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P16 immunostaining is considered a useful surrogate of transcriptionally active high-risk (hr) HPV infection. Only strong and widespread “block-like” immunoreactivity is considered specific, whereas weak/focal p16 positive immunostaining is considered not specific, and follow-up and HPV molecular detection is not indicated. The aim of the study was to evaluate the presence of HPV DNA and Ki67 immunostaining in 40 cervical atypical biopsies (CALs) with mild and focal histological features suggestive of HPV infection—20 cases with weak/focal p16 positive immunoreactivity and 20 cases negative for p16 expression. In 16/20 weak/focal p16 positive CALs (80%), the INNO-LiPA HPV genotyping detected hrHPV genotypes (HPV 31, 51, 56, 59, 26, 53, 66, 73, and 82). Co-infection of two or more hrHPV genotypes was often evidenced. HPV16 and 18 genotypes were never detected. Ki67 immunostaining was increased in 10/20 cases (50%). In 19/20 p16 negative CALs, hrHPV infection was absent and Ki67 was not increased. These results suggest that weak/focal p16 immunostaining represents the early stage of transcriptionally active infection, strongly related to the presence of less common hrHPV genotypes, probably with a slower transforming power, but with a potential risk of progression if the infection persists. HPV DNA genotyping and follow-up could be useful in these cases to verify if they are able to evolve into overt dysplastic changes and to improve knowledge of less common hrHPV genotypes.

Human behavior is influenced by both genetic and environmental factors. Monoamine oxidase A (MAOA) is among the most investigated genetic determinants of violent behaviors, while the monoamine oxidase B (MAOB) is explored in Parkinson's disease. We collected twenty-four post-mortem brain tissue datasets of 3871 and 1820 non-demented males and females, respectively, who died from causes not attributable to neurodegenerative diseases. The gene expressions of MAOA and MAOB (MAO genes) were analyzed in these subjects, who were further stratified according to age into eleven groups ranging from late Infancy (5-9 months) to centenarians (>100 years). MAO genes were differently expressed in brains during the entire life span. In particular, maximal and minimal expression levels were found in early life and around the teen years. Females tended to have higher MAO gene levels throughout their lives than those found in age-matched males, even when expressions were separately measured in different brain regions. We demonstrated the existence of age- and sex- related variations in the MAO transcript levels in defined brain regions. More in-depth protein studies are needed to confirm our preliminary results obtained only on messenger RNAs in order to establish the role played by MAO genes in human development.

Hepatocellular carcinoma (HCC) is one of the fastest-growing causes of cancer-related mortalities worldwide and this trend is mimicked by the surge of non-alcoholic fatty liver disease (NAFLD). Altered hepatic lipid metabolism promotes HCC development through inflammation and activation of oncogenes. GDF11 is a member of the TGF-β superfamily and recent data have implicated GDF11 as an anti-aging factor that can alleviate high-fat diet induced obesity, hyperglycemia, insulin resistance and NAFLD. However, its role in hepatic lipid metabolism is still not fully delineated. The aim of the present study was to characterize the role of GDF11 in hepatic and HCC cells lipid accumulation. To achieve this, we performed imaging, biochemical, lipidomic, and transcriptomic analyses in primary hepatocytes and in HCC cells treated with GDF11 to study the GDF11-activated signaling pathways. GDF11 treatment rapidly triggered ALK5-dependent SMAD2/3 nuclear translocation and elevated lipid droplets in HCC cells, but not in primary hepatocytes. In HCC cells, ALK5 inhibition hampered GDF11-mediated SMAD2/3 signaling and attenuated lipid accumulation. Using ultra-high-performance liquid chromatography/mass spectrometry, we detected increased accumulation of longer acyl-chain di/tri-acylglycerols and glycerophospholipids. Unbiased transcriptomic analysis identified TGF-β and PI3K-AKT signaling among the top pathways/cellular processes activated in GDF11 treated HCC cells. In summary, GDF11 supplementation promotes pro-lipogenic gene expression and lipid accumulation in HCC cells. Integration of our “omics” data pointed to a GDF11-induced upregulation of de novo lipogenesis through activation of ALK5/SMAD2/3/PI3K-AKT pathways. Thus, GDF11 could contribute to metabolic reprogramming and dysregulation of lipid metabolism in HCC cells, without effects on healthy hepatocytes.

A disruption to circadian rhythmicity and the sleep/wake cycle constitutes a major feature of Alzheimer's disease (AD). The maintenance of circadian rhythmicity is regulated by endogenous clock genes and a number of external Zeitgebers, including light. This study investigated the light induced changes in the expression of clock genes in a triple transgenic model of AD (3xTg-AD) and their wild type littermates (Non-Tg). Changes in gene expression were evaluated in four brain areas-suprachiasmatic nucleus (SCN), hippocampus, frontal cortex and brainstem-of 6- and 18-month-old Non-Tg and 3xTg-AD mice after 12 h exposure to light or darkness. Light exposure exerted significant effects on clock gene expression in the SCN, the site of the major circadian pacemaker. These patterns of expression were disrupted in 3xTg-AD and in 18-month-old compared with 6-month-old Non-Tg mice. In other brain areas, age rather than genotype affected gene expression; the effect of genotype was observed on hippocampal Sirt1 expression, while it modified the expression of genes regulating the negative feedback loop as well as Ror alpha, Csnk1 epsilon and Sirt1 in the brainstem. In conclusion, during the early development of AD, there is a disruption to the normal expression of genes regulating circadian function after exposure to light, particularly in the SCN but also in extra-hypothalamic brain areas supporting circadian regulation, suggesting a severe impairment of functioning of the clock gene pathway. Even though this study did not demonstrate a direct association between these alterations in clock gene expression among brain areas with the cognitive impairments and chrono-disruption that characterize the early onset of AD, our novel results encourage further investigation aimed at testing this hypothesis.

