Probable Three-Species In Vivo Transfer of blaNDM-1 in a Single Patient in Greece: Occurrence of NDM-1-Producing Klebsiella pneumoniae, Proteus mirabilis, and Morganella morganii.

NDM carbapenemase-encoding genes disseminate commonly among Enterobacterales through transferable plasmids carrying additional resistance determinants. Apart from the intra-species dissemination, the inter-species exchange of plasmids seems to play an additional important role in the spread of blaNDM. We here present the genetics related to the isolation of three species (Klebsiella pneumoniae, Proteus mirabilis, and Morganella morganii) harboring the blaNDM-1 gene from a single patient in Greece. Bacterial identification and antimicrobial susceptibility testing were performed using the Vitek2. Whole genome sequencing and bioinformatic tools were used to identify resistance genes and plasmids. BlaNDM-1 harboring plasmids were found in all three isolates. Moreover, the plasmid constructs of the respective incomplete or circular contigs showed that the blaNDM-1 and its neighboring genes form a cluster that was found in all isolates. Our microbiological findings, together with the patient's history, suggest the in vivo transfer of the blaNDM-1-containing cluster through three different species in a single patient.

16S-23S rRNA Internal Transcribed Spacer Region (ITS) Sequencing: A Potential Molecular Diagnostic Tool for Differentiating Lactococcus garvieae and Lactococcus petauri.

Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.

Biological Effects of "Inflammageing" on Human Oral Cells: Insights into a Potential Confounder of Age-Related Diseases.

OBJECTIVES: The term "inflammageing" describes the process of inflammation-induced aging that leads living cells to a state of permanent cell cycle arrest due to chronic antigenic irritation. This in vitro study aimed to shed light on the mechanisms of "inflammageing" on human oral cells. METHODS: Primary cultures of human gingival fibroblasts (hGFs) were exposed to variable pro-inflammatory stimuli, including lipopolysaccharide (LPS), Tumor Necrosis Factor-alpha (TNFa), and gingival crevicular fluid (GCF) collected from active periodontal pockets of systemically healthy patients. Inflammageing was studied through two experimental models, employing either late-passage ("aged") cells (p. 10) that were exposed to the pro-inflammatory stimuli or early-passage ("young") cells (p. 1) continuously exposed during a period of several passages (up to p. 10) to the above-mentioned stimuli. Cells were evaluated for the expression of beta-galactosidase activity (histochemical staining), senescence-associated genes (qPCR analysis), and biomarkers related to a Senescence-Associated Secretory Phenotype (SASP), through proteome profile analysis and bioinformatics. RESULTS: A significant increase (p < 0.05) in beta-galactosidase-positive cells was observed after exposure to each pro-inflammatory stimulus. The senescence-associated gene expression included upregulation for CCND1 and downregulation for SUSD6, and STAG1, a profile typical for cellular senescence. Overall, pro-inflammatory priming of late-passage cells caused more pronounced effects in terms of senescence than long-term exposure of early-passage cells to these stimuli. Proteomic analysis showed induction of SASP, evidenced by upregulation of several pro-inflammatory proteins (IL-6, IL-10, IL-16, IP-10, MCP-1, MCP-2, M-CSF, MIP-1a, MIP-1b, TNFb, sTNF-RI, sTNF-RII, TIMP-2) implicated in cellular aging and immune responses. The least potent impact on the induction of SASP was provoked by LPS and the most pronounced by GCF. CONCLUSION: This study demonstrates that long-term exposure of hGFs to various pro-inflammatory signals induced or accelerated cellular senescence with the most pronounced impact noted for the late-passage cells. The outcome of these analyses provides insights into oral chronic inflammation as a potential confounder of age-related diseases.

First detection of mcr-1-producing Escherichia coli in Greece.

OBJECTIVES: In this communication, we describe the emergence of the mcr-1 colistin resistance gene in a blaCTX-M-32 extended-spectrum-ß-lactamase-producing Escherichia coli isolate recovered from a pediatric patient in Greece. METHODS: Bacterial identification and antimicrobial susceptibility testing were performed with the VITEK2 automated system and broth microdilution. Detection of resistance genes, assignment to sequence type, in silico plasmid detection, and virulence factors were carried out using ResFinder, MLST 2.0, PlasmidFinder 2.1., and VirulenceFinder 2.0, respectively. PlasmidSPAdes v3.11.1 was used to assemble the plasmid contigs. The mcr-1.1-containing plasmid was analyzed for insertion sequence elements using ISfinder. Phylogenetically relevant sequences of the plasmid were identified using the Microbe BLASTN suite. RESULTS: The microorganism was assigned to sequence type 48 and carried four plasmids of different incompatibility groups. The specific mcr-1.1 allele was located in a 32.722 bp plasmid belonging to the IncX4 group with no additional resistance genes. CONCLUSION: To the best of our knowledge, this is the first detection of mcr-1 in a human specimen in our country. A potential spread of mcr-1 in Greece is concerning because of the existing high rates of carbapenem resistance and colistin usage as a last resort regimen.

