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Background: Hepatitis C virus infects ~3% of the population and it is a risk factor for hepatocarcinogenesis. The epigenetic mechanisms of HCV-induced hepatocyte transformation towards malignancy in this context are unclear.Aims: The purpose of this study was to evaluate the effect of HCV core proteins of different genotypes on DNA methyltransferases (DNMTs) induction.Materials/methods: We investigated DNMT1, DNMT3b and E-Cadherin (CDH1) mRNA and protein expression levels in an in vitro model of Huh-7 cells expressing the HCV core protein of different genotypes: 1b, 2a, 3a, 4h and 5a.Results: We found that both mRNA and protein expression levels of DNMT1 and 3b were upregulated in genotype 1b HCV core expressing cells as compared to control cells. DNMT3b mRNA levels did not change in genotypes 2a, 3a, 4h and 5a, but were upregulated at the protein level by genotype 1b, 2a, 3a. CDH1 mRNA expression was downregulated only in genotype 1b, whereas its protein expression resulted in downregulation by the HCV core of genotypes 1b, 2a and 3a. Conversely, no significant changes were observed for DNMTs and CDH1 investigated in Huh-7 cells expressing the genotypes 4h and 5a. Furthermore, we present evidence that HCV core 1b protein expression induces DNMTs overexpression through STAT3 protein as demonstrated by NSC74859 treatment. Moreover, SIRT1 inhibition affected DNMT1 and 3b expression only in HCV core protein genotype 1b expressing cells as demonstrated by treatment with its inhibitor sirtinol.Conclusions: Our findings suggest that HCV core protein could play a role in HCC development at least in part by altering DNMTs expression. [ABSTRACT FROM AUTHOR]

Several laboratories, including ours, have employed the Slc25a47 tm1c(EUCOMM)Hmgu mouse model to investigate the role of SLC25A47, a hepatocyte-specific mitochondrial carrier, in regulating hepatic metabolism and systemic physiology. In this study, we reveal that the hepatic and systemic phenotypes observed following recombination of the Slc25a47-Wars1 locus in hepatocytes are primarily driven by the unexpected downregulation of Wars1, the cytosolic tryptophan aminoacyl-tRNA synthetase located adjacent to Slc25a47. While the downregulation of Wars1 predictably affects cytosolic translation, we also observed a significant impairment in mitochondrial protein synthesis within hepatocytes. This disturbance in mitochondrial function leads to an activation of the mitochondrial unfolded protein response (UPR mt ), a critical component of the mitochondrial stress response (MSR). Our findings clarify the distinct roles of Slc25a47 and Wars1 in maintaining both systemic and hepatic metabolic homeostasis. This study sheds new light on the broader implications of aminoacyl-tRNA synthetases in mitochondrial physiology and stress responses., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Cellular metabolism is highly dynamic during hematopoiesis, yet the regulatory networks that maintain metabolic homeostasis during differentiation are incompletely understood. Herein, we have studied the grave immunodeficiency syndrome reticular dysgenesis caused by loss of mitochondrial adenylate kinase 2 (AK2) function. By coupling single-cell transcriptomics in samples from patients with reticular dysgenesis with a CRISPR model of this disorder in primary human hematopoietic stem cells, we found that the consequences of AK2 deficiency for the hematopoietic system are contingent on the effective engagement of metabolic checkpoints. In hematopoietic stem and progenitor cells, including early granulocyte precursors, AK2 deficiency reduced mechanistic target of rapamycin (mTOR) signaling and anabolic pathway activation. This conserved nutrient homeostasis and maintained cell survival and proliferation. In contrast, during late-stage granulopoiesis, metabolic checkpoints were ineffective, leading to a paradoxical upregulation of mTOR activity and energy-consuming anabolic pathways such as ribonucleoprotein synthesis in AK2-deficient cells. This caused nucleotide imbalance, including highly elevated adenosine monophosphate and inosine monophosphate levels, the depletion of essential substrates such as NAD+ and aspartate, and ultimately resulted in proliferation arrest and demise of the granulocyte lineage. Our findings suggest that even severe metabolic defects can be tolerated with the help of metabolic checkpoints but that the failure of such checkpoints in differentiated cells results in a catastrophic loss of homeostasis., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Susceptibility to metabolic syndrome (MetS) is dependent on genetics, environment, and gene-by-environment interactions, rendering the study of underlying mechanisms challenging. The majority of experiments in model organisms do not incorporate genetic variation and lack specific evaluation criteria for MetS. Here, we derived a continuous metric, the metabolic health score (MHS), based on standard clinical parameters and defined its molecular signatures in the liver and circulation. In human UK Biobank, the MHS associated with MetS status and was predictive of future disease incidence, even in individuals without MetS. Using quantitative trait locus analyses in mice, we found two MHS-associated genetic loci and replicated them in unrelated mouse populations. Through a prioritization scheme in mice and human genetic data, we identified TNKS and MCPH1 as candidates mediating differences in the MHS. Our findings provide insights into the molecular mechanisms sustaining metabolic health across species and uncover likely regulators. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inflammatory gut disorders, including inflammatory bowel disease (IBD), can be impacted by dietary, environmental, and genetic factors. While the incidence of IBD is increasing worldwide, we still lack a complete understanding of the gene-by-environment interactions underlying inflammation and IBD. Here, we profiled the colon transcriptome of 52 BXD mouse strains fed with a chow or high-fat diet (HFD) and identified a subset of BXD strains that exhibit an IBD-like transcriptome signature on HFD, indicating that an interplay of genetics and diet can significantly affect intestinal inflammation. Using gene co-expression analyses, we identified modules that are enriched for IBD-dysregulated genes and found that these IBD-related modules share cis -regulatory elements that are responsive to the STAT2, SMAD3 , and REL transcription factors. We used module quantitative trait locus analyses to identify genetic loci associated with the expression of these modules. Through a prioritization scheme involving systems genetics in the mouse and integration with external human datasets, we identified Muc4 and Epha6 as the top candidates mediating differences in HFD-driven intestinal inflammation. This work provides insights into the contribution of genetics and diet to IBD risk and identifies two candidate genes, MUC4 and EPHA6 , that may mediate IBD susceptibility in humans., Competing Interests: XL, JM, GB, JP, AB, AR, JS, EW, AP, KS, MB, JA No competing interests declared, (© 2023, Li et al.)

