Characterization of transcriptional profiles associated with stress-induced neuronal activation in Arc-GFP mice.

Chronic stress has become a predominant factor associated with a variety of psychiatric disorders, such as depression and anxiety, in both human and animal models. Although multiple studies have looked at transcriptional changes after social defeat stress, these studies primarily focus on bulk tissues, which might dilute important molecular signatures of social interaction in activated cells. In this study, we employed the Arc-GFP mouse model in conjunction with chronic social defeat (CSD) to selectively isolate activated nuclei (AN) populations in the ventral hippocampus (vHIP) and prefrontal cortex (PFC) of resilient and susceptible animals. Nuclear RNA-seq of susceptible vs. resilient populations revealed distinct transcriptional profiles linked predominantly with neuronal and synaptic regulation mechanisms. In the vHIP, susceptible AN exhibited increased expression of genes related to the cytoskeleton and synaptic organization. At the same time, resilient AN showed upregulation of cell adhesion genes and differential expression of major glutamatergic subunits. In the PFC, susceptible mice exhibited upregulation of synaptotagmins and immediate early genes (IEGs), suggesting a potentially over-amplified neuronal activity state. Our findings provide a novel view of stress-exposed neuronal activation and the molecular response mechanisms in stress-susceptible vs. resilient animals, which may have important implications for understanding mental resilience.

Nanopore adaptive sampling of a metagenomic sample derived from a human monkeypox case.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misannotated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic.

A Call for Gene Expression Analysis in Whole Blood of Patients With Rheumatoid Arthritis (RA) as a Biomarker for RA-Associated Interstitial Lung Disease.

OBJECTIVE: Rheumatoid arthritis (RA)-associated interstitial lung disease (ILD) is one of the most common and prognostic organ manifestations of RA. Therefore, to allow effective treatment, it is of crucial importance to diagnose RA-ILD at the earliest possible stage. So far, the gold standard of early detection has been high-resolution computed tomography (HRCT) of the lungs. This procedure involves considerable radiation exposure for the patient and is therefore unsuitable as a routine screening measure for ethical reasons. Here, we propose the analysis of characteristic gene expression patterns as a biomarker to aid in the early detection and initiation of appropriate, possibly antifibrotic, therapy. METHODS: To investigate unique molecular patterns of RA-ILD, whole blood samples were taken from 12 female patients with RA-ILD (n = 7) or RA (n = 5). The RNA was extracted, sequenced by RNA-Seq, and analyzed for characteristic differences in the gene expression patterns between patients with RA-ILD and those with RA without ILD. RESULTS: The differential gene expression analysis revealed 9 significantly upregulated genes in RA-ILD compared to RA without ILD: arginase 1 (ARG1), thymidylate synthetase (TYMS), sortilin 1 (SORT1), marker of proliferation Ki-67 (MKI67), olfactomedin 4 (OLFM4), baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5), membrane spanning 4-domains A4A (MS4A4A), C-type lectin domain family 12 member A (CLEC12A), and the long intergenic nonprotein coding RNA (LINC02967). CONCLUSION: All gene products of these genes (except for LINC02967) are known from the literature to be involved in the pathogenesis of fibrosis. Further, for some, a contribution to the development of pulmonary fibrosis has even been demonstrated in experimental studies. Therefore, the results presented here provide an encouraging perspective for using specific gene expression patterns as biomarkers for the early detection and differential diagnosis of RA-ILD as a routine screening test.

2-Propanol interacting with Co3O4(001): A combined vSFS and AIMD study.

The interaction of 2-propanol with Co3O4(001) was studied by vibrational sum frequency spectroscopy and ab initio molecular dynamics simulations of 2-propanol dissolved in a water film to gain an insight, at the molecular level, into the pathways of catalytic oxidation. The experimental study has been performed under near ambient conditions, where the presence of water vapor is unavoidable, resulting in a water film on the sample and, thereby, allowing us to mimic the solution-water interface. Both experiment and theory conclude that 2-propanol adsorbs molecularly. The lack of dissociation is attributed to the adsorption geometry of 2-propanol in which the O-H bond does not point toward the surface. Furthermore, the copresent water not only competitively adsorbs on the surface but also inhibits 2-propanol deprotonation. The calculations reveal that the presence of water deactivates the lattice oxygen, thereby reducing the surface activity. This finding sheds light on the multifaceted role of water at the interface for the electrochemical oxidation of 2-propanol in aqueous solution as recently reported [Falk et al., ChemCatChem 13, 2942-2951 (2021)]. At higher temperatures, 2-propanol remains molecularly adsorbed on Co3O4(001) until it desorbs with increasing surface temperature.

