'; ?> geneimprint : Hot off the Press http://www.geneimprint.com/site/hot-off-the-press Daily listing of the most recent articles in epigenetics and imprinting, collected from the PubMed database. en-us Thu, 18 Apr 2019 08:51:10 EDT Thu, 18 Apr 2019 08:51:10 EDT jirtle@radonc.duke.edu james001@jirtle.com The Genomic Distribution of Histone H3K4me2 in Spermatogonia is Highly Conserved in Sperm. Lambrot R, Siklenka K, Lafleur C, Kimmins S
Biol Reprod (Apr 2019)

Environmental exposures can alter the long-term health and development of offspring. How this environmental information is transmitted via the germline remains unknown, but it is thought to involve epigenetic inheritance. We recently determined that genetic disruption of histone H3 di-methylation at lysine 4 (H3K4me2) in sperm alters gene expression in the embryo and negatively impacts development across generations. However, little is known regarding when in spermatogenesis H3K4me2 methylation is established, and whether specific regions bearing H3K4me2 resist the epigenome remodelling that occurs throughout spermatogenesis. Our objective was to determine what genomic regions bearing histone H3K4me2 in spermatogonia are also present in sperm. Methods: Using transgenic mice expressing Oct4-GFP, we isolated an enriched spermatogonia population and performed ChIP-seq for H3K4me2, followed by downstream bioinformatics analysis. Using our epigenomic data and existing datasets, we compared the genomic distribution of H3K4me2 between spermatogonia and sperm. We also assessed the expression level of genes enriched in H3K4me2 in spermatogenic cell types and at specific embryonic developmental time-points. We observed that many regions of the sperm epigenome bearing H3K4me2 are already present in spermatogonia, suggesting an early establishment of this histone mark in spermatogenesis. Subsets of genes with a high enrichment in H3K4me2 in sperm are strongly expressed in spermatogenesis and others are associated with high gene expression during embryo development. These findings suggest that if epimutations in H3K4me2 are induced in spermatogonia they have the possibility to persist throughout spermatogenesis and may influence fertility by altering gene expression in spermatogenesis and in the embryo.]]>
Wed, 31 Dec 1969 19:00:00 EST
Differential methylation in rare ophthalmic disorders: a systematic review protocol. Kerr K, McAneney H, McKnight AJ
Syst Rev (Apr 2019)

Rare ophthalmic conditions often cause degenerative vision loss which leads to loss of independence, ability to work and ultimately quality life. Differential methylation is an epigenomic marker that is a feature of several diseases, including eye conditions. This review will aim to elucidate the extent to which differential methylation has been identified in rare ophthalmic conditions.]]>
Wed, 31 Dec 1969 19:00:00 EST
Multi-network approach to identify differentially methylated gene communities in cancer. R V, Nazeer KAA
Gene (May 2019)

High-throughput Next Generation Sequencing tools have generated immense quantity of genome-wide methylation and expression profiling data, resulting in an unprecedented opportunity to unravel the epigenetic regulatory mechanisms underlying cancer. Identifying differentially methylated regions within gene networks is an important step towards revealing the cancer epigenome blueprint. Approaches that integrate gene methylation and expression profiles assume their negative correlation and build a single scaffold network to cluster. However, the exact regulatory mechanism between gene expression and methylation is not precisely deciphered.]]>
Wed, 31 Dec 1969 19:00:00 EST
Backbone and side-chain resonance assignments of the methyl-CpG-binding domain of MBD6 from Arabidopsis thaliana. Iwakawa N, Mahana Y, Ono A, Ohki I, Walinda E, Morimoto D, Sugase K, Shirakawa M
Biomol NMR Assign (04 2019)