Non-alcoholic steatohepatitis (NASH) is a global health concern without treatment. The challenge in finding effective therapies is due to the lack of good mouse models and the complexity of the disease, characterized by gene-environment interactions. We tested the susceptibility of seven mouse strains to develop NASH. The severity of the clinical phenotypes observed varied widely across strains. PWK/PhJ mice were the most prone to develop hepatic inflammation and the only strain to progress to NASH with extensive fibrosis, while CAST/EiJ mice were completely resistant. Levels of mitochondrial transcripts and proteins as well as mitochondrial function were robustly reduced specifically in the liver of PWK/PhJ mice, suggesting a central role of mitochondrial dysfunction in NASH progression. Importantly, the NASH gene expression profile of PWK/PhJ mice had the highest overlap with the human NASH signature. Our study exposes the limitations of using a single mouse genetic background in metabolic studies and describes a novel NASH mouse model with features of the human NASH., (© 2023 Benegiamo et al.)

Objective: Improving mitochondrial function is a promising strategy for intervention in type 2 diabetes mellitus. This study investigated the preventive effects of sodium ferrous citrate (SFC) and 5-aminolevulinic acid phosphate (ALA) on several metabolic dysfunctions associated with obesity because they have been shown to alleviate abnormal glucose metabolism in humans., Methods: Six-week-old male C57BL/6J mice were fed with a normal diet, a high-fat diet, or a high-fat diet supplemented with SFC and ALA for 15 weeks., Results: The simultaneous supplementation of SFC + ALA to high-fat diet-fed mice prevented loss of muscle mass, improved muscle strength, and reduced obesity and insulin resistance. SFC + ALA prevented abnormalities in mitochondrial morphology and reverted the diet effect on the skeletal muscle transcriptome, including the expression of glucose uptake and mitochondrial oxidative phosphorylation-related genes. In addition, SFC + ALA prevented the decline in mitochondrial DNA copy number by enhancing mitochondrial DNA maintenance and antioxidant transcription activity, both of which are impaired in high-fat diet-fed mice during long-term fasting., Conclusions: These findings suggest that SFC + ALA supplementation exerts its preventive effects in type 2 diabetes mellitus via improved skeletal muscle and mitochondrial health, further validating its application as a promising strategy for the prevention of obesity-induced metabolic disorders., (© 2023 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society.)

Mitochondrial respiratory complexes form superassembled structures called supercomplexes. COX7A2L is a supercomplex-specific assembly factor in mammals, although its implication for supercomplex formation and cellular metabolism remains controversial. Here we identify a role for COX7A2L for mitochondrial supercomplex formation in humans. By using human cis-expression quantitative trait loci data, we highlight genetic variants in the COX7A2L gene that affect its skeletal muscle expression specifically. The most significant cis-expression quantitative trait locus is a 10-bp insertion in the COX7A2L 3' untranslated region that increases messenger RNA stability and expression. Human myotubes harboring this insertion have more supercomplexes and increased respiration. Notably, increased COX7A2L expression in the muscle is associated with lower body fat and improved cardiorespiratory fitness in humans. Accordingly, specific reconstitution of Cox7a2l expression in C57BL/6J mice leads to higher maximal oxygen consumption, increased lean mass and increased energy expenditure. Furthermore, Cox7a2l expression in mice is induced specifically in the muscle upon exercise. These findings elucidate the genetic basis of mitochondrial supercomplex formation and function in humans and show that COX7A2L plays an important role in cardiorespiratory fitness, which could have broad therapeutic implications in reducing cardiovascular mortality., (© 2022. The Author(s).)

