Pantelis Hatzis

Hepatitis B virus (HBV) drives hepatocellular carcinoma in part through the activity of its X protein (HBx), yet the mechanisms by which HBx alters hepatocyte function remain incompletely understood. Progress has been limited by the lack of relevant human models that support controlled HBx expression in mature hepatocytes. To address this limitation, here, we use an improved hepatocyte-like organoid (HLO) platform that supports enhanced hepatocyte maturation to investigate HBx function in a differentiated hepatocyte context. Adult stem cell-derived HLOs were differentiated using an optimized protocol to generate hepatocyte-like cells with enhanced maturation and transcriptional similarity to primary liver tissue. HBx function was interrogated using both cognate promoter-driven expression and doxycycline-inducible systems across multiple donor-derived organoid lines. Transcriptomic, pathway, and single-cell imaging analyses were performed to assess the impact of HBx expression on hepatocytes. HBx expression consistently suppressed apoptosis-associated transcripts and reduced expression of core hepatocyte identity genes, including CYP3A4. Pathway analysis revealed downregulation of liver-specific functions, including metabolism, detoxification, complement, and coagulation. At the single-cell level, higher HBx expression was associated with reduced caspase 3/7 activation following apoptotic challenge and decreased hepatocyte marker expression. Functionally, the expression of HBx increased resistance to apoptosis and enhanced the ability of differentiated hepatocyte-like cells to revert to a proliferative state. HBx expression in differentiated human liver organoids reduces apoptosis and impairs hepatocyte identity, consistently across donors and expression systems. Together, these findings support a model in which HBx promotes a survival-permissive state of reduced hepatocyte differentiation. This state may contribute to early HBV-driven tumorigenesis. This HLO platform provides a relevant system to dissect HBV-host interactions and reveals a mechanism by which HBV may prime the liver for malignant transformation. Impact and implications: Understanding how HBV promotes hepatocellular carcinoma remains a critical challenge, partly due to the lack of physiologically relevant human derived model systems to study HBx function. Using a differentiated adult human liver organoid system, we show that HBx simultaneously suppresses apoptosis and reduces hepatocyte differentiation, providing a mechanistic framework for how HBV may prime hepatocytes for malignant transformation. These findings are particularly relevant for researchers studying HBV pathogenesis and liver cancer, as well as for clinicians aiming to better understand early disease progression. While further validation in more complex multicellular systems is needed, this platform can support the identification of HBx-targeted therapeutic strategies and guide the development of improved adult human derived models for virus-host interaction studies.

Background: Preterm infants mount lower humoral responses to the 13-valent pneumococcal conjugate vaccine (PCV13) than full-term infants; however, the long-term impact of prematurity on vaccine-induced protection remains unclear. This study investigates the innate and adaptive immune responses to PCV13 in preterm versus full-term infants. Methods: Thirty-eight infants (19 preterm, 19 full-term) received PCV13 at 2, 4, 6, and 12 months. Polysaccharide-specific memory B cells (MBCs) were enumerated and phenotyped by flow cytometry, while PS-specific IgG concentrations were measured by ELISA before and after the third and booster doses. RNA sequencing was performed before and 3 days after the third dose. Results: Primary immunization induced a markedly broader, predominantly proinflammatory transcriptional response in preterm infants. Preterm infants exhibited higher frequencies of total and PS-specific extragerminal center (GC) MBCs but lower switched MBCs following primary immunization; these differences largely converged postbooster. Polysaccharide-specific IgG titers remained generally lower in preterm infants. Polysaccharide-specific switched MBCs postprimary correlated positively with antibody titers postbooster. The broad upregulation of proinflammatory genes in preterm infants was negatively correlated with antibody concentrations. Conclusions: PCV13 immunization elicits distinct immunological profiles in preterm versus full-term infants. Booster immunization largely harmonized MBC composition across gestational groups, while early GC-derived MBC populations emerged as candidate correlates of durable immunity.

