Epigenetic/genetic variations in CG-rich elements of immune-related genes contribute to food allergy development during childhood.

BACKGROUND: Genetic areas of FOXP3 TSDR, human leukocyte antigen-G (HLA-G) upstream of CpG island 96, CpG41 and CpG73 islands of the HLA-DRB1 and HLA-DQB1 genes respectively, previously documented to display immune-modulatory properties, were subjected to epigenetic/genetic analysis to assess their influence in IgE-mediated food allergy (FA) development in children. METHODS: Sixty-four orally challenged and IgE-tested food allergic subjects together with 44 controls were recruited. Targeted pyrosequencing analysis to detect DNA methylation status and genetic variations was utilized and experimental results obtained were analyzed by a statistical software platform and correlated to clinical data. Also, transcription factor (TF) binding sites in study areas were unmasked by the JASPAR prediction database. RESULTS: Parents' smoking was significantly correlated with aberrant methylation patterns, regardless of food allergic or control status. HLA-G promoter region showed a trend for hypomethylation in food allergic subjects, with one of the CG sites displaying significantly decreased methylation values. Rs1233333, residing within the HLA-G promoter region preserved a protective role toward DNA methylation. Variable methylation patterns were recorded for CpG41 of the HLA-DRB1 gene and hypermethylation of the region was significantly correlated with the presence of single nucleotide polymorphisms (SNPs). TFs' recognition sites, located in studied genetic areas and exerting pivotal regulatory biological roles, are potentially affected by divergent DNA methylation status. CONCLUSIONS: We propose that HLA-G expression is triggered by food-derived allergens, providing a TregFoxP3-/HLA-G+ subpopulation generation to promote direct immune tolerance. Furthermore, clear evidence is provided for the underlying co-operation of genetic polymorphisms with epigenetic events, mainly at the CpG41 island of the HLA-DRB1 gene, which needs an extended investigation and elucidation.

Contingent Synergistic Interactions between Non-Coding RNAs and DNA-Modifying Enzymes in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders with maturation and differentiation defects exhibiting morphological dysplasia in one or more hematopoietic cell lineages. They are associated with peripheral blood cytopenias and by increased risk for progression into acute myelogenous leukemia. Among their multifactorial pathogenesis, age-related epigenetic instability and the error-rate DNA methylation maintenance have been recognized as critical factors for both the initial steps of their pathogenesis and for disease progression. Although lower-risk MDS is associated with an inflammatory bone marrow microenvironment, higher-risk disease is delineated by immunosuppression and clonal expansion. "Epigenetics" is a multidimensional level of gene regulation that determines the specific gene networks expressed in tissues under physiological conditions and guides appropriate chromatin rearrangements upon influence of environmental stimulation. Regulation of this level consists of biochemical modifications in amino acid residues of the histone proteins' N-terminal tails and their concomitant effects on chromatin structure, DNA methylation patterns in CpG dinucleotides and the tissue-specific non-coding RNAs repertoire, which are directed against various gene targets. The role of epigenetic modifications is widely recognized as pivotal both in gene expression control and differential molecular response to drug therapies in humans. Insights to the potential of synergistic cooperations of epigenetic mechanisms provide new avenues for treatment development to comfort human diseases with a known epigenetic shift, such as MDS. Hypomethylating agents (HMAs), such as epigenetic modulating drugs, have been widely used in the past years as first line treatment for elderly higher-risk MDS patients; however, just half of them respond to therapy and are benefited. Rational outcome predictors following epigenetic therapy in MDS and biomarkers associated with disease relapse are of high importance to improve our efforts in developing patient-tailored clinical approaches.

LRF Promotes Indirectly Advantageous Chromatin Conformation via BGLT3-lncRNA Expression and Switch from Fetal to Adult Hemoglobin.

