Lytic infection of differentiated cell types with human cytomegalovirus (HCMV) results in the temporal expression of between 170–200 open reading frames (ORFs). A number of studies have demonstrated the temporal regulation of these ORFs and that this is orchestrated by both viral and cellular mechanisms associated with the co-ordinated recruitment of transcription complexes and, more recently, higher order chromatin structure. Importantly, HCMV, like all herpes viruses, establishes a lifelong latent infection of the host—one major site of latency being the undifferentiated haematopoietic progenitor cells resident in the bone marrow. Crucially, the establishment of latency is concomitant with the recruitment of cellular enzymes that promote extensive methylation of histones bound to the major immediate early promoter. As such, the repressive chromatin structure formed at the major immediate early promoter (MIEP) elicits inhibition of IE gene expression and is a major factor involved in maintenance of HCMV latency. However, it is becoming increasingly clear that a distinct subset of viral genes is also expressed during latency. In this review, we will discuss the mechanisms that control the expression of these latency-associated transcripts and illustrate that regulation of these latency-associated promoters is also subject to chromatin mediated regulation and that the instructive observations previously reported regarding the negative regulation of the MIEP during latency are paralleled in the regulation of latent gene expression. Human cytomegalovirus (HCMV) is an opportunistic pathogen that, like all herpes viruses, can establish a latent infection that persists for the lifetime of the host. In healthy individuals both primary infection and the reactivation of latent virus rarely causes any significant clinical symptoms due to a robust immune response in the host ,2. In coThe ‘histone code hypothesis’ predicts that the signature of the post-translational modifications of histone proteins directly impacts on the transcriptional activity of the cell ,11,12. IThe regulation of the major immediate early promoter (MIEP) during latency and reactivation has been the subject of a number of recent reviews and, thus, it will not be covered extensively, here. Suffice to say, the regulation of the MIEP during latency and reactivation exhibits many hallmarks associated with chromatin-mediated regulation of eukaryotic gene expression. The MIEP in latently infected CD34+ haematopoietic cells or circulating monocytes isolated from healthy seropositive donors is predominantly associated with HP1 —a markerIt is now well accepted that differentiation-dependent changes in the post-translational modifications of histones around the MIEP regulate latency-associated repression and reactivation of MIEP activity. However, much less is known about viral gene expression associated with latent infection and whether this is also regulated by histone modifications. With the advent of more sensitive high throughput screening techniques, the identification of a small number of latency associated transcripts  has been possible. These transcripts expressed during latent infection are from viral genes dispersed throughout the viral genome, suggesting that no one region is particularly ‘latently active’ but rather that latency-associated transcripts are regulated independently by specific promoters.In this short review, we will present the current status of our knowledge regarding the regulation of expression of the latency associated transcripts of HCMV and its relationship to post-translational modifications of histones.in vivo likely due to the robust immune response directed against EBV in the host promoting their elimination. Indeed, the most common phenotype (latency 0) is genome carriage in the absence of latent gene expression [in vivo [A key biological property of all herpes viruses is their ability to undergo latent infection during which time only a subset of viral genes are expressed. Latent herpes simplex virus (HSV) gene expression is predominantly restricted to one locus—the latency associated transcript (LAT) region . Alternapression —althoughpression . However[in vivo .In contrast to HSV and EBV, the analysis of latent HCMV gene expression is in its relative infancy. Latent transcripts arising from the major immediate early region had been identified in specific populations of granulocyte-macrophage progenitors by the Mocarski laboratory in the 1990s ,40 and UThe selective expression of viral genes during latent infection is likely to depend on similar mechanisms known to modulate the MIEP during latent and lytic infection. Importantly, aberrant expression of viral gene products not required during latency would likely risk triggering a robust host immune response, from the high memory T cell population known to recognise lytic antigens in normal HCMV carriers, with no