PARP1 orchestrates epigenetic events setting up chromatin domains.

Epigenetic events include reversible modifications of DNA and histone tails driving chromatin organization and thus transcription. The epigenetic regulation is a highly integrated process underlying the plasticity of the genomic information both in the context of complex physiological and pathological processes. The global regulatory aspects of epigenetic events are largely unknown. PARylation and PARP1 are recently emerging as multi-level regulatory effectors that modulate the topology of chromatin by orchestrating very different processes. This review focuses in particular on the role of PARP1 in epigenetics, trying to build a comprehensive perspective of its involvement in the regulation of epigenetic modifications of histones and DNA, contextualizing it in the global organization of chromatin domains in the nucleus.

Diagnosis of sphingolipidoses: a new simultaneous measurement of lysosphingolipids by LC-MS/MS.

BACKGROUND: Lysosphingolipids (LysoSLs) are derivatives of sphingolipids which have lost the amide-linked acyl chain. More recently, LysoSLs have been identified as storage compounds in several sphingolipidoses, including Gaucher, Fabry and Niemann-Pick diseases. To date, different methods have been developed to measure each individual lysosphingolipid in plasma. This report describes a rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of several LysoSLs in plasma. METHODS: We analyzed the following compounds: hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), lysosphingomyelin (LysoSM) and lysosphingomyelin-509 (LysoSM-509). The sample preparation requires only 100 µL of plasma and consists of an extraction with a mixture of MeOH/acetone/H2O (45:45:10, v/v). RESULTS: The method validation showed high sensitivity, an excellent accuracy and precision. Reference ranges were determined in healthy adult and pediatric population. The results demonstrate that the LC-MS/MS method can quantify different LysoSLs and can be used to identify patients with Fabry (LysoGb3), Gaucher and Krabbe (HexSph) diseases, prosaposine deficiency (LysoGb3 and HexSph), and Niemann-Pick disease types A/B and C (LysoSM and LysoSM-509). CONCLUSIONS: This LC-MS/MS method allows a rapid and simultaneous quantification of LysoSLs and is useful as a biochemical diagnostic tool for sphingolipidoses.

The epigenetic factor BORIS/CTCFL regulates the NOTCH3 gene expression in cancer cells.

Aberrant upregulation of NOTCH3 gene plays a critical role in cancer pathogenesis. However, the underlying mechanisms are still unknown. We tested here the hypothesis that aberrant epigenetic modifications in the NOTCH3 promoter region might account for its upregulation in cancer cells. We compared DNA and histone methylation status of NOTCH3 promoter region in human normal blood cells and T cell acute lymphoblastic leukemia (T-ALL) cell lines, differentially expressing NOTCH3. We found that histone methylation, rather than DNA hypomethylation, contributes towards establishing an active chromatin status of NOTCH3 promoter in NOTCH3 overexpressing cancer cells. We discovered that the chromatin regulator protein BORIS/CTCFL plays an important role in regulating NOTCH3 gene expression. We observed that BORIS is present in T-ALL cell lines as well as in cell lines derived from several solid tumors overexpressing NOTCH3. Moreover, BORIS targets NOTCH3 promoter in cancer cells and it is able to induce and to maintain a permissive/active chromatin conformation. Importantly, the association between NOTCH3 overexpression and BORIS presence was confirmed in primary T-ALL samples from patients at the onset of the disease. Overall, our results provide novel insights into the determinants of NOTCH3 overexpression in cancer cells, by revealing a key role for BORIS as the main mediator of transcriptional deregulation of NOTCH3.

ADP-ribose polymer depletion leads to nuclear Ctcf re-localization and chromatin rearrangement(1).