Epigenetic regulation is essential to various biological phenomena such as cell differentiation and cancer. DNA methylation is one of the most important epigenetic signals, as it is directly involved in gene silencing of transposable elements, genomic imprinting, and chromosome X inactivation. To mediate these processes, methyl-CpG-binding domain (MBD) proteins recognize specific signals encoded in the form of DNA methylation patterns. AtMBD6, one of the 12 MBD proteins in Arabidopsis thaliana, shares a high sequential homology in the MBD domain with mammalian MBD proteins, but a detailed characterization of its structural and functional properties remains elusive. Here, we report the H, C, and N resonance assignments of the isolated MBD domain of AtMBD6. Analysis of the chemical shift data implied that the MBD domain of AtMBD6 has a secondary structure similar to that of mammalian MeCP2, while the β-strands β1 and β3 of AtMBD6 were found to be longer than those of MeCP2. The structural differences provide insight into the different recognition mechanisms of methylated DNA by plant and mammalian MBDs. The assignments reported here will aid further analyses such as titration experiments and three-dimensional structure determination using NMR to yield a detailed characterization of the interaction between AtMBD6 and methylated DNAs.]]>
Wed, 31 Dec 1969 19:00:00 EST
The peculiar aging of human liver: A geroscience perspective within transplant context. Morsiani C, Bacalini MG, Santoro A, Garagnani P, Collura S, D'Errico A, de Eguileor M, Grazi GL, Cescon M, Franceschi C, Capri M
Ageing Res Rev (May 2019)

An appraisal of recent data highlighting aspects inspired by the new Geroscience perspective are here discussed. The main findings are summarized as follows: i) liver has to be considered an immunological organ, and new studies suggest a role for the recently described cells named telocytes; ii) the liver-gut axis represents a crucial connection with environment and life style habits and may influence liver diseases onset; iii) the physiological aging of liver shows relatively modest alterations. Nevertheless, several molecular changes appear to be relevant: a) an increase of microRNA-31-5p; -141-3p; -200c-3p expressions after 60 years of age; b) a remodeling of genome-wide DNA methylation profile evident until 60 years of age and then plateauing; c) changes in transcriptome including the metabolic zones of hepatocyte lobules; d) liver undergoes an accelerated aging in presence of chronic inflammation/liver diseases in a sort of continuum, largely as a consequence of unhealthy life styles and exposure to environmental noxious agents. We argue that chronic liver inflammation has all the major characteristics of "inflammaging" and likely sustains the onset and progression of liver diseases. Finally, we propose to use a combination of parameters, mostly obtained by omics such as transcriptomics and epigenomics, to evaluate in deep both the biological age of liver (in comparison with the chronological age) and the effects of donor-recipient age-mismatches in the context of liver transplant.]]>
Wed, 31 Dec 1969 19:00:00 EST
cisTopic: cis-regulatory topic modeling on single-cell ATAC-seq data. Bravo González-Blas C, Minnoye L, Papasokrati D, Aibar S, Hulselmans G, Christiaens V, Davie K, Wouters J, Aerts S
Nat Methods (Apr 2019)

We present cisTopic, a probabilistic framework used to simultaneously discover coaccessible enhancers and stable cell states from sparse single-cell epigenomics data ( http://github.com/aertslab/cistopic ). Using a compendium of single-cell ATAC-seq datasets from differentiating hematopoietic cells, brain and transcription factor perturbations, we demonstrate that topic modeling can be exploited for robust identification of cell types, enhancers and relevant transcription factors. cisTopic provides insight into the mechanisms underlying regulatory heterogeneity in cell populations.]]>
Wed, 31 Dec 1969 19:00:00 EST
miR-126 regulates glycogen trophoblast proliferation and DNA methylation in the murine placenta. Sharma A, Lacko LA, Argueta LB, Glendinning MD, Stuhlmann H
Dev Biol (May 2019)

