Human endogenous retrovirus W in multiple sclerosis: transcriptional activity is associated with decline in oligodendrocyte proportions in the white matter of the brain.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease. One of the basic mechanisms in this disease is the autoimmune response against the myelin sheet leading to axonal damage. There is strong evidence showing that this response is regulated by both genetic and environmental factors. In addition, the role of viruses has been extensively studied, especially in the case of human endogenous retroviruses (HERVs). However, although several associations with MS susceptibility, especially in the case of HERV-W family have been observed, the pathogenic mechanisms have remained enigmatic. To clarify these HERV-mediated mechanisms as well as the responsible HERV-W loci, we utilized RNA sequencing data obtained from the white matter of the brain of individuals with and without MS. CIBERSORTx tool was applied to estimate the proportions of neuronal, glial, and endothelial cells in the brain. In addition, the transcriptional activity of 215 HERV-W loci were analyzed. The results indicated that 65 HERV-W loci had detectable expression, of which 14 were differentially expressed between MS and control samples. Of these, 12 HERV-W loci were upregulated in MS. Expression levels of the 8 upregulated HERV-W loci had significant negative correlation with estimated oligodendrocyte proportions, suggesting that they are associated with the dynamics of oligodendrocyte generation and/or maintenance. Furthermore, Gene Set Enrichment Analysis (GSEA) results indicated that expression levels of three upregulated HERV-W loci: 2p16.2, 2q13, and Xq13.3, are associated with suppression of oligodendrocyte development and myelination. Taken together, these data suggest new HERV-W loci candidates that might take part in MS pathogenesis.

Human endogenous retroviruses of the HERV-K (HML-2) family are expressed in the brain of healthy individuals and modify the composition of the brain-infiltrating immune cells.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections in the human genome. RNA expression of individual HERVs has frequently been observed in various pathologic conditions, but some activity can also be seen in healthy individuals, e.g. in the blood. To quantitate the basal expression levels of HERVs in the brain, we now used high-throughput sequencing-based metagenomic analysis to characterize the expression profiles of the HERV-K (HML-2) family proviruses in different brain regions of healthy brain tissue. To this end, RNA-seq data from the Genotype-Tissue Expression (GTEx) project was used. The GTEx project is a public resource to study tissue-specific gene expression and regulation, consisting of a large selection of sequenced samples from different tissues. The GTEx data used in this study consisted of 378 samples taken from 13 brain regions from 55 individuals. The data demonstrated that out of 99 intact proviruses in the family 58 were expressed, but the expression profiles were highly divergent and there were no significant differences in the expression profiles between the various anatomic regions of the brain. It is known that the brain contains a variety of infiltrating immune cells, which are probably of great importance both in the normal defense mechanisms as well as in the various pathogenic processes. Digital cytometry (CIBERSORTx) was used to quantify the proportions of the infiltrating immune cells in the same brain samples. Six most abundant (>5 % of the total population) cell types were observed to be CD4 memory resting T cells, M0 macrophages, plasma cells, CD8 T cells, CD4 memory activated T cells, and monocytes. Analysis of the correlations between the individual HERVs and infiltrating cell types indicated that a cluster of 6 HERVs had a notable correlation signature between T cell type infiltrating cell proportions and HERV RNA expression intensity. The correlations between inflammatory type infiltrating cells were negative or weak. Taken together, these data indicate that the expression of HERVs is associated with a T cell type immunity.

Composition of the infiltrating immune cells in the brain of healthy individuals: effect of aging.

