Compulsive sexual behaviour (CSB) is relatively common and has been associated with significant distress and psychosocial impairments. CSB has been conceptualized as either an impulse control disorder or a non-substance ‘behavioural’ addiction. Substance use disorders are commonly associated with attentional biases to drug cues which are believed to reflect processes of incentive salience. Here we assess male CSB subjects compared to age-matched male healthy controls using a dot probe task to assess attentional bias to sexually explicit cues. We show that compared to healthy volunteers, CSB subjects have enhanced attentional bias to explicit cues but not neutral cues particularly for early stimuli latency. Our findings suggest enhanced attentional bias to explicit cues possibly related to an early orienting attentional response. This finding dovetails with our recent observation that sexually explicit videos were associated with greater activity in a neural network similar to that observed in drug-cue-reactivity studies. Greater desire or wanting rather than liking was further associated with activity in this neural network. These studies together provide support for an incentive motivation theory of addiction underlying the aberrant response towards sexual cues in CSB. Compulsive sexual behaviour (CSB), also termed hypersexual disorder or sexual addiction, is relatively common and associated with significant distress and psychosocial impairments Disorders of addiction are characterized by biases in selective attention towards drug cues We compared CSB subjects and matched healthy volunteers using a dot probe task to assess attentional biases to sexually explicit cues versus control stimuli and neutral cues versus control stimuli. As the latency of the stimulus has been shown to play a role in whether subjects engage in an early orienting facilitation response or a later inhibitory response CSB subjects were recruited via Internet-based advertisements and therapist referrals. Healthy volunteers were recruited from community-based advertisements in East Anglia. Screening of the CSB participants was conducted using the Internet Sex Screening Test (ISST) All CSB subjects and age-matched healthy volunteers were male and heterosexual given the nature of the cues. Healthy volunteers were matched in a 2∶1 ratio with CSB subjects. Exclusionary criteria included being under 18 years of age, history of substance use disorders, current regular user of illicit substances (including cannabis), and having a serious psychiatric disorder, including current moderate-severe major depression (Beck Depression Inventory >20) or obsessive-compulsive disorder, or history of bipolar disorder or schizophrenia  Subjects completed the UPPS-P Impulsive Behaviour Scale Subjects viewed a computer screen while placing their left and right index fingers of the letter ‘s’ and ‘l’ of the keyboard. Subjects were told that they would see two images (including explicit images) followed by a green dot . The goaThe primary outcomes were the difference in reaction time (RTdiff) between the cues  and paired neutral furniture cues /) for the three conditions. As the latency of the stimulus prior to the target  has been shown to play a role in whether subjects engage in an early orienting response or a later inhibitory response a priori hypothesis that attentional bias to early SOA would be higher to Explicit versus neutral cues but not to a Neutral person versus neutral Control cue in CSB subjects compared to healthy volunteers. P<0.05 was considered significant. Other analyses such as Erotic versus neutral Control cues for early SOA and analyses for late SOA were conducted on an exploratory basis. To assess the influence of SOA, we also compared early versus late SOA for Explicit person cues using related-samples Kruskal-Wallis tests for each group on an exploratory basis.Subject characteristics and questionnaire scores were compared using independent t-tests or Chi-square tests. The RTdiff data were inspected for outliers (scores>3 SD above group mean) and tests for normality were conducted using Shapiro-Wilkes . As the RTdiff scores for Explicit materials were not normally distributed , non-parametric analyses were conducted. We compared RTdiff between groups using Kruskal-Wallis test focusing on the early SOA. We focused on the a priori hypothesis, CSB subjects had greater attentional bias to Explicit stimuli (P = 0.022) but not to Neutral person cues (p = 0.495) for the early SOA  years) and 44 age-matched (mean age 24.16 (SD 5.14) years) heterosexual male healthy volunteers without CSB were assessed. Two of 22 CSB subjects were taking antidepressants or had comorbid generalized anxiety disorder and social phobia (N = 2) or social phobia (N = 1) or a childhood history of ADHD (N = 1). The characteristics of the CSB subjects are reported in arly SOA . In explarly SOA .In exploratory analyses, healthy volunteers had greater attentional bias to Explicit stimuli in the late compared to early SOA (p = 0.013) but there were no differences between latencies in CSB subjects (p = 0.601). Similarly there were no differences between SOAs for the Neutral cue comparing early versus late SOAs for either the healthy volunteers (p = 0.404) or CSB subjects (p = 0.550). There were also no significant differences between groups for all raw RTs to the cues or neutral Control stimuli for all conditions and stimuli SOAs  .CSB subjects  had similar ratings of attractiveness of the Neutral person cues relative to healthy volunteers . All subjects reported that they had not previously viewed the Explicit or Erotic stimuli.Using the dot probe task, one commonly used to assess attentional bias in disorders of addiction, we show that CSB subjects have enhanced attentional bias towards sexually explicit stimuli but not to neutral cues.in early SOAs. These findings suggest a role for an early attentional orienting response underlying the relationship between CSB and sexually explicit cues.The mechanisms underlying cue reactivity and attentional bias may reflect classical conditioning in which neutral stimuli (conditioned stimulus) are repeatedly paired with rewarding stimuli , such that the conditioned stimulus eventually elicits a conditioned response such as physiological arousal or craving. Following conditioning, these conditioned stimuli or drug cues acquire incentive-motivational properties thus acquiring salience, biasing attention and becoming ‘wanted’ This predictive conditioned stimulus is believed to elicit an early orienting attentional response. Our task makes some attempt to address this initial fast automatic shifting of attention. Visual cues presented for less than 200 msec are more likely to reflect an initial attentional bias. Subjects require at least 50 msec to shift attention to a cue Alternatively, the results may represent the effects of familiarity with the category of Explicit stimuli in CSB subjects. A possible role for use-independent exposure has been suggested based on the lack of difference between attentional bias using a Stroop task in patients and a control group of employees in a substance use facility That the early orienting response to erotic stimuli was similar between CSB subjects and healthy volunteers was not unexpected, highlighting the salience of sexually relevant stimuli. Healthy male volunteers have shown enhanced initial orientation and maintenance of attention as measured by the number of first fixations and relative fixation time during eye-tracking to sexually preferred stimuli compared to non-preferred stimuli Our current findings dovetail with our recent observation that CSB subjects have enhanced activity to sexually explicit cues in the ventral striatum, amygdala and dorsal anterior cingulate activity, the same network activated in drug cue reactivity in disorders of addiction The study has multiple limitations. Only heterosexual male subjects were studied, and future studies should examine individuals of various sexual orientations and females That attentional bias is a feature across drug and natural rewards suggests a potential role for attentional bias as an important construct in the dimensional approach towards disorders


Direct measurement of [Ca2+] distributions in dendritic spines is experimentally difficult but we can investigate this hypothesis using mathematical models of Ca2+ diffusion.Capocampus . However2+ diffusion in three dimensions. We then study our model using finite element methods. The model allows predictions of intracellular [Ca2+]-field responses to combinations of pre- and post-synaptic spikes with nanometre and millisecond spatio-temporal resolution. Our results so far indicate that Ca2+ signalling is highly spatially non-uniform and that Ca2+ signal differences between induction protocols is dependent on location within the spine. This has implications for the ultimate biological role of the Ca2+ signal given that the relevant receptors in the spine are organised inhomogeneously [We have developed a spatio-temporal model of Caeneously .


Sleep is known to be important for memory consolidation , and memDuring sleep, replay events are associated with specific patterns of neuronal activity. Replay is seen in cortex during sleep oscillation – a rhythmic (< 1Hz) state in which periods of activity (active or Up states) alternate with quiet periods (silent or Down states), while replay in the hippocampus is associated with sharp-wave ripple events – irregularly brief bouts of high frequency (>150 Hz) firing . It is bIn the present study, build on our previous research , we deveIn this study we designed a computational model of ripple generation that emphasizes the role of axo-axonic cells in selecting which pyramidal cells are participating in ripple activity, hence what sequence is replayed during a given ripple. In our model, high-frequency firing of the perisomatic interneurons mediate high-frequency LFP oscillations in pyramidal neurons while axo-axonic interneurons define specific sequence of pyramidal cell firing. Using such model, we investigate the timing relationship between cortical slow-wave oscillations and hippocampal ripples. Our study proposes a novel mechanism of hippocampal ripple generation and predicts how interactions between different electrographic events during sleep may contribute to coordinated replay in cortical and hippocampal networks.