The hemoglobin switch from fetal (HbF) to adult (HbA) has been studied intensively as an essential model for gene expression regulation, but also as a beneficial therapeutic approach for ß-hemoglobinopathies, towards the objective of reactivating HbF. The transcription factor LRF (Leukemia/lymphoma-related), encoded from the ZBTB7A gene has been implicated in fetal hemoglobin silencing, though has a wide range of functions that have not been fully clarified. We thus established the LRF/ZBTB7A-overexpressing and ZBTB7A-knockdown K562 (human erythroleukemia cell line) clones to assess fetal vs. adult hemoglobin production pre- and post-induction. Transgenic K562 clones were further developed and studied under the influence of epigenetic chromatin regulators, such as DNA methyl transferase 3 (DNMT3) and Histone Deacetylase 1 (HDAC1), to evaluate LRF's potential disturbance upon the aberrant epigenetic background and provide valuable information of the preferable epigenetic frame, in which LRF unfolds its action on the ß-type globin's expression. The ChIP-seq analysis demonstrated that LRF binds to γ-globin genes (HBG2/1) and apparently associates BCL11A for their silencing, but also during erythropoiesis induction, LRF binds the BGLT3 gene, promoting BGLT3-lncRNA production through the γ-δ intergenic region of ß-type globin's locus, triggering the transcriptional events from γ- to ß-globin switch. Our findings are supported by an up-to-date looping model, which highlights chromatin alterations during erythropoiesis at late stages of gestation, to establish an "open" chromatin conformation across the γ-δ intergenic region and accomplish ß-globin expression and hemoglobin switch.

Analgesic effect of paracetamol monotherapy vs. the combination of paracetamol/parecoxib vs. the combination of pethidine/paracetamol in patients undergoing thyroidectomy.

INTRODUCTION: The purpose of this study was to investigate the analgesic effect of 3 different regimens of combination analgesics administered to patients undergoing thyroidectomy. MATERIAL AND METHODS: A total of 152 patients undergoing total or subtotal thyroidectomy were enrolled. Patients allocated to group A received a combination of intravenous (IV) paracetamol and intramuscular (IM) pethidine, patients in group B received a combination of IV paracetamol and IV parecoxib, while patients in group C received IV paracetamol monotherapy. RESULTS: The analgesic regimens of groups A and B were found to be of equivalent efficacy (p-value = 1.000). In contrast, patients in group C (paracetamol monotherapy) had higher numerical rating scale scores, compared to both patients in groups A (p-value < 0.001) and B (p-value < 0.001). CONCLUSIONS: The combinations of IV paracetamol with either IM pethidine or IV parecoxib are superior to IV paracetamol monotherapy in achieving pain control in patients undergoing thyroid surgery.

The multi-faceted functioning portrait of LRF/ZBTB7A.

Transcription factors (TFs) consisting of zinc fingers combined with BTB (for broad-complex, tram-track, and bric-a-brac) domain (ZBTB) are a highly conserved protein family that comprises a multifunctional and heterogeneous group of TFs, mainly modulating cell developmental events and cell fate. LRF/ZBTB7A, in particular, is reported to be implicated in a wide variety of physiological and cancer-related cell events. These physiological processes include regulation of erythrocyte maturation, B/T cell differentiation, adipogenesis, and thymic insulin expression affecting consequently insulin self-tolerance. In cancer, LRF/ZBTB7A has been reported to act either as oncogenic or as oncosuppressive factor by affecting specific cell processes (proliferation, apoptosis, invasion, migration, metastasis, etc) in opposed ways, depending on cancer type and molecular interactions. The molecular mechanisms via which LRF/ZBTB7A is known to exert either physiological or cancer-related cellular effects include chromatin organization and remodeling, regulation of the Notch signaling axis, cellular response to DNA damage stimulus, epigenetic-dependent regulation of transcription, regulation of the expression and activity of NF-κB and p53, and regulation of aerobic glycolysis and oxidative phosphorylation (Warburg effect). It is a pleiotropic TF, and thus, alterations to its expression status become detrimental for cell survival. This review summarizes its implication in different cellular activities and the commonly invoked molecular mechanisms triggered by LRF/ZBTB7A's orchestrated action.

Impact of ZBTB7A hypomethylation and expression patterns on treatment response to hydroxyurea.

