An epigenome-wide study of body mass index and DNA methylation in blood using participants from the Sister Study cohort.

BACKGROUND/OBJECTIVES: The relationship between obesity and chronic disease risk is well-established; the underlying biological mechanisms driving this risk increase may include obesity-related epigenetic modifications. To explore this hypothesis, we conducted a genome-wide analysis of DNA methylation and body mass index (BMI) using data from a subset of women in the Sister Study. SUBJECTS/METHODS: The Sister Study is a cohort of 50 884 US women who had a sister with breast cancer but were free of breast cancer themselves at enrollment. Study participants completed examinations which included measurements of height and weight, and provided blood samples. Blood DNA methylation data generated with the Illumina Infinium HumanMethylation27 BeadChip array covering 27,589 CpG sites was available for 871 women from a prior study of breast cancer and DNA methylation. To identify differentially methylated CpG sites associated with BMI, we analyzed this methylation data using robust linear regression with adjustment for age and case status. For those CpGs passing the false discovery rate significance level, we examined the association in a replication set comprised of a non-overlapping group of 187 women from the Sister Study who had DNA methylation data generated using the Infinium HumanMethylation450 BeadChip array. Analysis of this expanded 450 K array identified additional BMI-associated sites which were investigated with targeted pyrosequencing. RESULTS: Four CpG sites reached genome-wide significance (false discovery rate (FDR) q<0.05) in the discovery set and associations for all four were significant at strict Bonferroni correction in the replication set. An additional 23 sites passed FDR in the replication set and five were replicated by pyrosequencing in the discovery set. Several of the genes identified including ANGPT4, RORC, SOCS3, FSD2, XYLT1, ABCG1, STK39, ASB2 and CRHR2 have been linked to obesity and obesity-related chronic diseases. CONCLUSIONS: Our findings support the hypothesis that obesity-related epigenetic differences are detectable in blood and may be related to risk of chronic disease.

The incidence of and risk factors for MRSA bacteraemia in an HIV-infected cohort in the HAART era.

OBJECTIVES: To define the incidence and risk factors for methicillin resistant Staphylococcus aureus (MRSA) bacteraemia in an HIV-infected population. METHODS: From January 1, 2000 to December 31, 2004, we conducted a retrospective cohort study. We identified all cases of Staphylococcus aureus bacteraemia (SAB), including MRSA, among patients enrolled in the Johns Hopkins Hospital out-patient HIV clinic. A conditional logistic regression model was used to identify risk factors for MRSA bacteraemia compared with methicillin-sensitive SAB and no bacteraemia in unmatched (1:1) and matched (1:4) nested case-control analyses, respectively. RESULTS: Of 4607 patients followed for a total of 11 020 person-years (PY) of follow-up, 216 episodes of SAB occurred (incidence: 19.6 cases per 1000 PY), including 94 cases (43.5%) which were methicillin-resistant. The incidence of MRSA bacteraemia increased from 5.3 per 1000 PY in 2000-2001 to 11.9 per 1000 PY in 2003-2004 (P=0.001). Multivariate analysis demonstrated that independent predictors of MRSA bacteraemia (vs. no bacteraemia) were injection drug use (IDU), end-stage renal disease (ESRD) and CD4 count <200 cells/microL. CONCLUSIONS: MRSA bacteraemia was an increasingly common diagnosis in our HIV-infected cohort, especially in patients with history of IDU, low CD4 cell count and ESRD.

Heartburn requiring frequent antacid use may indicate significant illness.