A functional placenta develops through a delicate interplay of its vascular and trophoblast compartments. We have identified a previously unknown expression domain for the endothelial-specific microRNA miR-126 in trophoblasts of murine and human placentas. Here, we determine the role of miR-126 in placental development using a mouse model with a targeted deletion of miR-126. In addition to vascular defects observed only in the embryo, loss of miR-126 function in the placenta leads to junctional zone hyperplasia at E15.5 at the expense of the labyrinth, reduced placental volume for nutrient exchange and intra-uterine growth restriction of the embryos. Junctional zone hyperplasia results from increased numbers of proliferating glycogen trophoblast (GlyT) progenitors at E13.5 that give rise to an expanded glycogen trophoblast population at E15.5. Transcriptomic profile of miR-126 placentas revealed dysregulation of a large number of GlyT (Prl6a1, Prl7c1, Pcdh12) and trophoblast-specific genes (Tpbpa, Tpbpb, Prld1) and genes with known roles in placental development. We show that miR-126 placentas, but not miR-126 embryos, display aberrant expression of imprinted genes with important roles in glycogen trophoblasts and junctional zone development, including Igf2, H19, Cdkn1c and Phlda2, during mid-gestation. We also show that miR126 placentas display global hypermethylation, including at several imprint control centers. Our findings uncover a novel role for miR-126 in regulating extra-embryonic energy stores, expression of imprinted genes and DNA methylation in the placenta.]]>
Wed, 31 Dec 1969 19:00:00 EST
Integrative Genomics Analysis Identifies ACVR1B as a Candidate Causal Gene of Emphysema Distribution. Boueiz A, Pham B, Chase R, Lamb A, Lee S, Naing ZZC, Cho MH, Parker MM, Sakornsakolpat P, Hersh CP, Crapo JD, Stergachis AB, Tal-Singer R, DeMeo DL, Silverman EK, Zhou X, Castaldi PJ,  
Am J Respir Cell Mol Biol (Apr 2019)

Genome-wide association studies (GWAS) have identified multiple associations with emphysema apicobasal distribution (EABD), but the biological functions of these variants are unknown. To characterize the functions of EABD-associated variants, we integrated GWAS results with 1) expression quantitative trait loci (eQTL) from the Genotype Tissue Expression (GTEx) project and subjects in the COPDGene (Genetic Epidemiology of COPD) study and 2) cell type epigenomic marks from the Roadmap Epigenomics project. On the basis of these analyses, we selected a variant near ACVR1B (activin A receptor type 1B) for functional validation. SNPs from 168 loci with P values less than 5 × 10 in the largest GWAS meta-analysis of EABD were analyzed. Eighty-four loci overlapped eQTL, with 12 of these loci showing greater than 80% likelihood of harboring a single, shared GWAS and eQTL causal variant. Seventeen cell types were enriched for overlap between EABD loci and Roadmap Epigenomics marks (permutation P < 0.05), with the strongest enrichment observed in CD4, CD8, and regulatory T cells. We selected a putative causal variant, rs7962469, associated with ACVR1B expression in lung tissue for additional functional investigation, and reporter assays confirmed allele-specific regulatory activity for this variant in human bronchial epithelial and Jurkat immune cell lines. ACVR1B expression levels exhibit a nominally significant association with emphysema distribution. EABD-associated loci are preferentially enriched in regulatory elements of multiple cell types, most notably T-cell subsets. Multiple EABD loci colocalize to regulatory elements that are active across multiple tissues and cell types, and functional analyses confirm the presence of an EABD-associated functional variant that regulates ACVR1B expression, indicating that transforming growth factor-β signaling plays a role in the EABD phenotype. Clinical trial registered with www.clinicaltrials.gov (NCT00608764).]]>
Wed, 31 Dec 1969 19:00:00 EST
Parent-of-origin effects on carcass traits in Japanese Black cattle. Okamoto K, Oishi K, Nakamura R, Abe A, Inoue K, Kumagai H, Hirooka H
J Anim Breed Genet (May 2019)

Variances caused by the differential expression of paternally and maternally imprinted genes controlling carcass traits in Japanese Black cattle were estimated in this study. Data on marbling score (BMS), carcass weight, rib thickness, rib-eye area (REA) and subcutaneous fat thickness (SFT) were collected from a total of 13,115 feedlot steers and heifers in a commercial population. A sire-maternal grandsire model was used to analyse the data, and then, imprinting parameters were derived by replacing the genetic effect of the dam with the effect of the maternal grandsire in the imprinting model to calculate the genetic parameter estimates. The proportions of the total genetic variance attributable to imprinted genes ranged from 8.7% (SFT) to 35.2% (BMS). The remarkably large imprinting variance of BMS was mainly contributed by maternally expressed inheritance because the maternal contribution of the trait was much larger than that of the paternal trait. The parent-of-origin effect originating from maternal gene expression was also observed for REA. The results suggested the existence of genomic imprinting effects on the traits of the Japanese Black cattle. Hence, the parent-of-origin effect should be considered for the genetic evaluation of Japanese Black cattle in breeding programmes.]]>
Wed, 31 Dec 1969 19:00:00 EST
Genetics and Epigenetics of Infertility and Treatments on Outcomes. Pisarska MD, Chan JL, Lawrenson K, Gonzalez TL, Wang ET
J Clin Endocrinol Metab (Jun 2019)