Objectives Chemotherapeutic drugs induce senescence in cancer cells but, unlike replicative senescence or oncogene‐induced senescence, do so rather inefficiently and depending on DNA damage. A thorough understanding of the biology of chemotherapy‐induced senescent cells requires their isolation from a mixed population of adjacent senescent and non‐senescent cancer cells. Materials and methods We have developed and optimized a rapid iodixanol (OptiPrep)‐based gradient centrifugation system to identify, isolate and characterize doxorubicin (DXR)‐induced senescent hepatocellular carcinoma (HCC) cells (HepG2 and Huh‐7) in vitro. Results After cellular exposure to DXR, we used iodixanol gradient‐based centrifugation to isolate and re‐plate cells on collagen‐coated flasks, despite their low or null proliferative capacity. The isolated cell populations were enriched for DXR‐induced senescent HCC cells, as confirmed by proliferation arrest assay, and β‐galactosidase and DNA damage‐dependent γH2A.X staining. Conclusions Analysing pure cultures of chemotherapy‐induced senescent versus non‐responsive cancer cells will increase our knowledge on chemotherapeutic mechanisms of action, and help refine current therapeutic strategies.

Background The benefits of aerobic exercise are well-studied; there is no consensus on the association between resistance training and major adverse cardiovascular outcomes. This systematic review and meta-analysis aimed to address this issue. Design and methods We searched for randomized trials and cohort studies that evaluated the association between resistance training and mortality and cardiovascular events. Two investigators screened the identified abstracts and full-texts independently and in duplicate. Cochrane tools were used to assess the risk of bias. We calculated hazard ratios and 95% confidence intervals using random effect models. Results From the 1430 studies identified, 11 (one randomized trial and 10 cohort studies) met the inclusion criteria, totaling 370,256 participants with mean follow-up of 8.85 years. The meta-analysis showed that, compared with no exercise, resistance training was associated with 21% (hazard ratio (95% confidence interval (CI)), 0.79 (0.69–0.91)) and 40% (hazard ratio (95% CI), 0.60 (0.49–0.72)) lower all-cause mortality alone and when combined with aerobic exercise, respectively. Furthermore, resistance training had a borderline association with lower cardiovascular mortality (hazard ratio (95% CI), 0.83 (0.67–1.03)). In addition, resistance training showed no significant association with cancer mortality. Risk of bias was low to intermediate in the included studies. One cohort study looked at the effect of resistance training on coronary heart disease events in men and found a 23% risk reduction (risk ratio, 0.77, CI: 0.61–0.98). Conclusion Resistance training is associated with lower mortality and appears to have an additive effect when combined with aerobic exercise. There are insufficient data to determine the potential beneficial effect of resistance training on non-fatal events or the effect of substituting aerobic exercise with resistance training.

Rationale: Loss of histone macroH2A1 induces appearance of cancer stem cells (CSCs)-like cells in hepatocellular carcinoma (HCC). How CSCs interact with the tumor microenvironment and the adaptive immune system is unclear. Methods: We screened aggressive human HCC for macroH2A1 and CD44 CSC marker expression. We also knocked down (KD) macroH2A1 in HCC cells, and performed integrated transcriptomic and secretomic analyses. Results: Human HCC showed low macroH2A1 and high CD44 expression compared to control tissues. MacroH2A1 KD CSC-like cells transferred paracrinally their chemoresistant properties to parental HCC cells. MacroH2A1 KD conditioned media transcriptionally reprogrammed parental HCC cells activated regulatory CD4+/CD25+/FoxP3+ T cells (Tregs). Conclusions: Loss of macroH2A1 in HCC cells drives cancer stem-cell propagation and evasion from immune surveillance.

Little is still known about the effect of dietary patterns on left ventricular hypertrophy (LVH). Here, we derived dietary patterns by principal component analysis (PCA) and evaluated their association with LV structure, function, and remodelling. Our cross-sectional study included 438 members (aged 25–65 years; 59.1% women) of the Kardiovize Brno 2030 with no history of cardiovascular disease. Two dietary patterns were derived using PCA, namely prudent and western. Primary outcomes were echocardiographic parameters and LV geometric patterns, such as concentric LV remodelling (cLVR), concentric LVH (cLVH), and eccentric LVH (eLVH). Interestingly, participants with high adherence to the prudent dietary pattern had decreased odds of cLVH after adjustment for socio-demographic, clinical and behavioral covariates (OR = 0.24, 95% CI = 0.08–0.88; p = 0.031). By contrast, several echocardiographic parameters increased with increasing adherence to the western dietary pattern, which resulted in higher odds of cLVH among participants with high adherence (OR = 5.38, 95% CI = 1.17–23.58; p = 0.035). Although our findings may have an immediate relevance for public-health strategies, further large-size prospective studies should be encouraged to better understand the observed association and their causality.