Immune cells infiltrating the central nervous system (CNS) are involved in the defense against invading microbes as well as in the pathogenesis of neuroinflammatory diseases. In these conditions, the presence of several types of immune and inflammatory cells have been demonstrated. However, some studies have also reported low amounts of immune cells that have been detected in the CNS of healthy individuals, but the cell types present have not been systematically analyzed. To do this, we now used brain samples from The Genotype- Tissue Expression (GTEx) project to analyze the relative abundance of 22 infiltrating leukocyte types using a digital cytometry tool (CIBERSORTx). To characterize cell proportions in different parts of the CNS, samples from 13 different anatomic brain regions were used. The data obtained demonstrated that several leukocyte types were present in the CNS. Six leukocyte types (CD4 memory resting T cells, M0 macrophages, plasma cells, CD8 T cells, CD4 memory activated T cells, and monocytes) were present with a proportion higher than 0.05, i.e. 5%. These six cell types were present in most brain regions with only insignificant variation. A consistent association with age was seen with monocytes, CD8 T cells, and follicular helper T cells. Taken together, these data show that several infiltrating immune cell types are present in the non-diseased CNS tissue and that the proportions of infiltrating cells are affected by age in a manner that is consistent with literature on immunosenecence and inflammaging.

Herpesviruses and their genetic diversity in the blood virome of healthy individuals: effect of aging.

BACKGROUND: As we age, the functioning of the human immune system declines. The results of this are increases in morbidity and mortality associated with infectious diseases, cancer, cardiovascular disease, and neurodegenerative disease in elderly individuals, as well as a weakened vaccination response. The aging of the immune system is thought to affect and be affected by the human virome, the collection of all viruses present in an individual. Persistent viral infections, such as those caused by certain herpesviruses, can be present in an individual for long periods of time without any overt pathology, yet are associated with disease in states of compromised immune function. To better understand the effects on human health of such persistent viral infections, we must first understand how the human virome changes with age. We have now analyzed the composition of the whole blood virome of 317 individuals, 21-70 years old, using a metatranscriptomic approach. Use of RNA sequencing data allows for the unbiased detection of RNA viruses and active DNA viruses. RESULTS: The data obtained showed that Epstein-Barr virus (EBV) was the most frequently expressed virus, with other detected viruses being herpes simplex virus 1, human cytomegalovirus, torque teno viruses, and papillomaviruses. Of the 317 studied blood samples, 68 (21%) had EBV expression, whereas the other detected viruses were only detected in at most 6 samples (2%). We therefore focused on EBV in our further analyses. Frequency of EBV detection, relative EBV RNA abundance and the genetic diversity of EBV was not significantly different between age groups (21-59 and 60-70 years old). No significant correlation was seen between EBV RNA abundance and age. Deconvolution analysis revealed a significant difference in proportions of activated dendritic cells, macrophages M1, and activated mast cells between EBV expression positive and negative individuals. CONCLUSIONS: As it is likely that the EBV RNA quantified in this work is derived from reactivation of the latent EBV virus, these data suggest that age does not affect the rate of reactivation nor the genetic landscape of EBV. These findings offer new insight on the genetic diversity of a persistent EBV infection in the long-term.

Composition of the whole transcriptome in the human plasma: Cellular source and modification by aging.

Plasma contains several bioactive molecules (RNA, DNA, proteins, lipids, and metabolites), which are well preserved in extracellular vesicles, that are involved in many types of cell-to-cell interactions, and are capable of modifying biological processes in recipient cells. To obtain information about the source of mRNA molecules present in the plasma, we analyzed the plasma extracellular RNA (exRNA) of healthy individuals using RNA-sequencing and compared it to that of the peripheral blood mononuclear cell (PBMCs) of the same individual. The resultant data indicates that large proportion of the transcripts in plasma are derived from cell types other than PBMCs. To assess aging-associated changes in the plasma exRNA composition, gene ontology enrichment analysis was performed, revealing a functional decline in biological processes as a result of aging. Additionally, plasma RNA levels were analyzed with differential expression analysis, revealing 10 transcripts with significant aging-associated changes. Thus, it seems that the plasma exRNA is not fully derived from the PBMCs. Instead, other cell types supply RNAs to constitute the plasma exRNA compartment. This was true in both the young and elderly individuals that were tested. Furthermore, the RNA content of the plasma showed significant changes due to aging, affecting important biological processes.