Infertility affects 10% of the reproductive-age population. Even the most successful treatments such as assisted reproductive technologies still result in failed implantation. In addition, adverse pregnancy outcomes associated with infertility have been attributed to these fertility treatments owing to the presumed epigenetic modifications of in vitro fertilization and in vitro embryo development. However, the diagnosis of infertility has been associated with adverse outcomes, and the etiologies leading to infertility have been associated with adverse pregnancy and long-term outcomes.]]>
Wed, 31 Dec 1969 19:00:00 EST
Detection and Application of 5-Formylcytosine and 5-Formyluracil in DNA. Wang Y, Zhang X, Zou G, Peng S, Liu C, Zhou X
Acc Chem Res (Apr 2019)

Nucleic acids contain a variety of different base modifications, such as decoration at the fifth position of cytosine, which is one of the most important epigenetic modifications. Nucleic acid epigenetics mediate a wide variety of biological processes, including embryonic development and gene regulation, genomic imprinting, differentiation, and X-chromosome inactivation. Furthermore, the modification level can be aberrantly expressed in distinct sets of tissue that can indicate different tumor onsets and canceration. Thus, the analysis of modified nucleobases may contribute to the understanding of epigenetic modification-related biological processes and the correlation of modified nucleobase patterns with disease states for clinical diagnosis and treatment. In addition to 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine are found in organisms at a low content but are nevertheless extremely important chemical modifications, and 5-hydroxyuracil and 5-formyluracil compounds are also present. 5-Formyluracil is found in bacteriophages, prokaryotes, and mammalian cells. The 5-formyluracil content is higher in certain cancer tissues than in the normal tissues adjacent to the tumor. The content of 5-formyluracil in different cell tissues may have cell type specificity. With the continuous use of chemical tools, new detection technologies have greatly advanced the research on natural pyrimidine modifications. These modifications dynamically regulate the gene expression in eukaryotes and prokaryotes and provide mechanistic insights into the occurrence of diseases. Natural pyrimidine modifications act not only as intermediates for DNA demethylation or oxidative damage products but also as modulators of gene expression. Therefore, the development of more effective chemical tools will help us better understand the dynamic changes of natural pyrimidine modifications in vivo. In this Account, we summarize the recent advanced techniques for the detection of 5-formylpyrimidine (5-formylcytosine and 5-formyluracil) and highlight their great potential as biomarkers in biomedical applications. Focusing on the great urgency for the detection of epigenetic modifications, our group developed a series of methods for the qualitative and quantitative analysis of 5-formylpyrimidine in the past few years, aiming at facilitating the accurate detection and mapping of these epigenetic modifications. By the construction of probes, 5-formylpyrimidine can be selectively labeled. Using mass spectrometry, the epigenetic modifications can be quantified. Upon treatment under specific conditions, 5-formylcytosine can be recognized at single-base resolution. With this Account, we anticipate providing chemical and biological researchers with some insight to unlock the complex mechanism involved in 5-formylpyrimidine-related biological processes and stimulate more collaborative research interests from the different fields of materials, biological, medicine, and chemistry to promote the translational research of epigenetics in tumor diagnosis and treatment.]]>
Wed, 31 Dec 1969 19:00:00 EST
Risk variants disrupting enhancers of T1 and T cells in type 1 diabetes. Gao P, Uzun Y, He B, Salamati SE, Coffey JKM, Tsalikian E, Tan K
Proc Natl Acad Sci U S A (Apr 2019)