BACKGROUND: Many otherwise healthy individuals with episodic heartburn self-medicate with over-the-counter antacids. We evaluated clinical characteristics of subjects who had never been medically diagnosed as having any upper gastrointestinal tract disorder and who used antacids for symptomatic relief of heartburn. SUBJECTS AND METHODS: Subjects with at least 3 months of frequent heartburn relieved by antacids, and with heartburn on at least 4 of 7 days during the week prior to study entry, had their medical history and gastrointestinal pathological characteristics recorded. Tests included esophagogastroduodenoscopy, esophageal motility and sensitivity studies, and 24-hour pH monitoring. RESULTS: Of 178 subjects screened, 13 were excluded on the basis of other gastrointestinal diseases at baseline, including diffuse esophageal spasm, peptic ulcer disease, dysplastic columnar metaplasia of the esophagus (Barrett's esophagus), and adenocarcinoma. Ten subjects were ineligible because of insufficient baseline heartburn. The remaining 155 eligible subjects had heartburn for an average of 11 years. Forty-seven percent had daily symptoms and 70% described heartburn severity as moderate, even though on endoscopy most (53%) had normal-appearing esophageal mucosa (grade 0 or 1). Esophageal acid sensitivity was present in 86% of subjects. Mean lower esophageal sphincter pressures and esophageal contractile amplitudes were at the lower limits of normal and total esophageal acid contact time was slightly increased. CONCLUSIONS: Chronic heartburn can reflect a wide range of diagnostic findings, including important underlying pathological features, and may warrant a full medical examination to detect such abnormal conditions and to permit selection of appropriate therapy.

Leftwards error in bisecting the gap between two points: stimulus quality and hand effects.

Subjects attempted to bisect the gap between two LEDs; these differed from each other in brightness, stimulus duration, onset/offset (a) synchronies, and in continuous or flashing mode of presentation. There was a systematic bias to the left, especially with the left hand, indicating a motor contribution to this perceptual task. A brief, faint or flashing LED attracted the subjective midpoint, perhaps because of increased processing demands; indeed the general leftwards bias was attributed to a greater visuospatial processing capacity in the right hemisphere. Leftwards bias was rather greater when central fixation was maintained, indicating that hemispatial mechanisms may be more important than anatomical connectivities. The technique may be useful for understanding and treating unilateral neglect.

Effects of GABA receptor agonists on [3H] flunitrazepam binding to rat cerebellar and hippocampal membranes.

The effects of GABA agonists on [3H] flunitrazepam binding were examined in membranes from CNS areas proposed to contain different populations of benzodiazepine binding site subtypes (BZ1/BZ2). Since these effects were broadly similar in both cerebellar and hippocampal membranes, it seems unlikely that GABA receptors interacting with the proposed BZ1/BZ2 binding sites have markedly different properties. Sodium chloride clearly facilitated the potentiating effect of muscimol on [3H] flunitrazepam binding but produced a complex interaction with the effects of GABA itself and the partial agonist imidazoleacetic acid in the phosphate buffer system used.

Identification of recombinant baculoviruses using green fluorescent protein as a selectable marker.

A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperda cells resulted in the synthesis of a 30-kDa protein, which was confirmed as GFP by Western blotting and by the emission of green fluorescence when illuminated with longwave UV light (495 or 365 nm). To use GFP as a marker for the selection of recombinant baculoviruses, we prepared a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of lacZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfection to prepare recombinant baculoviruses, the virus DNA was linearized with Bsu361 to improve the recovery of non-parental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with the non-parental phenotype. Plaques were rapidly identified by simply exposing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains.

Hypothyroidism alters diaphragm muscle development.

The impact of hypothyroidism (Hyp) on myosin heavy chain (MHC) isoform expression, maximum specific force (P0), fatigability, and maximum unloaded shortening velocity (V0) was determined in the rat diaphragm muscle (Dia) at 0, 7, 14, 21, and 28 days of age. Hyp was induced by treating pregnant rats with 6-n-propyl-2-thiouracil (0.05% in drinking water) beginning at gestational day 10 and was confirmed by reduced plasma levels of 3,5,3'-triiodothyronine and thyroxine. MHC isoforms were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by densitometry. Isometric P0 and fatigue resistance of the Dia were measured in vitro at 26 degrees C, and V0 was determined at 15 degrees C with the slack test. Compared with control muscles, expression of MHC-slow was higher and expression of adult fast MHC isoforms was lower in Hyp Dia at all ages. The neonatal isoform of MHC continued to be expressed in the Hyp Dia until day 28. At each age, P0 and fatigability were reduced and V0 was slower in the Hyp Dia. We conclude that Hyp-induced alterations in MHC isoform expression do not fully predict the changes in Dia contractile properties.