Spontaneous mesenteric haematoma is a rare condition that occurs due to localized bleeding in the mesenteric vascular tree of a bowel segment in the absence of an identifiable cause. Here we report a case of spontaneous mesenteric haematoma during an inflammatory exacerbation of Crohn’s disease. The patient underwent surgical management for small bowel obstruction secondary to Crohn’s disease, however the concurrent presence of a spontaneous mesenteric haematoma in the mid-jejunal mesentery was successfully managed conservatively. This case identifies the first association of spontaneous mesenteric haematoma with an exacerbation of Crohn’s disease and highlights the need to consider rare differential diagnoses such as SMH when performing radiological assessment of unexplained symptoms in inflammatory bowel disease patients. Spontaneous mesenteric haematoma (SMH) is a rare condition of unknown aetiology. It can be managed conservatively in the event that there is no associated mesenteric haemorrhage. It must be clinically distinguished from spontaneous mesenteric intraperitoneal haemorrhage  where there is rupture or bleeding from a specific mesenteric vessel due to an unknown cause that typically requires urgent surgical management due to its high mortality. This report presents the first case in the literature associating SMH with an inflammatory exacerbation of Crohn’s disease.9/L and a C-reactive protein (CRP) of 7 mg/L. Her symptoms initially resolved with intravenous hydrocortisone but promptly recurred with worsening malnutrition despite intensifying steroid therapy. She had not received any formal anticoagulation other than prophylactic subcutaneous low-molecular weight heparin (20 mg once daily) during the period of her hospitalization. Repeat CT scan  and purine analogue (azathioprine) therapy. She was a non-smoker and aside from well-controlled essential hypertension had no other significant co-morbidities. Her inflammatory bowel disease had already required two previous hospitalizations, and she had suffered from symptoms of intermittent pain and bloating with constipation throughout the 12 months since her diagnosis. In the preceding month a computed tomography (CT) scan revealed 3 segments of Crohn’s disease strictures with prestenotic dilatation and an inflammatory appearance  is a rare condition that occurs due to localized bleeding in the mesenteric vascular tree of a bowel segment with unknown aetiology -8. It caPatients with isolated SMH typically present with non-specific symptoms of generalised abdominal pain and as a result the haematoma is usually identified on imaging modalities such as CT. Although the cause is unknown, the disease process may be associated with unidentified anticoagulation mismanagement or even undiagnosed connective tissue disease -8.Our case demonstrates the presence of localised mid-jejunal mesenteric fat haematoma. Localised visceral inflammation is a recognised contributor to confined microvascular bleeding  so that Mesenteric fat has been recently recognised an essential location for the generation of Crohn’s gastrointestinal inflammation ,12. HereOur intraoperative findings of mesojejunal haematoma reflect the current underestimation of Crohn’s ileitis, demonstrating explicit involvement of the mesenteric fat with an associated mesenteric haematoma. This case also highlights how mesenteric abnormalities such as SMH may explain refractoriness or recurrence of symptoms despite intensifying steroid therapy. Indeed, it is possible that ileal compression caused by a mesenteric mass such as this may explain many of the symptoms accredited to Crohn’s strictures. As a result, we believe that an increased awareness of the role of the bowel mesentery may give new insights into Crohn’s disease pathogenesis. Furthermore, assessment for mesenteric abnormalities may guide future clinical and radiological assessment of unexplained symptoms in patients suffering form this multi-systemic inflammatory process, and identify rare differential diagnoses such as SMH.We describe a case of spontaneous mesenteric haematoma in a female patient suffering from an exacerbation of Crohn’s disease and intestinal stenosis. The mesenteric haematoma was identifiable both on pre-operative computerised tomography and intra-operatively. This case identifies the first association of spontaneous mesenteric haematoma with an exacerbation of Crohn’s disease and highlights the need to consider rare differential diagnoses such as SMH when performing radiological assessment of unexplained symptoms in inflammatory bowel disease patients.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.CRP: C-reactive protein; TLR: Toll-like receptor; NOD: Nucleotide-binding oligomerization domain; SMH: Spontaneous mesenteric haematoma; CT: Computerized tomography.The author(s) declare that they have no competing interests.PM carried out the procedure, assisted by HA and DB. HA, JM and AM drafted the manuscript. HA finalized the manuscript in agreement with the senior author PM. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2482/14/35/prepub


Dendritic cells (DCs) are central players of the immune response because they regulate both innate and adaptive immune cells. As part of the leukocyte infiltrate of various tumours they can initiate antitumour immune responses through the activation of tumour-specific T cells.However, tumours employ strategies to evade the immune response by manipulating the differentiation and activation of DCs.+CD209+ expressing myeloid cell type that displays characteristics of both DCs and macrophages. As one example, ercDCs showed good antigen cross presentation which is a prototypic DC function and a prerequisite for the activation of T effector cells in local tumour tissue. To better understand the relationship of ercDCs to other myeloid cell types and to get insight in functional pathways operative in ercDCs we performed transcriptome analysis using Affymetrix® gene arrays comparing ercDCs, cDCs, proinflammatory M1 and GM-CSF macrophages and alternatively activated M2 macrophages. Cell types were generated in parallel from monocytes of 15 healthy donors. RNA was extracted using the Qiagen® RNeasy Micro kit. To reduce population variability, RNA from each cell type- from 5 donors- was pooled and 3 biological replicates generated. Clustering of normalised values showed that ercDCs differ from the other myeloid cell types. Further analysis included heat mapping and creation of signaling pathways using pathvisio software. Focusing on the cross presentation pathway, a gene list was assembled through literature mining and each gene was assigned an expression value using microarray data. A comparison using the heat map representation revealed that the expression pattern of ercDCs was most similar to GM-CSF macrophages and least to M1 macrophages. Comparison based on fold changes generated by normalisation to cDCs revealed highest similarity between ercDCs and M2 macrophages. Particularly, transcription of genes involved in receptor-mediated endocytosis and the vacuolar pathway were upregulated in ercDCs indicative that cross presentation could be active in ercDCs.Previously we described 'enriched-in-renal-carcinoma DCs' (ercDCs) as an unusual CD14