Acral lamellar ichthyosis with amino acid substitution in the C-terminus of keratin 2.

BACKGROUND: Most cases of hereditary ichthyoses present with generalized scaling and skin dryness. However, in some cases skin involvement is restricted to particular body regions as in acral lamellar ichthyosis. OBJECTIVES: We report on the genetic basis of acral ichthyosis in two families presenting with a similar phenotype. METHODS: Genetic testing was performed by targeted next generation sequencing and whole-exome sequencing. For identity-by-descent analysis, the parents were genotyped and data analysis was performed with the Chromosome Analysis Suite Software. RT-PCR with RNA extracted from skin samples was used to analyse the effect of variants on splicing. RESULTS: Genetic testing identified a few heterozygous variants, but only the variant in KRT2 c.1912 T > C, p.Phe638Leu segregated with the disease and remained the strongest candidate. Pairwise identity-by-descent analysis revealed no indication of family relationship. Phenylalanine 638 is the second last amino acid upstream of the termination codon in the tail of K2, and substitution to leucine is predicted as probably damaging. Assessment of the variant is difficult, in part due to the lack of crystal structures of this region. CONCLUSIONS: Altogether, we show that a type of autosomal dominant acral ichthyosis is most probably caused by an amino acid substitution in the C-terminus of keratin 2.

Hypermethylation of RAD9A intron 2 in childhood cancer patients, leukemia and tumor cell lines suggest a role for oncogenic transformation.

Most childhood cancers occur sporadically and cannot be explained by an inherited mutation or an unhealthy lifestyle. However, risk factors might trigger the oncogenic transformation of cells. Among other regulatory signals, hypermethylation of RAD9A intron 2 is responsible for the increased expression of RAD9A protein, which may play a role in oncogenic transformation. Here, we analyzed the RAD9A intron 2 methylation in primary fibroblasts of 20 patients with primary cancer in childhood and second primary cancer (2N) later in life, 20 matched patients with only one primary cancer in childhood (1N) and 20 matched cancer-free controls (0N), using bisulfite pyrosequencing and deep bisulfite sequencing (DBS). Four 1N patients and one 2N patient displayed elevated mean methylation levels (≥ 10 %) of RAD9A. DBS revealed ≥ 2 % hypermethylated alleles of RAD9A, indicative for constitutive mosaic epimutations. Bone marrow samples of NHL and AML tumor patients (n=74), EBV (Epstein Barr Virus) lymphoblasts (n=6), tumor cell lines (n=5) and FaDu subclones (n=13) were analyzed to substantiate our findings. We find a broad spectrum of tumor entities with an aberrant methylation of RAD9A. We detected a significant difference in mean methylation of RAD9A for NHL versus AML patients (p ≤0.025). Molecular karyotyping of AML samples during therapy with hypermethylated RAD9A showed an evolving duplication of 1.8 kb on Chr16p13.3 including the PKD1 gene. Radiation, colony formation assays, cell proliferation, PCR and molecular karyotyping SNP-array experiments using generated FaDu subclones suggest that hypermethylation of RAD9A intron 2 is associated with genomic imbalances in regions with tumor-relevant genes and survival of the cells. In conclusion, this is the very first study of RAD9A intron 2 methylation in childhood cancer and Leukemia. RAD9A epimutations may have an impact on leukemia and tumorigenesis and can potentially serve as a biomarker.

A fresh look at the structure of aromatic thiols on Au surfaces from theory and experiment.

