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HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics. [ABSTRACT FROM AUTHOR]

Background: Epigenetic mechanisms, including DNA methylation, histone modifications, and chromatin remodeling, are highly involved in the regulation of hepatocyte viability, proliferation, and plasticity. We have previously demonstrated that repression of H3K27 methylation in differentiated hepatic HepaRG cells by treatment with GSK-J4, an inhibitor of JMJD3 and UTX H3K27 demethylase activity, changed their phenotype, inducing differentiated hepatocytes to proliferate. In addition to the epigenetic enzymatic role in the regulation of the retro-differentiation process, emerging evidence indicate that microRNAs (miRNAs) are involved in controlling hepatocyte proliferation during liver regeneration. Hence, the aim of this work is to investigate the impact of H3K27 methylation on miRNAs expression profile and its role in the regulation of the differentiation status of human hepatic progenitors HepaRG cells., Methods: A miRNA-sequencing was carried out in differentiated HepaRG cells treated or not with GSK-J4. Target searching and Gene Ontology analysis were performed to identify the molecular processes modulated by differentially expressed miRNAs. The biological functions of selected miRNAs was further investigated by transfection of miRNAs inhibitors or mimics in differentiated HepaRG cells followed by qPCR analysis, albumin ELISA assay, CD49a FACS analysis and EdU staining., Results: We identified 12 miRNAs modulated by GSK-J4; among these, miR-27a-3p and miR- 423-5p influenced the expression of several proliferation genes in differentiated HepaRG cells. MiR-27a-3p overexpression increased the number of hepatic cells reentering proliferation. Interestingly, both miR-27a-3p and miR-423-5p did not affect the expression levels of genes involved in the differentiation of progenitors HepaRG cells., Conclusions: Modulation of H3K27me3 methylation in differentiated HepaRG cells, by GSK-J4 treatment, influenced miRNA' s expression profile pushing liver cells towards a proliferating phenotype. We demonstrated the involvement of miR-27a-3p in reinducing proliferation of differentiated hepatocytes suggesting a potential role in liver plasticity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neuron function. Although ophthalmic deficits are not considered a classic symptom of ALS, recent studies suggest that changes in retinal cells, similar to those in the spinal cord motor neurons, have been observed in postmortem human tissues and animal models., Methods: In this study, we examined by immunofluorescence analysis the retinal cell layers of sporadic ALS patients in post-mortem retinal slices. We evaluated the presence of cytoplasmic TDP-43 and SQSTM1/p62 aggregates, activation of the apoptotic pathway, and microglia and astrocytes reactivity., Results: We found in the retinal ganglion cell layer of ALS patients the increase of mislocalized TDP-43, SQSTM1/p62 aggregates, activation of cleaved caspase-3, and microglia density, suggesting that retinal changes can be used as an additional diagnostic tool for ALS., Discussion: The retina is considered part of the central nervous system, and neurodegenerative changes in the brain may be accompanied by structural and possibly functional changes in the neuroretina and ocular vasculature. Therefore, using in vivo retinal biomarkers as an additional diagnostic tool for ALS may provide an opportunity to longitudinally monitor individuals and therapies over time in a noninvasive and cost-effective manner., Competing Interests: YG and MP are employed by D-Tails s.r.l. SDA is a scientific advisor of D-Tails s.r.l. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pediconi, Gigante, Cama, Pitea, Mautone, Ruocco, Ghirga and Di Angelantonio.)

'Dysbiosis' of the adult gut microbiota, in response to challenges such as infection, altered diet, stress, and antibiotics treatment has been recently linked to pathological alteration of brain function and behavior. Moreover, gut microbiota composition constantly controls microglia maturation, as revealed by morphological observations and gene expression analysis. However, it is unclear whether microglia functional properties and crosstalk with neurons, known to shape and modulate synaptic development and function, are influenced by the gut microbiota. Here, we investigated how antibiotic-mediated alteration of the gut microbiota influences microglial and neuronal functions in adult mice hippocampus. Hippocampal microglia from adult mice treated with oral antibiotics exhibited increased microglia density, altered basal patrolling activity, and impaired process rearrangement in response to damage. Patch clamp recordings at CA3-CA1 synapses revealed that antibiotics treatment alters neuronal functions, reducing spontaneous postsynaptic glutamatergic currents and decreasing synaptic connectivity, without reducing dendritic spines density. Antibiotics treatment was unable to modulate synaptic function in CX3CR1-deficient mice, pointing to an involvement of microglia-neuron crosstalk through the CX3CL1/CX3CR1 axis in the effect of dysbiosis on neuronal functions. Together, our findings show that antibiotic alteration of gut microbiota impairs synaptic efficacy, suggesting that CX3CL1/CX3CR1 signaling supporting microglia is a major player in in the gut-brain axis, and in particular in the gut microbiota-to-neuron communication pathway.