Background Epigenetic regulation is important in hematopoiesis, but the involvement of histone variants is poorly understood. Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis. MacroH2A1.1 is a histone H2A variant that negatively correlates with the self-renewal capacity of embryonic, adult, and cancer stem cells. MacroH2A1.1 is a target of the frequent U2AF1 S34F mutation in MDS. The role of macroH2A1.1 in hematopoiesis is unclear. Results MacroH2A1.1 mRNA levels are significantly decreased in patients with low-risk MDS presenting with chromosomal 5q deletion and myeloid cytopenias and tend to be decreased in MDS patients carrying the U2AF1 S34F mutation. Using an innovative mouse allele lacking the macroH2A1.1 alternatively spliced exon, we investigated whether macroH2A1.1 regulates HSC homeostasis and differentiation. The lack of macroH2A1.1 decreased while macroH2A1.1 haploinsufficiency increased HSC frequency upon irradiation. Moreover, bone marrow transplantation experiments showed that both deficiency and haploinsufficiency of macroH2A1.1 resulted in enhanced HSC differentiation along the myeloid lineage. Finally, RNA-sequencing analysis implicated macroH2A1.1-mediated regulation of ribosomal gene expression in HSC homeostasis. Conclusions Together, our findings suggest a new epigenetic process contributing to hematopoiesis regulation. By combining clinical data with a discrete mutant mouse model and in vitro studies of human and mouse cells, we identify macroH2A1.1 as a key player in the cellular and molecular features of MDS. These data justify the exploration of macroH2A1.1 and associated proteins as therapeutic targets in hematological malignancies. Electronic supplementary material The online version of this article (10.1186/s13148-019-0724-z) contains supplementary material, which is available to authorized users.

MacroH2A is a histone variant whose expression has been studied in several neoplasms, including cutaneous melanomas (CMs). In the literature, it has been demonstrated that macroH2A.1 levels gradually decrease during CM progression, and a high expression of macroH2A.1 in CM cells relates to a better prognosis. Although both uveal and cutaneous melanomas arise from melanocytes, uveal melanoma (UM) is biologically and genetically distinct from the more common cutaneous melanoma. Metastasis to the liver is a frequent occurrence in UM, and about 40%&ndash, 50% of patients die of metastatic disease, even with early diagnosis, proper treatment, and close follow-up. We wanted to investigate macroH2A.1 immunohistochemical expression in UM. Our results demonstrated that mH2A.1 expression was higher in metastatic UM (21/23, 91.4%), while only 18/32 (56.3%). UMs without metastases showed mH2A.1 staining. These data could suggest a possible prognostic role for mH2A.1 and could form a basis for developing new pharmacological strategies for UM treatment.

Obesity and hypertension independently promote pathological left ventricular remodelling (LVR) and left ventricular hypertrophy (LVH), but to what extent they do so when they do not coexist is unclear. We used data from the Cardiovision Brno 2030 study to assess&mdash, for the first time in a region where no investigations have been previously carried out&mdash, the independent association of obesity and hypertension with LV geometry, and to evaluate the effects of hypertension in normal weight patients and the effects of obesity in normotensive patients. Overall, 433 individuals, aged 25&ndash, 65 years, with no history of cardiovascular disease and/or antihypertensive treatment, were stratified into four groups according to BMI and hypertension: normal weight non-hypertensive (NWNH), normal weight hypertensive (NWH), overweight/obese non-hypertensive (ONH) and overweight/obese hypertensive (OH). LVR was classified as normal, concentric LVR (cLVR), concentric LVH (cLVH) or eccentric LVH (eLVH). Linear regression analysis demonstrated that body mass index (BMI) and systolic blood pressure (SBP) are the main predictors of LV mass and that they interact: SBP had a stronger effect in overweight/obese (&beta, = 0.195, p = 0.033) compared to normal weight patients (&beta, = 0.134, p = 0.048). Hypertension increased the odds of cLVR (OR = 1.78, 95%CI = 1.04&ndash, 3.06, p = 0.037) and cLVH (OR = 8.20, 95% CI = 2.35&ndash, 28.66, p = 0.001), independent of age, sex and BMI. Stratified analyses showed that NWH had a greater odd of cLVH (OR = 7.96, 95%CI = 1.70&ndash, 37.08, p = 0.008) and cLVR (OR = 1.62, 95%CI = 1.02&ndash, 3.34, p = 0.047) than NWNH. In the absence of hypertension, obesity was not associated with LVM and abnormal LV geometry, suggesting that it is not per se a determinant of LVR. Thus, antihypertensive therapy still remains the first-line approach against LVH in hypertensive patients, though weight loss interventions might be helpful in those who are obese.

Lipid catabolism and anabolism changes play a role in stemness acquisition by cancer cells, and cancer stem cells (CSCs) are particularly dependent on the activity of the enzymes involved in these processes. Lipidomic changes could play a role in CSCs&rsquo, ability to cause disease relapse and chemoresistance. The exploration of lipid composition and metabolism changes in CSCs in the context of hepatocellular cancer (HCC) is still incomplete and their lipidomic scenario continues to be elusive. We aimed to evaluate through high-throughput mass spectrometry (MS)-based lipidomics the levels of the members of the six major classes of sphingolipids and phospholipids in two HCC cell lines (HepG2 and Huh-7) silenced for the expression of histone variant macroH2A1 (favoring stemness acquisition), or silenced for the expression of focal adhesion tyrosine kinase (FAK) (hindering aggressiveness and stemness). Transcriptomic changes were evaluated by RNA sequencing as well. We found definite lipidomic and transcriptomic changes in the HCC lines upon knockdown (KD) of macroH2A1 or FAK, in line with the acquisition or loss of stemness features. In particular, macroH2A1 KD increased total sphingomyelin (SM) levels and decreased total lysophosphatidylcholine (LPC) levels, while FAK KD decreased total phosphatidylcholine (PC) levels. In conclusion, in HCC cell lines knocked down for specific signaling/epigenetic processes driving opposite stemness potential, we defined a lipidomic signature that hallmarks hepatic CSCs to be exploited for therapeutic strategies.