The concealed phase of arrhythmogenic right ventricular cardiomyopathy (ARVC) may initially manifest electrophysiologically. No studies have examined dynamic conduction/repolarization kinetics to distinguish benign right ventricular outflow tract ectopy (RVOT ectopy) from ARVC's early phase. We investigated dynamic endocardial electrophysiological changes that differentiate early ARVC disease expression from RVOT ectopy.1-S2 restitution protocol.22 ARVC  patients without right ventricular structural anomalies underwent high-density non-contact mapping of the right ventricle. These were compared to data from 14 RVOT ectopy and 12 patients with supraventricular tachycardias and normal hearts. Endocardial & surface ECG conduction and repolarization parameters were assessed during a standard SDefinite ARVC without RV structural disease could not be clearly distinguished from RVOT ectopy during sinus rhythm or during steady state pacing. Delay in Activation Times at coupling intervals just above the ventricular effective refractory period (VERP) increased in definite ARVC (43±20 ms) more than RVOT ectopy patients  or Normals  and a progressive separation of the repolarisation time curves between groups existed. Repolarization time increases in the RVOT were also greatest in ARVC . Surface ECG correlates of these intracardiac measurements demonstrated an increase of greater than 48 ms in stimulus to surface ECG J-point pre-ERP versus steady state, with an 88% specificity and 68% sensitivity in distinguishing definite ARVC from the other groups. This technique could not distinguish patients with genetic predisposition to ARVC only (probable ARVC) from controls.Significant changes in dynamic conduction and repolarization are apparent in early ARVC before detectable RV structural abnormalities, and were present to a lesser degree in probable ARVC patients. Investigation of dynamic electrophysiological parameters may be useful to identify concealed ARVC in patients without disease pedigrees by using endocardial electrogram or paced ECG parameters. Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a disease of cardiomyocyte adhesion; initial myocyte slippage and loss of gap junction integrity is followed by more overt structural changes, with characteristic fibrofatty replacement of cardiomyocytesPre-clinical work has highlighted putative mechanisms by which arrhythmias in ARVC may occur. Genetically modified mice deficient in plakophilin-2 exhibit slow conduction in the absence of significant histological anomalies and heterozygous desmoplakin knock out mice have impaired conduction reserve To date, clinical studies on the electrophysiology of patients with ARVC have overwhelmingly concentrated on patients with established ventricular macroscopic structural markers as a principle feature of their disease. These studies have demonstrated that patients with ARVC exhibit slow conduction within the right ventricle and low endocardial bipolar voltages, indicative of endocardial fibrosisonly because of a definite family history/mutation carrier status without evidence of significant structural disease such that they have earlier/milder disease than customarily reported in ARVC. Patients with benign RV outflow tract ectopy (RVOT ectopy) can be viewed as an important control population as they may be expected to exhibit secondary features & adaptations associated with frequent ectopy. Differentiation between ARVC and RVOT ectopy groups will highlight salient disease features rather than merely adaptations to ectopy.We have demonstrated significant dynamic conduction and repolarization differences between desmoplakin mutation carriers and controls, but there has been no direct comparison of the dynamic electrophysiological changes in early ARVC versus patients with outflow tract ectopy (RVOT ectopy)We hypothesized that patients with early ARVC would exhibit slowing of conduction & repolarization changes compared to RVOT ectopy patients under conditions of electrical stress, even prior to detectable structural disease. Furthermore, we hypothesized these myocardial electrophysiological changes would manifest on the surface ECG which ultimately may be of value in the development of a diagnostic test.The research was approved by University College London Hospitals Ethics Committee A (08/H0714/97), prior written informed consent to participate in this study was obtained from all participants.did not have major imaging criteria for ARVC Patients aged 18–65 years with definite or probable ARVC by modified task force criteria  were prospectively recruited to participate in the study and informed consent was obtained. All patients underwent detailed imaging assessment including echocardiography/MRI and were only included in this study if they 10 ml whole blood samples were obtained from ARVC patients and family members. Genomic DNA was extracted using a commercially available DNA extraction kit . Index cases were part of a larger patient cohort comprehensively screened for mutations in desmoplakin, plakoglobin, plakophilin-2, desmoglein-2 and desmocollin-2. Primer pairs for DSP exons were designed in flanking intronic sequences and are available on request. Polymerase chain reaction (PCR) amplification and direct sequencing on an ABI 3130 Genetic Analyzer were performed using standard protocols as previously described 1S2 restitution protocol. This protocol consisted of performing 8 beat trains of pulses (S1) at 400 ms coupling intervals followed by a single, premature stimulus (S2). The S1S2 coupling interval was reduced sequentially from 400 ms by 20 ms until 300 ms, then by 5 ms intervals until failure to elicit a ventricular response (refractoriness) was reached. The S1S2 interval was increased by 8 ms and then further reduced by 2 ms steps to establish the Ventricular Effective Refractory Period (VERP) and proximal end of the restitution curve. Two-second intervals were left between trains. 12 lead electrocardiograms were recorded throughout the procedure.The procedure for non-contact mapping has been previously described in detail elsewhere 24 “virtual” unipolar electrograms were placed in 4 columns of 6 across the RV chamber on the Ensite console. Global data was thus acquired from the entire geometry  and goodmin) was used as the local activation time  to the steepest negative deflection  method and the alternative method. Both have come under intense theoretical and experimental scrutiny, and here we present results using the classical method Fractionation of activation was determined by counting the number of deflections in the differential of the unipolar electrogram 2 .Mean Increase of Delay is a convenient surrogate measure of conduction velocity restitution. In brief, the activation time is plotted against coupling interval during a restitution curve pacing sequence. Mean Increase in Delay refers to the mean increase in activation time as the coupling interval is reduced to ERP, and is expressed as millisecond increase in delay per millisecond reduction in coupling interval (ms/ms). This was calculated as previously described 1-S2 protocol. Signals were filtered with bandpass settings of 0.1 – 50Hz. Measurements were performed manually using on-screen calipers at 200 mm/sec (MF & AS) blinded to the patient diagnosis. Timings were measured from the pacing stimulus to a) the earliest sharp component of the QRS complex, b) the peak of the QRS complex c) the end of the QRS complex (i.e. the J-point), d) the peak of the T-wave and e) the end of the T-wave (12-lead ECGs were recorded simultaneously during the Se T-wave . These m2) and mean increase in delay) were made using student's t-test with correction for multiple comparisons. Continuous parametric data derived from electrogram data were modeled using mixed-effects linear regression and statistical significance was inferred from the modelAll statistical computing was performed in R software  was applied to surface ECG data to determine optimum cut-offs attempting to distinguish ARVC from a mixed group of patients. This is a decision-learning technique that enables optimal splits in a group formed on different observations to occurRecursive partitioning (a.k.a. 22 patients with definite (12 pts) or probable (10 pts) ARVC were studied (mean age 46±11 years)25 consecutive patients with monomophic ventricular ectopy (9652±8444 beats/24 h), structural normal hearts and normal resting ECGs underwent non-contact mapping and EP studies. 14 of these patients had >18 m follow-up with no recurrence of ventricular ectopy off antiarrhythmic medication, and no features suggestive of ARVC. These patients were analyzed as the RVOT ectopy group (age 45±14 years). Twelve further patients undergoing electrophysiological studies ± ablation for supraventricular tachycardia with structurally normal hearts acted as Normal controls (age 43±18 years).No significant differences in RV activation times, ARI or repolarization time were observed between groups .2, a measure of uniformity of conduction, was higher in both ARVC groups and in normal controls than in the RVOT ectopy patients . There were no differences between either of the ARVC or normal control groups.Differences in slowest measured activation gradient  between groups did not reach statistical significance, although activation gradients in ARVC and RVOT ectopy groups were 20% lower than controls . rIn steady state pacing, RV activation took significantly longer in probable ARVC patients than in RVOT ectopy subjects , but this did not reach statistical significance in the definite ARVC group (91±21 ms) or normal controls (94±28 ms). There was a trend to longer repolarization times in the ARVC groups when compared to the RVOT ectopy group , but no group was significantly different from normal (303±42 ms).1S2 coupling intervals just longer than VERP (pre-VERP) than in other groups  than in RVOT ectopy patients  or Normal controls .The activation delay curves were plotted for each electrode in every patient. Mean increase in delay, a measure of cumulative delay in activation times over all coupling intervals, were greater in the ARVC patients .The maximum slopes of ARI restitution curves were marginally steeper in the probable ARVC and RVOT groups compared to normal, but there was no statistically significant difference between the definite ARVC group and any other group . There were no significant differences in the odds ratio of having a maximum ARI restitution slope of >1 in any segment between patient groups.In sinus rhythm, more fractionation was observed in RVOT ectopy patients than in definite or probable ARVC or normal controls. However, during pacing at steady state, RVOT ectopy and both ARVC groups had more fractionation than normal controls  & 7. At Single intracardiac electrophysiological predictors were generally poor at differentiating definite ARVC from RVOT ectopy, but an increase in the local activation time of >47 ms at ERP compared to in steady state pacing gave a sensitivity of 70% and a specificity of 74% in predicting definite ARVC over RVOT ectopy. A mixed-effects logistic regression model was created to explore the predictive potential of combining intracardiac variables. The resultant model has an area under the curve of 0.85 , Table 3increase in activation time at ERP by 36±13%compared to steady-state was observed in the definite ARVC group than in the other groups  gave a sensitivity of 67% and specificity of 88% in the diagnosis of definite ARVC . A reducThis is the first study to compare differences in conduction and repolarization kinetics in an ARVC population with earlier disease than customarily reported with benign RVOT ectopy cases. The key findings include: 1) Marked conduction delay in patients with early ARVC when pacing at short coupling intervals, which was not evident in sinus rhythm. This exists on a spectrum, with definite ARVC exhibiting more conduction delay than probable ARVC patients compared to normals. 2), Progressively longer local repolarization times as coupling intervals decrease towards VERP in definite ARVC patients versus normal controls & RVOT ectopy subjects. 3) Marked differences in fractionation at short coupling intervals pre-VERP throughout the right ventricle between patient groups, with more fractionation in both ARVC and RVOT ectopy patients. 4) Surface ECG biomarkers of conduction/repolarization dynamics provide diagnostic information - an increase in the time from pacing stimulus to full ventricular activation (defined as the end of the QRS complex i.e. the “J-point”) of greater than 48 ms gave an 88% specificity and 68% sensitivity of distinguishing definite ARVC from other groups.Previous clinical studies have demonstrated electrophysiological abnormalities in established ARVC with significant structural disease, including prolonged RV activation times in sinus rhythm and low endocardial bipolar electrogram voltages 1-S2 restitution protocol, particularly in the outflow tract compared to SVT controlsAn earlier study of desmoplakin mutation carriers demonstrated that these patients have significantly greater mean increases in delay during an SOur observation of an increase in induced conduction slowing with premature extrastimuli can be regarded as evidence of a lack of conduction reserve. This is consistent with data implying a sodium current deficit in murine models of ARVC Importantly, it was primarily the family history of ARVC or mutation carrier status that allocated these patients to the “Definite ARVC” group in our study. In the absence of gene testing, 4/12 of the definite ARVC cases would be regarded as borderline. This implies that in a population of combined probable and definite cases the predictive accuracy of this partitioning method may be increased. Indeed, when both groups are combined, the classification tree was able to distinguish any ARVC patient from a combined group of RVOT/Normals with a sensitivity of 67% and specificity of 84%.We can speculate why a disparity may exist in the degree of induced activation delays between these two groups. Six patients in the definite ARVC group exhibited a surface 12-lead ECG feature of disease at rest, versus none in the probable ARVC group . Lack ofEven the patients with more advanced disease did not demonstrate global RV activation delays in sinus rhythm, but it was more marked at short coupling intervals during RV pacing. The dynamics of repolarization were also significantly affected, amplifying effects of conduction delay in ARVC and creating significantly more prolonged repolarization times pre-VERP than steady state versus controls and RVOT ectopy patients. Digital examination of the signal averaged ECG may yet reveal previously hidden features of conduction delay in these patients, but this fell outside of the scope of this study.The partitioning tests identified stimulus to T-wave peak interval as a potential distinguishing feature between normal and probable ARVC patients. This measure reflects a composite of activation and repolarisation phenomena, and could be seen as a surface ECG marker of the intracardiac repolarisation time changes observed .2 values indicating uniformity of conduction were observed compared to RVOT ectopy cases. Similarly, we have reported more uniform conduction in Brugada Syndrome versus controls Two phenomena were observed in the RVOT ectopy patients: consistently shorter repolarisation times versus supraventricular tachycardia patients and ARVC, cases and increased fractionation compared to control cases. The shorter repolarisation times may reflect a memory phenomenon induced by the high ventricular ectopic burden. A shortening in ARI occurs and with it a reduction in refractory period, thus allowing local myocytes to be more susceptible to activation. The high degree of fractionation in benign RVOT ectopy patients similar to that seen in ARVC also deserves comment. Conduction delay and fractionation occurs in ARVC due to fibrofatty replacement and/or dissociation of preferentially conducting myocardial pathways through reduced gap junction coupling and Na channel downregulation Taken together, features of conduction delay, increased repolarization times and mild fractionation at short coupling intervals pre-VERP would support a diagnosis of early ARVC over that of a benign RVOT ectopy, with a logistic model demonstrating a positive predictive value of 80% from the presented data. These differences can also be identified simply using paced-QRS-T-wave parameters.dynamic surface ECG conduction-repolarization parameters may be of diagnostic value in early disease.This study confirms that reduced conduction reserve develops early in ARVC before detectable structural RV changes on conventional imaging ensue. This has important implications since sudden cardiac death can occur in patients with minimal histological changes in the sub-clinical phase of ARVC and thus a more sophisticated clinical evaluation using dynamic conduction-repolarization changes is required de novo population will be required for an impartial assessment of the diagnostic utility of paced J-point hysteresis. Logistical regression models can highlight the differences between groups in this study, but cannot be taken as a diagnostic test based on the data presented.The human mapping technique evaluates endocardial electrophysiology and thus mid-myocardial and epicardial effects of ARVC could not be assessed. This is important since structural changes manifest epicardially prior to progressing endocardially The probable ARVC group still represents a diagnostic dilemma as a gold standard test is lacking, yet our data has important pathophysiological implications for the understanding of the development of arrhythmias in this population. Disease progression in increasingly understood as initially an epicardial phenomenon, we were not able to examine epicardial effects with our mapping or pacing strategy. Non-contact mapping may be insensitive at identifying low-voltage fractionation in small areas.Early ARVC exhibits greater conduction delay and dispersion of repolarization, particularly at short coupling intervals, than either normal hearts or benign RVOT ectopy. Fractionation is mildly increased in early ARVC, but also significantly in benign RVOT ectopy. The dynamic differences in conduction, repolarization and fractionation are manifest on the surface paced ECG & could help refine the early identification of early concealed ARVC patients from those with benign outflow tract ectopy.Figure S1Examples of restitution curves from a patient with ARVC and from a normal control. Repolarisation time, ARI and activation time are plotted against coupling interval. Points represent means of four repeated measurements. Early activated sites are shown in red, late in blue.(TIFF)Click here for additional data file.