The sharp increase in obesity prevalence worldwide is mainly attributable to changes in physical activity and eating behavior but the metabolic and clinical impacts of these obesogenic conditions vary between sexes and genetic backgrounds. This warrants personalized treatments of obesity and its complications, which require a thorough understanding of the diversity of metabolic responses to high-fat diet intake. By analyzing nine genetically diverse mouse strains, we show that much like humans, mice exhibit a huge variety of physiological and biochemical responses to high-fat diet. The strains exhibit various degrees of alterations in their phenotypic makeup. At the transcriptome level, we observe dysregulations of immunity, translation machinery, and mitochondrial genes. At the biochemical level, the enzymatic activity of mitochondrial complexes is affected. The diversity across mouse strains, diets, and sexes parallels that found in humans and supports the use of diverse mouse populations in future mechanistic or preclinical studies on metabolic dysfunctions., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Skeletal muscle displays remarkable plasticity upon exercise and is also one of the organs most affected by aging. Despite robust evidence that aging is associated with loss of fast-twitch (type II) muscle fibers, the underlying mechanisms remain to be elucidated. Here, we identified an exercise-induced long noncoding RNA, CYTOR , whose exercise responsiveness was conserved in human and rodents. Cytor overexpression in mouse myogenic progenitor cells enhanced myogenic differentiation by promoting fast-twitch cell fate, whereas Cytor knockdown deteriorated expression of mature type II myotubes. Skeletal muscle Cytor expression was reduced upon mouse aging, and Cytor expression in young mice was required to maintain proper muscle morphology and function. In aged mice, rescuing endogenous Cytor expression using adeno-associated virus serotype 9 delivery of CRISPRa reversed the age-related decrease in type II fibers and improved muscle mass and function. In humans, CYTOR expression correlated with type II isoform expression and was decreased in aged myoblasts. Increased CYTOR expression, mediated by a causal cis–expression quantitative trait locus located within a CYTOR skeletal muscle enhancer element, was associated with improved 6-min walk performance in aged individuals from the Helsinki Birth Cohort Study. Direct CYTOR overexpression using CRISPRa in aged human donor myoblasts enhanced expression of type II myosin isoforms. Mechanistically, Cytor reduced chromatin accessibility and occupancy at binding motifs of the transcription factor Tead1 by binding, and hence sequestering, Tead1 . In conclusion, the long noncoding RNA Cytor was found to be a regulator of fast-twitch myogenesis in aging.

Asperuloside (ASP) is an iridoid glycoside that is extracted from Eucommia leaves. Eucommia is used in traditional Chinese medicine and has a long history of benefits on health and longevity. Here, we investigated the impact of ASP on obesity-related metabolic disorders and show that ASP reduces body weight gain, glucose intolerance, and insulin resistance effectively in mice fed with a high-fat diet (HFD). Intestinal dysbiosis is closely linked with metabolic disorders. Our data indicate that ASP achieves these benefits on metabolic homeostasis by reversing HFD-induced gut dysbiosis and by changing gut-derived secondary metabolites and metabolic signaling. Our results indicate that ASP may be used to regulate gut microbiota for the treatment of obesity and type 2 diabetes., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Mutations in nuclear-encoded mitochondrial genes are responsible for a broad spectrum of disorders among which Leigh syndrome is the most common in infancy. No effective therapies are available for this severe disease mainly because of the limited capabilities of the standard adeno-associated viral (AAV) vectors to transduce both peripheral organs and the CNS when injected systemically in adults. Here, we used the brain-penetrating AAV-PHP.B vector to reinstate gene expression in the Ndufs4 knockout mouse model of Leigh syndrome. Intravenous delivery of an AAV.PHP.B-Ndufs4 vector in 1-month-old knockout mice restored mitochondrial complex I activity in several organs including the CNS. This gene replacement strategy extended lifespan, rescued metabolic parameters, provided behavioural improvement, and corrected the pathological phenotype in the brain, retina, and heart of Ndufs4 knockout mice. These results provide a robust proof that gene therapy strategies targeting multiple organs can rescue fatal neurometabolic disorders with CNS involvement., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: Treatment of rectocele associated with prolapsed hemorrhoids is a debated topic. Transanal stapling achieved good midterm results in patients with symptoms of obstructed defecation, nevertheless a number of severe complications have been reported. The aim of this study was to evaluate the safety and efficacy of a new endorectal manual technique in patients with obstructed defecation due to the combination of muco-hemorrhoidal prolapse and rectocele., Methods: Patients enrolled after preoperative obstructed defecation syndrome (ODS) score, defecography and anoscopy were submitted to the novel Mucopexy-Recto Anal Lifting (MuRAL) combined with a modified Block procedure, and followed up by independent observers with digital exploration 3 weeks postoperatively, and digital exploration plus anoscopy at 3, 6, and 12 months. Operative time, hospital stay, numerating rating scale (NRS), ODS, satisfaction scores, and recurrence rate were recorded., Results: Mean operative time was 35.7 minutes. Fifty-six patients completed 1-year follow-up: 7.1% had acute urinary retention, NRS score was < 3 from the third postoperative day, mean time of daily activity resumption was 12 days, none had persistent fecal urgency, 82% declared excellent/good satisfaction score, significant improvement of 6- and 12-month ODS score, no recurrence of rectocele, and 7.1% recurrence of prolapsed hemorrhoids were observed., Conclusion: MuRAL associated with modified Block technique gave no severe complications and resulted in a safe and effective approach to symptomatic rectocele associated with muco-rectal prolapse. Further randomized studies, larger series, and longer follow-up are needed., Competing Interests: Conflict of Interest: No potential conflict of interest relevant to this article was reported., (Copyright © 2020, the Korean Surgical Society.)