Hepatic stellate cells (HSCs), also known as perisinusoidal cells, are pericytes found in the perisinusoidal space of the liver. HSCs are the major cell type involved in liver fibrosis, which is the formation of scar tissue in response to liver damage. When the liver is damaged, stellate cells can shift into an activated state, characterized by proliferation, contractility and chemotaxis. The activated HSCs secrete collagen scar tissue, which can lead to cirrhosis. Recent studies have shown that in vivo activation of HSCs by fibrogenic agents can eventually lead to senescence of these cells, which would contribute to reversal of fibrosis although it may also favor the insurgence of liver cancer. HSCs in their non-active form store huge amounts of retinoic acid derivatives in lipid droplets, which are progressively depleted upon cell activation in injured liver. Retinoic acid is a metabolite of vitamin A (retinol) that mediates the functions of vitamin A, generally required for growth and development. The precise function of retinoic acid and its alterations in HSCs has yet to be elucidated, and nonetheless in various cell types retinoic acid and its receptors (RAR and RXR) are known to act synergistically with peroxisome proliferator-activated receptor gamma (PPAR-gamma) signaling through the activity of transcriptional heterodimers. Here, we review the recent advancements in the understanding of how retinoic acid signaling modulates the fibrogenic potential of HSCs and proposes a synergistic combined action with PPAR-gamma in the reversal of liver fibrosis.

BACKGROUND: The parasympathetic nervous system (PNS), via neurotransmitter Acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear. AIMS: We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH). METHODS: Primary hHSC were analysed for synthesis of endogenous ACh and acetylcholinesterase (AChE), and gene expression of choline acetyltransferase (ChAT) and muscarinic acetylcholine receptors (mAChR). Cell proliferation and fibrogenic markers were analysed in hHSC exposed to ACh, Atropine (Atrop), Mecamylamine (Mec), methoctramine and 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP). MAChR expression was analysed in human NASH scored for fibrosis. RESULTS: We observed that hHSC synthesise ACh and AChE, and express ChAT and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and PI-3 K and MEK signalling pathways, as well as increased fibrogenic markers. CONCLUSION: We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the PI-3 K and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.

The Timeless (Tim) gene, originally identified in Drosophila melanogaster and subsequently in mammals, is involved in the molecular clockwork that drives 24h periodicity in physiology and behavior. The Tim protein is involved not only in circadian rhythmicity but also in embryonic development, cell cycle progression, DNA replication, and the DNA damage response (DDR). It is thus a multifaceted factor implicated in the maintenance of many cellular processes, tissue functions, and ultimately homeostasis of various organisms, from insects to humans. This review highlights the current knowledge of Tim functions, especially the most recent achievements, and illustrates the possible roles that this factor plays in the physiological preservation of health, as well as in the pathogenic mechanisms of related diseases.

Contains fulltext : 153710.pdf (Publisher’s version ) (Open Access) Despite numerous developed drugs based on glucose metabolism interventions for treatment of age-related diseases such as diabetes neuropathies (DNs), DNs are still increasing in patients with type 1 or type 2 diabetes (T1D, T2D). We aimed to identify novel candidates in adipose tissue (AT) and pancreas with T2D for targeting to develop new drugs for DNs therapy.AT-T2D displayed 15 (e.g. SYT4 up-regulated and VGF down-regulated) and pancreas-T2D showed 10 (e.g. BAG3 up-regulated, VAV3 and APOA1 down-regulated) highly differentially expressed genes with neuronal functions as compared to control tissues. ELISA was blindly performed to measure proteins of 5 most differentially expressed genes in 41 human subjects. SYT4 protein was upregulated, VAV3 and APOA1 were down-regulated, and BAG3 remained unchanged in 1- Obese and 2- Obese-T2D without insulin, VGF protein was higher in these two groups as well as in group 3- Obese-T2D receiving insulin than 4-lean subjects. Interaction networks analysis of these 5 genes showed several metabolic pathways (e.g. lipid metabolism and insulin signaling).Pancreas is a novel site for APOA1 synthesis. VGF is synthesized in AT and could be considered as good diagnostic, and even prognostic, marker for age-induced diseases obesity and T2D. This study provides new targets for rational drugs development for the therapy of age-related DNs.

In the European Union, Czech Republic ranks 3rd and 6th for the incidence of obesity and cardiovascular diseases, respectively. Worldwide, short sleep duration and excessive daytime sleepiness (EDS) characterize obese subjects, which in turn exhibit scarce physical activity and unhealthy diet. We aimed to understand the relationship between irregular sleep patterns, obesity and lifestyle factors, such as diet and physical activity, in a vulnerable Czech population. 1482 members of the Kardiovize cohort, a random sample of the Czech urban population, were included in a cross-sectional study. Exposure variables included self-reported sleep duration and EDS, assessed by the Epworth Sleepiness Scale. Primary outcomes were BMI and waist-to-hip ratio or prevalence of obesity and central obesity. Covariates included physical activity and diet. Associations and interactions between variables were evaluated using logistic regression analyses. After adjustment for covariates, short sleep duration (&lt, 7 h) was associated with greater odds of overweight (BMI &gt, 25, OR = 1.42, 95%CI = 1.06&ndash, 1.90, p = 0.020) and obesity (BMI &gt, 30, OR = 1.40, 95%CI = 1.02&ndash, 1.94, p = 0.047), while EDS was associated with greater odds of central obesity (OR = 1.72, 2.79, p = 0.030), independent of diet and physical activity. However, due to the cross-sectional nature of our study, further prospective, large-scale studies are needed to evaluate the etiological link and causality between sleep disturbances and obesity.