Unilateral renal agenesis with vesicoureteral reflux in the ipsilateral full length ureter is a rare phenomenon. Herein we report a case of 10-year old boy who presented with recurrent urinary tract infections. No renal tissue was identified on left side in various imaging studies. Micturating cystourethrogram (MCUG) showed left sided refluxing and blind ending ureter. Left ureterectomy was done because of recurrent UTI in the refluxing system. A 10-year-old male child presented with several episodes of hematuria associated with mild dysuria for the last two years. He was a proven case of urinary tract infections (UTI) and was on chemoprophylaxis with nitrofurantoin (50 mg/day) since last 6 months. General physical and systemic examinations were normal. Urine examination revealed pus cells but culture grew no organisms. Hemogram, blood urea and serum creatinine levels were in normal range. Ultrasonography (USG) did not show left kidney in lumbar and other ectopic locations. Right kidney was normal in shape, size and location with normal echotexture. No evidence of dilated ureter on either side was reported. CECT with CT urogram confirmed the USG findings . MCUG showed refluxing full length blind ending ureter on left side. Left ureter was minimally dilated without any tortuosity. Urinary bladder was normal . DMSA scan showed no functional left renal tissue. Right kidney was normal on radioisotope scan. The diagnosis of left renal agenesis with blind ending left ureter and ipsilateral vesicoureteric reflux (VUR) was made. Antibiotics were given empirically for two weeks. MCUG, repeated after resolution of clinical symptoms and normalization of urine report, showed left sided VUR. Cystoscopy showed normal lower tract including a normal trigone. Retrograde pyelography (RGP) showed full length left ureter with proximal blind end. Due to recurrent UTI with VUR in spite of chemoprophylaxis, exploration with excision of the whole ureter was undertaken. On exploration no evidence of left renal tissue was found. Postoperative period was smooth and the child is symptom free at follow-up.Unilateral renal agenesis (URA) is an uncommon condition. Most cases of URA are associated with absent ipsilateral ureter or if ureter is present it is small near bladder end. Normally ureter develops from a bud which arises from mesonephric duct. Kidney develops when there is interaction between ureteric bud and metanephric blastema. In renal agenesis either ureteric bud is absent or interaction of ureteric bud with metanephric blastema does not occur.[1] When ureteric bud fails to fuse with renal plate, it results in blind ending ureter.[2] URA may be due to in-utero regression of multidysplastic kidney.[3]Isolated URA is usually asymptomatic. A blind-ending ureteric bud has a higher than normal incidence of vesicoureteric reflux and therefore may get acutely inflamed. It may result in hematuria, calculus formation or recurrent UTI.[1] Systemic examination and routine urine examination may be normal as found in the index case.MCUG is the gold standard to demonstrate the presence of VUR. Length of blind ending ureter and grade of reflux can also be determined. In our case, MCUG showed full length refluxing left sided blind ending ureter. Management of these cases depends upon symptoms. Surgical excision of blind ending ureter is advised in case of non-resolving VUR or breakthrough UTI. Both open and laparoscopic retroperitoneal approach may be used.. [6] Unilateral renal agenesis may be associated with ipsilateral refluxing blind ending full length ureter which can lead to recurrent urinary tract infection and its removal is thus indicated.Source of Support: NilConflict of Interest: None declared


Pulmonary nodules are commonly observed in patients with cancer as well as in patients with no known malignancy particularly in heavy smokers. Most of these nodules are small (less than 8 mm). Even in cancer patients a large proportion of these small nodules are benign . The likNon-solid nodules (ground glass opacities) are more likely to represent lung cancer (adenocarcinoma) than solid nodules ,5. HowevConsolidation or diffuse ground glass usually represents benign disease such as pneumonia. However, in adenocarcinoma with predominantly lepidic growth consolidation or diffuse ground glass may be due to cancer spread in the lung.Pleural effusion may be due to malignant spread  or to several benign conditions . Malignancy is usually confirmed by cytologic analysis of pleura fluid. However, imaging may suggest malignancy if solid pleural lesions are demonstrated within the effusion particularly at the lung base. Unilateral effusion, particularly in the left hemithorax or on the side of the underlying malignancy  also suggests malignant effusion.Solid pleural lesions may be clearly benign such as pleural lipoma or calcified pleural plaques in patients with asbestos exposure. Non-calcified focal solid lesions may be benign  or malignant. Although pleural carcinomatosis is usually associated with effusion solid metastases without effusion can occur. Diagnosis usually requires histology .


We report the good outcome of combined intravitreal aflibercept injection and photodynamic treatment in a case with fundus flavimaculatus (FFM) and unilateral subretinal neovascular membrane (SRNM). A 57-year-old man with FFM and unilateral SRNM who was treated with two consecutive ranibizumab injections with no improvement at another institution was referred to us. He was treated successfully with three aflibercept injections three months apart and a single photodynamic treatment performed a week after the initial aflibercept injection. Six weeks after the last aflibercept injection visual acuity was improved and submacular exudation exhibited dramatic resolution with a moderate degree of residual scarring. SRNM formation is rarely observed in eyes with FFM and a satisfactory outcome can be achieved with a proper treatment. Fundus flavimaculatus (FFM) is characterised by the fishtail-like or round, white-yellowish flecks confined to the retinal pigment epithelium that are scattered diffusely throughout the posterior pole and extend out to the midperiphery and the related visual symptoms commence between the third and fourth decade of life and progress slowly –3.During the course of the disease progressive macular atrophy is a much more common finding than the rarely diagnosed subretinal neovascular membrane –14. HereA 57-year-old man had a nine-month history of visual decline in the right eye and had received two intravitreal ranibizumab injections for presumed exudative macular degeneration elsewhere. He was referred to us for further evaluation. His family and medical history were unremarkable. On our examination, his best-corrected visual acuity was counting fingers at 2 meters in OD and 20/25 in OS. Slit-lamp examination was normal OU. Fundus examination showed multiple widespread pisciform flecks throughout the posterior pole and midperipheral retina in OU with severe subfoveal exudation and intraretinal fluid in OD Figures . AutofluERG demonstrated subnormal dark adapted responses of rods while photopic cone responses were mildly reduced with prolonged implicit times. Both scotopic 30 Hz flicker and maximum responses were also decreased in amplitude . Our dia2 verteporfin injected intravenously over a period of 10 minutes followed by the occlusion of lesion area five minutes later with a 689 nm diode laser for 83 seconds with 50 J/cm2 at an intensity of 600 mw/cm2. A spot size of 3500 μm was used. Two additional 2 mg intravitreal aflibercept injections were given three months apart. Six weeks after the third injection, his best-corrected visual acuity was 20/200 and subretinal exudation showed dramatic resolution with a residual scar in the OD (Figures Due to unsatisfactory anatomic and visual outcome with two previous ranibizumab injections and the presence of large feeder vessel-like changes noted in the fluorescein and indocyanine green angiographies, we elected proceeding with a combination of photodynamic therapy and aflibercept injection. We preferred aflibercept over ranibizumab as the patient might not be followed up monthly due to his profession. Seven days after the administration of initial 2 mg aflibercept injection standard photodynamic treatment was performed in OD, namely, 6 mg/m Figures .There were anecdotal case reports discussing the place of several treatment modalities and describing the outcome of patients with SRNM in association with FFM. In earlier reports, either observation –6, 9 or Tejerina and friends  injectedIntravitreal aflibercept treatment has been proven to be efficacious in exudative age-related macular degeneration . Moreove


Cardiac magnetic resonance imaging (CMR) is routinely performed to monitor progression and response to interventions in congenital and acquired heart diseases. Cross-sectional CMR studies report normalized reference ranges for cardiac parameters according to age decile together with assessments of short-term inter-study variability; however, most clinical imaging is repeated at much greater intervals of time. There are no prospective CMR data defining long-term changes in cardiac structure and function among healthy subjects. Without such data, it is difficult to determine whether serial changes in cardiac parameters observed in patients reflect disease progression or are merely the consequence of normal ageing. We examined the "within patient" changes in left ventricular (LV) volumes, mass and function over 12 months in a well-characterized cohort of healthy adults.We identified 21 healthy controls  who did not proceed to uni-nephrectomy after recruitment into an observational study assessing the cardiovascular effects of live kidney donation (NCT01028703). All subjects were asymptomatic and had a 10-year risk of a cardiovascular event of <20% (QRISK-2), a normal stress echocardiogram and normal haematology and biochemistry profiles. Exclusion criteria included: any history of cardiovascular disease, including hypertension; diabetes; glucose intolerance; chronic kidney disease; first degree relative with a proven or potentially inheritable cardiac condition. Subjects underwent CMR imaging  at baseline and 12 months. Analysis of SSFP images was performed offline  by a single blinded observer (W.E.M.) for assessment of LV volumes, systolic function and mass. Baseline studies were repeat analyzed by the same trained observer to assess intra-observer variability. A subset of participants underwent repeat imaging within 1 hour to determine inter-study variability.There were no significant changes in any LV parameter indexed to body surface area at 12 months (Table These data have particular importance for both clinical practice and observational research: for patients undergoing annual CMR, changes in LV structure and function can be largely attributed to disease progression rather than ageing.WEM is supported by a British Heart Foundation Clinical Research Training Fellowship.


