There were 102 articles published in the Journal of Cardiovascular Magnetic Resonance (JCMR) in 2014, which is a 6 % decrease on the 109 articles published in 2013. The quality of the submissions continues to increase. The 2013 JCMR Impact Factor (which is published in June 2014) fell to 4.72 from 5.11 for 2012 (as published in June 2013). The 2013 impact factor means that the JCMR papers that were published in 2011 and 2012 were cited on average 4.72 times in 2013. The impact factor undergoes natural variation according to citation rates of papers in the 2 years following publication, and is significantly influenced by highly cited papers such as official reports. However, the progress of the journal’s impact over the last 5 years has been impressive. Our acceptance rate is <25 % and has been falling because the number of articles being submitted has been increasing. In accordance with Open-Access publishing, the JCMR articles go on-line as they are accepted with no collating of the articles into sections or special thematic issues. For this reason, the Editors have felt that it is useful once per calendar year to summarize the papers for the readership into broad areas of interest or theme, so that areas of interest can be reviewed in a single article in relation to each other and other recent JCMR articles. The papers are presented in broad themes and set in context with related literature and previously published JCMR papers to guide continuity of thought in the journal. We hope that you find the open-access system increases wider reading and citation of your papers, and that you will continue to send your quality papers to JCMR for publication. CMR of congenital heart disease is well established, although high quality acquisition and interpretation require training . CMR is In this observational study, 27 adults with prior arterial switch operation (ASO) for transposition of the great arteries (D-TGA) underwent CMR for biventricular systolic function, myocardial scar burden, coronary ostial assessment and myocardial perfusion during vasodilator stress . CMR pron = 22), higher LV end diastolic volume (minimum 22 mL/m2), higher LV to RV stroke volume ratio (minimum 0.19) and higher mitral to tricuspid inflow ratio were associated with successful biventricular conversions. In unbalanced atrioventricular septal defect with right ventricular dominance (n = 10), which was a smaller subgroup with only one death, no association of CMR parameters with transplant-free survival with a biventricular circulation were found. Despite that this is a difficult topic to study with heterogeneous patients, and small sample size these data suggest that CMR parameters can be used to predict likelihood of successful biventricular repair in children with hypoplastic left heart. CMR predictors were superior to conventional echocardiographic measures.This retrospective analysis reported CMR parameters associated with transplant free survival with a biventricular circulation after conversion of a univentricular circulation to a biventricular circulation in two patient groups . In bordThe authors studied 45 preterm infants of which 16 had a patent ductus arteriosus (PDA) . EDV, SVSeventeen young patients with sudden death  who died between 2010 and 2012 were studied with 1.5 T MR and CT prior to autopsy [This group performed CMR with LGE in a cohort of 30 patients with Eisenmenger syndrome, mean age 43 ± 13 years, 20 female followed up for a median 7 years . LGE wasThere is relative paucity of data related to expected haemodynamic changes in pregnancy assessed by CMR. The objective of the Cardiac Hemodynamic Imaging and Remodeling in Pregnancy (CHIRP) study was to compare transthoracic echocardiogram and CMR in the non-invasive assessment of maternal cardiac remodeling during the peripartum period . Thirty n = 116), of which 64 (55 %) had an inter-arterial course, passing between the aorta and pulmonary artery [This dual tertiary centre study retrospectively identified consecutive patients with an anomalous coronary artery originating from the opposite sinus of Valsalva  before and early after (mean 4.9 ± 2.5 months) coronary reimplantation surgery . The groLGE enhancement is associated with adverse clinical features in repaired tetralogy of Fallot (ToF) and RV scar is the substrate for ventricular arrhythmia. This study demonstrated high resolution 3D LGE at three Tesla findings in 15 patients using a whole heart, respiratory navigated technique and evaluated a semi-automated segmentation analysis . Total rThis is an insightful study of right ventricular adaptation to pressure loading conditions in 65 patients with chronic thromboembolic hypertension before and after pulmonary endarterectomy using CMR combined with haemodynamic measurements available from routine right heart catheterization . The autThe march of technological improvement never stops and the cardiovascular system inspires considerable activity, probably because it is challenging and ripe for continued development. Advances reported in the journal have included real-time imaging,  dual traThis paper looks more deeply into the underlying issues behind the background phase errors that still represent a major problem with regard to the accuracy of clinical blood flow measurement . The resThis work was also focused on the subject area of the accuracy of phase contrast flow measurement but in this case on confidence intervals and how these were affected by noise associated with accelerated imaging . The aut3D phase contrast CMR has traditionally been limited by long acquisition times. This study tested an 8-fold acceleration of Cartesian time-resolved 3D flow . Data acThis innovative sequence development for real-time interventional CMR uses the concept of positive contrast of paramagnetic materials obtained with dephasing gradients in a dual echo sequence that allows both the dephasing gradient image and a more standard bSSFP image to be acquired simultaneously at frame rates of 5–6 frames/s . The appThis state of the art review of T1 mapping discusses current methods for T1 quantification and provides comparison of the differing techniques for native and post-gadolinium measurement . Both T1In recent years there has been considerable interest in T1 mapping which has led to the development of a wide variety of methodology for measurement . This paThe group presented a novel method of 3D interleaved T1 and T2 mapping which allowed acquisition of the entire left ventricular myocardium in a single breath hold . When vaWith the massive potential in CMR, to implement differing sampling and reconstruction approaches there has been recent increased interest in simulators that can be used to help optimize sequence parameters and reduce artifacts for example . This paAnother example of a CMR simulator again made available online is this simulator using a high performance multi-GPU environment . The autIn this paper the authors have performed a prospective study on 40 consecutive CAD patients to investigate the feasibility of a 3D perfusion CMR technique for detection of significant coronary disease . The novDuring the last year there has been continued interest in the use of dual-source parallel RF transmission to improve CMR; in this case it has been applied to bSSFP myocardial perfusion scanning at 3 T . The parArterial spin labelling represents an alternative non contrast approach to perfusion measurement that has been more widely applied outside the heart . As withIn order to acquire sharp motion free images of details in the heart such as coronary arteries or small late gadolinium enhanced regions, it is necessary to accurately define the patient specific diastasis period of the heart cycle . This paThe DENSE method of measuring myocardial strain is becoming more established at least with the CMR clinical research community and the following two papers describe and test methods of simplifying ventricular segmentation for the strain analysis . In thisAnother approach to simplifying the analysis is described and evaluated in this paper . Using tDiffusion tensor imaging (DTI) gives information on the mean intravoxel alignments of tissue microstructures using diffusion weighted imaging. It is used in particular in the brain, where larger diffusion values occur along nerve fibres in comparison to across fibres. The technique can also be used with considerably increased difficulty (due to motion) for the heart, however most work has been done in animals, or in ex-vivo human hearts, because of the high difficulty of deriving DTI data from the moving heart. Recently a more robust imaging sequence for in-vivo human cardiac DTI has been described which makes human studies more approachable. Recent work has shown in-vivo reproducibility in humans in hypertrophic cardiomyopathy, , 37 and Cardiac DTI was performed at 3 T in HCM and control subjects and studied at end-systole and late diastole . In contThis editorial,  reviews The authors validated a stimulated-echo diffusion tensor imaging (DTI) sequence using phantoms and assessed its reproducibility between two CMR centres at 3 T . The samBeside its key role in research, CMR is most frequently used clinically for the carotid arteries where it can be used to characterize thrombus,  plaque cThree dimensional respiratory and end systolic ECG gated, gadolinium enhanced magnetic resonance angiography was performed on a 3 Tesla (3 T) scanner in 101 consecutive patients, 35 of them in AF, prior to ablation . Image aThis is a feasibility study assessing an accelerated electrocardiogram-triggered contrast enhanced pulmonary vein MRA with isotropic spatial resolution, compressed sensing and respiratory navigator to define pulmonary venous anatomy prior PV ablation for atrial fibrillation in 19 patients and compared with the conventional breath-hold first pass non-ECG triggered CE-MRA . The autThis was a head-to-head comparison between Multi-detector Computed Tomography Angiography (MDCTA) and black-blood 3 T-cardiovascular magnetic resonance (bb-CMR) with respect to their ability to identify symptomatic carotid plaques characteristics . Twenty-This mainly technical work aims to develop a diffusion-prepared turbo-spin-echo (DP-TSE) technique for carotid plaque characterization with 3D high resolution and improved image quality and applied to 15 healthy volunteers and six patients with carotid