Ctcf (CCCTC-binding factor) directly induces Parp [poly(ADP-ribose) polymerase] 1 activity and its PARylation [poly(ADPribosyl)ation] in the absence of DNA damage. Ctcf, in turn, is a substrate for this post-synthetic modification and as such it is covalently and non-covalently modified by PARs (ADP-ribose polymers). Moreover, PARylation is able to protect certain DNA regions bound by Ctcf from DNA methylation. We recently reported that de novo methylation of Ctcf target sequences due to overexpression of Parg [poly(ADP-ribose)glycohydrolase] induces loss of Ctcf binding. Considering this, we investigate to what extent PARP activity is able to affect nuclear distribution of Ctcf in the present study. Notably, Ctcf lost its diffuse nuclear localization following PAR (ADP-ribose polymer) depletion and accumulated at the periphery of the nucleus where it was linked with nuclear pore complex proteins remaining external to the perinuclear Lamin B1 ring. We demonstrated that PAR depletion-dependent perinuclear localization of Ctcf was due to its blockage from entering the nucleus. Besides Ctcf nuclear delocalization, the outcome of PAR depletion led to changes in chromatin architecture. Immunofluorescence analyses indicated DNA redistribution, a generalized genomic hypermethylation and an increase of inactive compared with active chromatin marks in Parg-overexpressing or Ctcf-silenced cells. Together these results underline the importance of the cross-talk between Parp1 and Ctcf in the maintenance of nuclear organization.

PAR level mediates the link between ROS and inflammatory response in patients with type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is characterized by disrupted glucose homeostasis and metabolic abnormalities, with oxidative stress and inflammation playing pivotal roles in its pathophysiology. Poly(ADP-ribosyl)ation (PARylation) is a post-translational process involving the addition of ADP-ribose polymers (PAR) to target proteins. While preclinical studies have implicated PARylation in the interplay between oxidative stress and inflammation in T2DM, direct clinical evidence in humans remains limited. This study investigates the relationship between oxidative stress, PARylation, and inflammatory response in T2DM patients. METHODS: This cross-sectional investigation involved 61 T2DM patients and 48 controls. PAR levels were determined in peripheral blood cells (PBMC) by ELISA-based methodologies. Oxidative stress was assessed in plasma and PBMC. In plasma, we monitored reactive oxygen metabolites (d-ROMs) and ferric-reducing antioxidant power. In PBMC, we measured the expression of antioxidant enzymes SOD1, GPX1 and CAT by qPCR. Further, we evaluated the expression of inflammatory mediators such as IL6, TNF-α, CD68 and MCP1 by qPCR in PBMC. RESULTS: T2DM patients exhibited elevated PAR levels in PBMC and increased d-ROMs in plasma. Positive associations were found between PAR levels and d-ROMs, suggesting a link between oxidative stress and altered PAR metabolism. Mediation analysis revealed that d-ROMs mediate the association between HbA1c levels and PAR, indicating oxidative stress as a potential driver of increased PARylation in T2DM. Furthermore, elevated PAR levels were found to be associated with increased expression of pro-inflammatory cytokines IL6 and TNF-α in the PBMC of T2DM patients. CONCLUSIONS: This study highlights that hyperactivation of PARylation is associated with poor glycemic control and the resultant oxidative stress in T2DM. The increase of PAR levels is correlated with the upregulation of key mediators of the inflammatory response. Further research is warranted to validate these findings and explore their clinical implications.

ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites.

PARylation [poly(ADP-ribosyl)ation] is involved in the maintenance of genomic methylation patterns through its control of Dnmt1 [DNA (cytosine-5)-methyltransferase 1] activity. Our previous findings indicated that Ctcf (CCCTC-binding factor) may be an important player in key events whereby PARylation controls the unmethylated status of some CpG-rich regions. Ctcf is able to activate Parp1 [poly(ADP-ribose) polymerase 1], which ADP-ribosylates itself and, in turn, inhibits DNA methylation via non-covalent interaction between its ADP-ribose polymers and Dnmt1. By such a mechanism, Ctcf may preserve the epigenetic pattern at promoters of important housekeeping genes. The results of the present study showed Dnmt1 as a new protein partner of Ctcf. Moreover, we show that Ctcf forms a complex with Dnmt1 and PARylated Parp1 at specific Ctcf target sequences and that PARylation is responsible for the maintenance of the unmethylated status of some Ctcf-bound CpGs. We suggest a mechanism by which Parp1, tethered and activated at specific DNA target sites by Ctcf, preserves their methylation-free status.

Quadrato motor training (QMT) influences IL-1ß expression and creativity: Implications for inflammatory state reduction and cognitive enhancement.