The biological clock controls at the molecular level several aspects of mammalian physiology, by regulating daily oscillations of crucial biological processes such as nutrient metabolism in the liver. Disruption of the circadian clock circuitry has recently been identified as an independent risk factor for cancer and classified as a potential group 2A carcinogen to humans. Hepatocellular carcinoma (HCC) is the prevailing histological type of primary liver cancer, one of the most important causes of cancer-related death worldwide. HCC onset and progression is related to B and C viral hepatitis, alcoholic and especially non-alcoholic fatty liver disease (NAFLD)-related milieu of fibrosis, cirrhosis, and chronic inflammation. In this review, we recapitulate the state-of-the-art knowledge on the interplay between the biological clock and the oncogenic pathways and mechanisms involved in hepatocarcinogenesis. Finally, we propose how a deeper understanding of circadian clock circuitry–cancer pathways’ crosstalk is promising for developing new strategies for HCC prevention and management.

Adipokines are important circulating factors mediating intertissue crosstalk throughout the body and thus playing a key role in maintaining endocrine homeostasis. So far, multiple associations of adipokines with widespread noncommunicable diseases, including cardiometabolic diseases, cancer, neurodegenerative diseases, and fertility problems, have been identified. However, the precise mechanisms underlying empirically observed associations are still rather poorly understood, and more research must be carried out in order to make sense of and deepen the already published data.

Hepatocellular carcinomas (HCCs) contain a sub-population of cancer stem cells (CSCs) that are responsible for tumor relapse, metastasis, and chemoresistance. We recently showed that loss of macroH2A1, a variant of the histone H2A and an epigenetic regulator of stem-cell function, in HCC leads to CSC-like features such as resistance to chemotherapeutic agents and growth of large and relatively undifferentiated tumors in xenograft models. These HCC cells silenced for macroH2A1 also exhibited stem-like metabolic changes consistent with enhanced glycolysis. However, there is no consensus as to the metabolic characteristics of CSCs that render them adaptable to microenvironmental changes by conveniently shifting energy production source or by acquiring intermediate metabolic phenotypes. Here, we assessed long-term proliferation, energy metabolism, and central carbon metabolism in human hepatoma HepG2 cells depleted in macroH2A1. MacroH2A1-depleted HepG2 cells were insensitive to serum exhaustion and showed two distinct, but interdependent changes in glucose and lipid metabolism in CSCs: (1) massive upregulation of acetyl-coA that is transformed into enhanced lipid content and (2) increased activation of the pentose phosphate pathway, diverting glycolytic intermediates to provide precursors for nucleotide synthesis. Integration of metabolomic analyses with RNA-Seq data revealed a critical role for the Liver X Receptor pathway, whose inhibition resulted in attenuated CSCs-like features. These findings shed light on the metabolic phenotype of epigenetically modified CSC-like hepatic cells, and highlight a potential approach for selective therapeutic targeting.

Maternal obesity increases offspring propensity to metabolic dysfunctions and to non-alcoholic fatty liver disease (NAFLD), which may lead to cirrhosis or liver cancer. The circadian clock is a transcriptional/epigenetic molecular machinery synchronising physiological processes to coordinate energy utilisation within a 24-h light/dark period. Alterations in rhythmicity have profound effects on metabolic pathways, which we sought to investigate in offspring with programmed NAFLD.Mice were fed a standard or an obesogenic diet (OD), before and throughout pregnancy, and during lactation. Offspring were weaned onto standard or an OD at 3 weeks postpartum and housed in 12:12 light/dark conditions. Biochemical and histological indicators of NAFLD and fibrosis, analysis of canonical clock genes with methylation status and locomotor activity were investigated at 6 months.We show that maternal obesity interacts with an obesogenic post-weaning diet to promote the development of NAFLD with disruption of canonical metabolic rhythmicity gene expression in the liver. We demonstrate hypermethylation of BMAL-1 (brain and muscle Arnt like-1) and Per2 promoter regions and altered 24-h rhythmicity of hepatic pro-inflammatory and fibrogenic mediators.These data implicate disordered circadian rhythms in NAFLD and suggest that disruption of this system during critical developmental periods may be responsible for the onset of chronic liver disease in adulthood.

Pancreatic cancer is a highly aggressive and chemotherapy-resistant malignant neoplasm. In basal condition, it is characterized by elevated autophagy activity, which is required for tumor growth and that correlates with treatment failure. We analyzed the expression of autophagy related genes in different pancreatic cancer cell lines. A correlation-based network analysis evidenced the sociality and topological roles of the autophagy-related genes after serum starvation. Structural and functional tests identified a core set of autophagy related genes, suggesting different scenarios of autophagic responses to starvation, which may be responsible for the clinical variations associated with pancreatic cancer pathogenesis.