PrecisionFDA Truth Challenge V2: Calling variants from short and long reads in difficult-to-map regions.

The precisionFDA Truth Challenge V2 aimed to assess the state of the art of variant calling in challenging genomic regions. Starting with FASTQs, 20 challenge participants applied their variant-calling pipelines and submitted 64 variant call sets for one or more sequencing technologies (Illumina, PacBio HiFi, and Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with updated Genome in a Bottle benchmark sets and genome stratifications. Challenge submissions included numerous innovative methods, with graph-based and machine learning methods scoring best for short-read and long-read datasets, respectively. With machine learning approaches, combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants.

Listeria monocytogenes isolates from Cornu aspersum snails: Whole genome-based characterization and host-pathogen interactions in a snail infection model.

Even though Listeria monocytogenes is an extensive-studied foodborne pathogen, genome analysis of isolates from snails that may represent a reservoir of L. monocytogenes are still scarce. Here, we use whole-genome sequencing (WGS) to assess the genomic diversity of hypervirulent, virulent and non-virulent phenotypes of 15 L. monocytogenes isolated from snails to unveil their survival, virulence, and host-pathogen mechanisms of interactions in a snail infection model. Most of isolates (66.7%) were characterized as multidrug resistant (MDR) and belonged to clonal complexes (CCs) which are strongly associated with cases of human infection. All isolates contained intact genes associated with invasion and infection while hypervirulent isolates are adapted to host environment, possessing genes which are involved in teichoic acid biosynthesis, peptidoglycan modification and biofilm formation, correlating with their tolerance to haemolymph plasma phenotype and biofilm formation ability. A snail infection model showed that hypervirulent isolates triggered programmed host cell death pathway by increasing up to 30% the circulating apoptotic hemocytes in combination with induced nitrate production and reactive oxygen species (ROS) generation in snails' haemolymph. In contrast, the administration of the non-virulent strain which possesses a truncated mogR gene that regulates flagellar motility gene expression led only to an increase of necrotic non-apoptotic cells. Overall, this study provides significant insights into the genetic diversity of L. monocytogenes from snails, the genomic features of them linked to their hypervirulent/non-virulent phenotype, and the mechanisms of host-pathogen interactions.

Dysregulation of Plasma miR-146a and miR-155 Expression Profile in Mycosis Fungoides Is Associated with rs2910164 and rs767649 Polymorphisms.

Diagnosis of Mycosis Fungoides (MF) may be challenging, due to its polymorphic nature. The use of miRNAs as biomarkers to assist in diagnosis has been investigated, mainly in skin lesion biopsies. The purpose of this study is to evaluate the plasma levels of miR-146a and miR-155 in MF patients and to investigate their association with SNPs of their genes. Plasma miRNAs were quantified by RT-qPCR. Genomic DNA was used for SNPs' genotyping by Sanger sequencing. Plasma levels of miR-146a and miR-155 were significantly higher in patients vs. controls, in early MF patients vs. controls, and in advanced vs. early MF patients. Both miRNAs' levels were significantly higher in stage IIB vs. early-stage patients. miR-155 plasma levels were significantly higher in patients with skin tumors or erythroderma. CC genotype (rs2910164 C>G) was significantly more frequent in healthy controls and associated with lower MF risk and lower miR-146a levels. The AA genotype (rs767649 T>A) was significantly more frequent in patients and correlated with increased MF risk and increased miR-155 levels. The combination of GG+AA was only detected in patients and was correlated with higher MF susceptibility. Increased mir-146a and mir-155 plasma levels in MF is an important finding to establish putative noninvasive biomarkers. The presence of SNPs is closely associated with miRs' expression, and possibly with disease susceptibility.

Staphylococcus aureus Isolated from Ruminants with Mastitis in Northern Greece Dairy Herds: Genetic Relatedness and Phenotypic and Genotypic Characterization.