Mind-body practices and meditation have been increasingly studied in recent years due to their beneficial effects on cognition, and physical and psychological health. Growing evidence suggests that these practices could be utilized as interventions to impact age-related biological processes, such as cognitive decline, inflammation, and homeostatic dysregulation. Indeed, it has been reported that mindful meditation may induce neuroplasticity in brain regions involved in control of attention, emotional regulation, and self-awareness. In the current research we studied the effects of a recently developed movement meditation, named the Quadrato Motor Training (QMT), on the proinflammatory cytokine Interleukin-1beta (IL-1ß), utilizing a pre-post design. In addition to its role in the immune system, IL-1ß is also an important mediator of neuroimmune responses related to sickness behavior, and plays a role in complex cognitive processes, such as synaptic plasticity, neurogenesis, and neuromodulation. Thirty healthy participants were divided in two groups, one performing QMT for 2 months, and one passive control group. Salivary IL-1ß expression was examined by ELISA to measure protein levels and by qRT-PCR to quantify mRNA. In addition, the methylation profile of the IL-1ß promoter was examined. All participants further conducted the Alternate Uses Task (AUT) and Hidden Figure Test (HFT), to measure their creativity and spatial cognition. The results showed that, following QMT practice, IL-1ß protein level decreased and creativity increased, compared to the control group. These data demonstrate that QMT may help reduce inflammatory states and promote cognitive improvement, highlighting the importance of non-pharmacological approaches to health and well-being.

Hyperthermia-induced changes in leukocyte survival and phagocytosis: a comparative study in bovine and buffalo leukocytes.

Heat stress negatively affects health, welfare, and livestock productivity by impairing immune function, increasing disease incidence. In recent years, there has been increasing interest in understanding the immune system of water buffalo due to the growing economic impact of this species for the high quality and nutritional value of buffalo milk. While there are common responses across bovine and buffalo species, there are also some species-specific variations in the physiological responses to heat stress, mainly attributed to differences in metabolism and heat dissipation efficiency. At cellular level, the exposure to thermal stress induces several anomalies in cell functions. However, there is limited knowledge about the differential response of bovine and buffalo leucocytes to early and late exposure to different degrees of thermal exposure. The aim of this study was to compare the in vitro effect of hyperthermia on apoptosis and phagocytosis in leukocytes from bovine and buffalo species. For this, whole blood samples of six bovines and nine buffaloes were incubated at 39°C (mimicking normothermia condition) or 41°C (mimicking heat stress condition) for 1, 2, and 4 h. Two flow cytometric assays were then performed to evaluate apoptosis and determine functional capacity of phagocytic cells (neutrophils and monocytes). The results showed that the viability of bovine and buffalo leukocytes was differently affected by temperature and time of in vitro exposure. A higher percentage of apoptotic leukocytes was observed in bovines than in buffaloes at 39°C (3.19 vs. 1.51, p < 0.05) and 41°C (4.01 vs. 1.69, p < 0.05) and for all incubation time points (p < 0.05). In contrast, no difference was observed in the fraction of necrotic leukocytes between the two species. In both species, lymphocytes showed the highest sensitivity to hyperthermia, showing an increased apoptosis rates along with increased incubation time. In bovine, apoptotic lymphocytes increased from 5.79 to 12.7% at 39°C (p < 0.05), in buffalo, this population increased from 1.50 to 3.57% at 39°C and from 2.90 to 4.99% at 41°C (p < 0.05). Although no significant differences were found between the two species regarding the percentage of phagocytic neutrophils, lower phagocytosis capacity values (MFI, mean fluorescence intensity) were found in bovines compared with buffaloes at 41°C (27960.72 vs. 53676.45, p > 0.05). However, for monocytes, the differences between species were significant for both phagocytosis activity and capacity with lower percentages of bovine phagocytic monocytes after 2 h at 39°C and after 1 h at 41°C. The bovine monocytes showed lower MFI values for all temperature and time variations than buffaloes (37538.91 vs. 90445.47 at 39°C and 33752.91 vs. 70278.79 at 41°C, p < 0.05). In conclusion, the current study represents the first report on the comparative analysis of the effect of in vitro heat stress on bovine and buffalo leukocyte populations, highlighting that the leukocytes of buffalo exhibit relatively higher thermal adaptation than bovine cells.

Simulated Microgravity Exposure Induces Antioxidant Barrier Deregulation and Mitochondria Enlargement in TCam-2 Cell Spheroids.