benefit to the virus. Consequently, tight control of viral gene expression during latency is likely to be of some import. Since, most of the viral genes expressed during lytic infection are regulated by the prodigious activity of the IE72 and IE86 proteins ,50,51,52et al. [et al. [et al. identified RNAs spanning the UL81 region in naturally latent monocytes and showed that transcription of this RNA actually occurs from the opposite strand encoding UL81 (hence the designation UL81-82ast), giving rise to a putative 133aa serine rich protein called LUNA (Latency Unique Nuclear Antigen). Although there is no known function attributed to LUNA during natural latency, there is some recent evidence that expression of LUNA may impact on HCMV carriage and reactivation in vitro [Of all the putative latent transcripts, the factors controlling expression of the UL81-82 antisense transcript (UL81-82ast) during latent and lytic infection are arguably the best understood ,47,54. Uet al.  whilst s [et al. . Bego etin vitro .Superficially, at least, the regulation of LUNA promoter activity during lytic  and lateAfter confirmation that the expression of LUNA could be detected in experimentally and naturally latent CD34+ cells, a very simple question was asked—is the LUNA promoter associated with histone proteins during latency and, if so, what post-translational modifications do these histones have? Chromatin Immunoprecipitation analyses of the LUNA promoter alongside the MIEP showed that the promoter is indeed associated with histones . FurtherAlthough classified as a latent transcript, LUNA, like other latency associated transcripts, is also expressed during lytic infection . BroadlyThese observations are consistent with the view that chromatin architecture, whilst providing the framework for controlling gene expression, is not the only factor involved in LUNA promoter regulation. Indeed, it is interesting to note that parallels occur in other herpes viruses. EBV encoded BNRF1 disrupts the hDaxx:ATRX interaction to relieve early gene expression from the transcriptional silencing imposed by this complex also .The obvious question that arises from these observations is what drives differential LUNA promoter activity during latent infection? During latency LUNA expression must be independent of IE72 (by definition) whereas LUNA gene expression during lytic infection is IE72-dependent. Clues to the regulation of the LUNA promoter during latency came from a bioinformatics approach identifying a number of putative transcription sites in the LUNA promoter, including the GATA family of transcription factors . GATA trInterestingly, the LUNA promoter is GATA-2 responsive in transfection assays ,47 and wHowever, we hypothesise that in early myeloid cells high levels of GATA-2  out-compete the repressive hDaxx:ATRX complex C. In conin vitro system [The UL144 gene product was originally identified as an orthologue of the tumour necrosis factor receptor superfamily member, herpes virus entry mediator (HVEM) ,91. As so system .et al. [In lytic infection, the UL144 protein is expressed with early kinetics reaching steady state levels by 48 hours post infection . Althouget al. . In contet al. , was UL1et al. [The observation by Poole et al.  that allet al. .Direct evidence for a physical interaction between GATA-2 and UL144 promoter correlating with gene expression was also obtained by performing chromatin immunoprecipitation assays on infected myeloid cells. Crucially, GATA-2 immuno-precipitation with the UL144 promoter was only observed in cells infected with strains of HCMV that supported UL144 expression . FurtherIt is becoming increasingly clear that the regulation of cytomegalovirus gene expression during all phases of lytic infection involves post-translational modification of histone proteins (reviewed in ). Work fin vitro [in vivo [i.e., hDaxx, PML and Sp100) that accumulated at ND10 bodies. Consequently, the virus, through the action of pp71 [Although the exact order of events controlling viral gene expression is still not fully understood, a model for the control of latent viral gene expression, based on a number of instructive observations, can be made. Firstly, the viral genome in the virion is naked—no histone proteins can be detected ,58 consiin vitro  and in v[in vivo . The ass[in vivo ). Indeed[in vivo ). Consis[in vivo  and that of pp71 ,65,108 a of pp71 ,111,112 During latent infection, however, it is likely that the same initial events occur—the genome is chromatinised immediately upon infection and that the same intrinsic immune defences are activated. However, the failure of pp71 to translocate to the nucleus and inactivate these defences upon infection of myeloid progenitors A has beeIt is also likely that these same mechanisms which promote silencing of the MIEP in latency are also important for driving viral latent gene expression. GATA-2 is, like YY1 and ERF, highly expressed in myeloid progenitor cells  and, whi