Phytophthora ramorum genetic lineage EU2. We sequenced genomes of seven isolates collected in Northern Ireland between 2010 and 2012. Multiple genome sequences from P. ramorum EU2 will be valuable for identifying genetic variation within the clonal lineage that can be useful for tracking its spread.Here we present draft-quality genome sequence assemblies for the oomycete  Finally, we generated scaffolds and performed gap-filling with SSPACE 3.0 We used the Illumina HiSeq to generate 100-bp paired sequence reads from genomic DNA prepared from seven isolates of EU2 collected from multiple sites in Northern Ireland between 2010 and 2012 . We filtP. ramorum Pr102 genome sequence We estimated the completeness of our assemblies with the CEGMA pipeline Phytophthora plant pathogens P. ramorumP. ramorum EU2 will be valuable for identifying genetic variation within the clonal lineage that can be useful for tracking its spread Genome sequences have been invaluable in the study of


Purpose. Pathophysiology of macular hole (MH) is not yet well defined but the advances of spectral domain optical coherence tomography (SD-OCT) give us access to further detailed imaging. We report a case with macular inner segment ellipsoid (ISe) band loss and cone outer segment tips (COST) line changes seen in SD-OCT preceding MH appearance in a young patient. Methods. 21-year-old woman presented with a partial central scotoma, metamorphopsia, and a 20/25 vision in her right eye. Past medical history was positive for laser assisted in situ keratomileusis (LASIK) surgery 7 months ago with no complications. Macular SD-OCT showed ISe band loss and COST line elevation. She was followed a month later with visual acuity deteriorating to 20/200 and a full thickness MH. Results. The patient underwent a pars plana vitrectomy with internal limiting membrane peeling. Her visual acuity 2 months later was 20/20. Conclusion. SD-OCT can identify preliminary changes, yet to be described, preceding MH formation. Our patient demonstrated ISe band loss and COST abnormalities on SD-OCT a month prior to MH development. SD-OCT should be considered in young patients with subtle visual symptoms and mild changes in visual acuity that are not readily explained by ophthalmological exam. Macular hole (MH) is an idiopathic condition that in most cases is related to posterior vitreous detachment (PVD) and is mainly seen in the elderly. The minority of cases are observed in younger patients with a history of blunt ocular trauma as well as in myopic patients after laser assisted in situ keratomileusis (LASIK) surgery , 2.Spectral domain optical coherence tomography (SD-OCT) provides further detailed imaging that allows the detection of early anatomical changes, enabling us to alter and/or refine current theories.Different pathophysiological mechanisms might be involved in MH formation depending on vitreous syneresis and posterior hyaloid status. Several theories have emerged involving tangential traction  and anteWe report a case of a young patient who at first presented with subtle visual symptoms on the right eye. Her SD-OCT showed changes in the inner segment (ISe) band and cone outer segment tips (COST) line preceding MH formation in the absence of any vitreomacular interface abnormality in the ophthalmological examination.A 21-year-old female patient presented to the retina clinic with a new onset of partial central scotoma and metamorphopsia in her right eye (RE). Best corrected visual acuity (BCVA) of the RE was 20/25. Left eye (LE) was asymptomatic with BCVA of 20/20. She had no history of trauma and reported having a successful LASIK surgery in both eyes seven months earlier with BCVA of 20/20 in both eyes after surgery. She had a positive ocular family history, with her father being operated for a unilateral idiopathic MH. Dilated fundus examination did not reveal any retinal findings in both eyes. Therefore, a SD-OCT  was performed and showed an ISe band loss and COST line changes at the subfoveal area with no other findings in the RE . She wasA 23-gauge pars plana vitrectomy was performed in the RE. The posterior hyaloid was found to be very adherent and the surgeon faced difficulties trying to induce a PVD using high aspiration with the vitrector probe. A brilliant blue stain was injected to facilitate the visualization of the internal limiting membrane (ILM) on the margins of the macula . The ILMGass studied the pathophysiology of MH and suggested that the majority of macular holes begin as a central dehiscence with little loss of retinal tissue following tangential traction . BiomicrStudies by SD-OCT showed minor changes in the outer foveolar structure, especially in the COST line, in either eyes with a foveolar yellow spot or eyes with vitreomacular traction , 10. We We speculate that the strong vitreomacular adherence observed during vitrectomy might have induced vitreoretinal interface changes that caused changes in the outer retina with no apparent signs of traction on ophthalmoscopy. In patients with a strong vitreomacular adhesion, ISe band loss and COST line changes on SD-OCT may be a harbinger for MH formation; these patients should be followed closely. We are unaware of any publications on OCT changes preceding MH formation in young patient as we described with normal fundus examination.There is a higher incidence of MH in patients with a positive familial history of MH, suggesting the involvement of genetic factors . Our patAlthough there are reports of MH cases after LASIK surgery , we beliIn conclusion, SD-OCT can identify preliminary changes at the level of the foveal ISe band and COST line preceding MH formation. Therefore, it should be considered in young patients with subtle visual symptoms and mild changes in visual acuity that are not readily explained by ophthalmological examination.


Acute respiratory distress syndrome (ARDS) is characterized by damage to the alveolar epithelial-endothelial barrier resulting in neutrophil influx and pulmonary oedema. The activation of platelet and secondary capture of neutrophils may play an important role in propagation of inflammation in ARDS . Variousin vivo human model of acute respiratory distress syndrome, induced by inhaled lipopolysaccharide (LPS).To test the hypothesis that aspirin reduces pulmonary inflammation in an Healthy subjects were enrolled in a double-blind, placebo-controlled study and were randomised to receive aspirin 75mg or aspirin 1200mg or placebo (1:1:1) for seven days prior to LPS inhalation. Measurements were performed in bronchoalveolar lavage (BAL) fluid obtained at 6 hours after inhaling 50 micrograms of LPS.33healthy subjects were enrolled. There was no significant difference between aspirin 75mg and aspirin 1200mg. Data for both aspirin groups were combined. Aspirin pre-treatment reduced LPS induced BAL neutrophilia (figure This study shows for the first time that aspirin can reduce neutrophilic inflammation in the lung in humans. Further clinical studies are warranted to assess its ability to reduce neutrophil mediated inflammation in ARDS.


Activity of GFR/PI3K/AKT pathway inhibitors in glioblastoma clinical trials has not been robust. We hypothesized variations in the pathway between tumors contribute to poor response. We clustered GBM based on AKT pathway genes and discovered new subtypes then characterized their clinical and molecular features. There are at least 5 GBM AKT subtypes having distinct DNA copy number alterations, enrichment in oncogenes and tumor suppressor genes and patterns of expression for PI3K/AKT/mTOR signaling components. Gene Ontology terms indicate a different cell of origin or dominant phenotype for each subgroup. Evidence suggests one subtype is very sensitive to BCNU or CCNU . AKT subtyping advances previous approaches by revealing additional subgroups with unique clinical and molecular features. Evidence indicates it is a predictive marker for response to BCNU or CCNU and PI3K/AKT/mTOR pathway inhibitors. We anticipate Akt subtyping may help stratify patients for clinical trials and augment discovery of class-specific therapeutic targets. WHO grade IV astrocytoma or glioblastoma (GBM) are the most common primary brain tumors and, unfortunately, the most aggressive. Median survival of patients harboring these tumors is approximately 14 months. Despite a committed effort to investigate new chemotherapies, molecularly targeted therapies, immunotherapies, surgical and radiological approaches, there has been little improvement over the last 30 years. Inadequate classification of GBM may have contributed to the difficulty of developing new therapies by decreasing power of clinical trials and underestimating benefit of class-specific drugs. It may also have confounded discovery of class-specific pathways and drug targets.We know GBM diagnosed by histopathology is a collection of molecular and clinical subtypes. For example, there are two classes of GBM based on clinical presentation However, molecular classification of GBM is still in its infancy. There is no consensus on the number of subtypes and which classifiers best identify them. In addition, there is considerable reassignment of tumors to different classes depending on classifier used. We also have little information on which oncogenic pathways are active in subtypes and how subtypes