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of reactive oxygen species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes in gravitational force, even though published data on germ cell models are scarce. We previously studied the effects of simulated microgravity (s-microgravity) on a 2D cultured TCam-2 seminoma-derived cell line, considered the only human cell line available to study in vitro mitotically active human male germ cells. In this study, we used a corresponding TCam-2 3D cell culture model that mimics cell-cell contacts in organ tissue to test the possible effects induced by s-microgravity exposure. TCam-2 cell spheroids were cultured for 24 h under unitary gravity (Ctr) or s-microgravity conditions, the latter obtained using a random positioning machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels was observed after RPM exposure. In line with these results, a trend of protein and lipid oxidation increase and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis via transmission electron microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, with GPX1, NCF1, and CYBB being downregulated, whereas NOX4 and HMOX1 are upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated with the observed mitochondria alteration. Altogether, these data demonstrate TCam-2 spheroid sensitivity to acute s-microgravity exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow them to overcome the exposure to altered gravitational force.

YY1 Knockdown Relieves the Differentiation Block and Restores Apoptosis in AML Cells.

In this study we analyzed the expression of Yin and Yang 1 protein (YY1), a member of the noncanonical PcG complexes, in AML patient samples and AML cell lines and the effect of YY1 downregulation on the AML differentiation block. Our results show that YY1 is significantly overexpressed in AML patient samples and AML cell lines and that YY1 knockdown relieves the differentiation block. YY1 downregulation in two AML cell lines (HL-60 and OCI-AML3) and one AML patient sample restored the expression of members of the CEBP protein family, increased the expression of extrinsic growth factors/receptors and surface antigenic markers, induced morphological cell characteristics typical of myeloid differentiation, and sensitized cells to retinoic acid treatment and to apoptosis. Overall, our data show that YY1 is not a secondary regulator of myeloid differentiation but that, if overexpressed, it can play a predominant role in myeloid differentiation block.

Quadrato Motor Training (QMT) is associated with DNA methylation changes at DNA repeats: A pilot study.

The control of non-coding repeated DNA by DNA methylation plays an important role in genomic stability, contributing to health and healthy aging. Mind-body practices can elicit psychophysical wellbeing via epigenetic mechanisms, including DNA methylation. However, in this context the effects of movement meditations have rarely been examined. Consequently, the current study investigates the effects of a specifically structured movement meditation, called the Quadrato Motor Training (QMT) on psychophysical wellbeing and on the methylation level of repeated sequences. An 8-week daily QMT program was administered to healthy women aged 40-60 years and compared with a passive control group matched for gender and age. Psychological well-being was assessed within both groups by using self-reporting scales, including the Meaning in Life Questionnaire [MLQ] and Psychological Wellbeing Scale [PWB]). DNA methylation profiles of repeated sequences (ribosomal DNA, LINE-1 and Alu) were determined in saliva samples by deep-sequencing. In contrast to controls, the QMT group exhibited increased Search for Meaning, decreased Presence of Meaning and increased Positive Relations, suggesting that QMT may lessen the automatic patterns of thinking. In the QMT group, we also found site-specific significant methylation variations in ribosomal DNA and LINE-1 repeats, consistent with increased genome stability. Finally, the correlations found between changes in methylation and psychometric indices (MLQ and PWB) suggest that the observed epigenetic and psychological changes are interrelated. Collectively, the current results indicate that QMT may improve psychophysical health trajectories by influencing the DNA methylation of specific repetitive sequences.

Flow cytometric detection of IFN-γ production and Caspase-3 activation in CD4+ T lymphocytes to discriminate between healthy and Mycobacterium bovis naturally infected water buffaloes.

