Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an Enzyme-based Immunoassay

BACKGROUND: Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis. METHODS: To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin-avidin mediated enzyme-based immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection. RESULTS: We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025±0.081, 0.023±0.006 and 0.001±0.0002, respectively. We observed a significant (p<0.05) decrease in the mean global percentage of 5hmC in blood of patients with malignant lung cancer (0.013±0.003%) in comparison to healthy controls. CONCLUSION: The precise biological roles of these epigenetic modifications in cancers are still unknown but in the past two years it has become evident that the global 5hmC content is drastically reduced in a variety of cancers. To the best of our knowledge, this is the first report of decreased 5hmC content in the blood of metastatic lung cancer patients and the clinical utility of this observation needs to be further validated in larger sample datasets

When evolution is the solution to pollution : key principles, and lessons from rapid repeated adaptation of killifish (Fundulus heteroclitus) populations

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Evolutionary Applications 10 (2017): 762–783, doi:10.1111/eva.12470.For most species, evolutionary adaptation is not expected to be sufficiently rapid to buffer the effects of human-mediated environmental changes, including environmental pollution. Here we review how key features of populations, the characteristics of environmental pollution, and the genetic architecture underlying adaptive traits, may interact to shape the likelihood of evolutionary rescue from pollution. Large populations of Atlantic killifish (Fundulus heteroclitus) persist in some of the most contaminated estuaries of the United States, and killifish studies have provided some of the first insights into the types of genomic changes that enable rapid evolutionary rescue from complexly degraded environments. We describe how selection by industrial pollutants and other stressors has acted on multiple populations of killifish and posit that extreme nucleotide diversity uniquely positions this species for successful evolutionary adaptation. Mechanistic studies have identified some of the genetic underpinnings of adaptation to a well-studied class of toxic pollutants; however, multiple genetic regions under selection in wild populations seem to reflect more complex responses to diverse native stressors and/or compensatory responses to primary adaptation. The discovery of these pollution-adapted killifish populations suggests that the evolutionary influence of anthropogenic stressors as selective agents occurs widely. Yet adaptation to chemical pollution in terrestrial and aquatic vertebrate wildlife may rarely be a successful “solution to pollution” because potentially adaptive phenotypes may be complex and incur fitness costs, and therefore be unlikely to evolve quickly enough, especially in species with small population sizes.National Science Foundation Grant Numbers: DEB-1265282, OCE-1314567, DEB-1120263; National Institutes of Environmental Health Sciences Grant Numbers: R01ES021934-01, P42ES007381; Postdoctoral Research Program at the US Environmental Protection (US EPA); Office of Research and Development; Oak Ridge Institute for Science and Education (ORISE) Grant Number: DW92429801; US Department of Energ

Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae

Abstract Background The zebrafish Danio rerio is an important model system for drug discovery and to study cardiovascular development. Using a laser-scanning confocal microscope, we have developed a non-invasive method of measuring cardiac performance in zebrafish embryos and larvae that obtains cardiovascular parameters similar to those obtained using Doppler echocardiography in mammals. A laser scan line placed parallel to the path of blood in the dorsal aorta measures blood cell velocity, from which cardiac output and indices of vascular resistance and contractility are calculated. Results This technique, called laser-scanning velocimetry, was used to quantify the effects of pharmacological, developmental, and genetic modifiers of cardiac function. Laser-scanning velocimetry was applied to analyze the cardiovascular effects of morpholino knockdown of osmosensing scaffold for MEKK3 (OSM), which when mutated causes the human vascular disease cerebral cavernous malformations. OSM-deficient embryos had a constricted aortic arch and markedly increased peak cell velocity, a characteristic indicator of aortic stenosis. Conclusion These data validate laser-scanning velocimetry as a quantitative tool to measure cardiovascular performance for pharmacological and genetic analysis in zebrafish, which requires no specialized equipment other than a laser-scanning confocal microscope