respond to standard and experimental therapeutics. These questions need to be addressed before molecular classification can be reliably incorporated into clinical trials and patient treatment.Alterations in the growth factor receptor/phosphatidylinositol 3-kinase/AKT (GFR/PI3K/AKT) pathway occur in most human cancers including at least 85% of GBM The GFR/PI3K/AKT pathway is complex and nonlinear having many inputs from other pathways The discovery dataset (GBM195) consisted of 181 GBM  from 3 datasets http://kinasource.co.uk/Database/substrateList.php) (3) evidence from Pubmed of phosphorylation by AKT , (4) evidence from Pubmed that a gene regulates or is regulated by AKT either directly or indirectly . Eliminating the genes with low variability across tumors within the discovery dataset left the 69 most variable genes used to classify AKT subgroups in the discovery dataset  enrichment analysis was dominated by generic GBM biological processes; therefore we used a single-sample approach analogous to the method used by Verhaak and Barbie http://www.sanger.ac.uk/research/areas/humangenetics/cnv/]. For experiments that compare broad CNA in the discovery and validation dataset we identified broad copy number alterations in the validation dataset as follows. Briefly, we found the average q value  for 15 genes spaced evenly across the region of interest. If > 50% of genes had a q value less than expected by chance after correcting for multiple testing (q < 0.25), that region was called as a copy number alteration.The GISTIC algorithm http://www.cbioportal.org/public-portal/). One hundred and eighty six of the 215 tumors with RPPA data could be assigned to an AKT class and were used for analysis. Correlation coefficients between two antibodies against the same protein were high indicating adequate antibody specificity and pre-processing of data .Level 3  reverse phase protein array (RPPA) data was downloaded from the cBio Cancer Genomics Portal  in AKT subgroups from discovery  and valiPhillips, TCGA and G-CIMP subgroups distributed non-randomly in AKT subgroups  and S4. AKT subgroups have different clinical characteristics  and S4. Consistent with the less aggressive character of SL tumors, there was a trend toward decreased endothelial proliferation , and palisading necrosis  in the validation dataset . There wSurvival differences between subgroups suggest AKT subtypes are either prognostic or predictive . Since AKT influences response to chemotherapy We used TCGA data to investigate how molecular alterations partition in subgroups. All subgroups had unique broad  and/or fAn integrated analysis of mutations, CNA and mRNA expression in glioma-associated genes shows some AKT subgroups had similar features as TCGA subgroups . The AKTWe find subgroups have distinct patterns of expression of mRNA , proteinWe used Gene Ontology (GO) to investigate the biological role of genes expressed in tumors and how terms partition in subgroups. Each subgroup, except CLAS, had a high percentage of tumors with functionally related terms that suggested a different dominate biological process . The CLAThe major finding is that AKT pathway genes classify GBM into at least five patient subgroups with unique clinical and molecular characteristics. The results were validated in an independent dataset of non-overlapping samples, suggesting AKT classes reflect underlying structure in the data and do not arise from chance or technical artifacts such as batch effects and patient sampling. Taken together these data add to previous results suggesting histopathologically diagnosed GBM is a collection of molecular subgroups with fundamental differences in biology and clinical behavior. This approach advances classification of GBM by splitting out groups not previously identified by other approaches and expands our understanding of molecular aberrations underlying subgroups.We interpret with caution the finding that SL patients treated with BCNU or CCNU have appreciably longer survival than SL patients receiving other treatments . Inhomogeneity between the cohorts (including treatment protocols and institution providing tumor) could impact survival. However, age and IDH1 mutation status clearly do not contribute since patients in the longer surviving cohort were older and had less IDH1 mutations. If validated these results suggest AKT classification is a predictive marker that identifies a subset of GBM patients with sensitivity to BCNU/CCNU. Interestingly, there is a subset of anaplastic oligodendroglial tumors characterized by 1p19q loss of heterozygosity (LOH) and IDH1 mutations that significantly benefits from procarbazine, CCNU, and vincristine (PCV) chemotherapy Mutations in IDH1 are a common and early event in low grade glioma, they are present in secondary GBM We found distinct patterns of expression for PI3K/AKT/mTOR components in subgroups. Our results suggest gene products that inhibit AKT and mTOR are important regulators of PI3K/AKT/mTOR/S6 axis output. In our model the loss of AKT and mTOR inhibitors  increases output of the AKT/mTOR/S6 axis in the MES subgroup. Conversely, increased expression of these inhibitors decreases output in the SL subgroup. In an apparent paradox, p-AKT expression is low in the MES subgroup. We suggest AKT phosphorylation is held in check in the MES subgroup by (1) heightened activity of an mTOR/S6K/IRS1 negative feedback loop How do these results compare with other approaches that use mRNA to classify GBM? AKT classification is complementary to previous classification methods but divides GBM into more subgroups. It gives patient subgroups with statistically significant differences in survival while Phillips Survival differences found in the discovery dataset were diminished in the validation dataset. Inhomogeneity's between datasets that could confound comparisons including (1) age , (2) patient populations , (3) treatment . In addition, performance of the validation dataset is typically not as robust as the discovery dataset One AKT subgroup was not found in the validation dataset (C1). We know morphological heterogeneity can result in inconsistent intra- and inter-observer diagnosis of grade and histological type  GO term analysis suggests different cells of origin/dominant biological processes for each subgroup . Red indicates total consensus (consensus index of 1) while white indicates no consensus (consensus index of 0).(TIF)Click here for additional data file.Figure S2Average expression of AKT pathway genes in subgroups. Hierarchical clustering using AKT pathway genes was used to group GBM patients and genes in the discovery (GBM 195) dataset. Tumors in the validation dataset were grouped by AKT class keeping the same order of genes. The expression of AKT pathway genes in each class was averaged and is shown as a heatmap; red and green is high and low expression respectively.(TIF)Click here for additional data file.Figure S3Log2  DNA copy number in subgroups. Amplifications (red) and deletions (blue) in subgroups (y axis) were determined by segmentation analysis of normalized signal intensities from TCGA SNP arrays using GISTIC and viewed with IGV by chromosomal location (x axis).(TIF)Click here for additional data file.Table S1Clinical information for tumors in GBM195.(XLSX)Click here for additional data file.Table S2AKT pathway gene classifiers used for the discovery and validation datasets.(XLSX)Click here for additional data file.Table S3Distribution of clinical and molecular information by subgroup in the discovery dataset (GBM195). The table lists the number of tumors with the specified feature in each subgroup in the discovery dataset. Features with statistically significant enrichment in a subgroup after Bonferroni correction (p < 0.05) are highlighted.(XLSX)Click here for additional data file.Table S4Distribution of clinical and molecular information by subgroup in the validation dataset (TCGA). The table lists the number of tumors with the specified feature in each subgroup in the validation dataset. Features with statistically significant enrichment in a subgroup after Bonferroni correction (p < 0.05) are highlighted in dark grey. Features with statistically significant enrichment in a subgroup before Bonferroni correction are highlighted in light grey.(XLSX)Click here for additional data file.Table S5GO term analysis of genes differentially expressed in subgroups. The % of individual tumors within a subgroup that are enriched for a specific GO term is shown, ordered by decreasing representation. GO terms enriched in ≥ 40% of tumors are highlighted light grey. Neurodevelopmental terms are highlighted with dark grey.(XLSX)Click here for additional data file.Table S6Focal DNA amplifications in subgroups. Copy number alterations in subgroups were evaluated using GISTIC and the q score for statistically significant focal DNA copy number gains (q score < 0.25) listed. Focal copy number changes common to all subgroups  are not reported.(XLSX)Click here for additional data file.Table S7Focal DNA deletions in subgroups. Copy number alterations in subgroups were evaluated using GISTIC and the q score for statistically significant focal DNA copy number losses (q score < 0.25) are listed. Focal copy number changes common to all subgroups  are not reported.(XLSX)Click here for additional data file.