artery disease . In thisCardiovascular events and cardiovascular related mortality increased in HIV-infected patients treated with effective antiretroviral therapy . IdentifAuthors investigate the signal to noise ratio (SNR) and blood suppression performance of improved motion-sensitized driven-equilibrium (iMSDE) preparations technique for carotid wall imaging using composite RF pulses and sinusoidal gradients . Twelve Atheromatous plaques neovascularization and inflammation are associated with clinical cardiovascular events. Detection and quantification of neovascularization and inflammation in-vivo are important. This study aims to investigate the inter-scan reproducibility of kinetic parameters in atherosclerotic plaque using dynamic contrast-enhanced (DCE) CMR in a multi-center setting using 3 T field strength . Study rA 3D CMR technique using Multi-contrast Atherosclerosis Characterization (MATCH) was developed to address some aspects of CMR limitation in this field including low slice resolution, long scan time, image mis-registration, and complex image interpretation . The advThe purpose of this study was to evaluate associations of CMR measures of total arterial compliance and two CMR measures of aortic stiffness with respect to future cardiovascular events in 2122 Dallas Heart Study participants without cardiovascular disease . Study rThe purpose of this work was to refine, implement, and test a robust, practical single-breathhold bSSFP coronary MRA sequence at 3.0 T and to test the reproducibility of the technique . ResultsThe ability of novel 3D T2W black-blood imaging sequence to detect oedema induced in porcine carotid arteries by acute balloon injury compared to conventional 2D T2-weighted black-blood CMR was investigated in a porcine model . The novCMR is regarded the standard of reference for clinical assessment of the aorta, but normal CMR dimensions in large size population study are not available. The aim of this study is to produce a normal CMR reference range of aortic diameters and to investigate the relationship between regional aortic size and body surface area (BSA) in a large group of healthy subjects with no vascular risk factors . A normaThe objective of this feasibility study is to investigate the feasibility of assessing asymmetric stretch in healthy and diseased ascending aortas using Displacement Encoding with Stimulated Echoes (DENSE) . FifteenThis study investigated the ability of 3 T CMR to determine the components of recurrent carotid artery disease and examined whether these differed from primary atherosclerotic plaque . Twenty-Paravalular leak (PVL) is common post transcatheter aortic valve implantation (TAVI). A population of patients post TAVI underwent CMR and echocardiographic assessment of the aortic valve for PVL . SeveritMeasuring cardiac function is a fundamental for CMR. Myocardial tagging remains a source of research,  and at 3In this study, the CMR cine images of ten healthy volunteers, ten patients with left bundle branch block and ten with hypertrophic cardiomyopathy, who had also had CMR tissue tagging performed, were analysed by feature tracking (FT) of both the endocardial and mid wall circumferences in short axis planes . The intThis study aimed to examine whether CMR based strain analysis using feature tracking (FT) could serve as a quantifiable measure to confirm global and regional ventricular dysfunction in ARVC patients and so support the early detection of ARVC . Groups Myocardial feature tracking (FT) was applied to the SSFP long axis cine images of ten healthy volunteers, ten patients with hypertrophic cardiomyopathy (HCM) and ten with heart failure and preserved ejection fraction (HFpEF) to quantify left atrial deformation . EvidencThis study assessed the value of feature tracking for assessment of myocardial ischaemia . CircumfThe phenotyping of cardiomyopathy is a primary clinical indication for CMR, and has become mainstream in hypertrophic cardiomyopathy. Recent attention has been focussed on T1 imaging, , 90 withRecent reports suggest that crypts, meaning discrete clefts or fissures in the myocardium of the left ventricle (LV), usually inferobasal, are prevalent in phenotypically positive or negative carriers of genes associated with hypertrophic cardiomyopathy (HCM). However, the retrospective study by Child et al. of 1020 consecutive patients or volunteers who had undergone CMR during a 12-month period also identified crypts in healthy controls , there being crypts identified in 64 (6.3 %) of all of the 1020 CMR studies . Crypts In this study, LV short-axis cine images were acquired in 138 healthy volunteers using standard 2D imaging and 3D high spatial resolution CMR . A multiNative T1 mapping was assessed using MOLLI and ShMOLLI sequences at 1.5 T in 63 patients with genetically-confirmed Fabry disease, 25 of whom did not have left ventricular hypertrophy (LVH) . Native This trial determined the reference range for T1 mapping through study of healthy controls and low risk subjects using a standardized sequence 3(3)5)) on 1.5 and 3 T Phillips platforms across multiple hospital sites . There wThe group compared semi-automated LGE quantification techniques using manual segmentation as the reference standard in a cohort of 76 HCM patients . QuantifPatients with incident heart failure or suspected cardiomyopathy in whom CMR yielded a diagnosis of LV non compaction were followed for a mean of 27 months . On multFlorian assessed 88 patients with Duchenne and Becker muscular dystrophies with CMR and followed them for a 47+/−18 months . During The group used CMR first pass myocardial perfusion to perform pixel wise quantitative perfusion analysis in 35 HCM patients . MyocardMyotonic dystrophy type 1 (DM1) is associated with increased cardiac morbidity and mortality. This CMR study of 30 DM1 patients revealed a high prevalence of myocardial fibrosis (40 %), most commonly of the RV insertion points and with a heterogenous distribution within the LV myocardium . PresencThirty carriers of the Duchenne muscular dystrophy (DMD) mutation were compared to gender and age matched controls . A typicLiu et al. evaluated the effect of the use of the revised 2010 Task Force Criteria for the diagnosis of ARVC through retrospective review of 968 consecutive patients referred for CMR with clinical suspicion of ARVC between 1995 and 2010 . ApplicaThis highly accessed Review article provides an update of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), its diagnostic criteria, implicated genes, CMR evaluation and current management considerations . ARVC isThis proof of concept paper described a novel CMR technique to evaluate mitral annual motion through tracking of the atrioventricular junction (AVJ) . This waBrouwer et al. compared T1 mapping derived ECV, acquired using a MOLLI sequence, in HCM and healthy controls . Areas oThe authors describe the imaging findings of radiation recall reaction causing cardiotoxicity . The patCMR was performed in 19 patients with systemic sclerosis and 20 controls at 1.5 T with cine imaging, tagging, T1-mapping, STIR, LGE imaging and ECV quantification . CardiacNinety-four consecutive asymptomatic cocaine users with a mean duration of 14+/−9 years of drug use underwent CMR . CompareThis ex-vivo case study compared synchrotron X-ray fluorescence microscopy (XFM) elemental iron maps with magnetic resonance transverse relaxation rate maps of cardiac tissue samples from an iron-loaded patient with Diamond Blackfan anaemia . This alEx-vivo iron loaded hearts were studied using R1 and R2 measurements (R1 = 1/T1 and R2 = 1/T2) at 1.5 Tesla and compared with myocardial iron concentration calculated using inductively coupled plasma atomic emission spectroscopy . There wLiver iron concentration studies based on early T2* sequences were limited by a requirement for short echo times . MR techThe use of animals to study cardiac physiology and metabolism continues to contribute a small but important section to the journal, often using MR spectroscopy,  and high1ρ) is a tissue marker to non-invasively measure collagen content, and may have the potential to detect fibrosis in the hearts of patients with chronic myocardial infarction (MI) without the need of exogenous contrast agents [1ρ-mapping for the detection of myocardial fibrosis in the clinical setting using a large animal model, and translates this technique into patients with chronic MI. While significantly higher T1ρ –values were found in the infarct tissue of MI patients compared to the remote zone, the sensitivity of detecting the injury was lower compared to the gold-standard late gadolinium enhancement (LGE) CMR. This work opens a new avenue for non-invasive tissue characterization and further work is required to improve sensitivity and specificity of this technique.The longitudinal relaxation time in the rotating frame  of the fiber architecture in fixed mouse hearts [The complex cardiac micro-structure and spatial arrangement of cardiomyocytes in laminar sheetlets is important for normal function of the heart. Cardiac diffusion tensor imaging (CDTI) of hearts e hearts . The excThis paper reports on the validation of quantitative cardiovascular Magnetic Resonance (CMR) myocardial perfusion imaging against fluorescently-labeled microspheres on an isolated perfused pig heart model in order to determine the accuracy of available algorithms for the quantification of myocardial blood flow (MBF) . PerfusiA comprehensive, multi-parametric CMR protocol, including cine-MRI, tagging, BOLD, stress-perfusion and 31P-MRS was used to address the question whether or not reduced myocardial perfusion reserve observed in patients with left-ventricular hypertrophy due to aortic stenosis (AS) but without obstructive coronary artery disease leads to tissue deoxygenation . This stThis study sought to simultaneously quantify cardiac function and cardiopulmonary hemodynamic parameters from a free-breathing 4D  flow CMR examination in a canine model of acute thromboembolic pulmonary hypertension (PH) . The accIn this study, arterial spin labeling (ASL)-cine MRI was used to quantify variations in myocardial blood flow during a cardiac cycle in healthy rats . The autIn this study, BOLD imaging was used in patients with renal artery stenosis (RAS) to characterize increases in renal oxygenation, as a marker of kidney viability, following the administration