A DNA methylation biomarker of alcohol consumption.

The lack of reliable measures of alcohol intake is a major obstacle to the diagnosis and treatment of alcohol-related diseases. Epigenetic modifications such as DNA methylation may provide novel biomarkers of alcohol use. To examine this possibility, we performed an epigenome-wide association study of methylation of cytosine-phosphate-guanine dinucleotide (CpG) sites in relation to alcohol intake in 13 population-based cohorts (ntotal=13 317; 54% women; mean age across cohorts 42-76 years) using whole blood (9643 European and 2423 African ancestries) or monocyte-derived DNA (588 European, 263 African and 400 Hispanic ancestry) samples. We performed meta-analysis and variable selection in whole-blood samples of people of European ancestry (n=6926) and identified 144 CpGs that provided substantial discrimination (area under the curve=0.90-0.99) for current heavy alcohol intake (⩾42 g per day in men and ⩾28 g per day in women) in four replication cohorts. The ancestry-stratified meta-analysis in whole blood identified 328 (9643 European ancestry samples) and 165 (2423 African ancestry samples) alcohol-related CpGs at Bonferroni-adjusted P<1 × 10-7. Analysis of the monocyte-derived DNA (n=1251) identified 62 alcohol-related CpGs at P<1 × 10-7. In whole-blood samples of people of European ancestry, we detected differential methylation in two neurotransmitter receptor genes, the γ-Aminobutyric acid-A receptor delta and γ-aminobutyric acid B receptor subunit 1; their differential methylation was associated with expression levels of a number of genes involved in immune function. In conclusion, we have identified a robust alcohol-related DNA methylation signature and shown the potential utility of DNA methylation as a clinically useful diagnostic test to detect current heavy alcohol consumption.

CPT1A methylation is associated with plasma adiponectin.

BACKGROUND AND AIMS: Adiponectin, an adipose-secreted protein that has been linked to insulin sensitivity, plasma lipids, and inflammatory patterns, is an established biomarker for metabolic health. Despite clinical relevance and high heritability, the determinants of plasma adiponectin levels remain poorly understood. METHODS AND RESULTS: We conducted the first epigenome-wide cross-sectional study of adiponectin levels using methylation data on 368,051 cytosine-phosphate-guanine (CpG) sites in CD4+ T-cells from the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN, n = 991). We fit linear mixed models, adjusting for age, sex, study site, T-cell purity, and family. We have identified a positive association (regression coefficient ± SE = 0.01 ± 0.001, P = 3.4 × 10-13) between plasma adiponectin levels and methylation of a CpG site in CPT1A, a key player in fatty acid metabolism. The association was replicated (n = 474, P = 0.0009) in whole blood samples from the Amish participants of the Heredity and Phenotype Intervention (HAPI) Heart Study as well as White (n = 592, P = 0.0005) but not Black (n = 243, P = 0.18) participants of the Bogalusa Heart Study (BHS). The association remained significant upon adjusting for BMI and smoking in GOLDN and HAPI but not BHS. We also identified associations between methylation loci in RNF145 and UFM1 and plasma adiponectin in GOLDN and White BHS participants, although the association was not robust to adjustment for BMI or smoking. CONCLUSION: We have identified and replicated associations between several biologically plausible loci and plasma adiponectin. These findings support the importance of epigenetic processes in metabolic traits, laying the groundwork for future translational applications.

Identification and validation of seven new loci showing differential DNA methylation related to serum lipid profile: an epigenome-wide approach. The REGICOR study.

Lipid traits (total, low-density and high-density lipoprotein cholesterol, and triglycerides) are risk factors for cardiovascular disease. DNA methylation is not only an inherited but also modifiable epigenetic mark that has been related to cardiovascular risk factors. Our aim was to identify loci showing differential DNA methylation related to serum lipid levels. Blood DNA methylation was assessed using the Illumina Human Methylation 450 BeadChip. A two-stage epigenome-wide association study was performed, with a discovery sample in the REGICOR study (n = 645) and validation in the Framingham Offspring Study (n = 2,542). Fourteen CpG sites located in nine genes (SREBF1, SREBF2, PHOSPHO1, SYNGAP1, ABCG1, CPT1A, MYLIP, TXNIP and SLC7A11) and 2 intergenic regions showed differential methylation in association with lipid traits. Six of these genes and 1 intergenic region were new discoveries showing differential methylation related to total cholesterol (SREBF2), HDL-cholesterol (PHOSPHO1, SYNGAP1 and an intergenic region in chromosome 2) and triglycerides (MYLIP, TXNIP and SLC7A11). These CpGs explained 0.7%, 9.5% and 18.9% of the variability of total cholesterol, HDL cholesterol and triglycerides in the Framingham Offspring Study, respectively. The expression of the genes SREBF2 and SREBF1 was inversely associated with methylation of their corresponding CpGs (P-value = 0.0042 and 0.0045, respectively) in participants of the GOLDN study (n = 98). In turn, SREBF1 expression was directly associated with HDL cholesterol (P-value = 0.0429). Genetic variants in SREBF1, PHOSPHO1, ABCG1 and CPT1A were also associated with lipid profile. Further research is warranted to functionally validate these new loci and assess the causality of new and established associations between these differentially methylated loci and lipid metabolism.