Tuberculosis has a negative economic impact on buffalo farming, and it poses a potential threat to human health. Interferon-gamma (IFN-γ) plays a central role in protection against mycobacterial diseases, illustrating the importance of T-cell mediated immune responses in tuberculosis infection. Recently, the expression of Caspase-3, a critical executor of apoptosis, in M. tuberculosis-specific IFN-γ+CD4+ T cells was used as a new marker to distinguish active from latent tuberculosis infection in humans. The aims of this work were to develop a whole blood flow cytometric assay to detect the production of IFN-γ and the activation of Caspase-3 by CD4+ T lymphocytes from water buffalo and to evaluate whether these parameters can discriminate between healthy and M. bovis naturally infected buffaloes. A total of 35 Italian Mediterranean buffaloes were grouped in two groups: uninfected and M. bovis infected (based on the results of antemortem diagnostic tests: single intradermal tuberculin (SIT) and ELISA IFN-γ tests). Whole blood was incubated for 6 h with tubercular antigens: PPD-B, PPD-A, ESAT-6/CFP-10 and a new mix of precocious secreted antigens (PA). Our results showed a significant increase in the percentage of IFN-γ+CD4+ T cells in infected compared to the uninfected animals after each stimulus. Improved sensitivity of the assay was obtained by including the stimulation with the new mix of PA. Interestingly, we observed a concomitant decrease in percentage of Caspase-3+CD4+ T cells in M. bovis infected animals compared to the control healthy ones, regardless of the stimulus used. Overall, these results showed that M. bovis infection activates CD4+ T lymphocytes to produce IFN-γ and at the same time causes a concomitant decrease of Caspase-3 activation in CD4+ T cells. This study for the first time in water buffalo describes the development of a whole blood flow cytometric assay for the detection of IFN-γ producing CD4+ T cells and proposes the expression of active Caspase-3 as an additional bovine TB biomarker. Although further studies are needed to better understand the mechanisms of Caspase-3-mediated cell death during tuberculosis, our data can help to better understand the cellular immune response to M. bovis infection in buffalo species.

Uncovering the Relationship between Selenium Status, Age, Health, and Dietary Habits: Insights from a Large Population Study including Nonagenarian Offspring from the MARK-AGE Project.

An inadequate selenium (Se) status can accelerate the aging process, increasing the vulnerability to age-related diseases. The study aimed to investigate plasma Se and Se species in a large population, including 2200 older adults from the general population (RASIG), 514 nonagenarian offspring (GO), and 293 GO Spouses (SGO). Plasma Se levels in women exhibit an inverted U-shaped pattern, increasing with age until the post-menopausal period and then declining. Conversely, men exhibit a linear decline in plasma Se levels with age. Subjects from Finland had the highest plasma Se values, while those from Poland had the lowest ones. Plasma Se was influenced by fish and vitamin consumption, but there were no significant differences between RASIG, GO, and SGO. Plasma Se was positively associated with albumin, HDL, total cholesterol, fibrinogen, and triglycerides and negatively associated with homocysteine. Fractionation analysis showed that Se distribution among plasma selenoproteins is affected by age, glucometabolic and inflammatory factors, and being GO or SGO. These findings show that sex-specific, nutritional, and inflammatory factors play a crucial role in the regulation of Se plasma levels throughout the aging process and that the shared environment of GO and SGO plays a role in their distinctive Se fractionation.

Methylation-dependent PAD2 upregulation in multiple sclerosis peripheral blood.

BACKGROUND: Peptidylarginine deiminase 2 (PAD2) and peptidylarginine deiminase 4 (PAD4) are two members of PAD family which are over-expressed in the multiple sclerosis (MS) brain. Through its enzymatic activity PAD2 converts myelin basic protein (MBP) arginines into citrullines - an event that may favour autoimmunity - while peptidylarginine deiminase 4 (PAD4) is involved in chromatin remodelling. OBJECTIVES: Our aim was to verify whether an altered epigenetic control of PAD2, as already shown in the MS brain, can be observed in peripheral blood mononuclear cells (PBMCs) of patients with MS since some of these cells also synthesize MBP. METHODS: The expression of most suitable reference genes and of PAD2 and PAD4 was assessed by qPCR. Analysis of DNA methylation was performed by bisulfite method. RESULTS: The comparison of PAD2 expression level in PBMCs from patients with MS vs. healthy donors showed that, as well as in the white matter of MS patients, the enzyme is significantly upregulated in affected subjects. Methylation pattern analysis of a CpG island located in the PAD2 promoter showed that over-expression is associated with promoter demethylation. CONCLUSION: Defective regulation of PAD2 in the periphery, without the immunological shelter of the blood-brain barrier, may contribute to the development of the autoimmune responses in MS.

Counteracting aged DNA methylation states to combat ageing and age-related diseases.

