Javier Ramón-Azcón

Background & Aims: YAQ001 is a novel, non‐absorbable, gut‐restricted, multiporous, carbon bead adsorbent. Pre‐clinical studies and a clinical trial suggested its therapeutic potential and safety in patients with cirrhosis. This substudy aimed to explore the effects of YAQ001 on the gut microbiome and their exploratory association with biomarkers of gut permeability and systemic inflammation. Methods: Twenty‐eight‐patients with cirrhosis were randomized (double‐blind) to receive YAQ001 (4 g once daily) or placebo. Gut microbiome and biomarkers of inflammation and gut permeability were assessed at baseline, after 4 and 12 weeks. Changes in biomarkers and relationship with microbiome composition and gene profiling were assessed. The effect of YAQ001 on Klebsiella pneumoniae biofilms was assessed in vitro. Results: Overall alpha and beta diversity remained unaltered. YAQ001 increased the abundance of bacteria associated with improved gut health such as Adlercreutzia equolifaciens , a bacterium commonly depleted in liver disease, and decreased the abundance of bacteria associated with infections and poor outcomes such as Klebsiella pneumonia , and Streptococcus mutans . YAQ001 impacted positively on virulence factors such as siderophores, fimbriae structures, and lipopolysaccharides that are associated with inflammation and invasion. Antibiotic resistance genes decreased significantly in the YAQ001 group. These changes were associated with a reduction in markers of gut permeability and systemic inflammation. YAQ001 prevented K. pneumoniae biofilms in vitro. Conclusion: YAQ001 was associated with favourable compositional shifts and trends towards reduced virulence‐related gene signatures in the gut microbiome, alongside trends towards improved inflammatory biomarkers. The in vitro anti‐biofilm effect of YAQ001 warrants further mechanistic investigation. The findings position YAQ001 as a potential microbiome therapeutic.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects >30% of the global population and is rising to become the most common liver disorder. MASLD can influence distal organs via secreted mediators, and up to 43% of patients show sarcopenia or related muscle-wasting phenotypes. We developed a modular human three-dimensional bioengineered liver–muscle platform to dissect causal liver–muscle crosstalk. Liver constructs recapitulated steatosis and transition to metabolic dysfunction-associated steatohepatitis (MASH), with inflammatory, apoptotic, immune cell-recruiting and pro-fibrotic signatures. Transferring liver-derived conditioned media to engineered muscles revealed severity-dependent impairment of contractile performance, altered protein balance and myogenic regulation. Contractility declined progressively from steatosis to MASH. Steatotic conditioned media reduced myotube size, whereas MASH conditioned media triggered a compensatory program that partially preserved size despite functional loss. Muscle exposure to MASLD mediators was associated with disrupted autophagic degradation, consistent with impaired flux and accumulation of autophagosomes and lysosomes. This scalable human platform enables systematic investigation of MASLD extrahepatic mechanisms and provides a testbed for perturbations and interventions targeting liver-driven muscle wasting.

The liver is frequently exposed to acute and chronic insults that disrupt its function, leading to inflammation, fibrosis, and serious conditions like cirrhosis and hepatocellular carcinoma. Fibrosis, driven by the activation of hepatic stellate cells is a key feature of chronic liver damage. Traditional 2D liver models face limitations in maintaining liver functions and accurately replicating chronic conditions like fibrosis. In response to these needs, 3D models have emerged as more physiologically relevant platforms for studying liver disease and drug testing. However, most existing 3D models still lack chronic injury features or immune elements. In this study, we developed a 3D human-liver model to investigate both acute and chronic drug-induced liver injury. The model consists of encapsulated human hepatocytes (HepaRG) and hepatic stellate cells (LX-2) within a gelatin methacryloyl (GelMA) and carboxymethyl cellulose methacrylate matrix. Over a 30-day culture period, the 3D liver constructs maintained physiologically relevant functions, including albumin secretion and cytochrome P450 activity, which are lost rapidly in conventional 2D models. The model enabled reliable simulation of low-level, long-term, and high-dose, short-term damage by administering lipopolysaccharide and Paracetamol. Chronic liver injury was characterized by progressive fibrosis, elevated expression of COL1A1, and persistent hyperammonemia. In contrast, acute models showed significant but transient injury. Moreover, dexamethasone treatment successfully reversed fibrotic markers and restored hepatocyte functionality, indicating the model’s predictive power for evaluating potential therapies. Incorporating human monocytes (THP-1) allowed investigation of the immune response and macrophage activation, which are critical contributors to liver disease pathogenesis. Overall, this 3D liver model offers a physiologically relevant and versatile platform for studying complex multi-cellular interactions under acute and chronic injury. It has broad implications for drug safety screening, disease modeling, and personalized therapy. The tri-culture design extends the model’s capacity to elucidate immune-driven hepatic pathology.