Cancer cell proliferation is inhibited by specific modulation frequencies.

BACKGROUND: There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. METHODS: To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100 Hz-21 kHz range as cancer-specific frequencies. RESULTS: The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle. CONCLUSION: These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology.

The genetic basis of divergent pigment patterns in juvenile threespine sticklebacks.

Animal pigment patterns are important for a range of functions, including camouflage and communication. Repeating pigment patterns, such as stripes, bars and spots have been of particular interest to developmental and theoretical biologists, but the genetic basis of natural variation in such patterns is largely unexplored. In this study, we identify a difference in a periodic pigment pattern among juvenile threespine sticklebacks (Gasterosteus aculeatus) from different environments. Freshwater sticklebacks exhibit prominent vertical bars that visually break up the body shape, but sticklebacks from marine populations do not. We hypothesize that these distinct pigment patterns are tuned to provide crypsis in different habitats. This phenotypic difference is widespread and appears in most of the freshwater populations that we sampled. We used quantitative trait locus (QTL) mapping in freshwater-marine F2 hybrids to elucidate the genetic architecture underlying divergence in this pigmentation pattern. We identified two QTL that were significantly associated with variation in barring. Interestingly, these QTL were associated with two distinct aspects of the pigment pattern: melanophore number and overall pigment level. We compared the QTL locations with positions of known pigment candidate genes in the stickleback genome. We also identified two major QTL for juvenile body size, providing new insights into the genetic basis of juvenile growth rates in natural populations. In summary, although there is a growing literature describing simple genetic bases for adaptive coloration differences, this study emphasizes that pigment patterns can also possess a more complex genetic architecture.

Recipients in need of ancillary services and their receipt of HIV medical care in California.

For many individuals with access to quality medical care, HIV disease is no longer a critical short term illness but a chronic condition giving rise to more clients requiring ongoing medical care. Programs funded by the federal Ryan White Comprehensive AIDS Resources Emergency Act not only provide essential medical care for these individuals but also facilitate access to medical care services. These programmes fund services, including case management, transportation, and translation assistance, that feature ongoing assistance and enable individuals to remain in the health care system. Because of the importance of maintaining the strict drug regimen, retention in care is also an important part of the overall HIV care component. This study analyzed the relationship of ancillary services and a federal health programme client's receipt of medical care and retention in the health care system. We defined a cohort in need of ancillary services in part by a questionnaire designed to identify factors relating to need. These factors included education, language, and substance use. By merging client level data files we were able to identify medical service utilization trends among the individuals in the cohort who received a high number of ancillary services (more than 11 ancillary service visits in the two-year study period, n = 138) and those who received few services (fewer than six ancillary service visits in the two-year study period, n = 132). Results suggest that the receipt of ancillary services is associated with receipt of and retention in primary medical care. We found that for federal health programme clients in need of ancillary services, a positive relationship existed between their receipt of ancillary services and their access to primary medical care (p

Lobar emphysema due to anomalous aortic origin of the left pulmonary artery.

The unusual case of an infant with aortic origin of the left pulmonary artery is presented. The patient developed a rare complication of lobar emphysema due to bronchial compression from the enlarged right pulmonary artery. Operative anastomosis of the left pulmonary artery to the pulmonary trunk was successful, with subsequent resolution of the lobar emphysema.

FMRP associates with polyribosomes as an mRNP, and the I304N mutation of severe fragile X syndrome abolishes this association.

Fragile X mental retardation is caused by the lack of FMRP, a selective RNA-binding protein associated with ribosomes. A missense mutation, I304N, has been found to result in an unusually severe phenotype. We show here that normal FMRP associates with elongating polyribosomes via large mRNP particles. Despite normal expression and cytoplasmic mRNA association, the I304N FMRP is incorporated into abnormal mRNP particles that are not associated with polyribosomes. These data indicate that association of FMRP with polyribosomes must be functionally important and imply that the mechanism of the severe phenotype in the I304N patient lies in the sequestration of bound mRNAs in nontranslatable mRNP particles. In the absence of FMRP, these same mRNAs may be partially translated via alternative mRNPs, although perhaps abnormally localized or regulated, resulting in typical fragile X syndrome.