Agricultural intensification has reduced biodiversity and weakened ecosystem functions essential for sustainable crop production. Agroforestry has been proposed as a regenerative strategy to restore these functions through vegetation diversification, yet its ecological effects at the farm scale remain insufficiently documented, particularly in Mediterranean perennial systems. Here, adaptive field monitoring and soil shotgun metagenomics were combined to investigate ecological responses across a 10-year agroforestry transition gradient in a Mediterranean citrus and olive farm. Mature agroforestry plots supported higher insect richness and functional diversity while maintaining stable pollinator communities. Plant–insect interaction analyses suggested that potential pest activity was largely associated with spontaneous and supportive vegetation rather than crops. In contrast, soil communities showed limited changes in overall richness but substantial compositional and functional restructuring, including enrichment of the arbuscular mycorrhizal fungus Rhizophagus and proteins with nutrient-cycling functions in advanced plots. Together, these findings suggest that agroforestry transition can promote functional diversification above and below ground and highlight the value of farmer-led regenerative transitions coupled with integrative ecological monitoring for the development of more resilient agricultural systems.

Diet strongly influences the gut microbiome, which in turn influences health, yet the effects of dietary patterns on microbiome composition and function in humans remain underexplored. A unique group of apparently healthy individuals from Greece, who alternate between omnivory and restriction of animal products for religious reasons (periodically restricted group, n = 200), has been profiled. Using 16S rRNA sequencing, plasma metabolomics, and proteomics, the impact of three‐to‐four weeks of dietary restriction on gut microbiome composition and function is assessed and associations with host plasma biology are explored. Findings are compared to a continuously omnivorous group profiled in parallel (non‐restricted group, n = 211). Animal product restriction reduced microbial diversity, primarily affecting rare taxa, and altered the abundance of nearly one‐third of bacterial genera. Inferred functional shifts included downregulation of pathways contributing to cholesterol biosynthesis and purine degradation, alongside upregulation of vitamin B2 and tryptophan biosynthesis, suggesting compensatory microbial responses to dietary nutrient depletion. Multi‐omics integration revealed four microbial‐metabolite‐protein clusters, including a diet‐responsive module associating Negativibacillus with metabolic regulator FGF21 and intermediate‐density lipoproteins. These findings demonstrate rapid adaptive plasticity of the human gut microbiome in response to short‐term dietary restriction and highlight candidate microbial and molecular pathways associated with animal product restriction and host biology.

Natural killer T (NKT) cells play a complex role in hepatocellular carcinoma (HCC) progression. Using a diethylnitrosamine (DEN)-induced HCC mouse model, we observed a significant reduction in NKT cells within malignant liver tissue due to apoptosis, with the remaining cells exhibiting impaired cytokine production and cytotoxic potential. CD1d-deficient mice, which lack NKT cells, showed delayed tumor initiation and fewer tumors, yet the tumors that did form were larger and exhibited enhanced proliferation and immunosuppression. Notably, adoptive transfer of healthy NKT cells after tumor establishment reduced tumor burden, suggesting a protective role in later disease stages. These findings indicate that NKT cells contribute to early tumor development but may help control tumor progression in later stages. Their functional impairment in HCC underscores the need for strategies to restore their anti-tumor activity. Understanding the dual role of NKT cells in liver carcinogenesis may pave the way for novel immunotherapeutic approaches to improve HCC treatment and patient outcomes.

Diet strongly influences the gut microbiome, which in turn influences health, yet the effects of dietary patterns on microbiome composition and function remain underexplored in humans. We profiled a unique group of apparently healthy individuals from Greece, who alternate between omnivory and restriction of animal products for religious reasons (periodically restricted group, N=200). Using 16S rRNA sequencing, plasma metabolomics, and proteomics, we assessed the impact of three-to-four weeks of dietary restriction on gut microbiome composition and function and also explored links with host plasma biology. We compared findings to a continuously omnivorous group profiled in parallel (non-restricted group, N=211). Animal product restriction was found to reduce microbial diversity, primarily affecting rare taxa, and altered the abundance of nearly one-third of bacterial genera. Functional shifts included downregulation of cholesterol biosynthesis and purine degradation pathways, alongside upregulation of microbial biosynthesis of vitamin B2 and tryptophan, suggesting compensatory microbial responses to dietary nutrient depletion. Multi-omic integration revealed four microbial-metabolite-protein clusters, including a diet-responsive module linking Negativibacillus with potent metabolic regulator FGF21 and intermediate-density lipoproteins. Our findings demonstrate rapid adaptive plasticity of the human gut microbiome in response to short-term dietary restriction and highlight candidate microbial and molecular pathways that may mediate effects of animal product restriction on health.