Staphylococcus aureus is the most common mastitis-related pathogen in dairy cattle, goats, and sheep worldwide. However, the population structure and genomic characteristics of mastitis-associated S. aureus in small ruminants are limited. Furthermore, the genotypic and phenotypic characteristics involved in the pathogenicity of S. aureus have been thoroughly defined, yet their association with the severity of mastitis is not fully established. Here, we performed genotyping by pulsed-field gel electrophoresis (PFGE) and spa analyses to assess the genetic diversity and relatedness of 162 S. aureus strains recovered from clinical mastitis (CM) and subclinical mastitis (SCM) cases from goats, sheep, and bovines. PFGE analysis revealed 108 distinguishable pulsotypes and 3 main clusters that comprised isolates from the three host species, while according to spa typing, 32 different spa types were identified. Genotypic analysis revealed a spreading of genetically related or indistinguishable S. aureus strains among ovine, caprine, and bovine farms of distant geographical regions. In total, 28 different staphylococcal enterotoxin (SE) gene profiles were observed, revealing a diverse range of SE genes among isolates. By evaluating the antimicrobial resistance, we found low phenotypic antimicrobial resistance among all ruminant isolates. We also performed multiple correspondence analysis, which indicated that the presence of the sec gene, biofilm production, and high autoaggregation ability are associated with CM cases.

In silico structural analysis of sequences containing 5-hydroxymethylcytosine reveals its potential as binding regulator for development, ageing and cancer-related transcription factors.

The presence of 5-hydroxymethyl cytosine in DNA has been previously associated with ageing. Using in silico analysis of normal liver samples we presently observed that in 5-hydroxymethyl cytosine sequences, DNA methylation is dependent on the co-presence of G-quadruplexes and palindromes. This association exhibits discrete patterns depending on G-quadruplex and palindrome densities. DNase-Seq data show that 5-hydroxymethyl cytosine sequences are common among liver nucleosomes (p < 2.2x10-16) and threefold more frequent than nucleosome sequences. Nucleosomes lacking palindromes and potential G-quadruplexes are rare in vivo (1%) and nucleosome occupancy potential decreases with increasing G-quadruplexes. Palindrome distribution is similar to that previously reported in nucleosomes. In low and mixed complexity sequences 5-hydroxymethyl cytosine is frequently located next to three elements: G-quadruplexes or imperfect G-quadruplexes with CpGs, or unstable hairpin loops (TCCCAY6TGGGA) mostly located in antisense strands or finally A-/T-rich segments near these motifs. The high frequencies and selective distribution of pentamer sequences (including TCCCA, TGGGA) probably indicate the positive contribution of 5-hydroxymethyl cytosine to stabilize the formation of structures unstable in the absence of this cytosine modification. Common motifs identified in all total 5-hydroxymethyl cytosine-containing sequences exhibit high homology to recognition sites of several transcription factor families: homeobox, factors involved in growth, mortality/ageing, cancer, neuronal function, vision, and reproduction. We conclude that cytosine hydroxymethylation could play a role in the recognition of sequences with G-quadruplexes/palindromes by forming epigenetically regulated DNA 'springs' and governing expansions or compressions recognized by different transcription factors or stabilizing nucleosomes. The balance of these epigenetic elements is lost in hepatocellular carcinoma.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: Triggering a Lethal Fight to Keep Control of the Ten-Eleven Translocase (TET)-Associated DNA Demethylation?

The extended and diverse interference of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in multiple host functions and the diverse associated symptoms implicate its involvement in fundamental cellular regulatory processes. The activity of ten-eleven translocase 2 (TET2) responsible for selective DNA demethylation, has been recently identified as a regulator of endogenous virus inactivation and viral invasion, possibly by proteasomal deregulation of the TET2/TET3 activities. In a recent report, we presented a detailed list of factors that can be affected by TET activity, including recognition of zinc finger protein binding sites and bimodal promoters, by enhancing the flexibility of adjacent sequences. In this review, we summarize the TET-associated processes and factors that could account for SARS-CoV-2 diverse symptoms. Moreover, we provide a correlation for the observed virus-induced symptoms that have been previously associated with TET activities by in vitro and in vitro studies. These include early hypoxia, neuronal regulation, smell and taste development, liver, intestinal, and cardiomyocyte differentiation. Finally, we propose that the high mortality of SARS-CoV-2 among adult patients, the different clinical symptoms of adults compared to children, the higher risk of patients with metabolic deregulation, and the low mortality rates among women can all be accounted for by the complex balance of the three enzymes with TET activity, which is developmentally regulated. This activity is age-dependent, related to telomere homeostasis and integrity, and associated with X chromosome inactivation via (de)regulation of the responsible XIST gene expression.