Effect of aging on the transcriptomic changes associated with the expression of the HERV-K (HML-2) provirus at 1q22.

BACKGROUND: The human genome contains remnants of ancient retroviral infections called human endogenous retroviruses (HERV). Their expression is often observed in several diseases of autoimmune or inflammatory nature. However, the exact biological mechanisms induced by HERVs are still poorly understood. We have previously shown that several HERVs of the HERV-K (HML-2) family are strongly transcribed in the peripheral blood mononuclear cells (PBMC) derived from young and old individuals. To examine the potential functional consequences of HERV-K (HML-2) expression, we have now analyzed the correlation of its expression with age-associated changes in the transcriptome using gene set enrichment analysis (GSEA). We focused our analysis on the HERV-K (HML-2) provirus at 1q22, also known as ERVK-7. RESULTS: The genes strongly correlating with the expression of HERV-K (HML-2) provirus at 1q22 expression were found to be almost entirely different in young and old individuals. The number of genes strongly correlating (Pearson correlation coefficient ≥ 0.7) with 1q22 expression was 946 genes in the old and 435 in the young, of which only 41 genes correlated strongly in both. Consequently, the related gene ontology (GO) biological processes were different. In the older individuals, many of the highest correlating processes relate to the function of neutrophils. CONCLUSIONS: The results of this work suggest that the biological processes associated with the expression of HERV-K (HML-2) provirus at 1q22 are different in the blood of young and old individuals. Specifically, a strong association was found in the older individuals between neutrophil activity and the expression of the HERV-K (HML-2) provirus at 1q22. These findings offer insight into potential effects of altered HERV expression in older individuals.

Evaluation of FASN inhibitors by a versatile toolkit reveals differences in pharmacology between human and rodent FASN preparations and in antiproliferative efficacy in vitro vs. in situ in human cancer cells.

De novo synthesis of fatty acids is essential to maintain intensive proliferation of cancer cells. Unlike normal cells that utilize food-derived circulating lipids for their fuel, cancer cells rely on heightened lipogenesis irrespective of exogenous lipid availability. Overexpression and activity of the multidomain enzyme fatty acid synthase (FASN) is crucial in supplying palmitate for protumorigenic activity. Therefore, FASN has been proposed as an attractive target for drug development. As an effort to set up an effective toolkit to study FASN inhibitors in human and rodent tissues, we validated activity-based protein profiling (ABPP) as a viable approach to unveil inhibitors targeting FASN thioesterase domain (FASN-TE). ABPP was combined with multi-well plate-assays designed for classical substrate-based FASN activity analysis together with powerful monitoring of cancer cell proliferation using IncuCyte® Live Cell Analyzing System. FASN-TE inhibitors were identified by competitive ABPP using HEK293 cell lysates in a screen of in-house compounds (200+) designed to target serine hydrolase (SH) family. The identified compounds were tested for their inhibitor potencies in vitro using a substrate-based activity assay monitoring FASN-dependent NADPH consumption in LNCaP prostate cancer cell preparation, in parallel with selected reference inhibitors, including orlistat (THL), GSK2194069, GSK837149A, platensimycin and BI-99179. LNCaP lysate supernatant was validated as a reliable native preparation to monitor FASN-dependent NADPH consumption as opposed to human glioma GAMG cells, whereas FASN enrichment was a prerequisite for accurate assays. While inhibitor pharmacology was identical between human prostate and glioma cancer cell FASN preparations, notable differences were revealed between human and rodent FASN preparations, especially for inhibitors targeting FASN-TE. ABPP combined with substrate-based assays facilitated identification of pan thiol-reactive inhibitor scaffolds, exemplified by the 1,2,4-thiadiazole moiety. Finally, selected compounds were evaluated for their antiproliferative efficacy in situ using GAMG cells. These studies revealed that while the tested compounds acted as potent FASN inhibitors in vitro, only a few showed antiproliferative efficacy in situ. To conclude, we describe a versatile toolkit to study FASN inhibitors in vitro and in situ using human cancer cells and reveal dramatic pharmacological differences between human and rodent FASN preparations.