DNA methylation (DNAm) overwrites information about multiple extrinsic factors on the genome. Age is one of these factors. Age causes characteristic DNAm changes that are thought to be not only major drivers of normal ageing but also precursors to diseases, cancer being one of these. Although there is still much to learn about the relationship between ageing, age-related diseases and DNAm, we now know how to interpret some of the effects caused by age in the form of changes in methylation marks at specific loci. In fact, these changes form the basis of the so called "epigenetic clocks", which translate the genomic methylation profile into an "epigenetic age". Epigenetic age does not only estimate chronological age but can also predict the risk of chronic diseases and mortality. Epigenetic age is believed to be one of the most accurate metrics of biological age. Initial evidence has recently been gathered pointing to the possibility that the rate of epigenetic ageing can be slowed down or even reversed. In this review, we discuss some of the most relevant advances in this field. Expected outcome is that this approach can provide insights into how to preserve health and reduce the impact of ageing diseases in humans.

DNA Methylation Analysis of Ribosomal DNA in Adults With Down Syndrome.

Control of ribosome biogenesis is a critical aspect of the regulation of cell metabolism. As ribosomal genes (rDNA) are organized in repeated clusters on chromosomes 13, 14, 15, 21, and 22, trisomy of chromosome 21 confers an excess of rDNA copies to persons with Down syndrome (DS). Previous studies showed an alteration of ribosome biogenesis in children with DS, but the epigenetic regulation of rDNA genes has not been investigated in adults with DS so far. In this study, we used a targeted deep-sequencing approach to measure DNA methylation (DNAm) of rDNA units in whole blood from 69 adults with DS and 95 euploid controls. We further evaluated the expression of the precursor of ribosomal RNAs (RNA45S) in peripheral blood mononuclear cells (PBMCs) from the same subjects. We found that the rDNA promoter tends to be hypermethylated in DS concerning the control group. The analysis of epihaplotypes (the combination of methylated and unmethylated CpG sites along the same DNA molecule) showed a significantly lower intra-individual diversity in the DS group, which at the same time was characterized by a higher interindividual variability. Finally, we showed that RNA45S expression is lower in adults with DS. Collectively, our results suggest a rearrangement of the epigenetic profile of rDNA in DS, possibly to compensate for the extranumerary rDNA copies. Future studies should assess whether the regulation of ribosome biogenesis can contribute to the pathogenesis of DS and explain the clinical heterogeneity characteristic of the syndrome.

New Insights into the Significance of PARP-1 Activation: Flow Cytometric Detection of Poly(ADP-Ribose) as a Marker of Bovine Intramammary Infection.

Bovine intramammary infections are common diseases affecting dairy cattle worldwide and represent a major focus of veterinary research due to financial losses and food safety concerns. The identification of new biomarkers of intramammary infection, useful for monitoring the health of dairy cows and wellness verification, represents a key advancement having potential beneficial effects on public health. In vitro experiments using bovine peripheral blood mononuclear cells (PBMC), stimulated with the bacterial endotoxin lipopolysaccharide (LPS) enabled a flow cytometric assay in order to evaluate in vivo poly-ADP-ribose (PAR) levels. Results showed a significant increase of PAR after 1 h of treatment, which is consistent with the involvement of PARP activity in the inflammatory response. This study investigated PARP-1 activation in leukocyte subpopulations from bovine milk samples during udder infection. A flow cytometric assay was, therefore, performed to evaluate the PAR content in milk leukocyte subsets of cows with and without intramammary infection (IMI). Results showed that milk lymphocytes and macrophages isolated from cows with IMI had a significant increase of PAR content compared to uninfected samples. These results suggest mastitis as a new model for the study of the role of PARP in zoonotic inflammatory diseases, opening a new perspective to the "One Health" approach.

Biliverdin reductase-A protein levels are reduced in type 2 diabetes and are associated with poor glycometabolic control.

AIM: Biliverdin reductase-A (BVR-A) other than its canonical role in the degradation pathway of heme as partner of heme oxygenase-1 (HO1), has recently drawn attention as a protein with pleiotropic functions involved in insulin-glucose homeostasis. However, whether BVR-A expression is altered in type 2 diabetes (T2D) has never been evaluated. MAIN METHODS: BVR-A protein levels were evaluated in T2D (n = 44) and non-T2D (n = 29) subjects, who underwent complete clinical workup and routine biochemistry. In parallel, levels HO1, whose expression is regulated by BVR-A as well as levels of tumor necrosis factor α (TNFα), which is a known repressor for BVR-A with pro-inflammatory properties, were also assessed. KEY FINDINGS: BVR-A levels were significantly lower in T2D subjects than in non-T2D subjects. Reduced BVR-A levels were associated with greater body mass, systolic blood pressure, fasting blood glucose (FBG), glycated hemoglobin (HbA1c), triglycerides, transaminases and TNFα, and with lower high-density lipoprotein (HDL) levels. Lower BVR-A levels are associated with reduced HO1 protein levels and the multivariate analysis showed that BVR-A represented the main determinant of HO1 levels in T2D after adjustment. In addition, reduced BVR-A levels were able to predict the presence of T2D with AUROC = 0.69. for potential confounders. SIGNIFICANCE: Our results demonstrate for the first time that BVR-A protein levels are reduced in T2D individuals, and that this alteration strictly correlates with poor glycometabolic control and a pro-inflammatory state. Hence, these observations reinforce the hypothesis that reduced BVR-A protein levels may represent a key event in the dysregulation of intracellular pathways finally leading to metabolic disorders.