First Report of Isolation and Genome Sequence of L. petauri Strain from a Rainbow Trout Lactococcosis Outbreak.

Lactococcosis is a disease encountered in a wide variety of fish species causing mortalities and having great economic impact on farmed fish. In this study, we report for the first time the isolation of a strain of the recently described novel species Lactococcus petauri, from rainbow trout suffering from lactococcosis. The aim of this study was to determine the complete genome sequence of L. petauri strain LG_SAV_20 and to characterize its antimicrobial resistance and virulence. The genome of L. petauri LG_SAV_20 consists of 2,078,949 base pair (bp) with a GC content of 38.05%, 1950 predicted coding sequence (CDS), and 60 RNAs (51 tRNAs, 3 ncRNAs, and 6 rRNAs). Phylogenetic analysis revealed that L. petauri LG_SAV_20 shares most of its genome with L. garvieae strains isolated from rainbow trout. Detection of genes associated with antimicrobial resistance indicated that the isolate possesses the multidrug transporter mdt(A) gene, while using comparative analysis we identified several genes that might be related to bacterial pathogenesis. This genomic information provides new insights into the role of this novel species as an etiological agent of lactococcosis.

DNA methylation profiles in chronic lymphocytic leukemia patients treated with chemoimmunotherapy.

BACKGROUND: In order to gain insight into the contribution of DNA methylation to disease progression of chronic lymphocytic leukemia (CLL), using 450K Illumina arrays, we determined the DNA methylation profiles in paired pre-treatment/relapse samples from 34 CLL patients treated with chemoimmunotherapy, mostly (n = 31) with the fludarabine-cyclophosphamide-rituximab (FCR) regimen. RESULTS: The extent of identified changes in CLL cells versus memory B cells from healthy donors was termed "epigenetic burden" (EB) whereas the number of changes between the pre-treatment versus the relapse sample was termed "relapse changes" (RC). Significant (p < 0.05) associations were identified between (i) high EB and short time-to-first-treatment (TTFT); and, (ii) few RCs and short time-to-relapse. Both the EB and the RC clustered in specific genomic regions and chromatin states, including regulatory regions containing binding sites of transcription factors implicated in B cell and CLL biology. CONCLUSIONS: Overall, we show that DNA methylation in CLL follows different dynamics in response to chemoimmunotherapy. These epigenetic alterations were linked with specific clinical and biological features.

MeinteR: A framework to prioritize DNA methylation aberrations based on conformational and cis-regulatory element enrichment.

DNA methylation studies have been reformed with the advent of single-base resolution arrays and bisulfite sequencing methods, enabling deeper investigation of methylation-mediated mechanisms. In addition to these advancements, numerous bioinformatics tools address important computational challenges, covering DNA methylation calling up to multi-modal interpretative analyses. However, contrary to the analytical frameworks that detect driver mutational signatures, the identification of putatively actionable epigenetic events remains an unmet need. The present work describes a novel computational framework, called MeinteR, that prioritizes critical DNA methylation events based on the following hypothesis: critical aberrations of DNA methylation more likely occur on a genomic substrate that is enriched in cis-acting regulatory elements with distinct structural characteristics, rather than in genomic "deserts". In this context, the framework incorporates functional cis-elements, e.g. transcription factor binding sites, tentative splice sites, as well as conformational features, such as G-quadruplexes and palindromes, to identify critical epigenetic aberrations with potential implications on transcriptional regulation. The evaluation on multiple, public cancer datasets revealed significant associations between the highest-ranking loci with gene expression and known driver genes, enabling for the first time the computational identification of high impact epigenetic changes based on high-throughput DNA methylation data.

Emergence of Klebsiella pneumoniae ST11 co-producing NDM-1 and OXA-48 carbapenemases in Greece.