CD27- IgD- B cell memory subset associates with inflammation and frailty in elderly individuals but only in males.

BACKGROUND: Immunosenescence, i.e. the aging-associated decline of the capacity of the immune system, is characterized by several distinct changes in the number and functions of the immune cells. In the case of B cells, the total number of CD19+ B cells is lower in the blood of elderly individuals than in the younger ones. CD19+ B cell population contains several subsets, which are commonly characterized by the presence of CD27 and IgD molecules, i.e. naïve B cells (CD27- IgD+), IgM memory (CD27+ IgD+), switched memory (CD27+ IgD-) and late memory (CD27- IgD-). This late memory, double negative, population represents cells which are nondividing, but are still able to produce inflammatory mediators and in this way maybe contributing to the aging-associated inflammation, inflammaging. Here we have focused on the role of these B cell subsets in elderly individuals, nonagenarians, in the regulation of inflammation and inflammation-associated decline of bodily functions. As the biological aging process demonstrates gender-specific characteristics, the analyses were performed separately in males and female. RESULTS: A subcohort of The Vitality 90+ study (67 nonagenarians, 22/45 males/females and 40 young controls, 13/27 males/females) was used. Flow cytometric analysis indicated that the total percentage of the CD19+ cells was ca. 50% lower in the nonagenarians than in the controls in both genders. The proportions of these four B cell subsets within the CD19+ populations were very similar in young and old individuals. Thus, it seems that the aging-associated decline of the total CD19+ cells affects similarly all these B cell subsets. To analyze the role of these subsets in the regulation of inflammation, the correlation with IL-6 levels was calculated. A significant correlation was observed only with the percentage of CD27- IgD- cells and only in males. As inflammation is associated with aging-associated functional performance and frailty, the correlations with the Barthel index and frailty score was analyzed. Again, only the CD27- IgD- population demonstrated a strong male-specific correlation. CONCLUSIONS: These data show that the CD27- IgD- B cell subset demonstrates a strong pro-inflammatory effect in nonagenarians, which significantly associates with the decline of the bodily functions. However, this phenomenon is only observed in males.

Aging-associated patterns in the expression of human endogenous retroviruses.

Human endogenous retroviruses (HERV) are relics of ancient retroviral infections in our genome. Most of them have lost their coding capacity, but proviral RNA or protein have been observed in several disease states (e.g. in inflammatory and autoimmune diseases and malignancies). However, their clinical significance as well as their mechanisms of action have still remained elusive. As human aging is associated with several biological characteristics of these diseases, we now analyzed the aging-associated expression of the individual proviruses of two HERV families, HERV-K (91 proviruses) and HERV-W (213 proviruses) using genome-wide RNA-sequencing (RNA-seq). RNA was purified from blood cells derived from healthy young individuals (n = 7) and from nonagenarians (n = 7). The data indicated that in the case of HERV-K (HML-2) 33 proviruses had a detectable expression but in only 3 of those the expression levels were significantly different between the young and old individuals. In the HERV-W family expression was observed in 45 loci and only in one case the young/old difference was significant. However, applying hierarchical clustering on the HERV expression data resulted in the formation of two distinct clusters, one containing the young individuals and another the nonagenarians. This suggests, that even though the aging-associated differences in the expression levels of individual proviruses are minor, there seems to be some underlying aging-related pattern. These data indicate that aging does not have a strong effect on the expression of individual HERV proviruses, but instead several proviruses are affected moderately, leading to age-dependent expression profiles.

Human endogenous retrovirus HERV-K(HML-2) env expression is not associated with markers of immunosenescence.