of a standard 20 mg intravenous furosemide stimulus . In partAlthough it is possible to get diagnostic images in the majority of patients referred for CMR, image quality can be compromised by patient difficulty performing breath holds and also by arrhythmia. We have received a number of papers validating sequences aimed at overcoming difficulties,  and alsop-values of 0.77, 0.82, and 0.97, respectively). Although there is a relatively lengthy calibration phase required for this technique, the scan time was significantly shorter for the highly accelerated real-time data collection (p < 0.001) and fewer artifacts were reported in the real-time images (p < 0.01). In the qualitative image analysis, reviewers marginally preferred the standard images although some features including cardiac motion were equivalently rated. The authors suggest that real-time functional CMR with through-time radial GRAPPA performed without ECG-gating under free-breathing can be considered as an alternative to gold-standard breathhold cine imaging for the evaluation of ejection fraction. As such, this can now be further evaluated in the more challenging groups to image.In this paper, the authors determined LV volumes and LVEF using a free breathing real-time radial GRAPPA sequence at 1.5 T in 63 subjects comparing results to a standard breath-hold SSFP sequence . The difThe authors presented data on the initial validation of a novel 3D cine volumetric acquisition using multiple acceleration techniques . A six-fr = 0.99, p < 0.001) as well as 3D CMR measured LV mass (301 ± 93 g) and the pathology measured LV mass . Strong positive correlations were demonstrated between the 3D CMR measured RV mass (149 ± 46 g) and the pathology measured RV mass . The mean bias between 3D-CMR and pathology measures for total mass, LV mass and RV mass were: 3.0, −16 and 19 g, respectively. This paper validates the accuracy of SSFP-CMR sequence to determine myocardial mass as a ‘gold standard’ in humans.This study has evaluated ex vivo LV and RV mass CMR assessment by SSFP sequence in comparison with autopsy mass of hearts from cardiac transplants patients in a single centre study of 55 explanted cardiomyopathy hearts which were successfully scanned just after surgery and then weighed by a pathologist . SignifiThe authors successfully constructed an atlas comprising 1991 CMR cases contributed from the Multi-Ethnic Study of Atherosclerosis baseline examination. From this collection of data from asymptomatic adult volunteers, they quantified the dominant components of global shape variation in sub-cohorts  . The datr = −0.41, p = 0.001) and stroke volume , as well as diastolic functional parameters including peak-early-filling rate  and early late filling ratio . LV hemodynamic and diastolic function was associated more with LV fat as compared to RV or total pericardial fat, but not with systemic inflammatory markers or adipokines. These findings suggest a site-specific influence of pericardial fat on LV function, which could imply local secretion of molecules into the underlying tissue or an anatomic effect.This paper explores whether the previously reported association between increased pericardial fat volume and cardiac dysfunction is due to a systemic process or due to a direct local interaction. 60 obese females underwent CMR to assess LV function and pericardial fat volumes . The autThe ability of echocardiography to rapidly and robustly evaluate diastolic function is one of its strengths. Although CMR techniques to reliably assess diastolic function have existed for many years, numerous methods for making this more routine within the CMR exam continue to materialise, , 154 oftn = 62) showed significantly different normalized MA sweep volume profiles compared to those with TTE-based normal diastolic function. Good correlations were observed between CMR early-to-late mitral sweep rate (PSRE/PSRA) and early-to-late diastolic annular velocity ratios (e’/a’) measured by TDI . 3D MA sweep volumes generated by semi-automated AVJ tracking in routinely acquired CMR images yielded diastolic parameters that were effective in identifying patients with diastolic dysfunction when correlated with TTE-based variables.In this study, the authors evaluate a semi-automated CMR technique for quantifying global LV diastolic function, using 3D volume tracking of the mitral annulus (MA) from conventional 2D cine-CMR images . One hunp < 0.001) in the hypertensive patients with concentric remodelling compared to controls and also correlated with E/A . The authors conclude that the normalised torsion shear angle volume loop incorporates both the active and passive recoil processes of LV diastolic and systolic phases, providing a new global description of LV diastolic function.In this paper, the authors propose a novel index for evaluating LV diastolic function utilizing the LV normalized torsion shear angle volume loop . They evThe use of CMR in the diagnosis and assessment of myocarditis has become commonplace with greater appreciation of the value of LGE and T2W oedema imaging in identifying this common but often challenging pathology, in addition to the merits of CMR for the assessment of ventricular remodelling.p = 0.0003). Based on their data, the authors conclude that patients with suspected myocarditis yet absence of LV dysfunction and absence of LGE images have a good prognosis independent of their clinical symptoms and other findings.In this paper, the authors assess the prognostic value of a normal CMR scan (defined by LV volumes and ejection fraction and the absence of LGE) in a single centre cohort of 405 consecutive patients with clinically suspected myocarditis followed up prospectively for median time of 1591 days . CMR diaT1 mapping continues to generate much additional data with a variety of sequences applied to a widened spectrum of myocardial pathologies in pursuit of identifying and demonstrating the areas where this technique has greatest clinical utility. In this paper, Ferreira et al. study 60 patients with suspected acute myocarditis and suggest that their technique based on native T1mapping (ShMOLLI) can reveal the typical non-ischemic regional patterns of disease, negating the need for Gadolinium contrast . A thresThe quest for enhanced predictors of response to cardiac resynchronisation therapy (CRT) and improved benefit from CRT implantation continues, with significant data assessing the role of CMR in this field.2 = 0.78 ± 0.23). They demonstrate that MRI can potentially be used to prospectively determine the ideal LV lead location, and show that within the coronary veins, the site of latest mechanical delay is the site of the latest electrical delay in the majority of patients (91 %). This study provides initial evidence that non-invasive mechanical activation patterns derived from CMR can accurately reflect the underlying electromechanical substrate of intraventricular dyssynchrony.This study compares mechanical delay times derived from high temporal resolution short-axis cine CMR data with intra-operative electrograms recorded at multiple locations throughout the coronary sinus and its tributaries . The autP = 0.003). SDI derived from myocardial strain was least predictive. Volume-change SDI was applied as a predictor of RR to a second population of 50 patients (mean QRSd 146 ± 21 ms). When compared to QRSd, LBBB morphology and scar burden, volume-change SDI was the only statistically significant predictor of RR in this group. The authors conclude that volume-change SDI is a highly reproducible measurement that can be derived from routinely acquired SSFP cine images and predicts reverse remodelling following CRT.In this study, the authors establish and then evaluate a systolic dyssynchrony index (SDI) derived from CMR-based data as a predictor of response to CRT. SSFP and CSPAMM images were performed at 1.5 T prior to CRT and processed using a novel framework to extract regional ventricular volume-change, thickening and deformation fields (strain) . A systoCMR is invaluable to the study of patients with ischaemic heart disease and has made a major impact on this field and is now widely used to delineate myocardial infarction, , 162 andThis study evaluated the relation between baseline markers of viability and long-term functional outcome in 42 patients with ischaemic heart disease after revascularisation . Both thA recently developed in vivo diffusion weighted CMR (dwCMR) technique was compared with LGE in 11 pigs with chronic MI . ApparenThis prospective longitudinal study evaluated the performance of multiparametric CMR for detecting acute cardiac allograft rejection (ACAR) and characterizing graft recovery following transplantation . SignifiThe authors sought to characterise microvascular obstruction (MVO) and infarct size (IS) in relation to the mode and timing of reperfusion in 94 patients with ST-Elevation Myocardial Infarction (STEMI) . IS was Despite increasing clinical use, there is limited data regarding regadenoson in stress perfusion CMR. Perfusion imaging was performed in 117 patients with suspected myocardial ischaemia and myocardial perfusion reserve (MPR) was estimated by coronary sinus flow measurements . A subgrCMR and fractional flow reserve (FFR) allow for a reliable assessment of ischaemia in combination with anatomical information provided by coronary angiography (CXA). The cost-effectiveness of these two strategies was compared . CMR + CThis study evaluated the effect of common methodological differences in CMR voxel-wise measurement of MBF, using position emission tomography (PET) as the reference standard . EighteeDetection of intramyocardial haemorrhage (IMH) by T2-weighted imaging or T2* may be limited by long breath holds and sensitivity to artefacts, particularly at 3 T. This paper evaluated susceptibility weighted imaging (SW MRI) for detection of IMH . Forty-nWith the approval of some makes of pacemakers and implantable defibrillators for use in the MR scanner, there has been considerable interest in reporting their use and safety, , 191 andThe purpose of this study was to analyse safety and potential alterations of electrical lead parameters in 36 patients implanted with the EnRhythm/Advisa MRI SureScan PM during and after CMR at 1.5 Tesla . No adveThis study evaluated the safety of conventional cardiac pacemakers undergoing cranial MRI at 1.5 Tesla . A totalThere are always papers which do not fall into simple categories. This section pulls together such varia which has included interventional CMR techniques, –196 reviThe paper presents recommendations for unified terms to describe CMR pulse sequences based on the purpose of each sequence rather than the technical approach . As wellThe authors describe a method where an oral gel solution of 0.7 mL gadobenate dimeglumine contrast was swallowed by patients immediately before a free-breathing 3D MRA acquisition . The oesImaging of LV diastolic vortex formation may allow valuable insights in evaluation of LV function and measures of LV filling efficiency. 4D Flow CMR was acquired in healthy volunteers during passive (E) and active (A) diastolic filling using retrospective cardiac gating and 3D vortex analysis . The oriStem cells for regeneration of the myocardium has received much attention over the past 10 years, with conflicting trial data regarding efficacy. This review focuses on the role of CMR in assessment of myocardial regeneration and for confirmation of successful cell delivery and engraftment . CellulaThe group assessed the impact of the intravascular compartment on native T1 values in a cohort of patients with severe aortic stenosis studied at rest and adenosine-induced hyperaemia . As expe