BACKGROUND: We aimed to clarify the emerging epigenetic landscape in a group of genes classified as "modifier genes" of the ß-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/ß-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented. RESULTS: We examined the CpG islands' DNA methylation profile of BCL11A, KLF1, MYB, MAP3K5, SIN3A, ZBTB7A, and GATA2, along with γ-globin and LRF/ZBTB7A expression levels. In vitro treatment of hematopoietic stem cells (HSCs) with HU induced a significant DNA hypomethylation pattern in ZBTB7A (p*, 0.04) and GATA2 (p*, 0.03) CpGs exclusively in the HU non-responders. Also, this group of patients exhibited significantly elevated baseline methylation patterns in ZBTB7A, before the HU treatment, compared to HU responders (p*, 0.019) and to control group of healthy individuals (p*, 0.021), which resembles a potential epigenetic barrier for the γ-globin expression. γ-Globin expression in vitro matched with detected HbF levels during patients' monitoring tests (in vivo) under HU treatment, implying a good reproducibility of the in vitro HU epigenetic effect. LRF/ZBTB7A expression was elevated only in the HU non-responders under the influence of HU. CONCLUSIONS: This is one of the very first pharmacoepigenomic studies indicating that the hypomethylation of ZBTB7A during HU treatment enhances the LRF expression, which by its turn suppresses the HbF resumption in the HU non-responders. Its role as an epigenetic regulator of hemoglobin switching is also supported by the wide distribution of ZBTB7A-binding sites within the 5' CpG sequences of all studied human HBB cluster "modifier genes." Also, the baseline methylation level of selective CpGs in ZBTB7A and GATA2 could be an indicator of the negative HU response among the ß-type hemoglobinopathy patients.

Role of Genomic Biomarkers in Increasing Fetal Hemoglobin Levels Upon Hydroxyurea Therapy and in ß-Thalassemia Intermedia: A Validation Cohort Study.

Hemoglobinopathies exhibit a remarkable phenotypic diversity in terms of disease severity, while individual genetic background plays a key role in differential response to drug treatment. In the last decade, genomic variants in genes located within, as well as outside the human ß-globin cluster have been shown to be significantly associated with Hb F increase, in relation to hydroxyurea (HU) therapy in patients with these diseases. Here, we aim to determine the effect of genomic variants located in genes, such as MAP3K5, ASS1, NOS2A, TOX, PDE7B, NOS1, FLT1 and ARG2, previously shown to modulate fetal hemoglobin (Hb F) levels in patients with ß type hemoglobinopathies and reflecting disease severity and response to HU therapy in an independent cohort of Greek patients with these diseases. We recruited and genotyped 45 ß-thalassemia patients (ß-thal), either transfusion-dependent (TDT) or non transfusion-dependent (NTDT), 42 Hb S (HBB: c.20A>T)-ß-thal compound heterozygotes, who were treated with HU, as well as 53 healthy individuals, all of Hellenic origin. Our study showed that genomic variants of the MAP3K5, NOS2A and ARG2 gene are associated with HU therapy efficacy in Hb S-ß-thal compound heterozygotes. We have also shown that FLT1 and ARG2 genomic variants are associated with the mild phenotype of NTDT patients. Our findings provide evidence that MAP3K5, NOS2A, ARG2 and FLT1 genomic variants could be considered as genomic biomarkers to predict HU therapy efficacy in Hb S-ß-thal compound heterozygotes and also to describe disease severity in patients with ß type hemoglobinopathies.

Mechanical stress affects methylation pattern of GNAS isoforms and osteogenic differentiation of hAT-MSCs.

Mechanical stress exerts a substantial role on skeletal-cell renewal systems, whereas accumulating evidence suggests that epigenetic mechanisms induce changes and differential gene expression. Although the underlying mechanisms remain to be fully elucidated, our study suggests that the influence of the long term mechanical stimulation elicits epigenetic modifications controlling osteogenic differentiation of human adipose tissue multipotential stromal cells (hAT-MSCs) and contributes to an accelerating in vitro osteogenesis. GNAS imprinting gene acts as a critical regulator of osteoblast differentiation and is implicated in human genetic disorders with pathological formation of ectopic-skeletal bone. Investigating a wide variety of stimuli, we showed that daily mechanical stretch on hAT-MSCs of 7th and 15th days' intervals induced a significant down-regulation in DNA methylation status of critical CpG sites of NESP and GNASXL isoforms, accompanied by up-regulation of the corresponding gene transcripts, and osteogenic differentiation earlier in culture. Importantly, methylation analysis of differentiating bone marrow-derived MSCs revealed similar methylation patterns. Bioinformatic analysis further showed that all CpG islands exhibiting significant methylation alterations encompassed transcriptional repressor CTCF binding sites. We hereby emphasize the need to investigate the epigenetic alterations on hAT-MSCs during environmental mechanical forces and to consider how the knowledge gained through these studies may foster new means of symptoms prevention and management of ectopic bone formation in the clinic.