Epigenetic factors, principally CpG methylation, are widely held to play a dominant regulatory role in controlling the locus' activity. The purpose of this study was to examine its behavior in ES cells and determine if this locus could be exploited for use in screen-based investigations. We have derived multiple vyA ES cell lines from the C57BL/6 strain and generated a cell line carrying a GFP-reporter gene (vyA/GFPA). Use of the DNA demethylating drug 5-azacitidine on various ES cell lines does not induce either agouti or GFP expression. Methylation analysis reveals that although most lines display normal methylation at IAP elements in general, the vyA IAP element is essentially unmethylated. In addition, we find that different repeat compartments are epigenetically unstable in a number of derived cell lines.The  Retrotransposons constitute a large proportion of the mammalian genome and pose a significant threat to its integrity vyA) mouse strain is one of the best-studied examples of how a retroelement can interfere with neighbouring gene expression resulting in a phenotypic consequence. Over 50 years ago, the allele arose spontaneously as a result of an IΔ1-class intracisternal A particle (IAP) retrotransposon inserting into an upstream non-coding exon (pseudoexon 1A) of the agouti gene, located about 100 kb from the first coding exon agouti expression agouti expression and such animals are termed pseudoagouti. The IAP is subject to epigenetic regulation and the activity state of the vyA allele is inversely correlated with DNA methylation at the IAP element The agouti viable yellow  cells derived from vyA animals and their utility.To date, all studies on the role of DNA methylation at the vyA animals . To increase the number of embryos, female animals received injections to stimulate superovulation (gonadotrophin from pregnant mare serum) followed by human chorionic gonadotrophin. 3 days after detection of a copulation plug, females were sacrificed and uterine horns dissected out. Embryos were flushed out and collected before being individually seeded onto mitomycin-C treated mouse embryonic fibroblasts in standard FBS-containing ES cell media supplemented with 2i (PD03259010 and CHIR99021) in 96-well plate format. 2i was only included during the initial derivation phase. Six days after initial seeding, wells were tryplated onto MMCT-MEFs and passaged every third day. Established ES cell clones were checked by morphology and expanded up for further experiments. All animals used for this study were covered by a Home Office Project License under The Animals (Scientific Procedures) Act 1986 to S.K.T.O. In addition, this research study was approved by the UCL Research Ethics Committee. Mice were humanely sacrificed at a designated establishment by cervical dislocation.Timed matings were set up between male and female vy-GFPA replacement construct was generated using a bacterial artificial chromosome (BAC) (RP23-466H11) containing approximately 180 kb of mouse chromosome 2 encompassing the agouti locus (CHORI). A 17.4 kb fragment of this was retrieved into the vector pL253 by BAC recombineering in the recombineering strain SW102 agouti locus. For the eGFP cassette, pEGFP-C1 (Clontech) was used as the template and PCR-amplified using primers spanning the Start ATG and SV40 early mRNA Polyadenylation Signal. The replacement construct was sequence verified before being used to target one of the vyA ES cell lines derived (clone B5). Targeting was performed as previously described vy-neoA), this was further expanded and the neo selection cassette was removed by transient transfection using the Cre expression plasmid pCAGGS-Cre neo cassette (vy/AGFPA) were expanded and used in the experiments described. Primer sequences (5′ to 3′) used to generate the amplicons shown in TTG GAG GCA GCC TAG ACA CT (Primer A); TTG GAG GCA GCC TAG ACA CT (Primer C); GAA TTC CTC TAA GAT ACA TTG ATG AGT (Primer D). Primer sequences used to sex derived ES cell lines: AGA GAT CAG CAA GCA GCT GG (sry_F); TCT TGC CTG TAT GTG ATG GC (sry_R).The agouti genotyping, the probe used is as described in Duhl et al. Kit extracted DNA  was digested using the appropriate enzyme. 10 µg and 1 µg of DNA was digested for genotyping and methylation analysis Southern blots, respectively, for 6 hours. Digested DNAs were resolved on 0.8% TAE gels by electrophoresis before image capture. Gels were then depurinated , denatured  and neutralized  before blotting onto a 0.45 mm Nylon membrane  overnight. The next day, transferred DNA was fixed by UV cross-linking then incubated in Prehybridization Solution  for at least two hours before overnight incubation with radiolabelled probes. For methylation analysis, probes used were described previously TGA CAG GAG TCT GCG GAG TA (qAgouti_F); TCT TGG ATT TCT TGT TCA GTG C (qAgouti_R); TTG GCA GAT AAG GCC ACC TA (qIAP_F); AAT TTC TTG GGC AGC CTC TAC (qIAP_R); TCG TGA CCA CCC TGA CCT AC (qGFP_F); AAG TCG TGC TGC TTC ATG TG (qGFP_R). UPL probes and reference genes used: Agouti , IAP , GFP .Total RNA was extracted using TRIZOL reagent according to the manufacturer's protocol (Life Technologies). Prior to use for PCR, RNAs were DNase I treated  to remove any residual contaminating DNA. Expression of the different sequences indicated was determined using the Roche Universal Probe Library System (Roche Diagnostics). To detect IAP, a suitable hydrolysis probe was generated using a full-length consensus IAP sequence (GenBank accession number M17551). Primer sequences (5′ to 3′) used: RIPA lysates were prepared from pelleted cells. Following lysis on ice, and centrifugation to clarify, samples were quantified and diluted into 1× Laemmli Buffer. Approximately 50 µg of lysate was loaded in each lane of a 10% Mini-Protean TGX Gel (Bio-rad). Following separation by electrophoresis, proteins were transferred onto PVDF membrane. Membranes were then blocked in 1× PBS+0.1% Tween-20 (PBST)+5% milk for one hour before incubating with anti-GFP antibody  overnight. The next day, membranes were washed in PBST , before incubation with secondary antibody  for one hour at room temperature. Membranes were then washed in PBST , before HRP signal was developed using Luminata Forte Western HRP Substrate (Millipore). Signals were detected by exposure onto Hyperfilm ECL (Amersham). Post-signal acquisition, membranes were washed in PSBT and re-probed using anti-tubulin antibody  using the same methods as described.GTA GAG GTT TAA GGA TTT AGA TTG GTG (Avy bis fwd); AAC CCA CAA AAC CAA AAT CTT CTA C (Avy bis rev).Kit-purified DNA was bisulphite converted using the EZ DNA Methylation Lightning Kit (Zymo Research) according to the manufacturer's protocol. Cloning and analysis were performed as previously described Prior to the start of drug treatment, ES cells were seeded and grown in culture for 42 hours. Drug-containing media was then added to cells and the cells left for a further 30 hours before harvesting for analysis.vyA locus as a ‘controlling element’ is that the associated IAP element aberrantly controls agouti expression. From the existing data in animals/differentiated cells, the vyA IAP is predicted to be regulated by DNA methylation at the 5′ LTR sequence. We therefore reasoned that this would be an ideal system from which to generate a GFP-based reporter cell line for use in screen-based approaches to identify novel regulators necessary for retrotransposon silencing. To that end, we derived ES cells from animals where the vyA IAP element has been inbred onto the C57BL/6 background. Images of representative derived lines are shown in vyA allele , we detected robust and ectopic agouti expression by qRT-PCR (vyA ES cells did not induce agouti expression (Oct4-GiP), we were unable to induce GFP expression in our targeted reporter cell line using either 5-aza or the histone deacetylase inhibitor Trichostatin A (TSA) , suggest it plays a major role in silencing multiple classes of retroelements including IAPs Under the prevailing model and combined with our observations we predicted that loss of DNA methylation at IAP elements, including the  qRT-PCR . Howeverpression . This wa A (TSA) . Therefoagouti and GFP expression. To that end, we examined the methylation status within the repeat compartment of eleven different vyA ES cell lines that were derived. Primers designed to detect the presence of the Sry locus by PCR confirmed that all the cell lines analysed were Sry+ and therefore XY , we asked whether the thylated .agouti/GFP expression. Furthermore, although we were unable to directly assess the expression status of the vyA IAP element, these data suggest that at least in pluripotent stem cells, regulation of the downstream agouti locus is uncoupled from the methylation state of this IAP element's LTR.This provides an obvious explanation as to why 5-aza-treatment failed to induce vyA IAP LTR in ES cells. Our initial intention was to generate a cell line that could be used in screen-based approaches to uncover novel epigenetic regulators of retrotransposon silencing. Our observation that the vyA IAP is apparently unmethylated offers insight into the unusual behaviour of the IAP at this particular locus. ES cells are derived from the inner cell mass of blastocyst embryos vyA locus from blastocysts derived from both pseudoagouti and yellow animals vyA IAP we observe in our various cell lines reflects a failure to correctly establish methylation following this erasure, or if methylation is correctly established but is rapidly lost upon ES cell derivation. Given that our bisulphite data indicate that methylation can (rarely) be detected at the locus, we favor the latter interpretation that the vyA IAP is inherently epigenetically unstable during the pluripotent state.To the best of our knowledge, this is the first study to examine the methylation state of the Genetic data have long supported the importance of DNA methylation in silencing IAP elements but the mechanism remains unclear vyA locus requires refinement. That is, control of the vyA IAP LTR cryptic promoter by DNA methylation and regulation of downstream agouti expression does not operate in pluripotent stem cells. Prior studies show that in pseudoagouti animals, where the vyA IAP is presumably silent, the IAP is significantly less methylated than other closely related IAPs vyA locus does not control promoter activity of the vyA IAP. A broader implication of our study is that although IAP activity is constrained by DNA methylation it is likely that not all IAP elements are dependent on this mode of regulation. It has been estimated that there are between 1,000 to 2,500 IAP elements present per haploid genome Our results also indicate that the prevailing model of the vyA locus demonstrate that DNA methylation-independent mechanisms ensure repression of the locus in pluripotent stem cells. A candidate pathway involves sequence specific KRAB-ZF proteins and KAP1, the latter being important in repression of endogenous retroviruses in murine ES cells Through interference with transcription of nearby genes, retrotransposon-derived controlling elements have the potential to affect gene expression. The mechanisms by which they are silenced are not fully understood. Our results on the well-studied vyA IAP in differentiated cells is a consequence of other signals that control the activity of the locus between generations. This has implications for the nascent field of environmental epigenetics and studies that attribute perturbations in DNA methylation, particularly in the early embryo, as the mechanism responsible for diet/environmentally induced alterations in gene expression.Finally, our data also suggests that the presence of DNA methylation at the