Contractile properties of the developing diaphragm correlate with myosin heavy chain phenotype.

The objective of this study was to determine the relationship between developmental transitions in myosin heavy chain (MHC) composition and changes in maximum unloaded shortening velocity (Vo) and maximum specific force (Po) of the rat diaphragm muscle. The diaphragm was excised at postnatal days 0, 3, 7, 14, 21, and 28 and in adults. MHC isoform expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and laser densitometry. In muscle fiber bundles, Vo was determined at 15 degrees C by use of the "slack" test. Isometric Po was determined at 15 and 26 degrees C. Simple and stepwise regressions were used to evaluate the correlations between Vo, Po, and MHC phenotype transitions and the various developmental ages. The progressive increases in Vo and Po with age were found to be inversely correlated to MHC-neonatal isoform expression (r2 = -0.84 and -0.63, respectively) and positively correlated to MHC-2X (r2 = 0.78 and 0.57) and MHC-2B (r2 = 0.51 and 0.40) isoform expression (P < 0.001). Changes in MHC-neonatal isoform expression contributed to most of the developmental variance in Vo and Po, with changes in MHC-2X and MHC-2B expression also contributing significant increments to total variance. The postnatal increase in Vo most likely relates to differences in the actomyosin adenosinetriphosphatase activity between neonatal and adult fast MHC phenotypes. The increase in Po may reflect inherent differences in myofibrillar density, cross-bridge cycling kinetics, and/or the force produced per cross bridge among fibers composed of the different MHC isoforms.

Dose-response relationship for radiation-induced mutations at micro- and minisatellite loci in human somatic cells in culture.

PURPOSE: The study was designed to determine the dose-response relationship for radiation induction of mutations at mini- and microsatellite loci in human somatic cells. Mutations induced by graded doses of gamma-irradiation were quantified by screening clones derived from single irradiated cells for micro- and minisatellite alterations following irradiation with 1, 2 or 3 Gy. MATERIALS AND METHODS: After irradiation, the moderately radioresistant glioma cell line UVW was seeded at low density into Petri dishes to allow formation of discrete colonies, 100 of which were examined at each dose. All the cells within a colony were presumed to have arisen from a single irradiated cell. Radiation-induced microsatellite alterations were determined at 16 different loci, by PCR amplification and visualization on polyacrylamide gels. Minisatellite alterations were identified at four different minisatellite loci by restriction enzyme digestion and Southern blotting. RESULTS: A dose-response curve for mutation frequency was obtained by analysis of 100 clones, yielding a minisatellite mutation rate of 5.5x10(-3) mutations/locus/Gy/cell and a microsatellite mutation rate of 8.75x10(-4) mutations/locus/ Gy/cell. At microsatellite loci, alterations were predominantly simple loss or gain of repeat units and loss of heterozygosity (LOH). The mutations in minisatellite loci resulted predominantly in LOH and variation in repeat number. The background instability at each locus was determined by analysis of non-irradiated clones. Only 2% and 1% of the micro-and minisatellite loci respectively showed altered bands. CONCLUSIONS: This is the first report of a dose-response relationship for radiation-induced micro- and minisatellite mutations in human somatic cells. Described is a sensitive method for analysis of low-dose radiation mutagenesis in somatic cells that may prove to be a useful tool for radiation protection and dosimetry.

Contraceptive outcomes among post-partum and post-abortal adolescents.