Eucalyptus camaldulensis Dehnh.) is a widespread Australian eucalypt inhabiting riverine and floodplain habitats which spans strong environmental gradients. We investigated the effects of adaptation to environment on population level genetic diversity of E. camaldulensis, examining SNP variation in candidate gene loci sampled across 20 climatically diverse populations approximating the species natural distribution. Genetic differentiation among populations was high (FST = 17%), exceeding previous estimates based on neutral markers. Complementary statistical approaches identified 6 SNP loci in four genes  which, after accounting for demographic effects, exhibited higher than expected levels of genetic differentiation among populations and whose allelic variation was associated with local environment. While this study employs but a small proportion of available diversity in the eucalyptus genome, it draws our attention to the potential for application of wide spread eucalypt species to test adaptive hypotheses.As an increasing number of ecosystems face departures from long standing environmental conditions under climate change, our understanding of the capacity of species to adapt will become important for directing conservation and management of biodiversity. Insights into the potential for genetic adaptation might be gained by assessing genomic signatures of adaptation to historic or prevailing environmental conditions. The river red gum ( Trees are foundation species in many terrestrial ecosystems and changes to local environment associated with natural and anthropogenic climate change are projected to impact, or are already impacting, the health of forest tree populations and the ecosystems they service worldwide Reversible plastic responses are important for short term adaptation of natural populations Insights into a species potential for genetic adaptation could be gained through characterisation of the relative abundance of adaptive versus neutral variation in response to historic or prevailing environmental conditions, which might be applied to empirically test hypotheses about environmental adaptation. Forest trees are tractable models for adaptive genetic studies owing to widespread populations traversing environmental gradients, high levels of diversity and low frequency of co-segregation among gene loci (linkage disequilibrium) Eucalyptus camaldulensis Dehnh.) is a large tree found in riparian zones and associated floodplains in arid and semi-arid regions throughout Australia Significant areas of forest and woodland growing in marginal or semi-arid regions of Australia are currently at risk of decline in response to climate change. The river red gum , and 2. xylem cell wall development . The first group have the potential to impact plant water use in response to climate, via directing the movement of water between cellular compartments and out of the leaf stomata, or acting as internal cellular stabilisers in response to dehydration Using single nucleotide polymorphisms (SNPs) we examined the variability of twelve genes in trees sampled across the natural range of E. camaldulensis individuals drawn from Murray-Darling Basin populations in the collection of Butcher et al. Eucalyptus grandis genome sequence  in CLC Genomics Workbench (CLCbio). A twelfth gene, ERECTA, was sequenced in 36 individuals drawn from four Murray-Darling Basin populations of E. camaldulensis , Menindee (8), Wenthworth (8) and Wilcannia (8)), which were sampled from the collection of Butcher et al. Polymorphism data was identified from eleven candidate genes for which amplicons representing the open reading frame had previously been sequenced on a pooled sample of 500 Single nucleotide polymorphisms were selected from DNA sequence alignments based on the following criteria: a base pair change was present, the base position was not an indel and the minor SNP allele occurred in at least three individuals. SNPs were selected to maximise coverage across the gene and avoid redundancy due to linkage disequilibrium, by selecting a SNP at least one to two hundred base pairs apart. Gene sequences were annotated, and for each SNP the base change, gene position (intron/exon) and amino acid substitution were recorded. Ten SNPs were chosen for each gene, with the exception of CAD (8), bZIP (7), Dehydrin (5) and Erecta (5). SNP genotyping was performed on genomic DNA samples using the Sequenom MassARRAY System at the Australian Research Genome Facility (AGRF) . GenotypLinkage disequilibrium (LD) in outcrossing forest tree species is typically low, and in eucalypts such as river red gum co-segregation of SNP loci along a gene decays within several hundred base pairs In total, 76 of the 105 selected SNPs were successfully genotyped. Monomorphic SNPs and trees with >20% missing data were omitted from the data set, resulting in 59 SNPs typed across 191 individuals. Observed and expected heterozygosity and tests for Hardy-Weinberg Equilibrium (HWE) were performed on individual SNPs, and for sets of SNPs representing whole genes, for each population separately and overall using GenAlEx 6.2 ST was also estimated in GenAlEx to obtain both overall and pairwise population estimates of genetic differentiation. Significance of the observed differentiation was tested by performing 1000 random permutations of the data. In the same way, FST was also estimated for these populations (with the exception of Wirrengren Plain) using polymorphism data for the 15 putatively neutral microsatellite loci (nuSSR) applied in Butcher et al.Genetic divergence among populations based on all SNP loci was investigated using several complementary approaches. Firstly, analysis of molecular variance (AMOVA) was performed in GenAlEx to partition genetic diversity residing within and among populations and individuals. Using the AMOVA framework FST were examined via a Mantel test implemented in GenAlEx, and significance was based on 1000 permutations of the data. Divergence was also compared via principal coordinate analysis (PCoA) of genetic differentiation among populations implemented in GenAlEx. Spatial autocorrelation (isolation by distance or IBD) of population pairwise genetic divergence based on SNP loci was assessed using the Mantel function in GenAlEx, and significance was assessed on 1000 permutations. The overall pattern of genetic structure reflected in the SNP dataset was summarised as a reduced set of orthogonal axes following principal component analyses (PCA) in the package StatistiXL. The first 20 principal components, which had an Eigen value ≥1, and cumulatively accounted for ≥50% of the genotypic variance in the SNP data set, were used to describe genetic structure in association tests.To compare patterns of genetic divergence inferred from the different marker systems correlations between matrices of pairwise population FE. camaldulensis. Genetic structure in this species will potentially confound tests aimed to detect adaptation based on co-variation of genetic markers and environmental parameters, in light of the fact that neutral genetic variation and environment are autocorrelated among populations In this study we investigate signatures of genetic adaptation in candidate genes that could underlie adaptive variation in environmentally contrasted populations of ST) at individual loci (genes or SNPs) with respect to a neutral model The SNP markers employed in this study reside within functional genes, and their allele frequencies may be subject to locus-specific effects such as selection. One approach to detect such effects is to assess observed differentiation (FST conditioned on heterozygosity (He) ranging from 0 to 1 was generated via coalescent simulation over 40000 loci applying a Hierarchical Island Model that accounts for user defined population structure ST for SNP loci were subsequently tested. The mean, 97.5% (upper) and 2.5% (lower) confidence intervals for the simulated FST distribution were calculated using ‘cplot’, which is distributed with the Fdist2 package ST and He for 59 SNP loci were generated using ‘datacal’, also distributed with Fdist2. Estimates of FST and He averaged over whole genes were generated using GenAlEx. Significance of marker FST was tested using the ‘pv’ program distributed with Fdist2. Taking a conservative approach, markers which exceeded the upper or lower confidence intervals (P≤0.01) were classed as outlier loci. Additional outliers were also considered between 0.01<P<0.05. To account for multiple testing the false discovery rate (FDR) and q-values for each locus was estimated Firstly, a neutral distribution of FST was interpreted from posterior odds using Jeffrey's scale A second approach based on the multinomical-Dirichlet model was applied to identify SNP loci that may be under selection using the program BayeScan The spatial analysis method (SAM) of Joost et al. E. camaldulensisTests of association between allelic variation and environment were also performed while attempting to account for potentially confounding population genetic structure using two alternative