Genome-wide association studies (GWASs) have revealed 59 genomic loci associated with type 1 diabetes (T1D). Functional interpretation of the SNPs located in the noncoding region of these loci remains challenging. We perform epigenomic profiling of two enhancer marks, H3K4me1 and H3K27ac, using primary T1 and T cells isolated from healthy and T1D subjects. We uncover a large number of deregulated enhancers and altered transcriptional circuitries in both cell types of T1D patients. We identify four SNPs (rs10772119, rs10772120, rs3176792, rs883868) in linkage disequilibrium (LD) with T1D-associated GWAS lead SNPs that alter enhancer activity and expression of immune genes. Among them, rs10772119 and rs883868 disrupt the binding of retinoic acid receptor α (RARA) and Yin and Yang 1 (YY1), respectively. Loss of binding by YY1 also results in the loss of long-range enhancer-promoter interaction. These findings provide insights into how noncoding variants affect the transcriptomes of two T-cell subtypes that play critical roles in T1D pathogenesis.]]>
Wed, 31 Dec 1969 19:00:00 EST
Robust CTCF-Based Chromatin Architecture Underpins Epigenetic Changes in the Heart Failure Stress-Gene Response. Lee DP, Tan WLW, Anene-Nzelu CG, Lee CJM, Li PY, Luu TDA, Chan CX, Tiang Z, Ng SL, Huang X, Efthymios M, Autio MI, Jiang J, Fullwood MJ, Prabhakar S, Lieberman Aiden E, Foo RS
Circulation (Apr 2019)

The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis-regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress-gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout.]]>
Wed, 31 Dec 1969 19:00:00 EST
Mechanisms linking preterm birth to onset of cardiovascular disease later in adulthood. Bavineni M, Wassenaar TM, Agnihotri K, Ussery DW, Lüscher TF, Mehta JL
Eur Heart J (Apr 2019)

Cardiovascular disease (CVD) rates in adulthood are high in premature infants; unfortunately, the underlying mechanisms are not well defined. In this review, we discuss potential pathways that could lead to CVD in premature babies. Studies show intense oxidant stress and inflammation at tissue levels in these neonates. Alterations in lipid profile, foetal epigenomics, and gut microbiota in these infants may also underlie the development of CVD. Recently, probiotic bacteria, such as the mucin-degrading bacterium Akkermansia muciniphila have been shown to reduce inflammation and prevent heart disease in animal models. All this information might enable scientists and clinicians to target pathways to act early to curtail the adverse effects of prematurity on the cardiovascular system. This could lead to primary and secondary prevention of CVD and improve survival among preterm neonates later in adult life.]]>
Wed, 31 Dec 1969 19:00:00 EST
Moderate maternal folic acid supplementation ameliorates adverse embryonic and epigenetic outcomes associated with assisted reproduction in a mouse model. Rahimi S, Martel J, Karahan G, Angle C, Behan NA, Chan D, MacFarlane AJ, Trasler JM
Hum Reprod (Apr 2019)

Could clinically-relevant moderate and/or high dose maternal folic acid supplementation prevent aberrant developmental and epigenetic outcomes associated with assisted reproductive technologies (ART)?]]>
Wed, 31 Dec 1969 19:00:00 EST
How does homeostasis happen? Integrative physiological, systems biological, and evolutionary perspectives. Goldstein DS
Am J Physiol Regul Integr Comp Physiol (Apr 2019)

Homeostasis is a founding principle of integrative physiology. In current systems biology, however, homeostasis seems almost invisible. Is homeostasis a key goal driving body processes, or is it an emergent mechanistic fact? In this perspective piece, I propose that the integrative physiological and systems biological viewpoints about homeostasis reflect different epistemologies, different philosophies of knowledge. Integrative physiology is concept driven. It attempts to explain biological phenomena by continuous formation of theories that experimentation or observation can test. In integrative physiology, "function" refers to goals or purposes. Systems biology is data driven. It explains biological phenomena in terms of "omics"-i.e., genomics, gene expression, epigenomics, proteomics, and metabolomics-it depicts the data in computer models of complex cascades or networks, and it makes predictions from the models. In systems biology, "function" refers more to mechanisms than to goals. The integrative physiologist emphasizes homeostasis of internal variables such as Pco and blood pressure. The systems biologist views these emphases as teleological and unparsimonious in that the "regulated variable" (e.g., arterial Pco and blood pressure) and the "regulator" (e.g., the "carbistat" and "barostat") are unobservable constructs. The integrative physiologist views systems biological explanations as not really explanations but descriptions that cannot account for phenomena we humans believe exist, although they cannot be observed directly, such as feelings and, ultimately, the conscious mind. This essay reviews the history of the two epistemologies, emphasizing autonomic neuroscience. I predict rapprochement of integrative physiology with systems biology. The resolution will avoid teleological purposiveness, transcend pure mechanism, and incorporate adaptiveness in evolution, i.e., "Darwinian medicine."]]>
Wed, 31 Dec 1969 19:00:00 EST
Making headway towards understanding how epigenetic mechanisms contribute to early-life effects. Vukic M, Wu H, Daxinger L
Philos Trans R Soc Lond B Biol Sci (Apr 2019)