Xanthomonas type III effector XopD resulting in suppression of AtMYB30-mediated plant defenses. Moreover, the function of AtMYB30 is also tightly controlled by plant cells through protein-protein interactions and post-translational modifications (PTMs). AtMYB30 studies highlight the importance of cellular dynamics for defense-associated gene regulation in plants. Finally, we discuss how AtMYB30 and other MYB TFs mediate the interplay between disease resistance and other stress responses.Transcriptional regulation in host cells plays a crucial role in the establishment of plant defense and associated cell death in response to pathogen attack. Here, we review our current knowledge of the transcriptional control of plant defenses with a focus on the MYB family of transcription factors (TFs). Within this family, the Arabidopsis MYB protein AtMYB30 is a key regulator of plant defenses and one of the best characterized MYB regulators directing defense-related transcriptional responses. The crucial role played by AtMYB30 in the regulation of plant disease resistance is underlined by the finding that AtMYB30 is targeted by the  As sessile organisms, plants must face the diversity of pathogens that they encounter in their habitat. Unlike mammals, plants rely on cell autonomous innate immunity and on systemic signals originating from infection sites  and Pseudomonas syringae pv. syringae avirulent bacteria. The Ntmyb1 protein binds to the promoter of the defense-related gene PR-1a suggesting a role in the regulation of immune responses  DC3000 but down regulates resistance to A. brassicicola via the WRKY70 TF -inoculated Arabidopsis cells . Here, we summarize our current knowledge of the AtMYB30 interaction and regulatory network involved in the control of plant defense responses. Additional roles of AtMYB30 during the integration of other environmental cues are also discussed.AtMYB30 activates genes encoding subunits of the acyl-coA elongase complex and alters the VLCFA content of Arabidopsis leaves. Furthermore, defense-related phenotypes of AtMYB30 transgenic plants are dependent on the VLCFA biosynthesis pathway, supporting the view that AtMYB30 modulates cell death-related lipid signaling by enhancing the synthesis of VLCFAs or VLCFA derivatives   is a modular type III effector protein of 801 amino acids that presents a modular structure and contains different domains with varied biochemical activities , that does not present the HLH domain and localizes homogenously within plant cell nuclei, is not able to interact with AtMYB30 and has no effect on AtMYB30 transcriptional activation. Considering the modular structure of XopD, it is likely that this type III effector mediates multiple molecular (protein-DNA and protein-protein) associations and that, depending on the Xanthomonas strain/host plant interaction, XopD is able to target different host components to subvert plant defense. For example, XopD from Xanthomonas euvesicatoria (Xcv) desumoylates the SlERF4 TF to suppress ethylene responses and promote pathogen growth in tomato  in challenged cells but not in peripheral cells, whereas Ats2-PLAα expression peaks 6 hpi in peripheral but not in challenged cells  as an AtMYB30 interactor in yeast  signaling /BZR1 (brassinazole-resistant1) family of TFs. BR treatment induces AtMYB30 gene expression in Arabidopsis seedlings and in bes1-D plants, that overexpress BES1, AtMYB30 expression is upregulated, indicating that AtMYB30 may function in the BR signaling pathway  signaling. BRs play important roles in several plant growth and developmental processes as well as during stress/disease resistance. BRs signal through the BES1 (Arabidopsis MYB regulator AtMYB30 is a multi-regulated protein that is involved in the integration of various environmental stimuli, including attack by microbes, abiotic stress and hormone signaling, likely through the activation of shared and specific sets of target genes. How simultaneous and diverse stress signals are integrated into a unified cellular response is a major unknown in cell signaling. The acceleration of large data set acquisition and the development of systems biology approaches promise to offer new insights into the functioning of such complex regulatory networks. The wealth of knowledge gained in recent years on Arabidopsis R2R3 MYB TFs provides an excellent framework toward this end.Cellular responses to environmental or physiological cues rely on transduction pathways that must discriminate between different signals and ensure a combinatorial regulation. Thus, combinations of different PTMs and protein-protein interactions provide different layers of information that may allow the integration of several transduction pathways and warrant highly specific cellular outputs. Accumulating evidence shows that the 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.