OBJECTIVES: Klebsiella pneumoniae is a well-known pathogen frequently implicated in serious life-threatening nosocomial infections. Here we present a K. pneumoniae isolate (AHEPA1046) co-harbouring blaNDM-1 and blaOXA-48 isolated from a blood sample of an inpatient in Thessaloniki, Greece. METHODS: Whole-genome sequencing (WGS) was performed using an Illumina MiniSeq Sequencing System. Multilocus sequence typing (MLST) was performed using a BLAST-based approach, and antimicrobial resistance genes and plasmid replicons were identified by ResFinder and PlasmidFinder, respectively. The Rapid Annotation using Subsystem Technology (RAST) v.2.0 server was used for genome annotation. RESULTS: WGS analysis revealed the complete resistome of K. pneumoniae AHEPA1046. The strain harboured blaNDM-1 and blaOXA-48 together with 16 additional antimicrobial resistance genes and was resistant to carbapenems, aminoglycosides, quinolones, macrolides, tetracyclines, trimethoprim, fosfomycin and phenicols. Moreover, it was classified as ST11. CONCLUSION: This is the first report of a K. pneumoniae clinical isolate from Greece co-producing NDM-1 and OXA-48 carbapenemases and is one of a few reported worldwide.

Linking Exome Sequencing Data with Drug Response Aberrations.

Next-generation sequencing has prompted the development of numerous -omics applications. Along with experimental procedures, various computational pipelines became available to address the inherent complexities concerning the volume and quality of data. These pipelines are effective and routinely applied; however, interpreting their outcomes into actionable evidence is still poorly addressed. In this context, this work proposes a method for translating patient genomic profiles to drug response aberrations by integrating pharmacogenomic data into sequencing data analysis pipelines.

Computational Advances in Drug Safety: Systematic and Mapping Review of Knowledge Engineering Based Approaches.

Drug Safety (DS) is a domain with significant public health and social impact. Knowledge Engineering (KE) is the Computer Science discipline elaborating on methods and tools for developing "knowledge-intensive" systems, depending on a conceptual "knowledge" schema and some kind of "reasoning" process. The present systematic and mapping review aims to investigate KE-based approaches employed for DS and highlight the introduced added value as well as trends and possible gaps in the domain. Journal articles published between 2006 and 2017 were retrieved from PubMed/MEDLINE and Web of Science® (873 in total) and filtered based on a comprehensive set of inclusion/exclusion criteria. The 80 finally selected articles were reviewed on full-text, while the mapping process relied on a set of concrete criteria (concerning specific KE and DS core activities, special DS topics, employed data sources, reference ontologies/terminologies, and computational methods, etc.). The analysis results are publicly available as online interactive analytics graphs. The review clearly depicted increased use of KE approaches for DS. The collected data illustrate the use of KE for various DS aspects, such as Adverse Drug Event (ADE) information collection, detection, and assessment. Moreover, the quantified analysis of using KE for the respective DS core activities highlighted room for intensifying research on KE for ADE monitoring, prevention and reporting. Finally, the assessed use of the various data sources for DS special topics demonstrated extensive use of dominant data sources for DS surveillance, i.e., Spontaneous Reporting Systems, but also increasing interest in the use of emerging data sources, e.g., observational healthcare databases, biochemical/genetic databases, and social media. Various exemplar applications were identified with promising results, e.g., improvement in Adverse Drug Reaction (ADR) prediction, detection of drug interactions, and novel ADE profiles related with specific mechanisms of action, etc. Nevertheless, since the reviewed studies mostly concerned proof-of-concept implementations, more intense research is required to increase the maturity level that is necessary for KE approaches to reach routine DS practice. In conclusion, we argue that efficiently addressing DS data analytics and management challenges requires the introduction of high-throughput KE-based methods for effective knowledge discovery and management, resulting ultimately, in the establishment of a continuous learning DS system.

Integrated epigenomic and transcriptomic analysis reveals TP63 as a novel player in clinically aggressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness.

Age-dependent methylation in epigenetic clock CpGs is associated with G-quadruplex, co-transcriptionally formed RNA structures and tentative splice sites.