MRI-guided Electrophysiology (EP) procedures integrate real-time MRI images with catheter position during Radiofrequency Ablation (RFA) of arrhythmias . Using sCatheter position and cardiovascular structure were updated from MR images Figure  in the fTen volunteers without training in EP procedures were recruited to participate in a simulated RFA procedure to evaluate the performance with and without the proposed endoscopic view and haptic interface. RFA targets were pre-defined at the left pulmonary veins inside a left atrium model reconstructed from the preoperative image data from a cardiac patient Figure . The subThe proposed image-based catheter haptic guidance and endoscopic view improved RFA procedural time and accuracy, and reduced the risk of perforation.


The following information is missing from the Acknowledgements section: The research leading to these results has partly received funding from the EU-FP7-SOCIOEC project under grant agreement No. 289192.


A small number of recent reports have suggested that altered placental DNA methylation may be associated with early onset preeclampsia. It is important that further studies be undertaken to confirm and develop these findings. We therefore undertook a systematic analysis of DNA methylation patterns in placental tissue from 24 women with preeclampsia and 24 with uncomplicated pregnancy outcome.We analyzed the DNA methylation status of approximately 27,000 CpG sites in placental tissues in a massively parallel fashion using an oligonucleotide microarray. Follow up analysis of DNA methylation at specific CpG loci was performed using the Epityper MassArray approach and high-throughput bisulfite sequencing.Preeclampsia-specific DNA methylation changes were identified in placental tissue samples irrespective of gestational age of delivery. In addition, we identified a group of CpG sites within specific gene sequences that were only altered in early onset-preeclampsia (EOPET) although these DNA methylation changes did not correlate with altered mRNA transcription. We found evidence that fetal gender influences DNA methylation at autosomal loci but could find no clear association between DNA methylation and gestational age.Preeclampsia is associated with altered placental DNA methylation. Fetal gender should be carefully considered during the design of future studies in which placental DNA is analyzed at the level of DNA methylation. Further large-scale analyses of preeclampsia-associated DNA methylation are necessary. Preeclampsia is a hypertensive disorder that affects 5–7% of all pregnancies and is one of the major obstetrical complications resulting in fetal morbidity and mortality Numerous studies have identified altered gene expression in the placentas of mothers affected by preeclampsia Although the relationship between DNA methylation and gene expression at the level of transcription is complex, there is strong evidence that altered DNA methylation, particularly within the regulatory regions of expressed genes, has a significant influence on transcription Given our relatively limited understanding of epigenetic alterations in preeclampsia, it is important that further studies are undertaken so that reported associations between DNA methylation and preeclampsia may be verified and expanded upon. Therefore we have undertaken an analysis of DNA methylation in the context of preeclampsia using a commercially available microarray that is able to simultaneously assess DNA methylation levels at 27,000 CpG dinucleotides.All patients provided written informed consent for use of their samples and de-identified clinical data under the umbrella of the Preeclampsia Program Project (PEPP study) and related ancillary studies. These women delivered at Magee-Womens Hospital within the years 1997–2007, and were recruited from clinics and private practices. This protocol was approved by the University of Pittsburgh Institutional Review Board (Protocol # IRB 0404159). Clinical and demographic characteristics of the individuals recruited to take part in this study are shown in A panel of clinicians met monthly to adjudicate the pregnancy outcome diagnosis of all the women. Preeclampsia was defined as gestational hypertension with proteinuria in accordance with the National High Blood Pressure Education Program Working Group Report on High Blood Pressure in Pregnancy 2]). Maternal race was by self-report at enrollment.All participants had no prior history of renal or vascular disease, and were non-smokers (<100 cigarettes in lifetime). Status of tobacco smoking was determined by self-report; these data were obtained during the pregnancy or immediately postpartum to minimize the potential problems caused by changing smoking habits during pregnancy. In a previous study of our patient population, there was no significant discordance between self-report of smoking status and maternal plasma cotinine concentration using a cutoff value of ≥2.0 ng/ml (P = 0.49), with 82% agreement and κ coefficient of 0.64 Placental biopsies were obtained immediately after delivery from the maternal side of the placenta, in regions free of infarcts between the placental margin and cord insertion. Decidua was removed by blunt dissection. The tissue was immediately transported in saline to the laboratory, flash frozen in liquid nitrogen and stored at −80°C for later use.To isolate DNA, 180-µL buffer ATL  and a 5-mm steel bead were added to each placenta sample. The samples were then homogenized in a TissueLyser (Qiagen) for 20 s at 30 Hz. The DNA was then purified using the DNeasy Blood and Tissue kit as per the manufacturer's protocol.DNA samples were prepared for microarray analysis and methylation status determined as previously described . P values were adjusted using Benjamini and Hochberg's method to control the false discovery rate (FDR) at 10%. DNA methylation data has been deposited at the Gene Expression Omnibus repository (GE59274).www.urogene.org/methprimer) or BiSearch (http://bisearch.enzim.hu). PCR products were amplified as separate reactions using Pfu Turbo Cx , Platinum Taq  or ZymoTaq (Zymo Research). Primer sequences, melting temperatures (Tm) and cycling conditions are shown in Tables S1A and S1B in DNA was bisulfite converted using Zymo's EZ DNA Methylation Gold kit as per manufacturer's protocol . Bisulfite specific primers were designed using Methprimer  on the methylation data to visualize the similarity of the methylation profiles across the samples. For hierarchical clustering, we used the Ward's minimum variance method, and chose respectively the Euclidean distance between the methylation rate of all probes, as well as 1– Pearson's correlation between the log ratio of the signal B to signal A of all probes, as the distance between two samples. The same two types of distance metric were also used in the multi-dimensional scaling analysis of the methylation data. Both types of unsupervised learning analyses were performed using the statistical computing program R.The clinical and demographic data for the 48 subjects who provided placental samples are presented in We first compared DNA methylation patterns in placental tissue samples from preeclampsia cases with normal controls by Infinium microarray. This identified a small number of differentially methylated CpG sites  when compared to normal controls who delivered at term. These previous studies, which controlled for gender bias by removing all X chromosome data points prior to analysis, reported no differences when comparing late onset preeclampsia samples (>34 weeks gestation) with normal pregnancy controls. To be consistent with these previous study designs we therefore reanalyzed our data, after excluding X chromosome-specific probe results, to compare EOPET (n = 9) samples with all normal controls (n = 24). As shown in Given previously published evidence that gestational age at time of delivery is a major contributor towards altered DNA methylation http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?targ=self&form=html&view=quick&acc=GSE44711). We hypothesized that reduced CpG methylation in a given sample cohort (preeclampsia or control) identified in our own data would correlate with an increase in transcription. We could find no overlap between genes containing differentially methylated CpG sites  samples.Furthermore, we identified a significant lack of correlation between multiple independent data sets from three separate studies.It is notable that we identified minimal overlap between our data and two previously published studies describing differences between normal controls and EOPET A number of notable differences in DNA methylation of specific genes were observed in our data. For example, the expression of Purinergic Receptor P2X, Ligand-Gated Ion Channel, 4 (P2RX4) has been shown to be elevated in both pre-term and term placental samples from preeclampsia cases compared to normal controls. CpG methylation within the P2RX4 promoter was reduced in preeclampsia samples relative to controls in our data, suggesting elevated transcription It is somewhat surprising that our gender-specific analysis of preeclampsia-related changes in DNA methylation  and 3 idIt is not surprising that the inclusion of X chromosome-specific data resulted in clear gender-dependent hierarchical clustering patterns. Notably, when X chromosome data were removed, samples were still broadly separated into gender-associated clusters and statistically significant gender-dependent autosomal alterations in DNA methylation were observed. These observations are worthy of further study at higher resolution with a larger sample size. The identification of autosomal origins of gender-specific differences is significant because previous studies of DNA methylation in preeclampsia have controlled for fetal gender by simply removing X chromosome specific probes from the analyses In summary, we have undertaken a systematic analysis of preeclampsia-associated DNA methylation changes in the placenta. We identified a number of differentially methylated CpG sites in tissues from preeclampsia patients including a number that were altered only in EOPET cases compared to controls.Figure S1Hierarchical clustering, with respect to gestational age at delivery, of DNA patterns in placental tissues from preeclampsia patients.(TIF)Click here for additional data file.Figure S2A. Hierarchical clustering, with respect to fetal gender, of DNA patterns in placental tissues from preeclampsia patients. B. MDS Analysis, with respect to fetal gender of DNA patterns, in placental tissues from preeclampsia patients. C. Hierarchical clustering with respect to fetal gender of DNA patterns in placental tissues from normal controls. D. MDS Analysis with respect to fetal gender of DNA patterns in placental tissues from normal controls.(TIFF)Click here for additional data file.File S1Tables S1–S8. Table S1A - Bisulfite Sequencing Primers and Tm's. Table S1B- PCR reaction conditions used. Table S1C. Epityper MassARRAY Primer Sequences. Table S2A-Preeclampsia-Associated Changes in Placental DNA Methylation Without Control for Fetal Gender: CpG sites at which samples from preeclampsia samples are more highly methylated than those from normal controls are shown. Table S2B- Preeclampsia-Associated Changes in Placental DNA Methylation Without Control for Fetal Gender: CpG sites at which samples from normal controls are more highly methylated than those from affected individuals are shown. Table S3A- Fetal Gender-Dependent Preeclampsia-Associated Changes in Placental DNA Methylation: CpG sites at which samples from normal controls are more highly methylated (<10%) than those from affected individuals are shown. Table S3B- Fetal Gender-Dependent Preeclampsia-Associated Changes in Placental DNA Methylation: CpG sites at which samples from preeclampsia samples are more highly methylated (<10%) than those from normal controls are shown. Table S4A- DNA Methylation Changes in Early Onset Preeclampsia (EOPET) Versus Normal Controls.% methylation levels for all significant differentially methylated CpG sites in EOPET  and normal controls . Adjusted p values are shown with FDR controlled at 10%. Table S4B- DNA Methylation Changes in Early Onset Preeclampsia (EOPET) Adjusted p values are shown with FDR controlled at 10%. Table S5A- Genes previously identified as containing differentially methylated CpG sites (reference 17) that were confirmed using our statistical/infomratic piepline. Table S5B- Genes identified from published data (reference 17) using our statistical/informatic pipeline containing differentially methylated CpG sites that were not previously identifed. Table S6A- Genomic Coordinates of Targeted DNA Methylation Analsysis Loci. Table S6B- Genomic Coordinates of Targeted DNA Methylation Analsysis Loci. Table S7- Gene expression differences between EOPET and normal placental tissue samples. Table S8A- Gender specific differences in DNA methylation at autosomal loci in normal samples.(XLSX)Click here for additional data file.


Grating-coupled propagating surface plasmons associated with silver-nanoparticle 2D crystalline sheets exhibit sensitive plasmonic resonance tuning. Multilayered silver-nanoparticle 2D crystalline sheets are fabricated on gold or silver grating surfaces by the Langmuir– Blodgett method. We show that the deposition of Ag crystalline nanosheets on Au or Ag grating surfaces causes a drastic change in propagating surface plasmon resonance (SPR) both in angle measurements at fixed wavelengths and in fixed incident-angle mode by irradiation of white light. The dielectric constant of the multilayered silver nanosheet is estimated by a rigorous coupled-wave analysis. We find that the dielectric constant drastically increases as the number of silver-nanosheet layers increases. The experimentally obtained SP dispersions of Ag crystalline nanosheets on Au and Ag gratings are compared with the calculated SP dispersion curves. The drastic change in the surface plasmon resonance caused by the deposition of Ag-nanoparticle 2D crystalline sheets on metal grating surfaces suggests the potential for applications in highly sensitive sensors or for plasmonic devices requiring greatly enhanced electric fields.The online version of this article (doi:10.1186/2193-1801-3-284) contains supplementary material, which is available to authorized users. In recent years, surface plasmon resonance (SPR) phenomena have attracted considerable attention because of the extremely strong enhancement and confinement of electric fields near metal surfaces Knoll . Two kinRecently, tuning of propagating SPR properties has attracted considerable interest from those seeking to develop applications for plasmonic devices . The grating samples were mounted on a θ-2θ goniometer. A halogen lamp as the white light source or a HeNe laser was used for the excitation of SPs. P-polarized light was collimated by objective lens and irradiated on the grating samples. The reflected zero-order light was detected by a spectrometer. SPR excitation experiments were carried out at a fixed incident angle as a function of wavelength or at a fixed wavelength as a function of incident angle. Grating-coupled SPR modeling was performed on G-Solver  using rigorous coupled-wave analysis.Silver nanoparticles were synthesized by thermal reduction of a silver acetate precursor in a melt of myristic acid, as described earlier  is the wavelength-dependent dielectric constant of silver given by the classical Drude’s free-electron model. As shown in Figure Here, We studied the SP excitation properties of Ag crystalline nanosheets on Au and Ag grating surfaces, and found a drastic change in SP excitation from angular measurements at fixed wavelength and from measurements at fixed incident angle under irradiation with white light. The SPR dips were drastically shifted when Ag crystalline nanosheets were deposited on the grating surfaces. The experimentally obtained SP dispersion data of the Ag crystalline nanosheets on Au and Ag gratings were compared to calculated SP dispersion curves. A large shift in the wavelength or dip angle by the deposition of Ag nanoparticle 2D crystalline sheets on a metal grating surface based on the drastic change in the surface plasmon resonance suggests the potential for applications in highly sensitive sensors or for plasmonic devices requiring greatly enhanced electric fields.Supporting Information. (PDF 384 KB)Additional file 1:


Cardiovascular Magnetic Resonance (CMR) with respiratory commands is the gold standard technique to measure cardiac volumes. Although cardiac volumes can be measured during free breathing in patients with congenital heart disease (CHD), its accuracy is unknown. Therefore, the aim of this study was to compare cardiac volumes acquired during free breathing with volumes acquired during breath hold commands.Cardiac volumes were measured in every patient using both free breathing and breath hold techniques, by a routine standard steady state free precession (SSFP) cine sequence in axial slice orientation. The sequence parameters were always identical besides the number of averages being three during free breathing and one during breath holds. Volumes were determined in a blinded fashion by endocardial contouring and then correlated using the coefficient of determination and compared by Bland Altman analysis. Eleven younger patients with CHD aged median 4 yrs, range 3 months-14 yrs, were examined under general anesthesia and intubation (intubated younger patient group). Twelve older patients with CHD aged median 20 yrs, range 11-61 yrs were examined consciously (conscious older patient group).The agreement of the end systolic volume (ESV) and end diastolic volumes (EDV) between scanning with and without respiratory commands in both ventricles was excellent in the intubated younger patients  and less acceptable in the consciously examined older patients  (see Figure Cardiac volumes can be quantified very accurately during free-breathing in young patients with congenital heart disease by CMR using standard routine imaging techniques. If needed, even in older patients cardiac volumes can be quantified reasonably accurate during free-breathing. Therefore, free-breathing is an alternative technique for patients not able to hold their breath and thus obliterating the need of anesthesia for many patients undergoing CMR. In addition it will provide a chance for the CMR to assess the hemodynamics in a resting physiologic condition.All authors have no conflict of interest.