Although cardiovascular magnetic resonance allows the non-invasive and radiation free visualization of both the coronary arteries and veins, coronary vessel wall imaging is still undergoing technical development to improve diagnostic quality. Assessment of the coronary vessels is a valuable addition to the analysis of cardiac function, cardiac anatomy, viability and perfusion which magnetic resonance imaging reliably allows. However, cardiac and respiratory motion and the small size of the coronary vessels present a challenge and require several technical solutions for image optimization. Furthermore, the acquisition protocols need to be adapted to the specific clinical question. This review provides an update on the current clinical applications of cardiovascular magnetic resonance coronary angiography, recent technical advances and describes the acquisition protocols in use. Despite substantial improvements in prevention and treatment , coronarCardiovascular magnetic resonance (CMR) theoretically provides a combined approach allowing the assessment of coronary arteries, cardiac function, viability, perfusion and cardiac anatomy. Moreover, magnetic resonance angiography (MRA) can potentially be used to directly visualize the coronary vessel wall , providiClinically accepted indications of coronary MRA are currently limited to the assessment of anomalies of the coronary arteries (class I indication) and aorto-coronary bypass grafts (class II indication). The use of MRA for the diagnosis of CAD on native coronary arteries has not yet entered clinical routine , 9.Coronary MRA can accurately visualize the origin and the path of anomalous coronary vessels, as well as the presence and location of coronary aneurysms. Aneurysms are found, for example, in Kawasaki disease. The usually larger caliber of the vessels and the location of the aneurysms in proximal or ectatic segments facilitate their visualization. An important added benefit of coronary MRA is the absence of ionizing radiation, particularly important in younger patients, children and young women , 10.Bypass grafts can be visualized by coronary MRA with good image quality, benefiting from their stationary position, straight and known course, and large diameter compared to the coronary arteries. Several different approaches for the visualization of coronary bypass grafts were published in the literature, including spin echo –14 and gHowever, the presence of metallic clips along the course of the graft, causing signal voids due to susceptibility artifacts, is a common limitation of coronary bypass MRA. According to current guidelines, coronary MRA may be used at specialized centers to identify stenoses in coronary arterial bypass grafts .Coronary MRA can visualize the proximal segments of the coronaries in nearly 100 % of cases. The best results are obtained with the left anterior descending (LAD) and the right coronary artery (RCA), while the left circumflex (LCX), which runs in the direct vicinity of the myocardium and at a larger distance from the coil elements, is frequently visualized with lower image quality and for a shorter course.Previous studies reported an average visible length of 50 mm for the LAD, 80 mm for the RCA and 40 mm for the LCX –23. TherThe spatial resolution of coronary MRA is still lower than that of invasive coronary angiography, which limits the visualization of small branches and affects diagnostic accuracy concerning stenosis detection. This limitation explains the low specificity demonstrated in a recent international multicenter study , wherebyA recent meta-analysis compared coronary MRA and multi-slice computed tomography (CT) for ruling out significant CAD in adults •. CT wasMoreover, a more recent multicenter study from Japan showed that non-contrast-enhanced whole-heart coronary MRA at 1.5 T can detect significant CAD with high sensitivity (88 %) and moderate specificity (72 %). In particular, a negative predictive value (NPV) of 88 % indicates that whole heart coronary MRA can effectively be used to rule out CAD ••. Of noIn a direct comparison between coronary MRA and CTA no significant difference was shown for the detection of coronary artery stenosis between 3 T MR and 64-slice CTA, although CTA showed a favorable trend toward higher diagnostic performance ••.With the advent of resynchronization therapy, the assessment of the anatomy of the coronary venous system has become increasingly important, particularly for the pre-interventional identification of optimal placement site for the left ventricular lead of resynchronization devices. The same techniques used for coronary (artery) MRA can be used to visualize the coronary veins. Three-dimensional MR coronary vein angiograms can be overlaid onto real-time time acquired x-ray images, to improve guidance for catheter implantation , 40.The integration of coronary venous anatomy and myocardial scar information may also guide left ventricular lead implantation remote from areas of scanned myocardium. Contrast agent enhanced MR can be used for the assessment of the course of the coronary sinus, the great cardiac vein, and their tributaries , 7, 41.The first magnetic resonance images of the coronary vessel wall were obtained by 2D fat saturated fast spin echo techniques , 43. A dRecently, the double inversion recovery prepulse has been combined with fast gradient echo readout techniques , with spClinical studies demonstrated the ability of vessel wall imaging to detect outward positive remodeling with relative lumen preservation in patients with CAD and increased vessel wall thickness in patients with type I diabetes and renal dysfunction , 49. As It is of particular interest