Horvath's epigenetic clock consists of 353 CpGs whose methylation levels can accurately predict the age of individuals. Using bioinformatics analysis, we investigated the conformation, energy characteristics and presence of tentative splice sites of the sequences surrounding the epigenetic clock CpGs, in relation to the median methylation changes in different ages, the presence of CpG islands and their position in genes. Common characteristics in the 100 nt sequences surrounding the epigenetic clock CpGs are G-quadruplexes and/or tentative splice site motifs. Median methylation increases significantly in sequences which adopt less stable structures during transcription. Methylation is higher when CpGs overlap with G-quadruplexes than when they precede them. Median methylation in epigenetic clock CpGs is higher in sequences expressed as single products rather than in multiple products and those containing single donors and multiple acceptors. Age-related methylation variation is significant in sequences without G-quadruplexes, particularly those producing low stability nascent RNA and those with splice sites. CpGs in sequences close to transcription start sites and those which are possibly never expressed (hypothetical proteins) undergo similar extent of age-related median methylation decrease and increase. Preservation of methylation is observed in CpG islands without G-quadruplexes, contrary to CpGs far from CpG islands (open sea). Sequences containing G-quadruplexes and RNA pseudoknots, determining the recognition by H3K27 histone methyltransferase, are hypomethylated. The presented structural DNA and co-transcriptional RNA analysis of epigenetic clock sequences, foreshadows the association of age-related methylation changes with the principle biological processes of DNA and histone methylation, splicing and chromatin silencing.

Association of two synonymous splicing-associated CpG single nucleotide polymorphisms in calpain 10 and solute carrier family 2 member 2 with type 2 diabetes.

Coding synonymous single nucleotide polymorphisms (SNPs) have attracted little attention until recently. However, such SNPs located in epigenetic, CpG sites modifying exonic splicing enhancers (ESEs) can be informative with regards to the recently verified association of intragenic methylation and splicing. The present study describes the association of type 2 diabetes (T2D) with the exonic, synonymous, epigenetic SNPs, rs3749166 in calpain 10 (CAPN10) glucose transporter (GLUT4) translocator and rs5404 in solute carrier family 2, member 2 (SLC2A2), also termed GLUT2, which, according to prior bioinformatic analysis, strongly modify the splicing potential of glucose transport-associated genes. Previous association studies reveal that only rs5404 exhibits a strong negative T2D association, while data on the CAPN10 polymorphism are contradictory. In the present study DNA from blood samples of 99 Greek non-diabetic control subjects and 71 T2D patients was analyzed. In addition, relevant publicly available cases (40) resulting from examination of 110 Personal Genome Project data files were analyzed. The frequency of the rs3749166 A allele, was similar in the patients and non-diabetic control subjects. However, AG heterozygotes were more frequent among patients (73.24% for Greek patients and 54.55% for corresponding non-diabetic control subjects; P=0.0262; total cases, 52.99 and 75.00%, respectively; P=0.0039). The rs5404 T allele was only observed in CT heterozygotes (Greek non-diabetic control subjects, 39.39% and Greek patients, 22.54%; P=0.0205; total cases, 34.69 and 21.28%, respectively; P=0.0258). Notably, only one genotype, heterozygous AG/CC, was T2D-associated (Greek non-diabetic control subjects, 29.29% and Greek patients, 56.33%; P=0.004; total cases, 32.84 and 56.58%, respectively; P=0.0008). Furthermore, AG/CC was strongly associated with very high (≥8.5%) glycosylated plasma hemoglobin levels among patients (P=0.0002 for all cases). These results reveal the complex heterozygotic SNP association with T2D, and indicate possible synergies of these epigenetic, splicing-regulatory, synonymous SNPs, which modify the splicing potential of two alternative glucose transport-associated genes.

Differential gene methylation in paired glioblastomas suggests a role of immune response pathways in tumor progression.

DNA and histone methylation are post-transcriptional modifications that have been recently described in gliomas. Indeed, glioma CpG island hypermethylated phenotype has been identified as prognostic biomarker and as a surrogate marker of IDH1/2 mutations. However, the role of DNA methylation in glioblastoma progression is unknown. We sought to analyze DNA methylation levels in paired (initial and recurrent) primary glioblastoma samples to identify candidate pathways that may prone to glioblastoma progression. We have analyzed 12 samples (5 paired samples, two of them with three surgeries) using methylation arrays. We have analyzed differential methylation at probe and at gene region level. Finally, pathway analysis has been performed using differentially methylated regions. All analysis has been performed with R and Bioconductor packages. Mean methylation level at initial sample compared to recurrence was strongly positively correlated (R(2) = 0.98). There was no differentially methylation at probe level. However, at gene level 3080 regions were differentially methylated. Interestingly, pathways analysis showed that the most differentially methylated genes are involved in cellular response to macrophage colony-stimulating factor stimulus (GO:0036006). Methylation levels were strongly conserved when comparing initial to recurrence in primary glioblastomas. Interestingly, differentially methylated pathway analysis suggests that a modulation of methylation in immune response genes may play a role in glioblastoma progression. Further studies are needed to validate the role of methylation of glioblastoma immune response genes in tumor progression.