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are indispensable for non-image-forming visual responses that sustain under prolonged illumination. For sustained signaling of ipRGCs, the melanopsin photopigment must continuously regenerate. The underlying mechanism is unknown. We discovered that a cluster of Ser/Thr sites within the C-terminal region of mammalian melanopsin is phosphorylated after a light pulse. This forms a binding site for β-arrestin 1 (βARR1) and β-arrestin 2. β-arrestin 2 primarily regulates the deactivation of melanopsin; accordingly, βαrr2 -/- mice exhibit prolonged ipRGC responses after cessation of a light pulse. β-arrestin 1 primes melanopsin for regeneration. Therefore, βαrr1 -/- ipRGCs become desensitized after repeated or prolonged photostimulation. The lack of either β-arrestin attenuates ipRGC response under prolonged illumination, suggesting that β-arrestin 2-mediated deactivation and β-arrestin 1-dependent regeneration of melanopsin function in sequence. In conclusion, we discovered a molecular mechanism by which β-arrestins regulate different aspects of melanopsin photoresponses and allow ipRGC-sustained responses under prolonged illumination., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Conservative surgery of hemorrhoidal disease is less painful than traditional hemorrhoidectomy, and mucopexy has less risk of serious postoperative complications than stapled hemorrhoidopexy. The aim of this study was to evaluate the safety and effectiveness of a standardized, modified hemorrhoidopexy, named Mucopexy-Recto Anal Lifting (MuRAL) with the HemorPex System (HPS) in patients with symptomatic III and IV degree hemorrhoids., Methods: Patients were enrolled from May 2013 to Dec 2015 and operated on with the MuRAL technique, based on arterial ligation and mucopexy at 6 locations, using a standardized clockwise/anti-clockwise rotation sequence of the HPS anoscope. Follow-up controls were carried out by independent observers, as follows: a digital exploration 3 weeks after the intervention, digital exploration plus proctoscopy at 3 and 12 months and repeated at a 12 months interval. Patients who did not strictly follow the postoperative controls were excluded from the study. Primary outcome measurement was the recurrence rate. Secondary measurements were: operative time, hospital stay, postoperative pain, postoperative symptoms and satisfaction score., Results: We operated on 126 patients (72 males, mean age 53.9, range 29-83): 87 (69.6%) with III degree and 39 with IV degree hemorrhoids; 13 patients had a MuRAL as a revisional procedure of a previous operation for hemorrhoids. Mean duration of follow-up was 554 days (range 281-1219). Four patients were excluded from the study. One-year recurrence rate was 4.1%. The mean duration of the intervention was 29.5 minutes (range 23-60) and 92 patients (73%) were discharged during the same day of the operation. Pain VAS Score in the first, second and third postoperative day was 3.9, 2.5, and 1.9, respectively. Twenty-two patients (18%), all submitted to spinal anesthesia, had postoperative acute urinary retention. Fecal urgency, observed in 18.8% of patients at the first control, disappeared within one year after the operation. Mean time to return to normal activity was 8 days (range 5 -10). The patient satisfaction scores at one-year follow up were 31.1% excellent, 57.4% good, 7.4% fairly good and 4.1% poor. In patients with III degree hemorrhoids operative time was significantly shorter, postoperative pain better and transient fecal urgency lower than in IV degree patients. In our experience the standardization of MuRAL operation with HPS, turned out to be a safe and effective minimally invasive approach in managing symptomatic III and IV degree hemorrhoids, avoiding the risk of severe complications, with the possibility to perform a redo-MuRAL in the event of recurrence., Conclusions: In our series up to 88% of the patients reported a good, or excellent one-year satisfaction score. Further comparative randomized studies with longer follow-up period are needed.