that there are approaches that may allow visualization of inflamed plaques by means of delayed gadolinium enhancement techniques. Clinically approved contrast agents showed non-specific uptake in plaques both in patients with chronic angina  and in pSeveral novel target specific contrast agents have been developed and tested in animal models. The accumulation of albumin binding blood-pool CA is associated with increased endothelial permeability and/or increased neovascularization , 54. FurSuch molecules and cells are providing targets for recently developed novel molecular contrast agents. These CA allow the selective visualization of inflammatory markers such as intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) or matrix metalloproteinase (MMP) , 58. AddThe small caliber of the coronary vessels, as well as the elevated anatomical variability, cardiac and respiratory motion pose major challenges to coronary MRA and require dedicated techniques for image quality optimization.The first approaches to coronary artery angiography were attempted by Edelman  and MannFor non-contrast enhanced imaging, spin preparation usually include fat suppression and T2-preparation. Fat saturation reduces the signal generated by the epicardial fat tissue, allowing a better delineation of the lumen of the coronaries , 64, 69.T2-preparation also suppresses deoxygenated venous blood due to the shorter T2 of deoxygenated hemoglobin. Contrast enhanced techniques are therefore preferred for coronary vein imaging.Other solutions, such as spin-locking  and magnDifferent types of CA have been tested for coronary MRA, ranging from extracellular CA  to bloodSpin preparation for CA enhanced MRA usually includes a saturation  or inverk-space has to be synchronized with the cardiac cycle and needs to be limited to periods of minimal cardiac movement [To freeze cardiac motion, the acquisition of the movement  occurrinThe choice of the trigger delay and the duration of the acquisition window depend on the patient’s heart rate, on the sequence used and on the structure to visualize (arteries or veins). A free breathing high temporal resolution cine scan in the 4-chamber view can be used to determine the resting period . This isSince three-dimensional (3D) acquisitions take a long period of time to complete, they require the synchronization of image acquisition with the respiratory cycle. Respiratory motion artifacts can be minimized by prospective real-time navigator gating and correction techniques , 83, in However, respiratory motion remains the major impediment in a substantial amount of patients undergoing coronary MRA. Usually, 1D navigator techniques assume a constant linear relationship between diaphragmatic and cardiac motion. This assumption is not correct in a significant percentage of subjects, leading to suboptimal results. Recently, the use of 2D navigator has been proposed, allowing for the prospective correction of translational motion both in craniocaudal and left-right direction  The use of image based navigators that directly track cardiac motion, navigators that monitor the movement of epicardial fat , scanninOf note, a lower sensitivity to motion and better image quality can also be obtained by improving the speed of image acquisition because of a shorter acquisition window or shorter overall data acquisition time . To thisParallel imaging techniques such as SENSE  or SMASHT2 preparation is not suitable for coronary vein imaging due to the shorter T2 values of deoxygenated venous blood. Current approaches to coronary vein MRA include non contrast-enhanced imaging by magnetization transfer (MTC) preparation , 98 or cThe optimal acquisition window for coronary vein imaging, as demonstrated by Nezafat and co-authors, is in end-systole, when the coronary vein diameter is maximal . HoweverUtilizing black-blood techniques allows the latest clinical MR scanners to provide a detailed visualization of the coronary artery wall, either in cross-section or along the path of the vessel. Partial volume effects are minimized in cross-sectional view, providing images suitable for accurate quantification of the vessel wall thickness. A long-axis view of the vessel wall provides instead a more extensive visualization and typically allows assessment of the proximal 5 cm .CA can be used for selective plaque visualization and delayed enhancement images can show focal or diffuse uptake of contrast agent indicating either a fibrous plaque or inflammation.The current application of coronary vessel wall imaging is restricted to research purposes but with developments such as plaque-targeting agents used for clinical purposes it may become part of routine CAD risk assessment and monitoring of treatment response, especially if plaque-targeting agents become available for clinical use.Cardiovascular magnetic resonance allows non-invasive and radiation free visualization of both the coronary arteries and veins, with the possibility of coronary vessel wall imaging. A major comparative advantage of MR is the possibility of a combined scanning protocol, investigating the anatomy of the coronaries as well as cardiac function, viability, stress perfusion and cardiac anatomy in the same study, providing valuable integrated information for patients with coronary artery disease and heart failure.Coronary MRA may be indicated for the visualization of anomalies of the origin and course of the coronaries (class I indication) as well as to visualize coronary bypass grafts (class II indication) and may potentially be used to exclude CAD in selected populations of patients.Ongoing technical developments continue to improve the robustness of the techniques.