Epigenetics in Prostate Cancer

Prostate cancer (PC) is the most commonly diagnosed nonskin malignancy and the second most common cause of cancer death among men in the United States. Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than changes in the underlying DNA sequences. Two common epigenetic mechanisms, DNA methylation and histone modification, have demonstrated critical roles in prostate cancer growth and metastasis. DNA hypermethylation of cytosine-guanine (CpG) rich sequence islands within gene promoter regions is widespread during neoplastic transformation of prostate cells, suggesting that treatment-induced restoration of a “normal” epigenome could be clinically beneficial. Histone modification leads to altered tumor gene function by changing chromosome structure and the level of gene transcription. The reversibility of epigenetic aberrations and restoration of tumor suppression gene function have made them attractive targets for prostate cancer treatment with modulators that demethylate DNA and inhibit histone deacetylases

Elevated Phospholipase A2 Activities in Plasma Samples from Multiple Cancers.

Only in recent years have phospholipase A2 enzymes (PLA2s) emerged as cancer targets. In this work, we report the first detection of elevated PLA2 activities in plasma from patients with colorectal, lung, pancreatic, and bladder cancers as compared to healthy controls. Independent sets of clinical plasma samples were obtained from two different sites. The first set was from patients with colorectal cancer (CRC; n = 38) and healthy controls (n = 77). The second set was from patients with lung (n = 95), bladder (n = 31), or pancreatic cancers (n = 38), and healthy controls (n = 79). PLA2 activities were analyzed by a validated quantitative fluorescent assay method and subtype PLA2 activities were defined in the presence of selective inhibitors. The natural PLA2 activity, as well as each subtype of PLA2 activity was elevated in each cancer group as compared to healthy controls. PLA2 activities were increased in late stage vs. early stage cases in CRC. PLA2 activities were not influenced by sex, smoking, alcohol consumption, or body-mass index (BMI). Samples from the two independent sites confirmed the results. Plasma PLA2 activities had approximately 70% specificity and sensitivity to detect cancer. The marker and targeting values of PLA2 activity have been suggested

Ryanodine receptor and FK506 binding protein 1 in the Atlantic killifish (Fundulus heteroclitus) : a phylogenetic and population-based comparison

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Aquatic Toxicology 192 (2017): 105-115, doi:10.1016/j.aquatox.2017.09.002.Non-dioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine receptors (RyR), microsomal Ca2+ channels of broad significance. Teleost fish may be important models for NDL PCB neurotoxicity, and we used sequencing databases to characterize teleost RyR and FK506 binding protein 12 or 12.6 kDa (genes FKBP1A; FKBP1B), which promote NDL PCB-triggered Ca2+ dysregulation. Particular focus was placed on describing genes in the Atlantic killifish (Fundulus heteroclitus) genome and searching available RNA-sequencing datasets for single nucleotide variants (SNV) between PCB tolerant killifish from New Bedford Harbor (NBH) versus sensitive killifish from Scorton Creek (SC), MA. Consistent with the teleost whole genome duplication (tWGD), killifish have six RyR genes, corresponding to a and b paralogs of mammalian RyR1, 2 and 3. The presence of six RyR genes was consistent in all teleosts investigated including zebrafish. Killifish have four FKBP1; one FKBP1b and three FKBP1a named FKBP1aa, FKBP1ab, likely from the tWGD and a single gene duplicate FKBP1a3 suggested to have arisen in Atherinomorphae. The RyR and FKBP1 genes displayed tissue and developmental stage-specific mRNA expression, and the previously uncharacterized RyR3, herein named RyR3b, and all FKBP1 genes were prominent in brain. We identified a SNV in RyR3b encoding missense mutation E1458D. In NBH killifish, 57% were heterozygous and 28% were homozygous for this SNV, whereas almost all SC killifish (94%) lacked the variant (n≥39 per population). The outlined sequence differences between mammalian and teleost RyR and FKBP1 together with outlined population differences in SNV frequency may contribute to our understanding of NDL PCB neurotoxicity.This research was supported by the KC Donnelly Research Externship made possible by the National Institute of Environmental Health Sciences’ Superfund Research Program (EBH) and the Superfund Research Programs at UC Davis (INP and EBH; P42ES004699) and Boston University (JJS, JVG, MEH, SIK; P42ES007381). Additional support was provided by the National Institute of Health (INP; R01 ES014901; and P01 AR052354) and by National Science Foundation collaborative research grants (MEH and SIK; DEB-1265282 and DEB-1120263). This research was also supported in part by an appointment (to BC) with the Postdoctoral Research Program at the U.S. Environmental Protection (US EPA) Office of Research and Development administered by the Oak Ridge Institute for Science and Education (ORISE) through Interagency Agreement No. DW92429801 between the U.S. Department of Energy and the US EPA