Whole transcriptome analysis of human erythropoietic cells during ontogenesis suggests a role of VEGFA gene as modulator of fetal hemoglobin and pharmacogenomic biomarker of treatment response to hydroxyurea in ß-type hemoglobinopathy patients.

BACKGROUND: Human erythropoiesis is characterized by distinct gene expression profiles at various developmental stages. Previous studies suggest that fetal-to-adult hemoglobin switch is regulated by a complex mechanism, in which many key players still remain unknown. Here, we report our findings from whole transcriptome analysis of erythroid cells, isolated from erythroid tissues at various developmental stages in an effort to identify distinct molecular signatures of each erythroid tissue. RESULTS: From our in-depth data analysis, pathway analysis, and text mining, we opted to focus on the VEGFA gene, given its gene expression characteristics. Selected VEGFA genomic variants, identified through linkage disequilibrium analysis, were explored further for their association with elevated fetal hemoglobin levels in ß-type hemoglobinopathy patients. Our downstream analysis of non-transfusion-dependent ß-thalassemia patients, ß-thalassemia major patients, compound heterozygous sickle cell disease/ß-thalassemia patients receiving hydroxyurea as fetal hemoglobin augmentation treatment, and non-thalassemic individuals indicated that VEGFA genomic variants were associated with disease severity in ß-thalassemia patients and hydroxyurea treatment efficacy in SCD/ß-thalassemia compound heterozygous patients. CONCLUSIONS: Our findings suggest that VEGFA may act as a modifier gene of human globin gene expression and, at the same time, serve as a genomic biomarker in ß-type hemoglobinopathy disease severity and hydroxyurea treatment efficacy.

Simple in vitro generation of human leukocyte antigen-G-expressing T-regulatory cells through pharmacological hypomethylation for adoptive cellular immunotherapy against graft-versus-host disease.

BACKGROUND: Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the "semi-allogeneic" fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs. METHODS: Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties. RESULTS: HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4+HLA-Gpos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-Gpos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD). DISCUSSION: We propose, in vitro generation of HLA-G-expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy.

Enhanced central nervous system transduction with lentiviral vectors pseudotyped with RVG/HIV-1gp41 chimeric envelope glycoproteins.

UNLABELLED: To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the "Kennedy" sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE: In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer of these vectors, depending on CT length and context. We also managed to achieve increased neuronal transduction in vivo in the rodent CNS, thus demonstrating that the efficiency of these vectors can be enhanced following merely CT manipulation. We believe that this paper is a novel contribution to the field and opens the way for further attempts to surface engineer lentiviral vectors and make them more amenable for applications in human disease.

"Crosstalk between non-coding RNAs and transcription factor LRF in non-small cell lung cancer".

Epigenetic approaches in direct correlation with assessment of critical genetic mutations in non-small cell lung cancer (NSCLC) are currently very intensive, as the epigenetic components underlying NSCLC development and progression have attained high recognition. In this level of research, established human NSCLC cell lines as well as experimental animals are widely used to detect novel biomarkers and pharmacological targets to treat NSCLC. The epigenetic background holds a great potential for the identification of epi-biomarkers for treatment response however, it is highly complex and requires precise definition as these phenomena are variable between NSCLC subtypes and systems origin. We engaged an in-depth characterization of non-coding (nc)RNAs prevalent in human KRAS-mutant NSCLC cell lines A549 and H460 and mouse KRAS-mutant NSCLC tissue by Next Generation Sequencing (NGS) and quantitative Real Time PCRs (qPCRs). Also, the transcription factor (TF) LRF, a known epigenetic silencer, was examined as a modulator of non-coding RNAs expression. Finally, interacting networks underlying epigenetic variations in NSCLC subtypes were created. Data derived from our study highlights the divergent epigenetic profiles of NSCLC of human and mouse origin, as well as the significant contribution of 12qf1: 109,709,060-109,747,960 mouse chromosomal region to micro-RNA upregulated species. Furthermore, the novel epigenetic miR-148b-3p/lncPVT1/ZBTB7A axis was identified, which differentiates human cell line of lung adenocarcinoma from large cell lung carcinoma, two characteristic NSCLC subtypes. The detailed recording of epigenetic events in NSCLC and combinational studies including networking between ncRNAs and TFs validate the identification of significant epigenetic features, prevailing in NSCLC subtypes and among experimental models. Our results enrich knowledge in the field and empower research on the epigenetic prognostic biomarkers of the disease progression, NSCLC subtypes discrimination and advancement to patient-tailored treatments.

