening studies. In conclusion, stem cell derived liver tissue has great potential for in vitro and in vivo endeavours. Our most recent advances will be presented at the meeting.