approaches. Firstly associations between 6 component traits and 59 SNP loci were tested across 191 individuals via a least-squares fixed effect general linear model implemented in Tassel The second method was used to validate environmental associations at six outlier loci identified following analyses with both MatSAM and Tassel. This approach utilised a Bayesian framework to test correlations between allele frequencies at these loci and six component environmental variables against a null model specified by the covariance structure of allele frequencies across populations Genetic relationships estimated upon loci that have been targets of local adaptation might be expected to reflect differences in local selection pressure between populations ST) at SNP loci identified as possible targets of diversifying selection  ranging from 0.19 at Normanby River (QLD) to 0.26 at Fortescue (WA). The overall level of LD between SNPs was low with only 0.6% of pairwise correlations between sites (R2) exceeding 0.2. This indicates that the majority of loci screened segregated independently across the 191 individuals sampled. Genetic differentiation (FST) estimated on all SNPs among populations  was greater than the estimate based on 15 nuSSR loci in the same populations (excluding Wirrengren plain) (FST = 0.08) SNP diversity generally conformed to Hardy Weinberg expectations within populations (55 out of 59 loci). When analysed as a combined sample, 76 percent of SNPs departed expectations, reflecting a Wahlund effect due to population structure. When estimated over all SNP loci, genetic diversity was moderate   and refulgens (pilbara) had greater affinity to subspecies minima when relationships were inferred from SNP markers than nuSSRs. Isolation by distance (IBD) was found to be significant when divergence at SNP loci among population pairs was compared with physical distance separating populations   , suggestp<0.001) . Differep<0.001) . This reST) at candidate loci (whole genes and SNPs) was compared to a neutral FST distribution simulated under a hierarchical island model ST outlier tests identified ten SNPs (from: PIP2 (4 SNPs), Dehydrin (3 SNPs), Erecta (1 SNP), COBL4 (1 SNP) and COMT1 (1 SNP)), as well as three genes , whose divergence was greater than expected under neutrality (p<0.01), and whose pattern of allelic diversity among populations might be accounted for by diversifying selection  to very strong (Log10Bayes factor >1<2) (10Bayes factor >0<0.5) based on Jeffrey's scale. Importantly, each of these loci were also identified by the first method employing coalescent simulations. BayeScan is expected to be a more conservative test for outliers than other methods, as it allows for deviations from a simple island model. Model averaging from the posterior distribution in BayeScan also tended to underestimate SNP differentiation (FST) compared to the method-of-moment estimates from allele frequency variance components in Fdist2 and GenAlEx.Outlier testing using an alternative Bayesian analyses identified three loci (from: Erecta (1 SNP) and PIP2 (2 SNPs)) where evidence in support of the model including diversifying selection was substantial (Logor >1<2) . Two adde) and environment among populations. Population level heterozygosity estimated from a reduced data set containing outlier SNP loci only was significantly correlated with environment , ecolPCA1  and geogPCA2 ) ) . CovariaCLIMPCA1 .To investigate further the adaptive evolutionary model suggested for some SNP loci following outlier tests, we looked for evidence of variation in selective constraints among populations that could explain allelic diversity. Tests of association between allelic variation and component environmental variables were performed using several alternative approaches. Firstly, logistic regression of allelic variation and component environmental variables was performed using MatSAM. This revealed associations between environment and allele frequency that were significant for both the LRT and Wald test following Bonferoni correction for number of loci, and supported a hypothesis of adaptive evolution at eleven outlier loci . HoweverST outliers were significantly associated with one or more environmental variable under the GLM , ranged between three and twelve percent, which at the upper end is large compared to those typically observed for quantitative traits in trees To correct for population structure SNP-environment associations were tested via two further approaches, a general linear model (GLM) implemented in the program Tassel to test for covariance of genotype and environmental scores across individuals,  the GLM . The inc10Bayes factor >0.5<1) based on Jeffrey's scale  . In totatructure . We alsoCLIMPCA1 .Eucalyptus camaldulensis across the species range. These populations are differentiated for climate, primarily reflecting variation in rainfall, evaporation, temperature and sunlight which potentially drive differences in water availability, a strong driver of adaptation in diverse plant species Natural populations occurring across environmental gradients offer opportunities to detect signatures of selection resulting from local adaptation, and test specific hypotheses about the spatial, environmental and temporal scales over which adaptive evolution is exerted E. camaldulensis has previously been suggested from variation in adaptive phenotypes which correspond to local environment including: morphological traits  2 assimilation) E. camaldulensis provenances approximating the species natural distribution. This approach provided for the first time a coarse indication of the distribution of potentially adaptive variation in this species at a broad landscape scale.Local adaptation among provenances and populations of ArabidopsisPopulus nigra . In a recent gene expression study in E. camaldulensis both PIP2 and ERECTA were found to be differentially expressed (down regulated) in droughted compared to well watered E. camaldulensis plants We identified six putative examples of adaptive evolution based on variation in SNP allele frequencies among populations and co-variation with environment . These Se) for outliers and climate initially suggested this (CLIMPCA1), was subsequently confirmed for several outlier loci using three different methods while accounting for demographic effects  and exhibited significant adaptive structure  in natural populations of river red gum CLIMPCA1). Tests of association indicate this cline in allele frequency reflects a greater proportion of T:T homozygotes at this locus in the driest populations. The heterozygote at this locus is associated with climates intermediate to the two homozygotes, suggesting an additive mode of gene action on an unknown adaptive trait. Similarly for ERECTA, an increase in the minor (G) allele frequency was associated with reduced values of CLIMPCA1 . The cline in allele frequency at this locus reflects a greater proportion of T:T homozygotes in the driest populations, and both the heterozygote G:T and homozygote G:G are associated with lower temperature and evaporation, suggesting a dominant mode of gene action  from Erecta (a putative leucine-rich repeat receptor-like kinase) and SNP58 (C/T) from PIP2 (an aquaporin), which were both associated with climate  among populations for several putatively adaptive loci was related to population level dissimilarity for CLIMPCA1 and GEOGPCA2 (CLIMPCA1 (2 = 0.008) co-variation of their inferred population genetic structure could point to concerted selection acting upon unlinked loci, both in genes influencing plant water relations, in response to a common selection pressure 2 = 0.41).Genetic divergence  is plotted on the x-axis and PCA casewise scores for populations on the y-axis in each case.(TIF)Click here for additional data file.Figure S2ST estimated on all SNP loci as compared to the 15 nuSSR loci from Butcher et al 2009 .Mantel correlation of pairwise population F(TIF)Click here for additional data file.Figure S3a) 59 SNP and (b) 15 nuSSR markers which indicate grouping by sub species: subsp. minima (•), subsp. obtusa (o), subsp. arida (♦), subsp. refulgens (Δ), subsp. simulata (◊) and subsp. camaldulensis (▴).Genetic relationships among populations, inferred from principal coordinate analysis (PCoA) for ((TIF)Click here for additional data file.Figure S4ST estimated from ANOVA variance components on all SNP loci as compared to physical distance between populations in kilometres (km) identified significant isolation by distance .Mantel correlation of pairwise population F(TIF)Click here for additional data file.Figure S5CLIMPCA1) for: a) Dehydrin , b) PIP2 , c) COMT .Heterozygosity (y-axis) estimated within populations for outlier genes plotted as a function of environment ((TIF)Click here for additional data file.Figure S6Variation in population level allele frequency (x-axis) for the six outlier SNP loci presented in (TIF)Click here for additional data file.Table S1Mean annual estaimtes for environmental variables applied in principal component analyses.(DOCX)Click here for additional data file.Table S2Correlations (loading) between environmental variables and principal components.(DOCX)Click here for additional data file.Table S3Summary of the 59 SNP loci used in this study. Minor allele frequency (MAF) was determined over all populations. Amino acid abbreviations according to IUPAC conventions.(DOCX)Click here for additional data file.Table S4E. camaldulensis individuals used in this study.Biallelic genotype calls for all SNP loci screened across each of the 191 (CSV)Click here for additional data file.Table S5Whole genes and SNP loci identified as having divergence more extreme than expected when compared to the neutral distribution simualted in Arlequin.(DOCX)Click here for additional data file.Table S6SNP loci for which a model including selection was supported following analyses with BayeScan.(DOCX)Click here for additional data file.Table S7SNP alleles exhibiting significant covariation (p≤0.05) with environment following bonferroni correction for both the Wald and likelihood ratio test implemented in SAM.(DOCX)Click here for additional data file.Table S8Significant assocaitions (p≤0.05) identified with component environemntal variables following adjustment for popualtion structure and multiple testing (permutation) in Tassel.(DOCX)Click here for additional data file.