The liver routinely encounters antigens from the gut, triggering pro- inflammatory responses. Unresolved inflammation can lead to liver damage, steatosis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCC is influenced by various immune cells, including invariant natural killer T (iNKT) cells, which exhibit both innate and adaptive immunity traits. Here, we examined iNKT cell dynamics in a diethyl-nitrosamine (DEN)-induced HCC mouse model. We observed a significant reduction in iNKT cell numbers in HCC livers due to apoptosis and impaired cytokine production. CD1d -deficient mice, which lack iNKT cells, displayed delayed tumor initiation and lower tumor and foci number. However, these tumors were larger in size and characterized by enhanced proliferation and immunosuppression. Interestingly, adoptive transfer of healthy iNKT cells post-tumor establishment reduced tumor burden, highlighting their potential therapeutic role. Our findings suggest that iNKT cells contribute to early HCC development, while in later stages they help to control tumor growth, thus underscoring their complex role in liver carcinogenesis. Further understanding of iNKT cell functions may inform novel immunotherapeutic strategies for HCC management.

The activation and accumulation of lung fibroblasts resulting in aberrant deposition of extracellular matrix components, is a pathogenic hallmark of Idiopathic Pulmonary Fibrosis, a lethal and incurable disease. In this report, increased expression of TKS5, a scaffold protein essential for the formation of podosomes, was detected in the lung tissue of Idiopathic Pulmonary Fibrosis patients and bleomycin-treated mice. Τhe profibrotic milieu is found to induceTKS5expression and the formation of prominent podosome rosettes in lung fibroblasts, that are retained ex vivo, culminating in increased extracellular matrix invasion.Tks5^+/-mice are found resistant to bleomycin-induced pulmonary fibrosis, largely attributed to diminished podosome formation in fibroblasts and decreased extracellular matrix invasion. As computationally predicted, inhibition of src kinase is shown to potently attenuate podosome formation in lung fibroblasts and extracellular matrix invasion, and bleomycin-induced pulmonary fibrosis, suggesting pharmacological targeting of podosomes as a very promising therapeutic option in pulmonary fibrosis.

Colorectal cancer (CRC) is one of the most prevalent cancers affecting humans, with a complex genetic and environmental aetiology. Unlike cancers with known environmental, heritable, or sex-linked causes, sporadic CRC is hard to foresee and has no molecular biomarkers of risk in clinical use. One in twenty CRC cases presents with an established heritable component. The remaining cases are sporadic and associated with partially obscure genetic, epigenetic, regenerative, microbiological, dietary, and lifestyle factors. To tackle this complexity, we should improve the practice of colonoscopy, which is recommended uniformly beyond a certain age, to include an assessment of biomarkers indicative of individual CRC risk. Ideally, such biomarkers will be causal to the disease and potentially modifiable upon dietary or therapeutic interventions. Multi-omics analysis, including transcriptional, epigenetic as well as metagenomic, and metabolomic profiles, are urgently required to provide data for risk analyses. The aim of this article is to provide a perspective on the multifactorial derailment of homeostasis leading to the initiation of CRC, which may be explored via multi-omics and Gut-on-Chip analysis to identify much-needed predictive biomarkers.

The rise of modern gene expression profiling techniques, such as RNA-Seq, has generated a wealth of high-quality datasets spanning all fields of current biological research. The large data sets and the continually expanding applications for which they can be mined, such as the investigation of alternative splicing and others, have created novel challenges for data management, exploration, analysis, and visualization. Although a large variety of RNA-Seq data analysis software packages has emerged, both open-source and commercial, most fail to simultaneously address the above challenges, while they lack obvious functionalities, such as estimating RNA abundance over non-annotated genomic regions of interest in real time. We have developed SeqCVIBE, an R Shiny web application for the interactive exploration, analysis, visualization, and genome browsing of large RNA-Seq datasets. SeqCVIBE allows for multiple on-the-fly visualizations and calculations, such as differential expression analysis, averaging genomic signals over specific regions of the genome, and calculating RNA abundances over custom, potentially non-annotated regions, such as novel long non-coding RNAs. In addition, SeqCVIBE comprises a database for pre-analyzed data, where users can navigate and explore results, as well as perform a variety of basic on-the-fly analyses and export the outcomes. Finally, we demonstrate the value of SeqCVIBE in the elucidation of the interplay of a novel lincRNA, WiNTRLINC1, and Wnt signaling in colon cancer.