Developmental Dyslexia: Insights from EEG-Based Findings and Molecular Signatures-A Pilot Study.

Developmental dyslexia (DD) is a learning disorder. Although risk genes have been identified, environmental factors, and particularly stress arising from constant difficulties, have been associated with the occurrence of DD by affecting brain plasticity and function, especially during critical neurodevelopmental stages. In this work, electroencephalogram (EEG) findings were coupled with the genetic and epigenetic molecular signatures of individuals with DD and matched controls. Specifically, we investigated the genetic and epigenetic correlates of key stress-associated genes (NR3C1, NR3C2, FKBP5, GILZ, SLC6A4) with psychological characteristics (depression, anxiety, and stress) often included in DD diagnostic criteria, as well as with brain EEG findings. We paired the observed brain rhythms with the expression levels of stress-related genes, investigated the epigenetic profile of the stress regulator glucocorticoid receptor (GR) and correlated such indices with demographic findings. This study presents a new interdisciplinary approach and findings that support the idea that stress, attributed to the demands of the school environment, may act as a contributing factor in the occurrence of the DD phenotype.

Fetal hemoglobin induction in azacytidine responders enlightens methylation patterns related to blast clearance in higher-risk MDS and CMML.

BACKGROUND: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients' epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. RESULTS: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24-1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, rs = - 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, rs = - 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, rs = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (pHBG2 = 0.011, rs = 0.480/pZBTB7A = 0.026, rs = 0.427) and showed prognostic value for PFS (pZBTB7A = 0.037, HR = 1.14, CI 0.34-3.8). CONCLUSIONS: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action.

Association of genome variations in the renin-angiotensin system with physical performance.

BACKGROUND: The aim of this study was to determine the genotype distribution and allelic frequencies of ACE (I/D), AGTR1 (A +1166 C), BDKRB2 (+9/-9) and LEP (G-2548A) genomic variations in 175 Greek athletes who excelled at a national and/or international level and 169 healthy Greek adults to identify whether some particular combinations of these loci might serve as predictive markers for superior physical condition. RESULTS: The D/D genotype of the ACE gene (p = 0.034) combined with the simultaneous existence of BDKRB2 (+9/-9) (p = 0.001) or LEP (G/A) (p = 0.021) genotypes was the most prevalent among female athletes compared to female controls. A statistical trend was also observed in BDKRB2 (+9/-9) and LEP (G-2548A) heterozygous genotypes among male and female Greek athletes, and in ACE (I/D) only in male athletes. Finally, both male and female athletes showed the highest rates in the AGTR1 (A/A) genotype. CONCLUSIONS: Our results suggest that the co-existence of ACE (D/D), BDKRB2 (+9/-9) or LEP (G/A) genotypes in female athletes might be correlated with a superior level of physical performance.

Transcriptional repression of lncRNA and miRNA subsets mediated by LRF during erythropoiesis.

Non-coding RNA (ncRNA) species, mainly long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been currently imputed for lesser or greater involvement in human erythropoiesis. These RNA subsets operate within a complex circuit with other epigenetic components and transcription factors (TF) affecting chromatin remodeling during cell differentiation. Lymphoma/leukemia-related (LRF) TF exerts higher occupancy on DNA CpG rich sites and is implicated in several differentiation cell pathways and erythropoiesis among them and also directs the epigenetic regulation of hemoglobin transversion from fetal (HbF) to adult (HbA) form by intervening in the γ-globin gene repression. We intended to investigate LRF activity in the evolving landscape of cells' commitment to the erythroid lineage and specifically during HbF to HbA transversion, to qualify this TF as potential repressor of lncRNAs and miRNAs. Transgenic human erythroleukemia cells, overexpressing LRF and further induced to erythropoiesis, were subjected to expression analysis in high LRF occupancy genetic loci-producing lncRNAs. LRF abundance in genetic loci transcribing for studied lncRNAs was determined by ChIP-Seq data analysis. qPCRs were performed to examine lncRNA expression status. Differentially expressed miRNA pre- and post-erythropoiesis induction were assessed by next-generation sequencing (NGS), and their promoter regions were charted. Expression levels of lncRNAs were correlated with DNA methylation status of flanked CpG islands, and contingent co-regulation of hosted miRNAs was considered. LRF-binding sites were overrepresented in LRF overexpressing cell clones during erythropoiesis induction and exerted a significant suppressive effect towards lncRNAs and miRNA collections. Based on present data interpretation, LRF's multiplied binding capacity across genome is suggested to be transient and associated with higher levels of DNA methylation. KEY MESSAGES: During erythropoiesis, LRF displays extensive occupancy across genetic loci. LRF significantly represses subsets of lncRNAs and miRNAs during erythropoiesis. Promoter region CpG islands' methylation levels affect lncRNA expression. MiRNAs embedded within lncRNA loci show differential regulation of expression.

Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the ß-Globin Gene into K562 and Human Haematopoietic Progenitor Cells.

ß-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPß-globin for the physiological ß-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the ß-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the ß-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce ß-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPß-globin provides the basis for the development of therapeutic EV for gene therapy of ß-thalassemias.

Arsenic exposure promotes the emergence of cardiovascular diseases.

A large number of studies conducted in the past decade 2010-2020 refer to the impact of arsenic (As) exposure on cardiovascular risk factors. The arsenic effect on humans is complex and mainly depends on the varying individual susceptibilities, its numerous toxic expressions and the variation in arsenic metabolism between individuals. In this review we present relevant data from studies which document the association of arsenic exposure with various biomarkers, the effect of several genome polymorphisms on arsenic methylation and the underling molecular mechanisms influencing the cardiovascular pathology. The corresponding results provide strong evidence that high and moderate-high As intake induce oxidative stress, inflammation and vessel endothelial dysfunction that are associated with increased risk for cardiovascular diseases (CVDs) and in particular hypertension, myocardial infarction, carotid intima-media thickness and stroke, ventricular arrhythmias and peripheral arterial disease. In addition, As exposure during pregnancy implies risks for blood pressure abnormalities among infants and increased mortality rates from acute myocardial infarction during early adulthood. Low water As concentrations are associated with increased systolic, diastolic and pulse pressure, coronary heart disease and incident stroke. For very low As concentrations the relevant studies are few. They predict a risk for myocardial infarction, stroke and ischemic stroke and incident CVD, but they are not in agreement regarding the risk magnitude.

HLA Polymorphisms and Food Allergy Predisposition.

Food allergy (FA) is a growing health problem that affects ∼8% of the children worldwide. Although the prevalence of FA is increasing, the underlying genetic mechanisms responsible for the onset of this immune disorder are not yet clarified. Genetic factors seem to play a leading role in the development of FA, though interaction with environmental factors cannot be excluded. The broader network of genetic loci mediating the risk of this complex disorder remains to be identified. The human leucocyte antigen (HLA) has been associated with various immune disorders, including FA. This review aims to unravel the potential associations between HLA gene functions and the manifestation and outcome of FA disorders. Exploring new aspects of FA development with the perspective to improve our understanding of the multifaceted etiology and the complex biological mechanisms involved in FA is essential.

Implementation and Evaluation of a Three-Dimensional Virtual Reality Biology Lab versus Conventional Didactic Practices in Lab Experimenting with the Photonic Microscope.

This study presents the integration of three different teaching scenarios, during biology laboratory lessons, with the overall aim of exploring the potential predominant effectiveness of teaching and improvement of students' learning, by the use of the three-dimensional virtual reality educational tool Onlabs, versus more traditional didactic practices. A sample of 83, fourth year, undergraduate students of the Primary Education Department of Patras' University in Greece, were equally separated into three cognitively balanced groups to be educated on the light microscopy experiment by three different educational scenarios. Students' conceptual understanding in the domain of microscopy, was evaluated during all learning procedure with Pre and Post tests, whereas their skill to handle properly a real light microscope in the wet biology lab was summatively assessed via a specially designed work sheet. Results of the present study provide evidence in favor of the virtual reality application. © 2019 International Union of Biochemistry and Molecular Biology, 48(1):21-27, 2020.