Pancreatic cancer (PC), the fourth leading cause of cancer-related deaths, is characterized by high aggressiveness and resistance to chemotherapy. Pancreatic carcinogenesis is kept going by derangement of essential cell processes, such as proliferation, apoptosis, metabolism and autophagy, characterized by rhythmic variations with 24-h periodicity driven by the biological clock. We assessed the expression of the circadian genes ARNLT, ARNLT2, CLOCK, PER1, PER2, PER3, CRY1, CRY2 and the starvation-activated histone/protein deacetylase SIRT1 in 34 matched tumor and non-tumor tissue specimens of PC patients, and evaluated in PC derived cell lines if the modulation of SIRT1 expression through starvation could influence the temporal pattern of expression of the circadian genes. We found a significant down-regulation of ARNLT (p = 0.015), CRY1 (p = 0.013), CRY2 (p = 0.001), PER1 (p 0.0001), PER2 (p 0.001), PER3 (p = 0.001) and SIRT1 (p = 0.017) in PC specimens. PER3 and CRY2 expression levels were lower in patients with jaundice at diagnosis ( 0.05). Having adjusted for age, adjuvant therapy and tumor stage, we evidenced that patients with higher PER2 and lower SIRT1 expression levels showed lower mortality (p = 0.028). Levels and temporal patterns of expression of many circadian genes and SIRT1 significantly changed upon serum starvation in vitro, with differences among four different PC cell lines examined (BXPC3, CFPAC, MIA-PaCa-2 and PANC-1). Serum deprivation induced changes of the overall mean level of the wave and amplitude, lengthened or shortened the cycle time and phase-advanced or phase-delayed the rhythmic oscillation depending on the gene and the PC cell line examined. In conclusion, a severe deregulation of expression of SIRT1 and circadian genes was evidenced in the cancer specimens of PC patients, and starvation influenced gene expression in PC cell lines, suggesting that the altered interplay between SIRT1 and the core circadian proteins could represent a crucial player in the process of pancreatic carcinogenesis.

The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variant macroH2A1 under alternative splicing gives rise to macroH2A1.1 and macroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histone marks on their promoters. This study underlines the importance of the replacement of noncanonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis.

Maternal obesity predisposes offspring to metabolic dysfunction and Non-Alcoholic Fatty Liver Disease (NAFLD). Melanocortin-4 receptor (Mc4r)-deficient mouse models exhibit obesity during adulthood. Here, we aim to determine the influence of the Mc4r gene on the liver of mice subjected to perinatal diet-induced obesity. Female mice heterozygous for Mc4r fed an obesogenic or a control diet for 5 weeks were mated with heterozygous males, with the same diet continued throughout pregnancy and lactation, generating four offspring groups: control wild type (C_wt), control knockout (C_KO), obese wild type (Ob_wt), and obese knockout (Ob_KO). At 21 days, offspring were genotyped, weaned onto a control diet, and sacrificed at 6 months old. Offspring phenotypic characteristics, plasma biochemical profile, liver histology, and hepatic gene expression were analyzed. Mc4r_ko offspring showed higher body, liver and adipose tissue weights respect to the wild type animals. Histological examination showed mild hepatic steatosis in offspring group C_KO. The expression of hepatic genes involved in regulating inflammation, fibrosis, and immune cell infiltration were upregulated by the absence of the Mc4r gene. These results demonstrate that maternal obesogenic feeding during the perinatal period programs offspring obesity development with involvement of the Mc4r system.

Background Atherosclerotic cardiovascular disease is highly prevalent in Eastern and Central Europe, where the incidence is the highest in the world. The Kardiovize Brno 2030 study was designed as a prospective cohort study to investigate the complex relationships of cardiovascular disease and outcomes with a range of biological, psychosocial, environmental, behavioral, and economic factors in an urban population of the Czech Republic. Methods We randomly selected a 1% sample of the city of Brno residents aged 25–64 years stratified by sex and age. The study assessed traditional and novel cardiovascular disease risk factors, including sociodemographic and smoking status, physical activity, diet, depression, stress, body fat, cardio-ankle vascular index, and intima media thickness, complemented by blood tests; biological samples were stored for future analyses. Results The study enrolled 2160 participants (54.8% women), with a mean age of 47 ± 11.3 years. They were mostly full-time employed (75.6%) and married (62.1%). Hyperlipidemia was highly prevalent (70.7% in men, and 67.1% in women, NS). Hypertension and diabetes mellitus were more prevalent in men than in women (54.3% vs. 38.7% and 7.1% vs. 3.5%, respectively, P Conclusions Cardiovascular risk factors are highly prevalent in the city of Brno, an urban population from Central Europe. The Kardiovize Brno 2030 study will provide unique multidimensional and longitudinal cardiovascular health data from a region where epidemiological studies are scarce.

ispartof: Progress in preventive medicine pages:1-2 status: published

Hepatocellular carcinomas (HCC) contain a subpopulation of cancer stem cells (CSCs), which exhibit stem cell–like features and are responsible for tumor relapse, metastasis, and chemoresistance. The development of effective treatments for HCC will depend on a molecular-level understanding of the specific pathways driving CSC emergence and stemness. MacroH2A1 is a variant of the histone H2A and an epigenetic regulator of stem-cell function, where it promotes differentiation and, conversely, acts as a barrier to somatic-cell reprogramming. Here, we focused on the role played by the histone variant macroH2A1 as a potential epigenetic factor promoting CSC differentiation. In human HCC sections we uncovered a significant correlation between low frequencies of macroH2A1 staining and advanced, aggressive HCC subtypes with poorly differentiated tumor phenotypes. Using HCC cell lines, we found that short hairpin RNA–mediated macroH2A1 knockdown induces acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolysis. The loss of macroH2A1 increased expression of a panel of stemness-associated genes and drove hyperactivation of the nuclear factor kappa B p65 pathway. Blocking phosphorylation of nuclear factor kappa B p65 on Ser536 inhibited the emergence of CSC-like features in HCC cells knocked down for macroH2A1. Conclusion: The absence of histone variant macroH2A1 confers a CSC-like phenotype to HCC cells in vitro and in vivo that depends on Ser536 phosphorylation of nuclear factor kappa B p65; this pathway may hold valuable targets for the development of CSC-focused treatments for HCC. (Hepatology 2018;67:636-650).