A detailed study of the adsorption structure of self-assembled monolayers of 4-nitrothiophenol on the Au(111) surface was performed from a theoretical perspective via first-principles density functional theory calculations and experimentally by Raman and vibrational sum frequency spectroscopy (vSFS) with an emphasis on the molecular orientation. Simulations-including an explicit van der Waals (vdW) description-for different adsorbate structures, namely, for (3×3), (2 × 2), and (3 × 3) surface unit cells, reveal a significant tilting of the molecules toward the surface with decreasing coverage from 75° down to 32° tilt angle. vSFS suggests a tilt angle of 50°, which agrees well with the one calculated for a structure with a coverage of 0.25. Furthermore, calculated vibrational eigenvectors and spectra allowed us to identify characteristic in-plane (NO2 scissoring) and out-of-plane (C-H wagging) modes and to predict their strength in the spectrum in dependence of the adsorption geometry. We additionally performed calculations for biphenylthiol and terphenylthiol to assess the impact of multiple aromatic rings and found that vdW interactions are significantly increasing with this number, as evidenced by the absorption energy and the molecule adopting a more upright-standing geometry.

Early life adversity targets the transcriptional signature of hippocampal NG2+ glia and affects voltage gated sodium (Nav) channels properties.

The precise mechanisms underlying the detrimental effects of early life adversity (ELA) on adult mental health remain still elusive. To date, most studies have exclusively targeted neuronal populations and not considered neuron-glia crosstalk as a crucially important element for the integrity of stress-related brain function. Here, we have investigated the impact of ELA, in the form of a limited bedding and nesting material (LBN) paradigm, on a glial subpopulation with unique properties in brain homeostasis, the NG2+ cells. First, we have established a link between maternal behavior, activation of the offspring's stress response and heterogeneity in the outcome to LBN manipulation. We further showed that LBN targets the hippocampal NG2+ transcriptome with glucocorticoids being an important mediator of the LBN-induced molecular changes. LBN altered the NG2+ transcriptome and these transcriptional effects were correlated with glucocorticoids levels. The functional relevance of one LBN-induced candidate gene, Scn7a, could be confirmed by an increase in the density of voltage-gated sodium (Nav) channel activated currents in hippocampal NG2+ cells. Scn7a remained upregulated until adulthood in LBN animals, which displayed impaired cognitive performance. Considering that Nav channels are important for NG2+ cell-to-neuron communication, our findings provide novel insights into the disruption of this process in LBN mice.

The DNA methylation profile of human spermatogonia at single-cell- and single-allele-resolution refutes its role in spermatogonial stem cell function and germ cell differentiation.

Human spermatogonial stem cells (hSSCs) have potential in fertility preservation of prepubertal boys or in treatment of male adults suffering from meiotic arrest. Prior to therapeutic application, in vitro propagation of rare hSSCs is mandatory. As the published data points to epigenetic alterations in long-term cell culture of spermatogonia (SPG), an initial characterisation of their DNA methylation state is important. Testicular biopsies from five adult normogonadotropic patients were converted into aggregate-free cell suspensions. FGFR3-positive (FGFR3+) SPG, resembling a very early stem cell state, were labelled with magnetic beads and isolated in addition to unlabelled SPG (FGFR3-). DNA methylation was assessed by limiting dilution bisulfite pyrosequencing for paternally imprinted (H19 and MEG3), maternally imprinted (KCNQ1OT1, PEG3, and SNRPN), pluripotency (POU5F1/OCT4 and NANOG), and spermatogonial/hSSC marker (FGFR3, GFRA1, PLZF, and L1TD1) genes on either single cells or pools of 10 cells. Both spermatogonial subpopulations exhibited a methylation pattern largely equivalent to sperm, with hypomethylation of hSSC marker and maternally imprinted genes and hypermethylation of pluripotency and paternally imprinted genes. Interestingly, we detected fine differences between the two spermatogonial subpopulations, which were reflected by an inverse methylation pattern of imprinted genes, i.e. decreasing methylation in hypomethylated genes and increasing methylation in hypermethylated genes, from FGFR3+ through FGFR3- SPG to sperm. Limitations of this study are due to it not being performed on a genome-wide level and being based on previously published regulatory gene regions. However, the concordance of DNA methylation between SPG and sperm implies that hSSC regulation and germ cell differentiation do not occur at the DNA methylation level.