Fibroadipogenic precursor cells (FAPs) are key contributors to the fibrotic and adipogenic remodeling observed in Duchenne muscular dystrophy (DMD), yet their precise role in muscle degeneration remains unclear. In this study, we investigated how FAPs from DMD muscle influence myogenesis by conducting co-culture experiments using healthy myoblasts with FAPs derived from either healthy or DMD biopsies, in both direct and indirect contact conditions. We observed that healthy FAPs enhance myogenic differentiation, increasing myotube area, while DMD FAPs significantly inhibit it. To identify underlying molecular mechanisms, we performed mass spectrometry of the FAP secretome and found that 368 of 760 detected proteins were upregulated in DMD FAPs, including C4b-binding protein alpha chain (C4BPA), which showed a notable increase. In vitro treatment of myoblasts with recombinant C4BPA led to severe impairment of myotube formation, evidenced by reduced myotube area, fewer nuclei per myotube, and smaller myotube size. C4BPA exposure also downregulated myogenic markers and upregulated genes associated with muscle atrophy. These findings were further validated in a 3D engineered muscle model, where C4BPA significantly reduced contractile function. Importantly, silencing C4BPA in DMD-derived cultures partially restored myogenic capacity, improving both the differentiation index and nuclear content per myotube. Together, our data demonstrate that DMD FAPs exert anti-myogenic effects, at least in part, through elevated secretion of C4BPA, which interferes with muscle differentiation and function. This highlights C4BPA as a novel effector of muscle degeneration and a promising therapeutic target for modulating the fibrotic environment in DMD. Targeting specific secreted factors from pathological FAPs may help preserve muscle regeneration and mitigate disease progression in dystrophic muscles.

Myotonic dystrophy type 1 (DM1) lacks human in vitro models that directly link RNA toxicity to mature skeletal muscle function, particularly myotonia. Here, we engineer contractile 3D human skeletal muscle tissues from immortalized myoblasts derived from three DM1 patients representing juvenile, adult, and late-onset subtypes. These tissues reproduce key molecular features of DM1, including nuclear RNA foci, MBNL1 sequestration, and widespread mis-splicing. Functionally, DM1 tissues exhibit impaired calcium handling, subtype-dependent weakness, rapid fatigue, and a fiber-type distribution characterized by increased slow type I fibers and pathological MyHC-I/IIx hybrids. Notably, the 3D environment enables expression and complete pathogenic mis-splicing of CLCN1 —undetectable in matched 2D cultures—accompanied by myotonia-like delayed relaxation. Using this model, we assessed therapeutic responses of candidate small-molecule modulators. Phenylbutazone reduced RNA foci and MBNL1 sequestration but failed to rescue spliceopathy or function. In contrast, calcitriol induced coordinated transcriptomic remodeling and robustly rescued myotonia-like relaxation despite persistent CLCN1 mis-splicing. These findings establish a functionally mature human DM1 muscle model and highlight compensatory network activation as a strategy to improve muscle function in DM1.