BACKGROUND: Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. METHODS: 24 hour fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. 24 hours fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. RESULTS: Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short-term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. CONCLUSIONS: Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use. This article is protected by copyright. All rights reserved

The liver plays a major role in lipid metabolism, importing free fatty acids (FFA) and manufacturing, storing and exporting lipids: derangements in any of these processes can lead to non-alcoholic fatty liver disease (NAFLD) [1]. NAFLD can be seen as the result of an imbalance between lipid availability and lipid disposal resulting in hepatic steatosis [2]. NAFLD is considered by many as the hepatic manifestation of insulin resistance (IR) and is strongly associated with the metabolic syndrome [3]. The rapid increase in obesity and diabetes mellitus (DM) during the last decade is associated with an increase in the prevalence of NAFLD, making it the most common cause of chronic liver disease in the Western countries with up to 30% of the population affected [4]. Histological evaluation is the gold standard for precisely estimating the degree of liver damage caused by simple steatosis or by non-alcoholic steatohepatitis (NASH).

Valproic acid (VPA) is an anti-epileptic and mood-stabilizing compound successfully used in the clinics since many decades. During the last few years, research on VPA revitalized. VPA has profound impact on nuclear chromatin structure in target cells by impinging on epigenetic mechanisms such as inhibition of histone deacetylase HDAC1, with implications for HIV and cancer treatment, and for the direct reprogramming in generation of induced pluripotent stem (iPS) cells. VPA can thus act at multiple levels and in several cellular systems. In addition to its established applications for the treatment of neurological and psychiatric disorders, and its newly discovered epigenetic mechanisms of action, the peripheral metabolic effects of VPA administration, in particular impinging on the heart and on the liver, are now starting to be understood. These have important consequences for the management of therapy and also for investigation purposes. The aim of this article is on one hand to summarize the emerging knowledge on the role of VPA during the occurrence of cardio-metabolic dysfunctions and on the other hand to describe concisely the VPA-induced epigenetic modifications in target cells.

Item does not contain fulltext Non-alcoholic-fatty-liver-disease (NAFLD) encompasses conditions associated to fat deposition in the liver, which are generally deteriorated during the aging process. MacroH2A1, a variant of histone H2A, is a key transcriptional regulator involved in tumorigenic processes and cell senescence, and featuring two alternatively splicing isoforms, macroH2A1.1 and macroH2A1.2. MacroH2A1.1 binds with high affinity O-acetyl ADP ribose, a small metabolite produced by the reaction catalysed by NAD+-dependent deacetylase SIRT1, whereas macroH2A1.2 is unable to do so. The functional significance of this binding is unknown. We previously reported that the hepatic levels of macroH2A1.1 and macroH2A1.2 are differentially expressed in mice models of NAFLD. Here we show that over-expression of macroH2A1.1, but not of macroH2A1.2, is able to protect hepatocytes against lipid accumulation. MacroH2A1.1 over-expressing cells display ameliorated glucose metabolism, reduced expression of lipidogenic genes and fatty acids content. SIRT1/macroH2A1.1-dependent epigenetic regulation of lipid metabolism may be relevant to NAFLD development. 01 januari 2014

We investigated the regulation of hepatic ER stress in healthy liver and adult or perinatally programmed diet-induced non-alcoholic fatty liver disease (NAFLD). Female mice were fed either obesogenic or control diet before mating, during pregnancy and lactation. Post-weaning, offspring from each maternal group were divided into either obesogenic or control diet. At six months, offspring were sacrificed at 4-h intervals over 24 h. Offspring fed obesogenic diets developed NAFLD phenotype, and the combination of maternal and offspring obesogenic diets exacerbated this phenotype. UPR signalling pathways (IREα, PERK, ATF6) and their downstream regulators showed different basal rhythmicity, which was modified in offspring exposed to obesogenic diet and maternal programming. The double obesogenic hit increased liver apoptosis measured by TUNEL staining, active caspase-3 and phospho-JNK and GRP78 promoter methylation levels. This study demonstrates that hepatic UPR is rhythmically activated. The combination of maternal obesity (MO) and obesogenic diets in offspring triggered altered UPR rhythmicity, DNA methylation and cellular apoptosis.

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-β1 in tubular epithelial cells and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS.