Diurnal gene expression patterns underlie time-of-the-day-specific functional specialization of tissues. However, available circadian gene expression atlases of a few organs are largely from nocturnal vertebrates. We report the diurnal transcriptome of 64 tissues, including 22 brain regions, sampled every 2 hours over 24 hours, from the primate Papio anubis (baboon). Genomic transcription was highly rhythmic, with up to 81.7% of protein-coding genes showing daily rhythms in expression. In addition to tissue-specific gene expression, the rhythmic transcriptome imparts another layer of functional specialization. Most ubiquitously expressed genes that participate in essential cellular functions exhibit rhythmic expression in a tissue-specific manner. The peak phases of rhythmic gene expression clustered around dawn and dusk, with a "quiescent period" during early night. Our findings also unveil a different temporal organization of central and peripheral tissues between diurnal and nocturnal animals., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The mechanisms by which feeding and fasting drive rhythmic gene expression for physiological adaptation to daily rhythm in nutrient availability are not well understood. Here we show that, upon feeding, the RNA-binding protein NONO accumulates within speckle-like structures in liver cell nuclei. Combining RNA-immunoprecipitation and sequencing (RIP-seq), we find that an increased number of RNAs are bound by NONO after feeding. We further show that NONO binds and regulates the rhythmicity of genes involved in nutrient metabolism post-transcriptionally. Finally, we show that disrupted rhythmicity of NONO target genes has profound metabolic impact. Indeed, NONO-deficient mice exhibit impaired glucose tolerance and lower hepatic glycogen and lipids. Accordingly, these mice shift from glucose storage to fat oxidation, and therefore remain lean throughout adulthood. In conclusion, our study demonstrates that NONO post-transcriptionally coordinates circadian mRNA expression of metabolic genes with the feeding/fasting cycle, thereby playing a critical role in energy homeostasis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The circadian oscillator is based on transcription-translation feedback loops that generate 24 h oscillations in gene expression. Although circadian regulation of mRNA expression at the transcriptional level is one of the most important steps for the generation of circadian rhythms within the cell, multiple lines of evidence point to a disconnect between transcript oscillation and protein oscillation. This can be explained by regulatory RNA-binding proteins acting on the nascent transcripts to modulate their processing, export, translation and degradation rates. In this chapter we will review what is known about the different steps involved in circadian gene expression from transcription initiation to mRNA stability and translation efficiency. The role of ribonucleoprotein particles in the generation of rhythmic gene expression is only starting to be elucidated, but it is likely that they cooperate with the basal transcriptional machinery to help to maintain the precision of the clock under diverse cellular and environmental conditions.

Introduction: We previously demonstrated that HER2/neu-driven mammary carcinogenesis can be prevented by an interleukin-12 (IL-12)-adjuvanted allogeneic HER2/neu-expressing cell vaccine. Since IL-12 can induce the release of interleukin-15 (IL-15), in the present study we investigated the role played by IL-15 in HER2/neu driven mammary carcinogenesis and in its immunoprevention., Methods: HER2/neu transgenic mice with homozygous knockout of IL-15 (here referred to as IL15KO/NeuT mice) were compared to IL-15 wild-type HER2/neu transgenic mice (NeuT) regarding mammary carcinogenesis, profile of peripheral blood lymphocytes and splenocytes and humoral and cellular responses induced by the vaccine., Results: IL15KO/NeuT mice showed a significantly earlier mammary cancer onset than NeuT mice, with median latency times of 16 and 20 weeks respectively, suggesting a role for IL-15 in cancer immunosurveillance. Natural killer (NK) and CD8+ lymphocytes were significantly lower in IL15KO/NeuT mice compared to mice with wild-type IL-15. The IL-12-adjuvanted allogeneic HER2/neu-expressing cell vaccine was still able to delay mammary cancer onset but efficacy in IL-15-lacking mice vanished earlier: all vaccinated IL15KO/NeuT mice developed tumors within 80 weeks of age (median latency of 53 weeks), whereas more than 70 % of vaccinated NeuT mice remained tumor-free up to 80 weeks of age. Vaccinated IL15KO/NeuT mice showed less necrotic tumors with fewer CD3+ lymphocyes and lacked perforin-positive infiltrating cells compared to NeuT mice. Concerning the anti-vaccine antibody response, antibody titer was unaffected by the lack of IL-15, but less antibodies of IgM and IgG1 isotypes were found in IL15KO/NeuT mice. A lower induction by vaccine of systemic interferon-gamma (IFN-γ) and interleukin-5 (IL-5) was also observed in IL15KO/NeuT mice when compared to NeuT mice. Finally, we found a lower level of CD8+ memory cells in the peripheral blood of vaccinated IL15KO/NeuT mice compared to NeuT mice., Conclusions: We demonstrated that IL-15 has a role in mammary cancer immunosurveillance and that IL-15-regulated NK and CD8+ memory cells play a role in long-lasting immunoprevention, further supporting the potential use of IL-15 as adjuvant in immunological strategies against tumors.