Ageing of the human immune system, or immunosenescence, is characterised by distinct changes in the proportion of the various cell types, e.g., increase of the CD14+ monocytic cells, decrease of CD19+ B lymphocytes, and changes in T cell subpopulations, namely increase of CD4+ and CD8+ cells which have lost the costimulatory CD28 antigen. Currently, it is believed that the lifelong antigenic burden may be one of the inducers of immunosenescence. Thus far, only one exogenous stimulus, cytomegalovirus infection, has shown to be a major factor in this respect. To find other possible candidates, we evaluated the role of the evolutionary youngest group of human endogenous retroviruses, namely HERV-K(HML-2), on immunosenescence. HERVs exist in the genome as proviruses, but their activation has been detected in several immunopathologic conditions. The expression of HERV-K(HML-2) env was observed to be lower in the peripheral blood mononuclear cells of nonagenarians (n=61) than in those of young controls (n=37). These mRNA levels did not correlate with the age-associated differences in the proportions of CD14+, CD4+CD28- and CD8+CD28- cells, but in the case of CD19+ B cells a strong positive correlation was observed in the nonagenarians. Thus, these data suggest that HERVs do not function as antigenic drivers of immunosenescence. On the contrary, expression of HERV-K(HML-2) env is associated with more youthful levels of B cells.

Design, synthesis, and biological evaluation of 2,4-dihydropyrano[2,3-c]pyrazole derivatives as autotaxin inhibitors.

Inhibition of Autotaxin (ATX) is a potential treatment strategy for several diseases, including tumors with elevated ATX-lysophosphatidic acid (LPA) signaling. Combining structure-based virtual screening together with hen egg-white Autotaxin (ewATX) activity assays enabled the discovery of novel small-molecule ATX inhibitors with a 2,4-dihydropyrano[2,3-c]pyrazole scaffold. These compounds are suggested to bind to the lipophilic pocket, leaving the active site unrestrained. Our most potent compound, (S)-6-amino-4-(3,4-dichlorophenyl)-3-(4-[(4-fluorobenzyl)oxy]phenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile [(S)-25], inhibited human ATX (hATX) with an IC50-value of 134nM. It also blocked ATX-evoked but not LPA-mediated A2058 melanoma cell migration. Noteworthy, molecular modeling correctly predicted the biologically active enantiomer of 2,4-dihydropyrano[2,3-c]pyrazoles, as verified by compound crystallization and activity assays. Our study established the ewATX activity assay as a valid and affordable tool in ATX inhibitor discovery. Overall, our study offers novel insights and approaches into design of novel ATX inhibitors targeting the hydrophobic pocket instead of the active site.

Obesity accelerates epigenetic aging in middle-aged but not in elderly individuals.

BACKGROUND: Human aging is associated with profound changes in one of the major epigenetic mechanisms, DNA methylation. Some of these changes occur in a clock-like fashion, i.e., correlating with the calendar age of an individual, thus providing a new aging biomarker. Some reports have identified factors associated with the acceleration of the epigenetic age. However, it is also important to analyze the temporal changes in the epigenetic age, i.e., the duration of the observed acceleration, and the effects of the possible therapeutic and lifestyle modifications. METHODS: To address this issue, we determined the epigenetic age for a cohort of 183 healthy individuals using blood samples derived from two time points that were 25 years apart (between 15-24 and 40-49 years of age). Additionally, we also determined the epigenetic ages of 119 individuals in a cohort consisting of 90-year-old participants (nonagenarians). These were determined by using the Horvath algorithm based on the methylation level of 353 CpG sites. The data are indicated as the deviation of the epigenetic age from the calendar age (calendar age minus epigenetic age = delta age, ΔAGE). As obesity is often associated with accelerating aging and degenerative phenotypes, the correlation of the body mass index (BMI) with the ΔAGE was analyzed in the following three age groups: young adults, middle-aged, and nonagenarian. RESULTS: The data showed that BMI is associated with decreased ΔAGE, i.e., increased epigenetic age, in middle-aged individuals. This effect is also seen during the 25-year period from early adulthood to middle age, in which an increase in the BMI is significantly associated with a decrease in the ΔAGE. We also analyzed the association between BMI and epigenetic age in young and elderly individuals, but these associations were not significant. CONCLUSION: Taken together, the main finding on this report suggests that association between increased BMI and accelerated epigenetic aging in the blood cells of middle-aged individuals can be observed, and this effect is also detectable if the BMI has increased in adulthood. The fact that the association between BMI and epigenetic age can only be observed in the middle-aged group does not exclude the possibility that this association could be present throughout the human lifespan; it might just be masked by confounding factors in young adults and nonagenarian individuals.