Between January 1992 and January 1993, there were 280 teens (ages 13-18) who either delivered a baby or terminated a pregnancy at Johns Hopkins Bayview Medical Center. Of these, 92 chose to contracept with Norplant implants, and 188 chose another method including "no" method. In July 1993, telephone interviews were conducted with 37 of those who chose Norplant implants and 41 of the non-Norplant implants users. After 1 year, 47% of oral contraceptive (COC) users had discontinued the method compared to only 16% of Norplant implants users (P < 0.03). Reasons for discontinuation centered on side effects for both groups but with some COC and condom users, discontinuing use due to "forgetfulness" or failure (pregnancy). Among the COC group (which was the most common choice after Norplant implant), 25% of the adolescents had experienced a subsequent unplanned pregnancy compared to 0% of the Norplant implant group (P < 0.01). Norplant implants were clearly an acceptable and effective contraceptive for these post-partum and post-abortal teens, who articulated a high motivation to avoid a subsequent unplanned pregnancy. However, it is clearly not the only method teens will choose to use, and more attention must be paid to adequate counseling of those choosing another method.

Expression of the green fluorescent protein carried by Autographa californica multiple nucleopolyhedrovirus in insect cell lines.

A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID50 titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to AcMNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401 + 10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401 + 10% FBS (37.8 x 10% TCID50/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4 x 10(6) TCID50/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9 x 10(6) TCID50/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.

A long-term follow-up study of children with supracondylar fractures of the humerus.

A retrospective study to assess the long-term results of the treatment of supracondylar fractures of the humerus was undertaken to provide guidance on the management of these troublesome injuries. In our experience as long as there was less than 25% displacement on an anteroposterior (A/P) or lateral x-ray, and less than 10 degrees angulation on an A/P or lateral x-ray, it is not essential to achieve an anatomical reduction, and good elbow function could be expected. Although children do not appear to correct for valgus or varus deformity there was no functional deficit from this deformity in this series. It was found that major displacements were more likely to have a worse result than undisplaced or minimally displaced fractures. These results would support a conservative approach to the management of these fractures--closed reduction followed by three weeks in a collar and cuff. If the position proved unstable, closed reduction was re-attempted or internal fixation performed. Conservative treatment was safe and effective, and the results comparable with other series that advocate internal fixation.

Investigation of a Q fever outbreak in a Scottish co-located slaughterhouse and cutting plant.

Outbreaks of Q fever are rare in the UK. In 2006, the largest outbreak of Q fever in Scotland occurred at a co-located slaughterhouse and cutting plant with 110 cases. Preliminary investigations pointed to the sheep lairage being the potential source of exposure to the infective agent. A retrospective cohort study was carried out among workers along with environmental sampling to guide public health interventions. A total of 179 individuals were interviewed of whom 66 (37%) were migrant workers. Seventy-five (41.9%) were serologically confirmed cases. Passing through a walkway situated next to the sheep lairage, a nearby stores area, and being male were independently associated with being serologically positive for Q fever. The large proportion of migrant workers infected presented a significant logistical problem during outbreak investigation and follow up. The topic of vaccination against Q fever for slaughterhouse workers is contentious out with Australasia, but this outbreak highlights important occupational health issues.

Topical ophthalmic exposure to rattlesnake venom.

An 18-year-old woman was struck in the eye from a distance of about 1 m by a stream of venom projected by a western diamond-back rattlesnake. She experienced an immediate burning sensation in the eye. The eye was irrigated first with tap water and later with a commercial eye wash solution. The patient's symptoms abated and no further eye injury was noted. Topical ophthalmic exposures to North American snake venom usually produce only transient symptoms, but the venom from tropical snakes has produced significant eye injury.

Lateralized recognition of incomplete figures in a prolonged display controlled by eye movements.