Colorectal carcinogenesis relies on loss of homeostasic mechanisms regulating cell proliferation, differentiation and survival. These cell processes have been reported to be influenced independently by transcription factors activated downstream of the Wnt pathway, such as SOX9 and β-catenin, and by the nuclear receptor PPARγ. The purpose of this study was to explore the expression levels and functional link between SOX9, β-catenin and PPARγ in the pathogenesis of colorectal cancer (CRC). We evaluated SOX9, β-catenin and PPARγ expression levels on human CRC specimens by qPCR and immunoblot detection. We tested the hypothesis that PPARγ activation might affect SOX9 and β-catenin expression using four colon cancer cell lines (CaCo2, SW480, HCT116, and HT29 cells). In CRC tissues SOX9 resulted up-regulated at both mRNA and protein levels when compared to matched normal mucosa, β-catenin resulted up-regulated at protein levels, while PPARG mRNA and PPARγ protein levels were down-regulated. A significant relationship was observed between high PPARG and SOX9 expression levels in the tumor tissue and female gender (p=0.005 and p=0.04, respectively), and between high SOX9 expression in the tumor tissue and age (p=0.04) and microsatellite instability (MSI), in particular with MSI-H (p=0.0002). Moreover, treatment with the synthetic PPARγ ligand rosiglitazone induced different changes of SOX9 and β-catenin expression and subcellular localization in the colon cancer cell lines examined. In conclusion, SOX9, β-catenin and PPARγ expression levels are deregulated in the CRC tissue, and in colon cancer cell lines ligand-dependent PPARγ activation unevenly influences SOX9 and β-catenin expression and subcellular localization, suggesting a variable mechanistic role in colon carcinogenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hepatitis delta virus (HDV) is a small, defective RNA virus that can infect only individuals carrying hepatitis B virus. HBV/HDV co-infection results in more severe liver disease than HBV single infection and more rapid progression to cirrhosis and hepatocellular carcinoma (HCC). The epigenetic events involved in hepatocyte transformation towards malignancy in this context are poorly known. Here we report that, in Huh-7 cells, HDV induces DNMT3b expression and is associated to E2F1 transcription factor hypermethylation. Moreover our cell cycle analysis showed that HDV induces G2/M arrest. These findings suggest that HDV could play a role in HCC development at least in part by altering DNA methylation events. A better understanding of the molecular mechanisms involved in HDV-related carcinogenesis could help to identify new therapeutic targets., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background: Hepatitis C virus (HCV) infects approximately 3% of the world population and is the leading cause of liver disease, impacting hepatocyte metabolism, depending on virus genotype. Hepatic metabolic functions show rhythmic fluctuations with 24-h periodicity (circadian), driven by molecular clockworks ticking through translational-transcriptional feedback loops, operated by a set of genes, called clock genes, encoding circadian proteins. Disruption of biologic clocks is implicated in a variety of disorders including fatty liver disease, obesity and diabetes. The relation between HCV replication and the circadian clock is unknown., Methods: We investigated the relationship between HCV core infection and viral replication and the expression of clock genes (Rev-Erbα, Rorα, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2) in two cellular models, the Huh-7 cells transiently expressing the HCV core protein genotypes 1b or 3a, and the OR6 cells stably harboring the full-length hepatitis C genotype 1b replicon, and in human liver biopsies, using qRT-PCR, immunoblotting, luciferase assays and immunohistochemistry., Results: In Huh-7 cells expressing the HCV core protein genotype 1b, but not 3a, and in OR6 cells, transcript and protein levels of PER2 and CRY2 were downregulated. Overexpression of PER2 led to a consistent decrease in HCV RNA replicating levels and restoration of altered expression pattern of a subset of interferon stimulated genes (ISGs) in OR6 cells. Furthermore, in liver biopsies from HCV genotype 1b infected patients, PER2 was markedly localized to the nucleus, consistent with an auto-inhibitory transcriptional feedback loop., Conclusions: HCV can modulate hepatic clock gene machinery, and the circadian protein PER2 counteracts viral replication. Further understanding of circadian regulation of HCV replication and rhythmic patterns of host-hosted relationship may improve the effectiveness of HCV antiviral therapy. This would extend to hepatic viral infections the current spectrum of chronotherapies, implemented to treat metabolic, immune related and neoplastic disease.

The rhythmic recurrence of biological processes is driven by the functioning of cellular circadian clocks, operated by a set of genes and proteins that generate self-sustaining transcriptional-translational feedback loops with a free-running period of about 24 h. In the gastrointestinal apparatus, the functioning of the biological clocks shows distinct patterns in the different organs. The aim of this study was to evaluate the time-related variation of clock gene expression in mouse liver and stomach, two components of the digestive system sharing vascular and autonomic supply, but performing completely different functions. The authors analyzed the periodicity by cosinor analysis and the dynamics of variation by computing the fractional variation to assess the rate of change in gene expression. Five-week-old male Balb/c mice were exposed to 2 wks of 12-h light/12-h dark cycles, then kept in complete darkness for 3 d as a continuation of the dark span of the last light-dark cycle. The authors evaluated the expression of Bmal1, Clock, Cry1, Cry2, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, Timeless, Dbp, Csnk1d, and Csnk1e by using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in mouse liver and stomach. A significant 24-h rhythmic component was found for 10 genes in the liver (Bmal1, Clock, Cry1, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, and Dbp), and for 9 genes in the stomach (Bmal1, Cry1, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, and Dbp). In particular, Clock showed marked rhythm differences between liver and stomach, putatively due to some compensation by Npas2. The acrophase of the original values of Bmal1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, and Dbp expression was delayed in the stomach, and the average delay expressed as mean ± SD was 14.30 ± 7.94 degrees (57.20 ± 31.78 minutes). A statistically significant difference was found in the acrophases of Bmal1 (p = .015) and Npas2 (p = .011). Fractional variations provided significant circadian rhythms for nine genes in the liver (Bmal1, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, Timeless, and Dbp), and for seven genes in the stomach (Bmal1, Clock, Per2, Rev-erbα, Npas2, Dbp, and Csnk1e). The acrophase of the fractional variations of Bmal1, Per2, Per3, Rev-erbα, Rev-erbβ, and Dbp expression was delayed in the stomach, and the average delay expressed as mean ± SD was 19.10 ± 9.39 degrees (76.40 ± 37.59 minutes). A significantly greater fractional variation was found in the liver for Clock at 06:00 h (p = .034), Per1 at 02:00 h (p = .037), and Per3 at 02:00 h (p = .029), whereas the fractional variation was greater in the stomach for Clock at 10:00 h (p = .016), and for Npas2 at 02:00 h (p = .029) and at 06:00 h (p = .044). In conclusion, liver and stomach show different phasing and dynamics of clock gene expression, which are probably related to prevailing control by different driving cues, and allow them to keep going the various metabolic pathways and diverse functional processes that they manage.