Electrical conductivity of silver nanoparticle doped carbon nanofibres measured by CS-AFM.

In this work, a pioneering study on the electrical properties of composite carbon nanofibres (CNFs) using current-sensitive atomic force microscopy (CS-AFM) has been demonstrated. CNFs are highly interesting materials which are usable in a wide array of applications e.g. electrode materials for biosensors, lithium ion batteries, fuel cells and supercapacitors. CNFs offer a high specific surface area and thus have a high contact area for charge transfer. CNFs can be produced using spinnable polyacrylonitrile (PAN) as a precursor for carbonisation. For the purpose of developing efficient CNFs with high conductivity and power density, silver nanoparticle (AgNPs)-containing PAN solutions were electrospun to form composite nanofibres which was followed by heat treatment. The applied voltage of the spinning setup and the content of both PAN and the silver nanoparticles in the spinning solution were varied in order to study their influence on the morphology and the electrical properties of the nanofibres. The resultant morphologies and fibre diameters were determined by scanning electron microscopy (SEM). The formation of silver nanoparticles was characterised in solution by UV-visible absorption spectroscopy and dynamic light scattering (DLS), while energy-dispersive X-ray spectroscopy (EDX) and transmission electron microscopy (TEM) were carried out to investigate the presence as well as the average diameter of the AgNPs. The electrical properties of the CNFs were investigated using CS-AFM. This technique gives us the possibility to explore the electrical properties of single fibers and hence derive relationships between the structural features and the electrical properties. Our results show that the composite CNFs have a higher electrical conductivity than the neat CNFs and both the average diameter of the fibers and the electrical conductivity increase with an increasing AgNP content.

Promoter methylation status of ASC/TMS1/PYCARD is associated with decreased overall survival and TNM status in patients with early stage non-small cell lung cancer (NSCLC).

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide, with 5-year overall survival less than 15%. Therefore, it is essential to find biomarkers for early detection and prognosis. Aberrant DNA methylation is a common feature of human cancers and its utility is already recognized in cancer management. The aim of this study was to explore the diagnostic and prognostic value of the promoter methylation status of the ASC/TMS1/PYCARD and MyD88 genes, key adaptor molecules in the activation of the innate immune response and apoptosis pathways. METHODS: A total of 50 non-small cell lung cancer (NSCLC) patients were enrolled in the study. Methylation of bisulphite converted DNA was quantified by pyrosequencing in fresh frozen malignant tissues and adjacent non-malignant tissues. Associations between methylation and lung function, tumor grade and overall survival were evaluated using receiver-operating characteristics (ROC) analysis and statistical tests of hypothesis. RESULTS: Methylation level of tested genes is generally low but significantly decreased in tumor tissues (ASC/TMS1/PYCARD, P<0.0001; MyD88, P<0.0002), which correlates with increased protein expression. Three CpG sites were identified as promising diagnostic marker candidates; CpG11 (-63 position) in ASC/TMS1/PYCARD and CpG1 (-253 position) and 2 (-265 position) in MyD88. The association study showed that the methylation status of the ASC/TMS1 CpG4 site (-34 position) in malignant and non-malignant tissues is associated with the overall survival (P=0.019) and the methylation status of CpG8 site (-92 position) is associated with TNM-stage (P=0.011). CONCLUSIONS: The methylation status of the ASC/TMS1/PYCARD and MyD88 promoters are promising prognostic biomarker candidates. However, presented results should be considered as a preliminary and should be confirmed on the larger number of the samples.

Superovulation Influences Methylation Reprogramming and Delays Onset of DNA Replication in Both Pronuclei of Mouse Zygotes.