A left field superiority is typically reported for processing a variety of simple and complex non-verbal and patterned visual stimuli, including pictures, though stimulus clarity, degradation and exposure duration may be a significant determinant. Clinical studies indicate deficits in the ability to recognize incomplete figures with right hemisphere damage. However when normal subjects attempted to recognize 80 series of progressively-less-incomplete common objects (each series graded in 10 steps) presented for prolonged inspection to one or other visual field via a display driven by an infrared eyemovement transducer, either field (hemisphere) proved to be equally competent. We review evidence to suggest that such "ecologically-more-valid" techniques may not in fact be intrinsically able to tap minor hemisphere function.

Mechanisms of the relaxant action of ketamine on isolated porcine trachealis muscle.

Ketamine is a potent bronchodilator, but its mode of action is unclear. We have studied the effect of ketamine on the peripheral vagus nerve motor pathway of isolated porcine trachealis muscle. Postsynaptic nicotinic cholinergic receptors of the intramural ganglia were stimulated selectively with 1,1-dimethyl-4-phenyl-piperazinium iodide, post-ganglionic nerve fibres with electrical field stimulation (in the presence of hexamethonium) and muscarinic cholinergic receptors with acetylcholine (in the presence of tetrodotoxin). Ketamine 10(-4) mol litre-1 significantly shifted the concentration-response curves of acetylcholine (P < 0.02) and electrical field stimulation (P < 0.001) to the right and abolished the response to 1,1-dimethyl-4-phenyl-piperazinium iodide (P < 0.02). Ketamine also caused a concentration-dependent relaxation of muscle strips precontracted with acetylcholine. This was unaffected by propranolol. Ketamine relaxed muscle strips precontracted with potassium chloride, in the absence and presence of atropine. We conclude that ketamine interacts with the peripheral vagus nerve by decreasing the excitability of the postsynaptic nicotinic receptors of the intramural ganglia, and by affecting the muscarinic receptor, smooth muscle, or both. Beta-2 adrenoceptors are not involved in the mechanism of relaxation.

Postnatal adrenoreceptor maturation in porcine intrapulmonary arteries.

The effect of postnatal age on norepinephrine-induced alpha 2-adrenoreceptor-mediated release of endothelium-derived relaxing factor in porcine intramuscular pulmonary arteries was studied. Rings of pulmonary artery from fetal, newborn, 3-d-, 10-d-, 9-wk-, and 15-wk-old pigs with and without endothelium were suspended for isometric force measurement in Krebs-Ringer bicarbonate solution (37 degrees C, 95% O2-5% CO2). In 15- and 10-wk-old pigs, norepinephrine increased tone in arteries with and without endothelium but produced relaxations at high concentrations only in arteries with endothelium. These relaxations were not inhibited by the alpha 1-antagonist prazosin but were completely abolished by the alpha 2-antagonist yohimbine and the inhibitor of nitric oxide release N-omega-nitro-L-arginine methyl ester. This confirms that the norepinephrine-induced relaxations were due to alpha 2-mediated release of endothelium-derived relaxing factor. Arteries from only 50% of the 10-d-old animals showed endothelium-dependent relaxations, and at 3 d of age and younger no relaxations were seen. In animals less than 10 d old, only some of the vessels contracted to norepinephrine and the contractile response was diminished compared with 15-wk-old animals, whereas the response to prostaglandin F2 alpha and histamine was similar in the neonatal group (newborn, 3 d old, and 10 d old). This group showed a dose-dependent relaxation to nitric oxide, with 10-d-old animals more sensitive to nitric oxide than newborn animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Line bisection by left-handed preschoolers: a phenomenon of symmetrical neglect.

When a normal dextral attempts to bisect a horizontal line placed across his/her midline, there is a tendency for the transection to be placed slightly to the left of true center, a tendency which may be a little more pronounced when the left hand is used. However, left-handed children at or younger than 5 years of age err substantially to the left with the left hand and to the right with the right hand. This symmetrical neglect of extents contralateral to the hand currently in use, which may possibly reflect relatively greater commissural immaturity in left-handers, was not seen in dextral children of comparable ages, whose performance closely followed adult patterns.