Increased Paternal Age at Conception Is Associated with Transcriptomic Changes Involved in Mitochondrial Function in Elderly Individuals.

The increased paternal age at conception (PAC) has been associated with autism spectrum disorder (ASD), schizophrenia and other neurodevelopmental disorders, thus raising questions that imply, potential health concerns in the offspring. As opposed to female oogonia, the male germ cells undergo hundreds of cell divisions during the fertile years. Thus, the advanced paternal age is associated with increase of point mutations in the male spermatogonia DNA, implying that this could be the major driving mechanism behind the paternal age effect observed in the offspring. In addition to replication errors, DNA replication fidelity and inefficient DNA repair machinery in the spermatogonia also contribute to the mutagenic load. Our study population consisted of 38 nonagenarians, participants in the Vitality 90+ Study, born in the year 1920 (women n = 25, men n = 13), for whom the parental birth dates were available. The gene expression profile of the study subjects was determined with HumanHT-12 v4 Expression BeadChip from peripheral blood mononuclear cells. We used Spearman's rank correlation to look for the associations of gene expression with paternal age at conception. Associated transcripts were further analyzed with GOrilla and IPA to determine enriched cellular processes and pathways. PAC was associated with the expression levels of 648 transcripts in nonagenarian subjects. These transcripts belonged to the process of mitochondrial translational termination and the canonical pathway of Mitochondrial dysfunction, more specifically of Oxidative phosphorylation. The observed systematic down-regulation of several mitochondrial respiratory chain components implies compromised function in oxidative phosphorylation and thus in the production of chemical energy.

Chemoproteomic, biochemical and pharmacological approaches in the discovery of inhibitors targeting human α/ß-hydrolase domain containing 11 (ABHD11).

ABHD11 (α/ß-hydrolase domain containing 11) is a non-annotated enzyme belonging to the family of metabolic serine hydrolases (mSHs). Its natural substrates and products are unknown. Using competitive activity-based protein profiling (ABPP) to identify novel inhibitors of human (h)ABHD11, three compounds from our chemical library exhibited low nanomolar potency towards hABHD11. Competitive ABPP of various proteomes revealed fatty acid amide hydrolase (FAAH) as the sole off-target among the mSHs. Our fluorescent activity assays designed for natural lipase substrates revealed no activity of hABHD11 towards mono- or diacylglycerols. A broader profiling using para-nitrophenyl (pNP)-linked substrates indicated no amidase/protease, phosphatase, sulfatase, phospholipase C or phosphodiesterase activity. Instead, hABHD11 readily utilized para-nitrophenyl butyrate (pNPC4), indicating lipase/esterase-type activity that could be exploited in inhibitor discovery. Additionally, a homology model was created based on the crystal structure of bacterial esterase YbfF. In contrast to YbfF, which reportedly hydrolyze long-chain acyl-CoA, hABHD11 did not utilize oleoyl-CoA or arachidonoyl-CoA. In conclusion, the present study reports the discovery of potent hABHD11 inhibitors with good selectivity among mSHs. We developed substrate-based activity assays for hABHD11 that could be further exploited in inhibitor discovery and created the first homology-based hABHD11 model, offering initial insights into the active site of this poorly characterized enzyme.