Although an essential component of assisted reproductive technologies, ovarian stimulation, or superovulation, may interfere with the epigenetic reprogramming machinery during early embryogenesis and gametogenesis. To investigate the possible impact of superovulation particularly on the methylation reprogramming process directly after fertilization, we performed immunofluorescence staining of pronuclear (PN) stage embryos with antibodies against 5mC and 5hmC. PN stage embryos obtained by superovulation displayed an increased incidence of abnormal methylation and hydroxymethylation patterns in both maternal and paternal pronuclear DNA. Subsequent single-cell RT-qPCR analyses of the Tet1, Tet2, and Tet3 genes revealed no significant expression differences between PN stage embryos from spontaneously and superovulated matings that could be causative for the abnormal methylation and hydroxymethylation patterns. To analyze the possible contribution of TET-independent replication-associated demethylation mechanisms, we then determined the 5mC and 5hmC levels of PN stage mouse embryos using immunofluorescence analyses after inhibition of DNA replication with aphidicolin. Inhibition of DNA replication had no effect on abnormal methylation and hydroxymethylation patterns that still persisted in the superovulated group. Interestingly, the onset of DNA replication, which was also analyzed in these experiments, was remarkably delayed in the superovulated group. Our findings imply an impact of superovulation on both replication-dependent and -independent or yet unknown demethylation mechanisms in PN stage mouse embryos. In addition, they reveal for the first time a negative effect of superovulation on the initiation of DNA replication in PN stage mouse embryos.

Early-life adversity selectively impairs α2-GABAA receptor expression in the mouse nucleus accumbens and influences the behavioral effects of cocaine.

Haplotypes of the Gabra2 gene encoding the α2-subunit of the GABAA receptor (GABAAR) are associated with drug abuse, suggesting that α2-GABAARs may play an important role in the circuitry underlying drug misuse. The genetic association of Gabra2 haplotypes with cocaine addiction appears to be evident primarily in individuals who had experienced childhood trauma. Given this association of childhood trauma, cocaine abuse and the Gabra2 haplotypes, we have explored in a mouse model of early life adversity (ELA) whether such events influence the behavioral effects of cocaine and if, as suggested by the human studies, α2-GABAARs in the nucleus accumbens (NAc) are involved in these perturbed behaviors. In adult mice prior ELA caused a selective decrease of accumbal α2-subunit mRNA, resulting in a selective decrease in the number and size of the α2-subunit (but not the α1-subunit) immunoreactive clusters in NAc core medium spiny neurons (MSNs). Functionally, in adult MSNs ELA decreased the amplitude and frequency of GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs), a profile similar to that of α2 "knock-out" (α2-/-) mice. Behaviourally, adult male ELA and α2-/- mice exhibited an enhanced locomotor response to acute cocaine and blunted sensitisation upon repeated cocaine administration, when compared to their appropriate controls. Collectively, these findings reveal a neurobiological mechanism which may relate to the clinical observation that early trauma increases the risk for substance abuse disorder (SAD) in individuals harbouring haplotypic variations in the Gabra2 gene.

Dppa3 expression is critical for generation of fully reprogrammed iPS cells and maintenance of Dlk1-Dio3 imprinting.

Reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) often generates partially reprogrammed iPSCs (pre-iPSCs), low-grade chimera forming iPSCs (lg-iPSCs) and fully reprogrammed, high-grade chimera production competent iPSCs (hg-iPSCs). Lg-iPSC transcriptome analysis revealed misregulated Dlk1-Dio3 cluster gene expression and subsequently the imprinting defect at the Dlk1-Dio3 locus. Here, we show that germ-cell marker Dppa3 is present only in lg-iPSCs and hg-iPSCs, and that induction with exogenous Dppa3 enhances reprogramming kinetics, generating all hg-iPSCs, similar to vitamin C (Vc). Conversely, Dppa3-null fibroblasts show reprogramming block at pre-iPSCs state and Dlk1-Dio3 imprinting defect. At the molecular level, we show that Dppa3 is associated with Dlk1-Dio3 locus and identify that Dppa3 maintains imprinting by antagonizing Dnmt3a binding. Our results further show molecular parallels between Dppa3 and Vc in Dlk1-Dio3 imprinting maintenance and suggest that early activation of Dppa3 is one of the cascades through which Vc facilitates the generation of fully reprogrammed iPSCs.

The disease-specific phenotype in cardiomyocytes derived from induced pluripotent stem cells of two long QT syndrome type 3 patients.