Background: Autosomal dominant hypocalcemia (ADH) is an endocrine disorder caused by activating mutations of the calcium-sensing receptor (CASR) gene which plays a major role in maintaining calcium homeostasis. Biochemical features of ADH are hypocalcemia and hypercalciuria with inappropriately low levels of parathyroid hormone (PTH). We report on two four-generation families affected by ADH., Aim: To identify mutations of CASR gene in subjects affected by familial idiopathic hypoparathyroidism. To perform functional assays of identified CASR variants by transient transfection on HEK293 cells., Results: We identified two CASR variants (Q681R and P221L): the Q681R variant was novel while the P221L had been previously published. Functional assays on the Q681R variant showed that it did not alter the whole expression nor the correct plasmamembrane localization, but enhanced the signaling function, increasing the sensitivity of the receptor as compared to the WT., Conclusions: We report two activating CASR mutations in two families affected by ADH and the functional assays performed on the novel variant Q681R. Our work enlarged the spectrum of mutations of the CASR and contributed to a better elucidation of the protein function., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background and Aim: Kidney cancer is associated with alteration in the pathways regulated by von Hippel-Lindau protein and hypoxia inducible factor α. Tight interrelationships have been evidenced between hypoxia response pathways and circadian pathways. The dysregulation of the circadian clock circuitry is involved in carcinogenesis. The aim of our study was to evaluate the clock gene machinery in kidney cancer., Methods: mRNA expression levels of the clock genes ARNTL1, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1, CRY2, TIMELESS, TIPIN and CSNK1E and of the clock controlled gene SERPINE1 were evaluated by DNA microarray assays and by qRT-PCR in primary tumor and matched nontumorous tissue collected from a cohort of 11 consecutive kidney cancer patients., Results: In kidney tumor tissue, we found down-regulation of PER2 (median=0.658, Q1-Q3=0.562-0.744, P<0.01), TIMELESS (median=0.705, Q1-Q3=0.299-1.330, P=0.04) and TIPIN (median=0.556, Q1-Q3=0.385-1.945, P=0.01), up-regulation of SERPINE1 (median=1.628, Q1-Q3=0.339-4.071, P=0.04), whereas the expression of ARNTL2 (median=0.605, Q1-Q3=0.318-1.738, P=0.74) and CSNK1E (median=0.927, Q1-Q3=0.612-2.321, P=0.33) did not differ. A statistically significant correlation was evidenced between mRNA levels of PER2 and CSNKIE (r=0.791, P<0.01), PER2 and TIPIN (r=0.729, P=0.01), PER2 and SERPINE1 (r=0.704, P=0.01), TIMELESS and TIPIN (r=0.605, P=0.04), TIMELESS and CSNKIE (r=0.637, P=0.03), TIPIN and CSNKIE (r=0.940, P<0.01)., Conclusion: In kidney cancer, the circadian clock circuitry is deregulated and the altered expression of the clock genes might be involved in disease onset and progression., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Molecular clocks drive circadian rhythmicity of cellular functions in peripheral tissues and organs, kidney included, whereas in the testis this clockwork seems constitutively active. We have evaluated the periodicity and the dynamics of expression of the clock genes BMAL1, CLOCK, PER1, PER2, CRY1, CRY2 and REV ERBalpha over 24 h in the kidney and testis using a mouse model. The periodicity was explored by single cosinor, and dynamics were explored by calculation of fractional variations of gene expression related to time intervals. Kidney and testis were harvested at 4-h intervals over a 24-h period from eight-week-old C57BL/6 male mice housed individually on a 12 h light (L)-dark (D) cycle (lights on at 08:00 h; lights off at 20:00 h) and mRNA was extracted and analyzed by Quantitative Real-time Reverse Transcription PCR. A statistically significant difference was evidenced between kidney and testis for the original values of expression level of BMAL1, PER1, PER2 CRY1, CRY2 and REV ERB&#945;. A statistically significant difference was evidenced between kidney and testis for the fractional variation of BMAL1, PER2, CRY1, CRY2 and REV ERB&#945;. A significant 24-h rhythmic component was found for BMAL1, CLOCK, PER1, PER2, CRY1, CRY2 and REV ERB&#945; in the kidney, whereas no core clock gene showed circadian rhythmicity in the testis. Fractional variations provided significant circadian rhythms for BMAL1, PER2, CRY, CRY2 and REV ERB&#945; in the kidney, whereas in the testis the fractional variation calculations showed no circadian rhythmicity, but quantitative comparison showed statistically significant differences in only 16.7 percent of the time points studied. In conclusion, in the kidney the clock gene machinery shows circadian oscillation of mRNA levels and time-related variations in the rate of change of clock gene expression. In the testis the clock genes do not show circadian rhythmicity of expression and the dynamics of variation are not characterized by a periodical pattern, but are quantitatively similar to those observed in the kidney. These data suggest that in the testis the clock gene machinery shows a tissue-specific pattern of function and clock genes may play a different role in the testis with regard to other peripheral tissues, maybe in relation to the presence of developmental and differentiation phenomena.