Althea officinalis  and Astragalus membranaceus, compared with commercial, field-grown roots. Hydroponically grown root extracts from both plant species were found to significantly reduce UVA-induced DNA damage in cultured human lung and skin fibroblasts, although initial Astragalus experimentation detected some genotoxic effects, indicating that Althea root extracts may be better suited as potential constituents of dermatological formulations. Glasshouse-grown soil and hydroponic Althea root extracts afforded lung fibroblasts with statistically significant protection against UVA irradiation for a greater period of time than the commercial field-grown roots. No significant reduction in DNA damage was observed when total ultraviolet irradiation (including UVB) was employed (data not shown), indicating that the extracted phytochemicals predominantly protected against indirect UVA-induced oxidative stress. Althea phytochemical root extracts may therefore be useful components in dermatological formulations. As lifetime exposure to ultraviolet (UV) radiation has risen, the deleterious effects have also become more apparent. Numerous sunscreen and skincare products have therefore been developed to help reduce the occurrence of sunburn, photoageing, and skin carcinogenesis. This has stimulated research into identifying new natural sources of effective skin protecting compounds. Alkaline single-cell gel electrophoresis (comet assay) was employed to assess aqueous extracts derived from soil or hydroponically glasshouse-grown roots of Skin cancer incidence is known to have increased significantly in the last 20 years, despite nonmelanoma skin cancer (NMSC) being greatly under reported –4. As li in vitro studies that UVA irradiation has a more significant role in skin carcinogenesis than previously thought .The root crop subsequently harvested was dried gently at 40°C in a forced air flow oven until reaching constant temperature before processing using a novel extraction method using superheated water  to optimize the production and extraction of UV protecting molecules and polysaccharides . Ten andThese processes produced consistent, pure samples of the roots of each plant species ready for testing (or use in a sunscreen product). All samples for comet assay analysis were supplied in phosphate buffered saline (PBS) at various concentrations (mg/mL) and were confirmed to be sterile solutions via microbiological analysis.2 in Eagles modified medium (EMEM) supplemented with 10 or 15% fetal calf serum (FCS), respectively, 200 mM L-glutamine and 2% penicillin/streptomycin solution (1000 iu penicillin and 1 g streptomycin). All reagents were supplied by Sigma (UK) unless otherwise stated. Monolayers of cells were grown aseptically in 25 cm2 vented tissue culture flasks until they were 70% confluent and were then washed twice with PBS to remove the spent medium. Fresh medium was added (10 mL) and supplemented with extract (which had been passed through a 0.22 μm filter to maintain sterility) at a dilution factor of 1 : 100  and incubated for one hour before harvesting   derived from the normal lung tissue of a 14-week-old male. Human skin fibroblasts (84BR)  derived from a biopsy of a radiosensitive female were used for subsequent experimentation. Fibroblasts were cultured at 37°C with 5% CO studies ). Cells μL of cell suspension was mixed with 500 μL premolten (43°C) 0.5% low melting point agarose . Aliquots (75 μL) of this cell/LMAgarose mixture were then transferred to each of the two circular sample areas of CometSlide™ glass microscope slides . Slides were left to set at 4°C for 15 minutes before irradiation. Following irradiation (described below), slides were immediately immersed in lysis solution  to prevent cellular repair and kept at 4°C for 1 hour. The DNA was allowed to unwind in an alkaline solution (pH > 13)  for 1 hour at room temperature before carrying out electrophoresis at  for 24 minutes. Following electrophoresis, the slides were rinsed with ethanol and then left to dry at room temperature before analysis. The DNA was stained using ethidium bromide (10 μg/mL) and DNA migration (% tail DNA) analysed using a fluorescence microscope connected to specialist image analysis software .Alkaline single-cell gel electrophoresis (comet assay), described by Singh et al. , can assIrradiation was administered using a 200 W xenon-mercury UV light source  with a four-furcated liquid light guide directed towards test areas on four separate comet slides simultaneously. Test areas were exposed to uniform, stable light intensity within the same wavelength range as that of terrestrial solar radiation, with (UVA + visible irradiation) or without (UVB + UVA + visible irradiation) the presence of a 320 nm cut-on filter  to remove wavelengths below 320 nm. UVA + visible light exposure was carried out for a minimum of 12 minutes, UVA + UVB + visible light exposure for 60 seconds (data not shown). These irradiation levels were determined as those required to reliably initiate sufficient DNA damage (circa 50%) in this test system and observe any significant changes (positive or negative) attributable to the presence of phytochemicals in the extracts being tested.The potential effects of the presence of antioxidants on cellular DNA in this test system were initially assessed using N-acetylcysteine , a low molecular weight antioxidant linked to free radical scavenging and singlet oxygen quenching as a positive test control substance (data not shown).U test. Box-whisker plots were produced using SigmaPlot 11.0 to indicate the median (solid line), ±25% of the data (box), and 10% to 90% spread of the data (whisker).Statistical analysis was conducted on median values utilising the nonparametric Mann-Whitney p < 0.001). These extracts were also found to increase levels of DNA damage on exposure to UVA irradiation (p < 0.001) (MRC-5 cells (human lung fibroblasts) were incubated for one hour with or without timed extracts from AL or AS and then they are exposed to either 16 minutes of filtered xenon-mercury irradiation (UVA + visible light) or no irradiation (dark control) . No incr< 0.001) . These rp < 0.05) and ultrafiltrated extracts R2, F2, R3, and F3 (p < 0.001) significantly reduced UVA-induced DNA damage. The change in DNA damage in cells following UVA irradiation incubated with AS whole extract W1 was not found to be of statistical significance and extract W3 was found to statistically increase the level of DNA damage observed (p < 0.001). With AL phytochemical exposure followed by irradiation (p < 0.001). So, although it would appear that extracts from the AS species could potentially be effective against UVA exposure, their capacity to induce UVA damage cannot be ignored, particularly if considering its use in emollients in future human studies. Future studies therefore concentrated on Althea, whose retained and filtered extracts all significantly reduced the UVA-induced genotoxicity produced in this cell type  when using extracts W2 from both plant species . This wap < 0.001) when compared to the dark control. This genotoxic damage was reduced in the presence of whole extract W2 from either AS or AL (p < 0.001), although the responses observed with each species at 15 and 18 minutes of exposure were not significantly different from one another (p > 0.05). Although these results cannot be directly extrapolated to those of an in vivo skin system which has vasculature and immunological factors to consider, there does appear to be target compound or compounds present in the extracts, which is combating the oxidative stress-induced genotoxic damage being produced by UVA radiation. Furthermore, as these positive results were obtained using “whole” root extracts, it appears that the extra processing step of ultrafiltration was not essential for efficacy.Experimentation was also conducted to consider the effect of UVB on the more robust and UV-sensitive human lung fibroblasts (MRC-5). None of the whole or ultrafiltrated extracts of AL or AS were found to reduce the levels of DNA damage observed using UVB + UVA + visible light with the experimental conditions employed . This was not unexpected as UVB is able to damage DNA directly and would suggest that either the components of the extracts were not able to absorb the UVB or there was insufficient amount of effective material present. This also indicated that the protection observed against UVA + visible irradiation in Figures Due to the novel extraction system used, the exact constituents of the test extracts employed and their concentration were unknown. Chemical analysis  established that AS extracts contained simple phenolics , various flavonoids, isoflavones, and saponins . AL extracts were found to predominantly contain carbohydrates and simple phenolics as well as 8-hydroxyflavones including luteolin (the latter found more so in extract W2). Several unidentified flavonoid-like compounds were also detected. Althea plants that had experienced different growing conditions. Due to the potential genotoxicity of extracts from AS . All AL extracts (independent of source) significantly reduced the effect of UVA-induced DNA damage with 12 minutes of UVA + visible light (p < 0.001) (p < 0.001) as did the glasshouse soil-grown root extract (p < 0.001), although in each case the protection afforded diminished with the continued light exposure. The extract from the commercially derived Althea roots offered the least period of protection (Increasing periods of irradiation without extract incubation resulted in increased DNA damage (< 0.001) . Hydropo< 0.001)  continue extract   over and above that achieved via more traditional growing methods. No significant reduction in DNA damage was observed when total ultraviolet irradiation (including UVB) was employed, indicating that it is most likely that the extracted phytochemicals predominantly protected against indirectly produced UVA-induced oxidative stress. This factor could be considered in more detail within future experimentation employing the enzyme formamidopyrimidine DNA glycosylase (FPG) to modify the comet assay protocol to reveal oxidised bases.From the point of view of preventing photoageing and/or potential skin carcinogenesis, the inclusion of such compounds in formulations designed to protect the skin may with further investigation prove to be beneficial. The conclusions that can be drawn from the data presented here in this particular respect, however, are somewhat limited. This is because the alkaline comet assay is only detecting genotoxic DNA damage and does not indicate where in the genome the damage is occurring or whether this damage may potentially be mutagenic or carcinogenic. It cannot therefore be concluded definitively that by preventing this genotoxic damage with phytochemical containing root extracts this would prevent or reduce cancer development, although this may be feasible. Additionally the cells were lysed immediately following light irradiation and so they were given no opportunity to repair the light-induced damage sustained or alternatively to trigger apoptotic cell death, and future investigations should consider these aspects.So, in conclusion, this investigation has demonstrated that phytochemical containing root extracts do have the potential to be useful natural components in dermatological formulations where a reduction in oxidative stress-induced damage is desired, with the glasshouse-grown soil derived AL roots producing the greatest level of protection against UVA-induced DNA damage observed. Additionally, more extensive chemical analysis of the extracts may be able to identify the individual phytochemical effector(s) involved in the protection afforded by these plants and further research may indicate whether these compounds do indeed have the potential to prevent some of the carcinogenetic processes known to be induced by sunlight.