In Vivo Characterization of the Ultrapotent Monoacylglycerol Lipase Inhibitor {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048).

Monoacylglycerol lipase (MAGL) is a serine hydrolase that acts as a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). In addition to terminating the signaling function of 2-AG, MAGL liberates arachidonic acid to be used as a primary source for neuroinflammatory prostaglandin synthesis in the brain. MAGL activity also contributes to cancer pathogenicity by producing precursors for tumor-promoting bioactive lipids. Pharmacological inhibitors of MAGL provide valuable tools for characterization of MAGL and 2-AG signaling pathways. They also hold great therapeutic potential to treat several pathophysiological conditions, such as pain, neurodegenerative disorders, and cancer. We have previously reported piperidine triazole urea, {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048), to be an ultrapotent and highly selective inhibitor of MAGL in vitro. Here, we characterize in vivo effects of JJKK-048. Acute in vivo administration of JJKK-048 induced a massive increase in mouse brain 2-AG levels without affecting brain anandamide levels. JJKK-048 appeared to be extremely potent in vivo. Activity-based protein profiling revealed that JJKK-048 maintains good selectivity toward MAGL over other serine hydrolases. Our results are also the first to show that JJKK-048 promoted significant analgesia in a writhing test with a low dose that did not cause cannabimimetic side effects. At a high dose, JJKK-048 induced analgesia both in the writhing test and in the tail-immersion test, as well as hypomotility and hyperthermia, but not catalepsy.

Methylomic predictors demonstrate the role of NF-κB in old-age mortality and are unrelated to the aging-associated epigenetic drift.

Changes in the DNA methylation (DNAm) landscape have been implicated in aging and cellular senescence. To unravel the role of specific DNAm patterns in late-life survival, we performed genome-wide methylation profiling in nonagenarians (n=111) and determined the performance of the methylomic predictors and conventional risk markers in a longitudinal setting. The survival model containing only the methylomic markers was superior in terms of predictive accuracy compared with the model containing only the conventional predictors or the model containing conventional predictors combined with the methylomic markers. At the 2.55-year follow-up, we identified 19 mortality-associated (false-discovery rate <0.5) CpG sites that mapped to genes functionally clustering around the nuclear factor kappa B (NF-κB) complex. Interestingly, none of the mortality-associated CpG sites overlapped with the established aging-associated DNAm sites. Our results are in line with previous findings on the role of NF-κB in controlling animal life spans and demonstrate the role of this complex in human longevity.

Potent and selective N-(4-sulfamoylphenyl)thiourea-based GPR55 agonists.

To date, many known G protein-coupled receptor 55 (GPR55) ligands are those identified among the cannabinoids. In order to further study the function of GPR55, new potent and selective ligands are needed. In this study, we utilized the screening results from PubChem bioassay AID 1961 which reports the results of Image-based HTS for Selective Agonists of GPR55. Three compounds, CID1792579, CID1252842 and CID1011163, were further evaluated and used as a starting point to create a series of nanomolar potency GPR55 agonists with N-(4-sulfamoylphenyl)thiourea scaffold. The GPR55 activity of the compounds were screened by using a commercial ß-arrestin PathHunter assay and the potential compounds were further evaluated by using a recombinant HEK cell line exhibiting GPR55-mediated effects on calcium signalling. The designed compounds were not active when tested against various endocannabinoid targets (CB1R, CB2R, FAAH, MGL, ABHD6 and ABHD12), indicating compounds' selectivity for the GPR55. Finally, structure-activity relationships of these compounds were explored.

Inhibitory effects and oxidation of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin via human CYP2A6 and its mouse and pig orthologous enzymes.