The NGC 672 and NGC 784 Galaxy Groups: Evidence for Galaxy Formation and Growth Along a Nearby Dark Matter Filament

(Abridged): We present U, B, V, R, I, H-alpha and NUV photometry of 14 galaxies in the very local Universe (within 10 Mpc that are dwarf irregular galaxies (dIrr), are at low redshift (51<v<610 km/s), and appear as a six degree long linear filament.. We examine the star formation (SF) properties of individual objects with the current SF rate (SFR) derived directly from the H-alpha line flux and compare the multi-band photometry with results of galaxy evolution assuming short SF bursts separated by long quiescence periods. Most objects contain at least one "old" stellar population (>1-10 Gyr) and one "young" population (<30 Myr) with the recent SF bursts occurring a few to a few 10s of Myr ago, arguing for synchronicity in star formation in these objects. We propose that the ~synchronous star formation in all objects is caused by the accretion of cold gas from intergalactic space onto dark matter haloes arranged along a filament threading the void where these dwarf galaxies reside and point out this galaxy sample as an ideal target to study hierarchical clustering and galaxy formation among very nearby objects.Comment: 29 pages, five figures. MNRAS, in press. This version with no galaxy images to reduce file size. A full version (.pdf) can be downloaded cia anonymous ftp from ftp://wise-gate.tau.ac.il/ftp/pub/noah/AdiPaper_accepted.pd

The landscape of extreme genomic variation in the highly adaptable Atlantic killifish

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Genome Biology and Evolution 9 (2017): 659-676, doi:10.1093/gbe/evx023.Understanding and predicting the fate of populations in changing environments require knowledge about the mechanisms that support phenotypic plasticity and the adaptive value and evolutionary fate of genetic variation within populations. Atlantic killifish (Fundulus heteroclitus) exhibit extensive phenotypic plasticity that supports large population sizes in highly fluctuating estuarine environments. Populations have also evolved diverse local adaptations. To yield insights into the genomic variation that supports their adaptability, we sequenced a reference genome and 48 additional whole genomes from a wild population. Evolution of genes associated with cell cycle regulation and apoptosis is accelerated along the killifish lineage, which is likely tied to adaptations for life in highly variable estuarine environments. Genome-wide standing genetic variation, including nucleotide diversity and copy number variation, is extremely high. The highest diversity genes are those associated with immune function and olfaction, whereas genes under greatest evolutionary constraint are those associated with neurological, developmental, and cytoskeletal functions. Reduced genetic variation is detected for tight junction proteins, which in killifish regulate paracellular permeability that supports their extreme physiological flexibility. Low-diversity genes engage in more regulatory interactions than high-diversity genes, consistent with the influence of pleiotropic constraint on molecular evolution. High genetic variation is crucial for continued persistence of species given the pace of contemporary environmental change. Killifish populations harbor among the highest levels of nucleotide diversity yet reported for a vertebrate species, and thus may serve as a useful model system for studying evolutionary potential in variable and changing environments.This work was primarily supported by a grant from the National Science Foundation (collaborative research grants DEB-1265282, DEB-1120512, DEB-1120013, DEB-1120263, DEB-1120333, DEB-1120398 to J.K.C., D.L.C., M.E.H., S.I.K., M.F.O., J.R.S., W.W., and A.W.). Further support was provided by the National Institute of Environmental Health Sciences (1R01ES021934-01 to A.W., P42ES7373 to T.H.H., P42ES007381 to M.E.H., and R01ES019324 to J.R.S.), the National Institute of General Medical Sciences (P20GM103423 and P20GM104318 to B.L.K.), and the National Science Foundation (DBI-0640462 and XSEDE-MCB100147 to D.G.)