TSHB, TRHR, IGSF1) in isolated TSH deficiency, or early  or late  pituitary transcription factors in combined hormone deficits. Since TSH cannot be used as an indicator of euthyroidism, adequacy of treatment can be difficult to monitor due to a paucity of alternative biomarkers. This review will summarize the normal physiology of pituitary development and the hypothalamic–pituitary–thyroid axis, then describe known genetic causes of isolated central hypothyroidism and combined pituitary hormone deficits associated with TSH deficiency. Difficulties in diagnosis and management of these conditions will then be discussed.Central congenital hypothyroidism (CCH) may occur in isolation, or more frequently in combination with additional pituitary hormone deficits with or without associated extrapituitary abnormalities. Although uncommon, it may be more prevalent than previously thought, affecting up to 1:16 000 neonates in the Netherlands. Since TSH is not elevated, CCH will evade diagnosis in primary, TSH-based, CH screening programs and delayed detection may result in neurodevelopmental delay due to untreated neonatal hypothyroidism. Alternatively, coexisting growth hormones or ACTH deficiency may pose additional risks, such as life threatening hypoglycaemia. Genetic ascertainment is possible in a minority of cases and reveals mutations in genes controlling the TSH biosynthetic pathway ( TRHR), thyroid stimulating hormone β subunit (TSHB) and the more recently-described immunoglobulin superfamily member 1 gene (IGSF1)  or late  transcription factors may cause central hypothyroidism with or without associated extrapituitary abnormalities. However, in these cases, central hypothyroidism does not occur in isolation, but is one of the evolving pituitary hormone deficiencies  is a rare disorder in which inadequate thyroid hormone biosynthesis occurs due to defective stimulation of a normal thyroid gland by thyroid stimulating hormone (TSH). The underlying molecular basis is often undefined, but hypothalamic or pituitary pathology contributes to a qualitative or quantitative deficit in TSH synthesis or secretion . In a miSince TSH is not elevated in CCH, this entity will evade diagnosis in TSH-based, primary CH screening programs, and patients are at risk of neurodevelopmental delay if severe CCH remains untreated postnatally. Additionally, the delayed diagnosis of other pituitary hormone deficiencies  may pose significant risks, such as life threatening hypoglycaemia.In spite of current knowledge regarding genetic causes of CCH, the majority of cases do not have an identifiable molecular defect in known causative genes. This review will summarize our current understanding of the molecular genetics underlying CCH as well as highlighting controversies in treatment and diagnosis.The development of the anterior pituitary requires the sequential temporal and spatial expression of a cascade of signaling molecules and transcription factors that are important for organ commitment, cell proliferation, patterning and terminal differentiation. Human pituitary gland development in humans largely mirrors that in rodents  Fig. 1)Fig. 1). Cga) in a restricted patch of cells in the ventral region of Rathke's pouch. These αGSU positive cells will express the transcription factor Islet-1 (Isl1) and will differentiate at E12.5 by initiating the expression of thyroid stimulating hormone subunit-β (Tshb) (Pomc (GH and Prolactin (Prl) by E15.5 is the hallmark of the differentiation of somatotrope and lactotrope lineages respectively. Whilst gonadotropes are the last cell type to emerge, beginning at E16.5 with the onset of luteinizing hormone (LH) subunit β (Lhb) expression, followed by follicle-stimulating hormone (FSH) subunit β (Fshb) a day later  . The defAlthough the classic description of cell differentiation is based on the sequential appearance of differentiating markers, recent birthdating studies imply that endocrine cells may be specified earlier and migrate some distance before they can be characterized by their differentiated markers. In fact, most of the hormone expressing cell types appear to differentiate between E11.5 and E13.5, denoting a broader range of specification rather than a sequential pattern of discrete times  of which GATA2, PITX1/2, PROP1 and POU1F1 are most critical for the differentiation of thyrotropes.Signalling molecules from the ventral diencephalon , the oral ectoderm , the surrounding mesenchyme  and the pouch itself  contribute to establish signalling gradients and the expression of transcription factors which will determine the positional identity of ventral pituitary cells . TerminaExpression of Pitx1 is first detected in the anterior ectoderm at E8.0, then expressed throughout the oral ectoderm and in Rathke's pouch by E 9.5, and maintained throughout anterior pituitary development in all hormone-producing cell types. In the adult pituitary, Pitx1 expression is highest in thyrotropes expressing αGSU and in gonadotropes with lower levels in other hormone-producing cell types  is a pituitary-specific paired-like homeodomain transcription factor initially detected in the dorsal portion of Rathke's pouch at E10-10.5; its expression peaks at E12 and becomes undetectable by E15.5 dpc  axis and centrally regulated by TRH (Circulating concentrations of Td by TRH . TRH is d by TRH . Recent CGA and TSHB), TRH mediates conjugation of TSH α and β subunits and regulates glycosylation and secretion of heterodimeric TSH. Incorporation of oligosaccharide determines TSH bioactivity, which is principally defined by the type and conformation of carbohydrate chain added at Asn-76 and -102 of the α subunit and Asn-43 on the β subunit such that TSH with a high sialic acid content exhibits decreased bioactivity and increased half life.  and β subunit genes (4). Deiodination of T4 in peripheral tissues yields the active hormone, 3,5,3′-triodothyronine (T3) which binds thyroid hormone receptors (TR's) to exert its transcriptional effects. TR isoforms TRα1, β1, and β2 exhibit tissue-specific expression patterns and heterodimerize with retinoid X receptor (RXR) on specific Thyroid Receptor DNA response elements (TREs), usually located in promoter regions of target genes. With upregulated genes, unliganded TRs mediate repression of basal gene transcription by binding the TRE together with other proteins, eg corepressors (NCOR1 and SMRT) and histone deacetylases (HDAC3) , stimulating follicular cell growth and thyroid hormone synthesis and release, predominantly in the form of the prohormone thyroxine  for uptake from tanycytes into neuronal cells and by organic anion-transporting polypeptide 1c1 (OATP1C1) for transport across the blood-brain barrier , and the influence of feeding behaviour, glucocorticoids, severe illness, cold, and circadian rhythm  shared with other glycoprotein hormone  family members and a hormone-specific β-subunit (TSHB) in which naturally-occurring mutations either truncate the protein, or perturb key structural features required for heterodimeric integrity . In commTSHB mutations have been described, including missense , nonsense and frameshift mutations   leading to a cysteine 125 to valine change (p.C125V) and subsequent frameshift and premature stop codon at position 134 (p.C125Vfs*10) , which disrupt heterodimer formation between TSHα and β polypeptides generally result in unmeasurable serum TSH concentrations, whereas mutations , which preserve formation of some mutant, heterodimeric TSH expressing the epitopes recognized by the anti-TSH monoclonal antibody, enables TSH to be detected in an immunoassay-dependent manner. Thus, some cases may have detectable levels of immunoreactive TSH, but these species will lack normal bioactivity , but circulating T4 levels rose appropriately following levothyroxine withdrawal, indicating that synthesis of bioactive TSH could occur in the absence of TRH signalling , was only diagnosed with central hypothyroidism following family screening at the age of 33, having previously achieved two normal pregnancies with subsequent lactation. Although TRHR is expressed in lactotrophs as well as thyrotrophs, and stimulates prolactin secretion in response to i.v. TRH administration, its physiological role in these cells is unclear. Both male and female individuals with biallelic TRHR mutations had absent TSH and prolactin responses to TRH administration. However, the obstetric history in the p.R17* homozygous female suggests that TRH action is not obligatory for pregnancy and lactation in humans , the structure of TRHR includes an extracellular N-terminus and intracellular C-terminus, flanking seven transmembrane domains connected by three intracellular and three extracellular loops, which form a ligand-binding pocket within the plasma membrane. TRH-binding results in a conformational change involving transmembrane helices 5 and 6 and the third intracellular loop, with subsequent carboxyterminal domain-mediated G-protein coupling . In bothTrhr1 who also exhibit subnormal T4 and T3 levels with inappropriately normal TSH  gene are the most recently identified cause of central hypothyroidism, with an estimated incidence of up to 1:100 000 . The variable phenotypes, associations and MRI findings are summarized in Central congenital hypothyroidism in association with combined pituitary hormone deficiencies may be i) syndromic, resulting from mutations in early transcription factors, and associated with developmental abnormalities  or ii) non syndromic resulting from mutations in late transcription factors  have now been implicated in their aetiology. In this respect, the TSH deficiency may be part of the initial presentation or evolving phenotype of these patients (Septo-optic dysplasia (SOD) is defined by the combination of two of the following i) optic nerve hypoplasia (ONH) ii) midline forebrain defects  and iii) pituitary hypoplasia with variable hypopituitarism. The commonest endocrine defect is GH deficiency followed by TSH and ACTH deficiency, whilst gonadotropin secretion may be retained. Mutations in LHX3, LHX4) lead to multiple pituitary hormone deficiencies, including TSH deficiency. Expression of Lhx3 is initially detected uniformly within Rathke's pouch from E9.5 and by E16.5 it is expressed in the developing anterior and intermediate pituitary where its expression persists into adulthood. Mice with a targeted homozygous disruption of Lhx3 die shortly after birth and exhibit pituitary aplasia. Although Rathke's pouch is initially formed, its expansion is arrested by E12.5 and lacks almost all of the hormone-secreting cell types, containing only a small population of corticotropes  is a transcription factor required for the formation of anterior structures and maintenance of the forebrain and has been implicated in 2–3% of anophthalmia/microphthalmia syndromes in humans  results in a diagnosis of CCH in as many as 1 in 16 000 newborns, the majority of whom will have additional pituitary hormone deficits , declining fT4 concentrations may be an early indicator of CCH, with a time-related decline in fT4 of >20% quoted diagnostically in individuals with acquired pituitary disease  and TRHR mutation (blunted responses)  will have additional pituitary hormone deficiencies  require long-term surveillance for evolving ACTH and other pituitary hormone deficiencies. Conversely, identification of a genetic defect specific to the TSH biosynthetic pathway  enables reassurance that additional hormone deficits will not develop. Reports of families with TRHR or IGSF1 mutations also highlight the fact that family screening following diagnosis in a young proband, may identify apparently healthy first, second or third-generation family members with hitherto undiagnosed central hypothyroidism. Untreated subclinical hypothyroidism is associated with adverse cardiometabolic risk, thus these individuals, who have overt central hypothyroidism may benefit from levothyroxine treatment both for cardiovascular health and for quality of life  has some utility, but detects overtreatment more readily than undertreatment. SHBG, bone turnover markers, and cholesterol are all sensitive to changes in fT4, but the fact that they are also influenced by GH and gonadal status, renders them unreliable in combined pituitary hormone deficits  −/− null mice in whom TSH, Tin vitro and in vivo studies are required to delineate its mechanistic role in the anterior pituitary and its effects on testicular growth. Although current data suggest it is required for adequate TRHR mRNA expression, the expression of IGSF1 in all cells of the POU1F1 lineage and observed abnormalities of other hormone levels  in deficient humans suggest that its role is more complex. Other cell surface immunoglobulin superfamily members bind specific antigens and interact with other receptors, such that a role for IGSF1 in paracrine signaling in the pituitary is plausible , and delayed diagnosis may result in neurodevelopmental delay.CCH will evade diagnosis on TSH-based screening programmes, therefore a high index of suspicion is needed for prompt detection. The mainstay of biochemical diagnosis is the presence of subnormal T4 with inappropriately normal/low TSH, after exclusion of assay interference.CCH may be isolated or occur as a component of combined pituitary hormone deficiencies (majority of cases). Genetic ascertainment may predict evolution of other hormone deficits and enable prompt diagnosis and treatment of affected siblings.In combined pituitary deficits, phenotypes can be highly variable, and can evolve, therefore careful ongoing assessment is required in these patients.Family screening in cases with IGSF1 or TRHR mutations is important to identify apparently healthy relatives with hitherto undiagnosed central hypothyroidism who may benefit from levothyroxine treatment.Levothyroxine is an effective treatment for CCH; fT4 levels should be maintained in the mid-upper part of the normal range.Cortisol deficiency should be corrected before initiation of levothyroxine and the clinician should be alert to the fact that other hormone replacements  may alter levothyroxine requirements.