Long QT syndromes (LQTS) are heritable diseases characterized by prolongation of the QT interval on an electrocardiogram, which often leads to syncope and sudden cardiac death. Here we report the generation of induced pluripotent stems (iPS) cells from two patients with LQTS type 3 carrying a different point mutation in a sodium channel Nav1.5 (p.V240M and p.R535Q) and functional characterization of cardiomyocytes (CM) derived from them. The iPS cells exhibited all characteristic properties of pluripotent stem cells, maintained the disease-specific mutation and readily differentiated to CM. The duration of action potentials at 50% and 90% repolarization was longer in LQTS-3 CM as compared to control CM but this difference did not reach statistical significance due to high variations among cells. Sodium current recordings demonstrated longer time to peak and longer time to 90% of inactivation of the Na(+) channel in the LQTS-3 CM. This hints at a defective Na(+) channel caused by deficiency in open-state inactivation of the Na(+) channel that is characteristic of LQTS-3. These analyses suggest that the effect of channel mutation in the diseased CM is demonstrated in vitro and that the iPS cell-derived CM can serve as a model system for studying the pathophysiology of LQTS-3, toxicity testing and design of novel therapeutics. However, further improvements in the model are still required to reduce cell-to-cell and cell line-to-cell line variability.

Stochastic loss of silencing of the imprinted Ndn/NDN allele, in a mouse model and humans with prader-willi syndrome, has functional consequences.

Genomic imprinting is a process that causes genes to be expressed from one allele only according to parental origin, the other allele being silent. Diseases can arise when the normally active alleles are not expressed. In this context, low level of expression of the normally silent alleles has been considered as genetic noise although such expression has never been further studied. Prader-Willi Syndrome (PWS) is a neurodevelopmental disease involving imprinted genes, including NDN, which are only expressed from the paternally inherited allele, with the maternally inherited allele silent. We present the first in-depth study of the low expression of a normally silent imprinted allele, in pathological context. Using a variety of qualitative and quantitative approaches and comparing wild-type, heterozygous and homozygous mice deleted for Ndn, we show that, in absence of the paternal Ndn allele, the maternal Ndn allele is expressed at an extremely low level with a high degree of non-genetic heterogeneity. The level of this expression is sex-dependent and shows transgenerational epigenetic inheritance. In about 50% of mutant mice, this expression reduces birth lethality and severity of the breathing deficiency, correlated with a reduction in the loss of serotonergic neurons. In wild-type brains, the maternal Ndn allele is never expressed. However, using several mouse models, we reveal a competition between non-imprinted Ndn promoters which results in monoallelic (paternal or maternal) Ndn expression, suggesting that Ndn allelic exclusion occurs in the absence of imprinting regulation. Importantly, specific expression of the maternal NDN allele is also detected in post-mortem brain samples of PWS individuals. Our data reveal an unexpected epigenetic flexibility of PWS imprinted genes that could be exploited to reactivate the functional but dormant maternal alleles in PWS. Overall our results reveal high non-genetic heterogeneity between genetically identical individuals that might underlie the variability of the phenotype.

Sirtuin-6-dependent genetic and epigenetic alterations are associated with poor clinical outcome in hepatocellular carcinoma patients.