It has become clear that in addition to the DNA sequence there is another layer of information, termed epigenetic modifications, that can influence phenotypes and traits. In particular, environmental epigenomics, which addresses the effect of the environment on the epigenome and human health, is becoming an area of great interest for many researchers working in different scientific fields. In this review, we will consider the current evidence that early-life environmental signals can have long-term effects on the epigenome. We will further evaluate how recent technological advances may enable us to unravel the molecular mechanisms underlying these phenomena, which will be crucial for understanding heritability in health and disease. This article is part of the theme issue 'Developing differences: early-life effects and evolutionary medicine'.]]>
Wed, 31 Dec 1969 19:00:00 EST
Genomic consequences of sleep restriction: the devil is in the details. Fuller PM, Eikermann M
Anaesthesia (Apr 2019)

Wed, 31 Dec 1969 19:00:00 EST
Syndromic Disorders Caused by Disturbed Human Imprinting Carli D, Riberi E, Ferrero GB, Mussa A
J Clin Res Pediatr Endocrinol (Apr 2019)

Imprinting disorders are a group of congenital diseases caused by dysregulation of genomic imprinting and affecting prenatal and postnatal growth, neurocognitive development, metabolism and cancer predisposition. Aberrant expression of imprinted genes can be achieved through different mechanisms, classified into epigenetic --- if not involving DNA sequence change --- or genetic --- in case of altered genomic sequence. Despite the underlying mechanism, the phenotype depends on the parental allele affected and opposite phenotypes may result in case of involvement of the maternal or the paternal chromosome. Imprinting disorders are largely underdiagnosed because of the broad range of clinical signs, the presentation overlap among different disorders, the presence of mild phenotypes, the mitigation of the phenotype with age and the limited availability of the molecular techniques employed for the diagnosis. This review briefly illustrates the currently known human imprinting disorders highlighting endocrinological aspects of pediatric interest.]]>
Wed, 31 Dec 1969 19:00:00 EST
Simultaneous Targeted Methylation Sequencing (sTM-Seq). Asmus N, Papale LA, Madrid A, Alisch RS
Curr Protoc Hum Genet (04 2019)

Mapping patterns of DNA methylation throughout the epigenome are critical to our understanding of several important biological and regulatory functions, such as transcriptional regulation, genomic imprinting, and embryonic development. The development and rapid advancement of next-generation sequencing (NGS) technologies have provided clinicians and researchers with accurate and reliable read-outs of genomic and epigenomic information at the nucleotide level. Such improvements have significantly lowered the cost required for genome-wide sequencing, facilitating the vast acquisition of data that has led to many improvements in patient care. However, the torrid rate of NGS data generation has left targeted validation approaches behind, including the confirmation of epigenetic marks such as DNA methylation. To overcome these shortcomings, we present a rapid and robust protocol for the parallel examination of multiple methylated sequences that we have termed simultaneous targeted methylation sequencing (sTM-Seq). Key features of this technique include the elimination of the need for large amounts of high-molecular weight DNA and the nucleotide specific distinction of both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Moreover, sTM-Seq is scalable and can be used to investigate multiple loci in dozens of samples within a single sequencing run. By utilizing freely available web-based software and universal primers for multipurpose barcoding, library preparation, and customized sequencing, sTM-Seq is affordable, efficient, and widely applicable. Together, these features enable sTM-Seq to have wide-reaching clinical applications that will greatly improve turnaround rates for same-day procedures and allow clinicians to collect high-resolution data that can be used in a variety of patient settings. © 2019 by John Wiley & Sons, Inc.]]>
Wed, 31 Dec 1969 19:00:00 EST