Escherichia, Salmonella, Klebsiella, and Enterobacter genera do not encode an AHL synthase but they do encode an AHL receptor of the LuxR family, SdiA. Instead of detecting their own AHL synthesis, these organisms use SdiA to detect the AHLs synthesized by other bacterial species. In this study, we used a genetic screen to identify AHL-responsive genes in a commensal Enterobacter cloacae strain that was isolated from a laboratory mouse. The genes include a putative type VI secretion system, copA (a copper transporter), and fepE (extends O-antigen chain length). A new transposon mutagenesis strategy and suicide vectors were used to construct an sdiA mutant of E. cloacae. The AHL-responsiveness of all fusions was entirely sdiA-dependent, although some genes were regulated by sdiA in the absence of AHL.Many bacteria determine their population density using quorum sensing. The most intensively studied mechanism of quorum sensing utilizes proteins of the LuxI family to synthesize a signaling molecule of the acylhomoserine lactone (AHL) type, and a protein of the LuxR family to bind AHL and regulate transcription. Genes regulated by quorum sensing often encode functions that are most effective when a group of bacteria are working cooperatively . Bacteria in the  Vibrio fischeri, in which luminescence is most effective when the entire population participates  as one of many inputs to gene regulation. The genes regulated by quorum sensing are often those that provide maximal benefit when expressed simultaneously throughout a population  type  the rck (resistance to complement killing) operon located on the virulence plasmid, pSLT , a single gene horizontal acquisition that encodes an effector protein that is secreted by type III secretion system 2 (T3SS2)  and has also been found to repress the expression of flagella genes and the enterocyte effacement (LEE) locus  -L-homoserine lactone (oxoC6) was obtained from Sigma-Aldrich and dissolved in ethyl acetate that had been acidified by the addition of 0.1 ml glacial acetic acid per liter (EA)  plate reader. Those wells that had greater than 3-fold difference after 9 h were streaked for isolation on LB kan nal plates at 37°C overnight. For confirmation, one colony from each plate was inoculated into LB kan nal broth or 0.3% motility agar in 96-well format in the presence of oxoC6, or the solvent control, EA. Plates were read on the Victor plate reader every 3 h. Those fusions that demonstrated greater than 2.5-fold AHL-dependent induction after 9 h were saved for future studies.Transposon mutagenesis was performed by mating BW20767+pUTmTnluxC coding region oriented out of the transposon. Oligonucleotides were synthesized by Integrated DNA Technologies . DNA sequencing was performed by the Plant Microbe Genomics Facility at The Ohio State University. The sequence adjacent to the transposon insertion site was used for BLASTN searches using the BLAST program at the National Center for Biotechnology Information (NCBI).Genomic DNA was isolated from overnight cultures of the transposon insertion mutants using the GenElute™ Bacterial Genomic DNA Isolation kit . The transposon insertion site in the genomic DNA was sequenced using Sanger sequencing with two different primers, BA247 and BA1090 (Table NlaIII (NEB) for 3 h at 37°C. The enzyme was inactivated for 20 min at 65°C, then T4 DNA ligase (NEB) was added to the digestion reaction in a total reaction volume of 200 μL at 16°C overnight. The ligation reaction was purified using the QiaQuick PCR purification kit (Qiagen), digested with XmnI for 3 h at 37°C, then heat inactivated at 65°C for 20 min. This digest was used in a PCR reaction using the primers IPCRF and IPCRR (Table Taq DNA polymerase (NEB). The PCR product was sequenced using the IPCRF primer at the Plant Microbe Genomics Facility at The Ohio State University.Not all insertion points were identified using Sanger genomic DNA sequencing. The rest were identified using inverse PCR. Genomic DNA was digested with sdiA+ strains or LB kan cam for sdiA mutants, and grown at 37°C shaking overnight. They were then subcultured 1:100 in triplicate into either LB broth or LB motility agar containing the appropriate antibiotics and either 1 μM oxoC6 or 0.1% EA as the solvent control and then placed in the well of a black clear bottom 96-well plate. The plate was grown with shaking at 37°C and time points were taken by placing the 96-well plate in the Wallac Victor plate reader. For broth cultures, both OD590 and luminescence were measured. For the motility agar assays, only the luminescence was measured. For the AHL concentration sensitivity assay, an sdiA-regulated fusion was assayed in LB broth or motility agar with 10-fold dilutions of either oxoC6 or oxoC8 starting at a concentration of 1 μM. AHL-dependent changes in gene expression were analyzed using a Two-Way ANOVA over the time course. P-values were marked accordingly . The sdiA-dependent changes in gene expression were larger than the AHL-dependent changes and were statistically significant (p ≤ 0.00005) for all fusions in all growth conditions for at least one time point.Strains were grown in LB kan for in vitro recombination reactions. To modify these vectors, a blunt-ended DNA fragment (Reading Frame Cassette C) obtained from Invitrogen containing attR-camr-ccdB-attR was ligated into the SmaI site of pGP704 and pDMS197, resulting in pMO704 and pMO197, respectively , was then used in an “LR” cloning reaction to recombine the mutated sdiA gene into the new pMO197 suicide vector by transforming BW20767, creating pASD708. This vector was then mobilized into each of the E. cloacae strains, selecting for replacement of the wild-type sdiA allele with the mutant allele, which was verified by PCR using primers BA2343 and BA1598.With the suicide vectors completed, the m Figure . EssentiEnterobacter cloacae strain, JLD400, was found to be genetically tractable and sensitive to all antibiotics tested except ampicillin  using the transposon mTn5-luxCDABE . Seventeen insertions were identified that demonstrated an increase in luminescence greater than 2.5-fold.In order to identify genes that are regulated in response to AHLs in i Ahmer, . TherefoEnterobacter cloacae isolate is not known, but BLAST searches revealed that the majority of these sequences were most similar to the genome sequence of Enterobacter cloacae subspecies cloacae NCTC 9394 (FP929040). The transposon insertions of six strains  were within ENC_40870, which encodes a hypothetical protein only present in Enterobacter cloacae. JLD501 and JLD511 each contained an insertion in ENC_10940, which encodes a hypothetical protein with a secretion signal predicted by SignalP within the first 20 amino acids  sequencing genomic DNA using two different sequencing primers that bind within the transposon sequence and are oriented outward, or 2) inverse PCR and subsequent sequencing of the product. After identification of the transposon insertion sites, a confirmatory PCR was performed using a primer within the transposon and another within the putative AHL-responsive gene. A positive PCR reaction confirms that the transposon insertion is in the correct location, but we also sequenced the resulting PCR product to further define the transposon insertion site. All 17 AHL-responsive fusions were located within 8 unique genes . Indeed, mutation of sdiA in each fusion strain eliminated any responsiveness to AHL in both liquid culture and motility agar   for the SdiA regulon in this isolate of  Figures , 10. Thee Figure . It woulE. cloacae that promotes the growth of rice roots, SdiA represses biofilm formation and rice root colonization (Shankar et al., sdiA-dependent repression of the genes encoding curli fimbriae (Shankar et al., E. coli as well (Lee et al., sdiA in this isolate, or if we simply missed them, either by chance or due to growth conditions. It would be interesting to determine if the genes identified in this study are regulated by sdiA in the E. cloacae plant isolate and to determine if the sdiA regulon has diverged between the plant and mouse isolates, or if the regulon has remained largely the same.In an isolate of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.