Angiotensin-(1-7) binding at angiotensin II receptors in the rat brain.

Angiotensin-(1-7) (Ang-(1-7)) is reported to be equipotent with angiotensin II (AII) in producing some central biological effects but the receptors responsible for these actions have not been defined. Three classes of receptor have been proposed: AT1, AT2, and a putative Ang-(1-7) selective receptor. This study specifically evaluates Ang-(1-7) competition at AII binding sites (AT1 and AT2) in the rat brain. 125I Sar1 Ile8 AII (269-312 pM) was used to conduct receptor autoradiographic binding assays in brain sections. Competition with Ile5 AII and Val5 AII was similar at nuclei in which either AT1 or AT2 receptor subtypes predominate (Ki = 11-18 nM). Ang-(1-7) competed 150-fold less effectively than native AII at AT1 predominant brain nuclei (Ki = 2.4 microM). At brain regions where AT2 receptors predominate, Ang-(1-7) showed a very low affinity (Ki = 104 microM) for the majority of the 125I Sar1 Ile8 AII binding sites (AT2). A small proportion of 125I Sar1 Ile8 AII binding sites showed an affinity of 2.0 microM, presumably AT1 receptors present in those brain regions. For biological responses where Ang-(1-7) is reported to be equipotent with AII, it is unlikely that these actions are mediated by the widely distributed AT1 or AT2 receptor subtypes which recognize 125I Sar1 Ile8 AII.

Gq- and ras-dependent pathways mediate hypertrophy of neonatal rat ventricular myocytes following alpha 1-adrenergic stimulation.

alpha 1-Adrenergic agonists activate a hypertrophic response in cultured neonatal ventricular myocytes, which include an increase in cell size, organization of contractile proteins into sarcomeric units, and the induction of the atrial natriuretic factor (ANF) gene. Previous findings have supported a role for ras in this signaling pathway. Utilizing microinjection techniques to delivery affinity-purified neutralizing antibodies to G alpha q,11 into cultured ventricular myocytes, the current studies demonstrate a functional requirement for the heterotrimeric G protein, Gq, in the alpha 1-adrenergic induction of the ANF gene, changes in cell size, organization of myofilaments, and phosphoinositide hydrolysis. Expression of a constitutively active mutant of G alpha q leads to the expression of ANF protein in these cells. Taken together, these data suggest that G q-dependent pathways are necessary and sufficient to activate defined features of the hypertrophic response. In attempts to further delineate the relative roles of ras and Gq in this pathway, we found that G alpha q is required for alpha 1-adrenergic phosphoinositide hydrolysis, though ras does not appear to be necessary for this response. In addition, we coexpressed an inhibitory ras mutant, along with the constitutively active G alpha q. Expression of ANF protein stimulated by the G alpha q mutant was not inhibited. Thus, both ras- and Gq-dependent pathways are necessary to fully transduce defined features of alpha 1-adrenergic-stimulated hypertrophy of neonatal cardiac ventricular myocytes, but activated Gq may be able to induce ANF expression independent of inhibitory ras.

Angiotensin II binding sites in the hamster brain: localization and subtype distribution.

This study was designed to characterize the distribution of angiotensin II (AII) binding sites in the hamster brain. Brain sections were incubated with [125I][sar1,ile8]-angiotensin II in the absence and presence of angiotensin II receptor subtype selective compounds, losartan (AT1 subtype) and PD123177 (AT2 subtype). Binding was quantified by densitometric analysis of autoradiograms and localized by comparison with adjacent thionein stained sections. The distribution of AII binding sites was similar to that found in the rat, with some exceptions. [125I][sar1,ile8]-angiotensin II binding was not evident in the subthalamic nucleus and thalamic regions, inferior olive, suprachiasmatic nucleus, and piriform cortex of the hamster, regions of prominent binding in the rat brain. However, intense binding was observed in the interpeduncular nucleus and the medial habenula of the hamster, nuclei void of binding in the rat brain. Competition with receptor subtype selective compounds revealed a similar AII receptor subtype profile in brain regions where binding is evident in both species. One notable exception is the medial geniculate nucleus, predominately AT1 binding sites in the hamster but AT2 in the rat. Generally, the AII binding site distribution in the hamster brain parallels that of the other species studied, particularly in brain regions associated with cardiovascular and dipsogenic functions. Functional correlates for AII binding sites have not been elucidated in the majority of brain regions and species mismatches might provide clues in this regard.