The circadian system controls the behavior and multiple physiological functions. In mammals, the suprachiasmatic nucleus (SCN) acts as the master pacemaker and regulates the circadian clocks of peripheral tissues. The SCN receives information regarding the light-dark cycle and is thus synchronized to the external 24-hour environment. In contrast, peripheral clocks, such as the liver clock, receive information from the SCN and other factors; in particular, food intake which leads to insulin secretion induces strong entrainment of the liver clock. On the other hand, the liver clock of insulin-depleted mice treated with streptozotocin (STZ) has been shown to be entrained by scheduled feeding, suggesting that insulin is not necessary for entrainment of the liver clock by feeding. In this study, we aimed to elucidate additional mechanism on entraining liver clock by feeding a protein-only diet and/or amino-acid administration which does not increase insulin levels. We demonstrated that protein-only diet and cysteine administration elicit entrainment of the liver clock via glucagon secretion and/or insulin-like growth factors (IGF-1) production. Our findings suggest that glucagon and/or IGF-1 production are additional key factors in food-induced entrainment., Highlights • Dietary protein or cysteine increase serum glucagon and hepatic IGF-1 levels, and entrain liver circadian rhythm. • Increasing IGF-1 levels is an additional entrainment factor of liver circadian rhythm. • Hepatic IGF-1 production is found to be a key factor in the entrainment of liver circadian rhythm in STZ-treated mice. Disruption of the circadian rhythm leads to multiple disorders; thus the maintenance of circadian oscillation is necessary for maintaining normalized physiological functions. Postprandial insulin secretion is known as an entraining factor of peripheral circadian rhythm; however, this pathway is not appropriate for diabetes patients in whom insulin signaling is disrupted. Here we report that both dietary protein and cysteine alone entrain liver circadian rhythm by increasing glucagon and/or IGF-1 levels independently of insulin. Findings indicate an additional entrainment factor that can be applied to chronotherapy by controlling food content or by supplementation in peoples with diabetes, circadian rhythm disorders.

Gastrointestinal cancers (GC) are malignancies involving the gastrointestinal (GI) tract and accessory organs of the digestive system, including the pancreas, liver, and gall bladder. GC is one of the most common cancers and contributes to more cancer-related deaths than cancers of any other system in the human body. Causative factors of GC have been consistently attributed to infections, smoking, an unhealthy diet, obesity, diabetes, and genetic factors. More recently, aberrant epigenetic regulation of gene expression has emerged as a new, fundamental pathway in GC pathogenesis. In this review, we summarize the role of the macroH2A histone family in GI cell function and malignant transformation, and highlight how this histone family may open up novel biomarkers for cancer detection, prediction, and response to treatment.

The liver is the only internal human organ capable of natural regeneration of lost tissue, as little as 25% of a liver can regenerate into a whole liver. The process of aging predisposes to hepatic functional and structural impairment and metabolic risk. Therefore, understanding how aging could affect the molecular pathology of liver diseases is particularly important, and few studies to date have tackled this complex process. The most common liver disease, affecting one-third of the overall population, is nonalcoholic fatty liver disease (NAFLD), characterized by an intrahepatic accumulation of lipids. NAFLD can evolve into nonalcoholic steatohepatitis (NASH) in the presence of oxidative stress and inflammation. NASH is a serious risk factor for disabling and deadly liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). Old age seems to favor NAFLD, NASH, and ultimately HCC, in agreement with the inflamm-aging theory, according to which aging accrues inflammation. However, the incidence of HCC drops significantly in the very elderly (individuals aged more than 70) and the relationship between the progression of NAFLD/NASH/HCC and very old age is obscure. In this review, we discuss the literature and we argue that there might be an age window in which the liver becomes resistant to the development of injury; this needs to be studied to understand fully the interaction between age and liver diseases from a therapeutic perspective.

Chronic hepatitis C patients display many genotype-specific clinical features of HCV infection. The core proteins encoded by different genotypes dysregulate numerous sets of distinct host genes. In this study we tested the hypothesis that HCV core proteins 1b and 3a would actually act on a limited number of independent cellular players, as well as on several functionally linked gene products. Structural and functional tests identified a core set of host genes dysregulated by HCV core genotypes 1b and 3a. The core proteins of HCV genotypes 1b and 3a target specifically limited sets of functionally related gene products, which may be responsible for the variations in the clinical spectra associated with HCV infection.

The physiological processes that maintain body homeostasis oscillate during the day. Diurnal changes characterize kidney functions, comprising regulation of hydro-electrolytic and acid-base balance, reabsorption of small solutes and hormone production. Renal physiology is characterized by 24-h periodicity and contributes to circadian variability of blood pressure levels, related as well to nychthemeral changes of sodium sensitivity, physical activity, vascular tone, autonomic function and neurotransmitter release from sympathetic innervations. The circadian rhythmicity of body physiology is driven by central and peripheral biological clockworks and entrained by the geophysical light/dark cycle. Chronodisruption, defined as the mismatch between environmental-social cues and physiological-behavioral patterns, causes internal desynchronization of periodic functions, leading to pathophysiological mechanisms underlying degenerative, immune related, metabolic and neoplastic diseases. In this review we will address the genetic, molecular and anatomical elements that hardwire circadian rhythmicity in renal physiology and subtend disarray of time-dependent changes in renal pathology.

The circadian clock machinery orchestrates organism metabolism in order to ensure that development, survival and reproduction are attuned to diurnal environmental variations. For unknown reasons, there is a decline in circadian rhythms with age, concomitant with declines in the overall metabolic tissues homeostasis and changes in the feeding behavior of aged organisms. This disruption of the relationship between the clock and the nutrient sensing networks might underlie age-related diseases; overall, greater knowledge of the molecular mediators of and variations in clock networks during lifespan may shed light on the aging process and how it may be delayed. In this review we address the complex links between the circadian clock, metabolic (dys)functions and aging in different model organisms.