The degeneration of the muscle in muscle dystrophies involves complex interactions among the different cell types. Here, we have used datasets from single-nuclei RNA sequencing (snRNAseq) of Duchenne Muscular Dystrophy (DMD) muscle samples to study the dysregulation of molecular pathways compared to healthy control muscle. We have observed that the epidermal growth factor (EGF) signaling is upregulated in DMD by an increase of the ligands EGF and EGF containing fibulin extracellular matrix protein 1 (EFEMP1). This study explores the role of EGF and EFEMP1 in FAPs and myoblasts in vitro. We provide evidence that EFEMP1 is secreted by FAPs in DMD and is mainly involved with increased myotube size but without enhancing muscle strength. Conversely, EGF enhances fibrotic differentiation in FAPs and promote smaller, proliferative myotubes in myoblasts, aligning with a fibrotic and dysfunctional muscle phenotype in DMD. The cellular differences from both ligands can be explained by the interactions with the receptor type, with EGF activating both EGFR and ErbB2, while EFEMP1 selectively maintained ErbB4 in an inactive state but promoting EGFR-ErbB2 and ErbB2-ErbB4 heterodimerization, potentially enhancing EGF signaling. Consequently, this study examinates the alteration of the EGF signalling in DMD and provides new molecular interactions in muscle that can be useful for targeted therapies of muscle degeneration.

Background and aims: YAQ001 is a novel, highly engineered, non-absorbable, gut-restricted, multiporous, carbon bead adsorbent. Pre-clinical studies and a clinical trial confirmed its therapeutic potential and safety in patients with cirrhosis. The aims of this sub study were to evaluate the effect of YAQ001 on the gut microbiome and its association with biomarkers of gut permeability and systemic inflammation. Methods: In total, 28-patients with cirrhosis were randomized (double-blind) to receive YAQ001 (4g once daily) or Placebo. Gut microbiome and biomarkers of inflammation and gut permeability were assessed at baseline, after 4 and 12-weeks. Changes in biomarkers and relationship with microbiome composition and gene profiling were assessed. The effect of YAQ001 on Klebsiella pneumoniae bacterial biofilms was also assessed using an in vitro gut epithelial model. Results: Alpha and beta diversity at different time points were unaltered. YAQ001 increased the abundance of bacteria associated with improved gut health such as Adlercreutzia equolifaciens (pKlebsiella pneumonia and Streptococcus mutans (pKlebsiella pneumoniae biofilms in vitro . Conclusions: The results show that YAQ001 impacts positively on the composition of the microbiome, significantly reduces its virulence, antibiotic resistance genes and biofilm formation, which is associated with modulation of gut permeability and systemic inflammation. Impact and implications: YAQ001 exerts its therapeutic effect by favorably modulating the gut ecosystem in cirrhosis. It selectively increases beneficial gut bacteria while suppressing pathogenic taxa linked to adverse outcomes. Beyond composition, YAQ001 reduces microbial virulence by limiting the production of invasive toxins and lowers the abundance of antibiotic-resistance genes. In vitro models also demonstrated its efficacy in inhibiting biofilm formation by key pathogens. In summary, this multifaceted modulation of the microbiome led to tangible clinical improvements, notably enhanced gut barrier function and reduced systemic inflammation. The data support further development of YAQ001 as a novel microbiome therapeutic. Highlights: In patients with cirrhosis, YAQ001 specifically enhanced beneficial gut bacteria while suppressing pathogenic bacteria linked to adverse clinical outcomes. YAQ001 significantly reduced the genetic expression of critical virulence factors, including siderophores, fimbriae, and lipopolysaccharides, thereby diminishing the potential for microbial invasion and inflammation. YAQ001 administration led to a significant decrease in the abundance of antibiotic resistance genes within the gut microbiome, potentially restoring therapeutic efficacy of antibiotics. In vitro studies demonstrated that YAQ001 effectively prevents the formation of Klebsiella pneumoniae biofilms, a key mechanism of bacterial persistence and chronic infection. The beneficial microbial shifts were directly associated with clinically relevant improvements in biomarkers of gut permeability and systemic inflammation.

Myo-MOVES is a versatile platform for electrical pulse stimulation (EPS) that mimics motor neuron activation in muscle tissue and enables a wide range of in vitro muscle contraction studies.