1. Information about the metabolism of compounds is essential in drug discovery and development, risk assessment of chemicals and further development of predictive methods. 2. In vitro and in silico methods were applied to evaluate the metabolic and inhibitory properties of 6-methylcoumarin, 7-methylcoumarin and 7-formylcoumarin with human CYP2A6, mouse CYP2A5 and pig CYP2A19. 3. 6-Methylcoumarin was oxidized to fluorescent 7-hydroxy-6-methylcoumarin by CYP2A6 (Km: 0.64-0.91 µM; Vmax: 0.81-0.89 min(-1)) and by CYP2A5 and CYP2A19. The reaction was almost completely inhibited at 10 µM 7-methylcoumarin in liver microsomes of human and mouse, but in pig only 40% inhibition was obtained with the anti-CYP2A5 antibody or with methoxsalen and pilocarpine. 7-Methylcoumarin was a mechanism-based inhibitor for CYP2A6, but not for the mouse and pig enzymes. 7-Formylcoumarin was a mechanism-based inhibitor for CYP2As of all species. 4. Docking and molecular dynamics simulations of 6-methylcoumarin and 7-methylcoumarin in the active sites of CYP2A6 and CYP2A5 demonstrated a favorable orientation of the 7-position of 6-methylcoumarin towards the heme moiety. Several orientations of 7-methylcoumarin were possible in CYP2A6 and CYP2A5. 5. These results indicate that the active site of CYP2A6 has unique interaction properties for ligands and differs in this respect from CYP2A5 and CYP2A19.

Cytomegalovirus infection accelerates epigenetic aging.

Epigenetic mechanisms such as DNA methylation (DNAm) have a central role in the regulation of gene expression and thereby in cellular differentiation and tissue homeostasis. It has recently been shown that aging is associated with profound changes in DNAm. Several of these methylation changes take place in a clock-like fashion, i.e. correlating with the calendar age of an individual. Thus, the epigenetic clock based on these kind of DNAm changes could provide a new biomarker for human aging process, i.e. being able to separate the calendar and biological age. Information about the correlation of the time indicated by this clock to the various aspects of immunosenescence is still missing. As chronic cytomegalovirus (CMV) infection is probably one of the major driving forces of immunosenescence, we now have analyzed the correlation of CMV seropositivity with the epigenetic age in the Vitality 90+cohort 1920 (122 nonagenarians and 21 young controls, CMV seropositivity rates 95% and 57%, respectively). The data showed that CMV seropositivity was associated with a higher epigenetic age in both of these age groups (median 26.5 vs. 24.0 (p < 0.02,Mann­Whitney U-test) in the young controls and 76.0 vs. 70.0 (p < 0.01) in the nonagenarians). Thus, these data provide a new aspect to the CMV associated pathological processes.

Length of paternal lifespan is manifested in the DNA methylome of their nonagenarian progeny.

The heritability of lifespan is 20-30%, but only a few genes associated with longevity have been identified. To explain this discrepancy, the inheritance of epigenetic features, such as DNA methylation, have been proposed to contribute to the heritability of lifespan.We investigated whether parental lifespan is associated with DNA methylation profile in nonagenarians. A regression model, adjusted for differences in blood cell proportions, identified 659 CpG sites where the level of methylation was associated with paternal lifespan. However, no association was observed between maternal lifespan and DNA methylation. The 659 CpG sites associated with paternal lifespan were enriched outside of CpG islands and were located in genes associated with development and morphogenesis, as well as cell signaling. The largest difference in the level of methylation between the progeny of the shortest-lived and longest-lived fathers was identified for CpG sites mapping to CXXC5. In addition, the level of methylation in three Notch-genes (NOTCH1, NOTCH3 and NOTCH4) was also associated with paternal lifespan.There are implications for the inheritance of acquired traits via epigenetic mechanisms in mammals. Here we describe DNA methylation features that are associated with paternal lifespan, and we speculate that the identified CpG sites may represent intergenerational epigenetic inheritance.