UNLABELLED: Sirtuin 6 (SIRT6) is a member of the sirtuin family of NAD+-dependent deacetylases. Genetic deletion of Sirt6 in mice results in a severe degenerative phenotype with impaired liver function and premature death. The role of SIRT6 in development and progression of hepatocellular carcinoma is currently unknown. We first investigated SIRT6 expression in 153 primary human liver cancers and in normal and cirrhotic livers using microarray analysis. SIRT6 was significantly down-regulated in both cirrhotic livers and cancer. A Sirt6 knockout (KO) gene expression signature was generated from primary hepatoctyes isolated from 3-week-old Sirt6-deficient animals. Sirt6-deficient hepatocytes showed up-regulation of established hepatocellular carcinoma (HCC) biomarkers alpha-fetoprotein (Afp), insulin-like growth factor 2 (Igf2), H19, and glypican-3. Furthermore, decreased SIRT6 expression was observed in hepatoma cell lines that are known to be apoptosis-insensitive. Re-expression of SIRT6 in HepG2 cells increased apoptosis sensitivity to CD95-stimulation or chemotherapy treatment. Loss of Sirt6 was characterized by oncogenic changes, such as global hypomethylation, as well as metabolic changes, such as hypoglycemia and increased fat deposition. The hepatocyte-specific Sirt6-KO signature had a prognostic impact and was enriched in patients with poorly differentiated tumors with high AFP levels as well as recurrent disease. Finally, we demonstrated that the Sirt6-KO signature possessed a predictive value for tumors other than HCC (e.g., breast and lung cancer). CONCLUSION: Loss of SIRT6 induces epigenetic changes that may be relevant to chronic liver disease and HCC development. Down-regulation of SIRT6 and genes dysregulated by loss of SIRT6 possess oncogenic effects in hepatocarcinogenesis. Our data demonstrate that deficiency in one epigenetic regulator predisposes a tumorigenic phenotype that ultimately has relevance for outcome of HCC and other cancer patients.

Induced pluripotent mesenchymal stromal cell clones retain donor-derived differences in DNA methylation profiles.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is an epigenetic phenomenon. It has been suggested that iPSC retain some tissue-specific memory whereas little is known about interindividual epigenetic variation. We have reprogrammed mesenchymal stromal cells from human bone marrow (iP-MSC) and compared their DNA methylation profiles with initial MSC and embryonic stem cells (ESCs) using high-density DNA methylation arrays covering more than 450,000 CpG sites. Overall, DNA methylation patterns of iP-MSC and ESC were similar whereas some CpG sites revealed highly significant differences, which were not related to parental MSC. Furthermore, hypermethylation in iP-MSC versus ESC occurred preferentially outside of CpG islands and was enriched in genes involved in epidermal differentiation indicating that these differences are not due to random de novo methylation. Subsequently, we searched for CpG sites with donor-specific variation. These "epigenetic fingerprints" were highly enriched in non-promoter regions and outside of CpG islands-and they were maintained upon reprogramming. In conclusion, iP-MSC clones revealed relatively little intraindividual variation but they maintained donor-derived epigenetic differences. In the absence of isogenic controls, it would therefore be more appropriate to compare iPSC from different donors rather than a high number of different clones from the same patient.

Transcriptome fingerprint of bovine 2-cell stage blastomeres is directly correlated with the individual developmental competence of the corresponding sister blastomere.

To date, gene expression profiles of bovine preimplantation embryos have only been indirectly related to developmental potential due to the invasive nature of such procedures. This study sought to find a direct correlation between transcriptome fingerprint of blastomeres of bovine 2-cell stage embryos with developmental competence of the corresponding sister blastomeres. Isolated blastomeres were classified according to the sister blastomere's development into three groups: two groups displayed developmental incompetency, including those blastomeres whose corresponding sister blastomeres either stopped cleaving after separation (2CB) or were blocked after two additional cleavages before embryonic genome activation (8CB). In the third group were competent blastomeres, which were defined as those whose sister blastomeres developed to the blastocyst stage (BL). As a result, developmental capacity of corresponding sister blastomeres was highly similar. Microarray analysis revealed 77 genes to be commonly differentially regulated among competent and incompetent blastomeres as well as blocked blastomeres. Clustering of differentially expressed genes according to molecular functions and pathways revealed antioxidant activity, NRF2-mediated oxidative stress response, and oxidative phosphorylation to be the main ontologies affected. Expression levels of selected candidate genes were further characterized in an independent model for developmental competence based on the time of first cleavage postfertilization. Moreover, overall results of this study were confirmed by higher developmental rates and more beneficial expression of CAT and PRDX1 when cultured in an antioxidative environment. These results will help us to understand molecular mechanisms defining developmental destination of individual bovine preimplantation embryos.

In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.

BACKGROUND/AIMS: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. METHODS: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. RESULTS: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca(2+) release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca(2+)-induced Ca(2+)-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. CONCLUSION: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.