Increased PARylation impacts the DNA methylation process in type 2 diabetes mellitus.

BACKGROUND: Epigenetic modifications, such as DNA methylation, can influence the genetic susceptibility to type 2 diabetes mellitus (T2DM) and the progression of the disease. Our previous studies demonstrated that the regulation of the DNA methylation pattern involves the poly(ADP-ribosyl)ation (PARylation) process, a post-translational modification of proteins catalysed by the poly(ADP-ribose) polymerase (PARP) enzymes. Experimental data showed that the hyperactivation of PARylation is associated with impaired glucose metabolism and the development of T2DM. Aims of this case-control study were to investigate the association between PARylation and global and site-specific DNA methylation in T2DM and to evaluate metabolic correlates. RESULTS: Data were collected from 61 subjects affected by T2DM and 48 healthy individuals, recruited as controls. Global levels of poly(ADP-ribose) (PAR, a surrogate of PARP activity), cytosine methylation (5-methylcytosine, 5mC) and de-methylation intermediates 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were determined in peripheral blood cells by ELISA-based methodologies. Site-specific DNA methylation profiling of SOCS3, SREBF1 and TXNIP candidate genes was performed by mass spectrometry-based bisulfite sequencing, methyl-sensitive endonucleases digestion and by DNA immuno-precipitation. T2DM subjects presented higher PAR levels than controls. In T2DM individuals, increased PAR levels were significantly associated with higher HbA1c levels and the accumulation of the de-methylation intermediates 5hmC and 5fC in the genome. In addition, T2DM patients with higher PAR levels showed reduced methylation with increased 5hmC and 5fC levels in specific SOCS3 sites, up-regulated SOCS3 expression compared to both T2DM subjects with low PAR levels and controls. CONCLUSIONS: This study demonstrates the activation of PARylation processes in patients with T2DM, particularly in those with poor glycaemic control. PARylation is linked to dysregulation of DNA methylation pattern via activation of the DNA de-methylation cascade and may be at the basis of the differential gene expression observed in presence of diabetes.

Ageing affects subtelomeric DNA methylation in blood cells from a large European population enrolled in the MARK-AGE study.

Ageing leaves characteristic traces in the DNA methylation make-up of the genome. However, the importance of DNA methylation in ageing remains unclear. The study of subtelomeric regions could give promising insights into this issue. Previously reported associations between susceptibility to age-related diseases and epigenetic instability at subtelomeres suggest that the DNA methylation profile of subtelomeres undergoes remodelling during ageing. In the present work, this hypothesis has been tested in the context of the European large-scale project MARK-AGE. In this cross-sectional study, we profiled the DNA methylation of chromosomes 5 and 21 subtelomeres, in more than 2000 age-stratified women and men recruited in eight European countries. The study included individuals from the general population as well as the offspring of nonagenarians and Down syndrome subjects, who served as putative models of delayed and accelerated ageing, respectively. Significant linear changes of subtelomeric DNA methylation with increasing age were detected in the general population, indicating that subtelomeric DNA methylation changes are typical signs of ageing. Data also show that, compared to the general population, the dynamics of age-related DNA methylation changes are attenuated in the offspring of centenarian, while they accelerate in Down syndrome individuals. This result suggests that subtelomeric DNA methylation changes reflect the rate of ageing progression. We next attempted to trace the age-related changes of subtelomeric methylation back to the influence of diverse variables associated with methylation variations in the population, including demographics, dietary/health habits and clinical parameters. Results indicate that the effects of age on subtelomeric DNA methylation are mostly independent of all other variables evaluated.