Purpose: Cathepsin and plasmin may favor cancer cell invasion degrading extracellular matrix. Plasmin formation from plasminogen is regulated by plasminogen activator inhibitor type-1 (PAI-1). ARNTL2 activates the promoters of the PAI-1 gene, officially called SERPINE1, driving the circadian variation in circulating PAI-1 levels., Methods: We evaluated ARNTL2 and SERPINE1 expression in 50 colorectal cancer specimens and adjacent normal tissue and in colon cancer cell lines., Results: We found up-regulation of ARNTL2 (P = 0.004) and SERPINE1 (P = 0.002) in tumor tissue. A statistically significant association was found between high ARNTL2 mRNA levels and vascular invasion (P < 0.0001), and between high SERPINE1 mRNA levels and microsatellite instability (MSI-H and MSI-L, P = 0.025). Sorting the subjects into quartile groups, a statistically significant association was found between high ARNTL2 expression and lymph node involvement (P < 0.001), between high SERPINE1 expression and grading (P < 0.001) and between high SERPINE1 expression and MSI H-L (P < 0.0001). In SW480 cells, a more proliferative model compared to CaCo2 cells, there were higher mRNA levels of ARNTL2 (P < 0.001) and SERPINE1 (P = 0.001)., Conclusion: ARNTL2 and SERPINE1 expression is increased in colorectal cancer and in a highly proliferative colon cancer cell line and is related to tumor invasiveness and aggressiveness.

SIRT1 and the clock genes are involved in carcinogenesis. We evaluated SIRT1 expression in 19 human colorectal cancer (CRC) specimens and clock gene expression in SIRT1-overexpressing CaCo2 and SW480 cells. In CRC, SIRT1 mean expression level was decreased. Compared to CaCo2 cells, SW480 cells displayed lower levels of SIRT1 and PER3 and higher levels of ARNTL1, CLOCK, PER1, PER2, CRY1, TIPIN, and CSNKIE. SIRT1 overexpression induced PER1 upregulation in CaCo2 and downregulation in SW480 cells. SIRT1 expression was heterogeneous in human CRC and in CRC cell lines. These results might have relevant implications for a better understanding of colorectal carcinogenesis.

Carcinogenesis is related to the loss of homeostatic control of cellular processes regulated by transcriptional circuits and epigenetic mechanisms. Among these, the activities of peroxisome proliferator-activated receptors (PPARs) and DNA methyltransferases (DNMTs) are crucial and intertwined. PPARγ is a key regulator of cell fate, linking nutrient sensing to transcription processes, and its expression oscillates with circadian rhythmicity. Aim of our study was to assess the periodicity of PPARγ and DNMTs in pancreatic cancer (PC). We investigated the time-related patterns of PPARG, DNMT1, and DNMT3B expression monitoring their mRNA levels by qRT-PCR at different time points over a 28-hour span in BxPC-3, CFPAC-1, PANC-1, and MIAPaCa-2 PC cells after synchronization with serum shock. PPARG and DNMT1 expression in PANC-1 cells and PPARG expression in MIAPaCa-2 cells were characterized by a 24 h period oscillation, and a borderline significant rhythm was observed for the PPARG, DNMT1, and DNMT3B expression profiles in the other cell lines. The time-qualified profiles of gene expression showed different shapes and phase relationships in the PC cell lines examined. In conclusion, PPARG and DNMTs expression is characterized by different time-qualified patterns in cell lines derived from human PC, and this heterogeneity could influence cell phenotype and human disease behaviour.

Emerging evidence indicates that peroxisome proliferator-activated receptor γ (PPARγ) and DNA methyltransferases (DNMTs) play a role in carcinogenesis. In this study we aimed to evaluate the expression of PPARγ, DNMT1, and DNMT3B and their correlation with clinical-pathological features in patients with pancreatic cancer (PC), and to define the effect of PPARγ activation on DNMTs expression in PC cell lines. qRT-PCR analysis showed that DNMT3B expression was downregulated in tumors compared to normal tissues (P = 0.03), whereas PPARγ and DNMT1 levels did not show significant alterations in PC patients. Expression levels between PPARγ and DNMT1 and between DNMT1 and DNMT3B were highly correlated (P = 0.008 and P = 0.05 resp.). DNMT3B overexpression in tumor tissue was positively correlated with both lymph nodes spreading (P = 0.046) and resection margin status (P = 0.04), and a borderline association with perineural invasion (P = 0.06) was found. Furthermore, high levels of DNMT3B expression were significantly associated with a lower mortality in the whole population (HR = 0.485; 95%CI = 0.262-0.895, P = 0.02) and in the subgroup of patients without perineural invasion (HR = 0.314; 95%CI = 0.130-0.758; P = 0.01), while such association was not observed in patients with tumor invasion into perineural structures (P = 0.70). In conclusion, in vitro and in vivo PPARγ and DNMTs appear interrelated in PC, and this interaction might influence cell phenotype and disease behavior.