Gene expression regulation by small interfering RNA (siRNA) holds promise in treating a wide range of diseases through selective gene silencing. However, successful clinical application of nucleic acid-based therapy requires novel delivery options. Herein, to achieve efficient delivery of negatively charged siRNA duplexes, the internal cavity of "humanized" chimeric Archaeal ferritin (HumAfFt) was specifically decorated with novel cationic piperazine-based compounds (PAs). By coupling these rigid-rod-like amines with thiol-reactive reagents, chemoselective conjugation was efficiently afforded on topologically selected cysteine residues properly located inside HumAfFt. The capability of PAs-HumAfFt to host and deliver siRNA molecules through human transferrin receptor (TfR1), overexpressed in many cancer cells, was explored. These systems allowed siRNA delivery into HeLa, HepG2, and MCF-7 cancer cells with improved silencing effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression with respect to traditional transfection methodologies and provided a promising TfR1-targeting system for multifunctional siRNA delivery to therapeutic applications.

Persistent mortality rates of medulloblastoma (MB) and severe side effects of the current therapies require the definition of the molecular mechanisms that contribute to tumor progression. Using cultured MB cancer stem cells and xenograft tumors generated in mice, we show that low expression of miR-326 and its host gene β-arrestin1 (ARRB1) promotes tumor growth enhancing the E2F1 pro-survival function. Our models revealed that miR-326 and ARRB1 are controlled by a bivalent domain, since the H3K27me3 repressive mark is found at their regulatory region together with the activation-associated H3K4me3 mark. High levels of EZH2, a feature of MB, are responsible for the presence of H3K27me3. Ectopic expression of miR-326 and ARRB1 provides hints into how their low levels regulate E2F1 activity. MiR-326 targets E2F1 mRNA, thereby reducing its protein levels; ARRB1, triggering E2F1 acetylation, reverses its function into pro-apoptotic activity. Similar to miR-326 and ARRB1 overexpression, we also show that EZH2 inhibition restores miR-326/ARRB1 expression, limiting E2F1 pro-proliferative activity. Our results reveal a new regulatory molecular axis critical for MB progression., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Tau is a microtubule-associated protein (MAPT) that is highly expressed in neurons and implicated in several cellular processes. Tau misfolding and self-aggregation give rise to proteinaceous deposits known as neuro-fibrillary tangles. Tau tangles play a key role in the genesis of a group of diseases commonly referred to as tauopathies; notably, these aggregates start to form decades before any clinical symptoms manifest. Advanced imaging methodologies have clarified important structural and functional aspects of tau and could have a role as diagnostic tools in clinical research. In the present review, recent progresses in tau imaging will be discussed. We will focus mainly on super-resolution imaging methods and the development of near-infrared fluorescent probes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Pizzarelli, Pediconi and Di Angelantonio.)

Alzheimer's disease (AD), a primary cause of dementia in the aging population, is characterized by extracellular amyloid-beta peptides aggregation, intracellular deposits of hyperphosphorylated tau, neurodegeneration and glial activation in the brain. It is commonly thought that the lack of early diagnostic criteria is among the main causes of pharmacological therapy and clinical trials failure; therefore, the actual challenge is to define new biomarkers and non-invasive technologies to measure neuropathological changes in vivo at pre-symptomatic stages. Recent evidences obtained from human samples and mouse models indicate the possibility to detect protein aggregates and other pathological features in the retina, paving the road for non-invasive rapid detection of AD biomarkers. Here, we report the presence of amyloid beta plaques, tau tangles, neurodegeneration and detrimental astrocyte and microglia activation according to a disease associated microglia phenotype (DAM). Thus, we propose the human retina as a useful site for the detection of cellular and molecular changes associated with Alzheimer's disease.

