The HBV HBx regulatory protein is required for transcription from the covalently closed circular DNA (cccDNA) minichromosome and affects the epigenetic control of both viral and host cellular ...chromatin.
We explored, in relevant cellular models of HBV replication, the functional consequences of HBx interaction with DLEU2, a long non-coding RNA (lncRNA) expressed in the liver and increased in human hepatocellular carcinoma (HCC), in the regulation of host target genes and the HBV cccDNA.
We show that HBx binds the promoter region, enhances the transcription and induces the accumulation of DLEU2 in infected hepatocytes. We found that nuclear DLEU2 directly binds HBx and the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic active subunit of the polycomb repressor complex 2 (PRC2) complex. Computational modelling and biochemical evidence suggest that HBx and EZH2 share two preferential binding sites in DLEU2 intron 1. HBx and DLEU2 co-recruitment on the cccDNA displaces EZH2 from the viral chromatin to boost transcription and viral replication. DLEU2-HBx association with target host promoters relieves EZH2 repression and leads to the transcriptional activation of a subset of EZH2/PRC2 target genes in HBV-infected cells and HBV-related HCCs.
Our results highlight the ability of HBx to bind RNA to impact on the epigenetic control of both viral cccDNA and host genes and provide a new key to understand the role of DLEU2 and EZH2 overexpression in HBV-related HCCs and HBx contribution to hepatocytes transformation.
Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs ...have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches.
Human pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells, represent powerful tools for disease modeling and for therapeutic applications. PSCs are ...particularly useful for the study of development and diseases of the nervous system. However, generating in vitro models that recapitulate the architecture and the full variety of subtypes of cells that make the complexity of our brain remains a challenge. In order to fully exploit the potential of PSCs, advanced methods that facilitate the identification of molecular signatures in neural differentiation and neurological diseases are highly demanded. Here, we review the literature on the development and application of digital color-coded molecular barcoding as a potential tool for standardizing PSC research and applications in neuroscience. We will also describe relevant examples of the use of this technique for the characterization of the heterogeneous composition of the brain tumor glioblastoma multiforme.
The HBV covalently closed circular DNA (cccDNA) is organized as a mini-chromosome in the nuclei of infected hepatocytes by histone and non-histone proteins. Transcription from the cccDNA of the RNA ...replicative intermediate termed pre-genome (pgRNA), is the critical step for genome amplification and ultimately determines the rate of HBV replication. Multiple evidences suggest that cccDNA epigenetic modifications, such as histone modifications and DNA methylation, participate in regulating the transcriptional activity of the HBV cccDNA. Inflammatory cytokines (TNFα, LTβ) and the pleiotropic cytokine interleukin-6 (IL6) inhibit hepatitis B virus (HBV) replication and transcription. Here we show, in HepG2 cells transfected with linear HBV monomers and HBV-infected NTCP-HepG2 cells, that IL6 treatment leads to a reduction of cccDNA-bound histone acetylation paralleled by a rapid decrease in 3.5kb/pgRNA and subgenomic HBV RNAs transcription without affecting cccDNA chromatinization or cccDNA levels. IL6 repressive effect on HBV replication is mediated by a loss of HNF1α and HNF4α binding to the cccDNA and a redistribution of STAT3 binding from the cccDNA to IL6 cellular target genes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in RNA binding proteins (RBPs) have been linked to the motor neuron disease amyotrophic lateral sclerosis (ALS). Extensive auto-regulation, cross-regulation, cooperation and competition ...mechanisms among RBPs are in place to ensure proper expression levels of common targets, often including other RBPs and their own transcripts. Moreover, several RBPs play a crucial role in the nervous system by localizing target RNAs in specific neuronal compartments. These include the RBPs FUS, FMRP, and HuD. ALS mutations in a given RBP are predicted to produce a broad impact on such delicate equilibrium. Here we studied the effects of the severe FUS-P525L mutation on common FUS and FMRP targets. Expression profiling by digital color-coded molecular barcoding in cell bodies and neurites of human iPSC-derived motor neurons revealed altered levels of transcripts involved in the cytoskeleton, neural projection and synapses. One of the common targets is HuD, which is upregulated because of the loss of FMRP binding to its 3'UTR due to mutant FUS competition. Notably, many genes are commonly altered upon FUS mutation or HuD overexpression, suggesting that a substantial part of the effects of mutant FUS on the motor neuron transcriptome could be due to HuD gain-of-function. Among altered transcripts, we also identified other common FUS and FMRP targets, namely MAP1B, PTEN, and AP2B1, that are upregulated upon loss of FMRP binding on their 3'UTR in FUS-P525L motor neurons. This work demonstrates that the impairment of FMRP function by mutant FUS might alter the expression of several genes, including new possible biomarkers and therapeutic targets for ALS.
Individuals exposed to Mycobacterium tuberculosis (Mtb) may be infected and remain for the entire life in this condition defined as latent tuberculosis infection (LTBI) or develop active tuberculosis ...(TB). Among the multiple factors governing the outcome of the infection, dendritic cells (DCs) play a major role in dictating antibacterial immunity. However, current knowledge on the role of the diverse components of human DCs in shaping specific T-cell response during Mtb infection is limited. In this study, we performed a comparative evaluation of peripheral blood circulating DC subsets as well as of monocyte-derived Interferon-α DCs (IFN-DCs) from patients with active TB, subjects with LTBI and healthy donors (HD). The proportion of circulating myeloid BDCA3+ DCs (mDC2) and plasmacytoid CD123+ DCs (pDCs) declined significantly in active TB patients compared to HD, whereas the same subsets displayed a remarkable activation in LTBI subjects. Simultaneously, the differentiation of IFN-DCs from active TB patients resulted profoundly impaired compared to those from LTBI and HD individuals. Importantly, the altered developmental trait of IFN-DCs from active TB patients was associated with down-modulation of IFN-linked genes, marked changes in molecular signaling conveying antigen (Ag) presentation and full inability to induce Ag-specific T cell response. Thus, these data reveal an important role of IFN-α in determining the induction of Mtb-specific immunity.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-α (IFN-α DC) ...show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-α in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-α in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-α-treated pDC. A specific miRNA signature was induced in IFN-α DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-α-driven DC differentiation. Of note, monocyte-derived IFN-α DC and in vitro IFN-α-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-α and identify Blimp-1 as an IFN-α-mediated key regulator potentially accounting for shared functional features between IFN-α DC and pDC.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hepatitis B virus (HBV)-specific CD8 T cells are functionally exhausted in chronic hepatitis B infection, and this condition can be corrected only partially through the modulation of inhibitory ...pathways, which suggests that a more complex molecular interplay underlies T cell exhaustion. To gain broader insight into this process and identify additional targets for the restoration of T cell function, we compared the transcriptome profiles of HBV-specific CD8 T cells from patients with acute and chronic disease with those of HBV-specific CD8 T cells from patients able to resolve HBV infection spontaneously and influenza (FLU)-specific CD8 T cells from healthy participants. The results indicate that exhausted HBV-specific CD8 T cells are markedly impaired at multiple levels and show substantial downregulation of various cellular processes centered on extensive mitochondrial alterations. A notable improvement of mitochondrial and antiviral CD8 functions was elicited by mitochondrion-targeted antioxidants, which suggests a central role for reactive oxygen species (ROS) in T cell exhaustion. Thus, mitochondria represent promising targets for novel reconstitution therapies to treat chronic hepatitis B infection.
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