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD),  which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

Modification of histones by lysine methylation plays a role in many biological processes, and it is dynamically regulated by several histone methyltransferases and demethylases. The polycomb repressive complex contains the H3K27 methyltransferase EZH2 and controls dimethylation and trimethylation of H3K27 (H3K27me2/3), which trigger gene suppression. JMJD3 and UTX have been identified as H3K27 demethylases that catalyze the demethylation of H3K27me2/3, which in turns lead to gene transcriptional activation. EZH2, JMJD3 and UTX have been extensively studied for their involvement in development, immune system, neurodegenerative disease, and cancer. However, their role in molecular mechanisms underlying the differentiation process of hepatic cells is yet to be elucidated. Here, we show that EZH2 methyltransferase and JMJD3/UTX demethylases were deregulated during hepatic differentiation of human HepaRG cells resulting in a strong reduction of H3K27 methylation levels. Inhibition of JMJD3 and UTX H3K27 demethylase activity by GSK-J4 epi-drug reverted phenotype of HepaRG DMSO-differentiated cells and human primary hepatocytes, drastically decreasing expression of hepatic markers and inducing cell proliferation. In parallel, inhibition of EZH2 H3K27me3 activity by GSK-126 epi-drug induced upregulation of hepatic markers and downregulated the expression of cell cycle inhibitor genes. To conclude, we demonstrated that modulation of H3K27 methylation by inhibiting methyl-transferase and dimethyl-transferase activity influences the differentiation status of hepatic cells, identifying a possible new role of EZH2, JMJD3 and UTX epi-drugs to modulate hepatic cell plasticity.

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease. Although genetic predisposition and epigenetic factors contribute to the development of NAFLD, our understanding of the molecular mechanism involved in the pathogenesis of the disease is still emerging. Here we investigated a possible role of a microRNAs-STAT3 pathway in the induction of hepatic steatosis. Differentiated HepaRG cells treated with the fatty acid sodium oleate (fatty dHepaRG) recapitulated features of liver vesicular steatosis and activated a cell-autonomous inflammatory response, inducing STAT3-Tyrosine-phosphorylation. With a genome-wide approach (Chromatin Immunoprecipitation Sequencing), many phospho-STAT3 binding sites were identified in fatty dHepaRG cells and several STAT3 and/or NAFLD-regulated microRNAs showed increased expression levels, including miR-21. Innovative CARS (Coherent Anti-Stokes Raman Scattering) microscopy revealed that chemical inhibition of STAT3 activity decreased lipid accumulation and deregulated STAT3-responsive microRNAs, including miR-21, in lipid overloaded dHepaRG cells. We were able to show in vivo that reducing phospho-STAT3-miR-21 levels in C57/BL6 mice liver, by long-term treatment with metformin, protected mice from aging-dependent hepatic vesicular steatosis. Our results identified a microRNAs-phosphoSTAT3 pathway involved in the development of hepatic steatosis, which may represent a molecular marker for both diagnosis and therapeutic targeting.

Metformin is a commonly prescribed type II diabetes medication that exhibits promising anticancer effects. Recently, these effects were found to be associated, at least in part, with a modulation of microRNA expression. However, the mechanisms by which single modulated microRNAs mediate the anticancer effects of metformin are not entirely clear and knowledge of such a process could be vital to maximize the potential therapeutic benefits of this safe and well-tolerated therapy. Our analysis here revealed that the expression of miR-21-5p was downregulated in multiple breast cancer cell lines treated with pharmacologically relevant doses of metformin. Interestingly, the inhibition of miR-21-5p following metformin treatment was also observed in mouse breast cancer xenografts and in sera from 96 breast cancer patients. This modulation occurred at the levels of both pri-miR-21 and pre-miR-21, suggesting transcriptional modulation. Antagomir-mediated ablation of miR-21-5p phenocopied the effects of metformin on both the clonogenicity and migration of the treated cells, while ectopic expression of miR-21-5p had the opposite effect. Mechanistically, this reduction in miR-21-5p enhanced the expression of critical upstream activators of the AMP-activated protein kinase, calcium-binding protein 39-like and Sestrin-1, leading to AMP-activated protein kinase activation and inhibition of mammalian target of rapamycin signaling. Importantly, these effects of metformin were synergistic with those of everolimus, a clinically relevant mammalian target of rapamycin inhibitor, and were independent of the phosphatase and tensin homolog status. This highlights the potential relevance of metformin in combinatorial settings for the treatment of breast cancer., Competing Interests: The authors declare no conflict of interest.