Currently, there is a remarkable lack of genetic epidemiological studies on alpha 1-antitrypsin (AAT) deficiency in about half of the 193 countries of the World. This fact impedes the establishment of a true prevalence pattern of this deleterious hereditary disorder in extensive regions of human population.The aim of the present study was to generate detailed maps of the frequency distribution of the two most frequent AAT deficiency alleles  in all areas of the World.Available data provided by epidemiological studies performed in 94 of 193 countries worldwide was used to develop detailed maps of these two alleles, We employed an informatics mathematical approach, namely: the ArcMap , based on the inverse distance weighting (IDW) multivariate interpolation method, which creates new numerical points from known data, using a simple logarithm based in the distance existing between themIn this method, PI*S and PI*Z frequencies were represented by colored scales, where qualitative colors were converted into quantitative data, providing information on their distribution in all parts of the world. This approach not only confirmed our previous data, but also provided digital images of the remaining regions of all continents.By using this approach, striking differences were found among regions, and unsuspected significant values of the PI*S and PI*Z alleles frequencies were obtained for several geographic regions where have not been studied yet. In fact, some of these regions might be considered as priority targets for further screening studies on AAT deficiency, in order to identify, and properly manage, individuals at risk for the diverse adverse health effects associated with AAT deficiency. Alpha1-antitrypsin (AAT) deficiency is a recessive hereditary metabolic disorder which results in the synthesis and secretion of defective AAT. Up to now, about 100 genetic variants of AAT have been recognized. Protease inhibitor (PI) M (medium mobility) is the normal allele, while the two most frequent deficient alleles are PI*S and PI*Z. The PI*ZZ genotype results in very low AAT serum concentrations 10–15%), and PI*SZ and PI*MZ phenotypes result in low to intermediate serum AAT concentrations from 35% to 70%. Other 30 variants affect the amount or the function of the AAT molecule, but clinically, most 96%) AAT deficiency-related patients are linked with the PI*Zz type, and the remaining 4% to PI*SZ, null and about other 30 rare or null phenotypes . Individ%, and PI6% AAT deFrom the available data on PI*S and PI*Z AAT deficiency gene frequencies, colored maps introducing new data on PI*S and PI*Z frequencies were obtained in each region and country of the World.The articles used in the present study were obtained through a variety of sources which have been discussed in earlier publications , 11-16. In general, interpolation is a method of constructing new data points within the range of a set of known data points obtained by sampling or experimentation. Inverse distance weighting (IDW) is a process of assigning values to unknown points by using values from scattered set of known points. In a digital image (or bitmap) each pixel has a value to construct an image , 19. AccIn In AAT deficiency is an underdiagnosed disorder, partly because it is thought to be a rare condition which practically only affects Caucasians (Whites) of Northern European heritage . Unfortu


Lab studies have suggested that ubiquitous phthalate exposures are related to obesity, but relevant epidemiological studies are scarce, especially for children.To investigate the association of phthalate exposures with body mass index (BMI) and waist circumference (WC) in Chinese school children.A cross-sectional study was conducted in three primary and three middle schools randomly selected from Changning District of Shanghai City of China in 2011–2012. According to the physical examination data in October, 2011, 124 normal weight, 53 overweight, and 82 obese students 8–15 years of age were randomly chosen from these schools on the basis of BMI-based age- and sex-specific criterion. First morning urine was collected in January, 2012, and fourteen urine phthalate metabolites (free plus conjugated) were determined by ultra-performance liquid chromatography coupled to tandem mass spectrometry. Multiple linear regression was used to explore the associations between naturally log-transformed urine phthalate metabolites and BMI or WC.The urine specific gravity-corrected concentrations of nine urine phthalate metabolites and five molar sums were positively associated with BMI or WC in Chinese school children after adjustment for age and sex. However, when other urine phthalate metabolites were included in the models together with age and sex as covariables, most of these significant associations disappeared except for mono (2-ethylhexyl) phthalate (MEHP) and monoethyl phthalate (MEP). Additionally, some associations showed sex- or age-specific differences.Some phthalate exposures were associated with BMI or WC in Chinese school children. Given the cross-sectional nature of this study and lack of some important obesity-related covariables, further studies are needed to confirm the associations. Phthalates, the diesters of phthalic acid, are extensively used as plasticizers and solvents in industrial, medical, and consumer products There is increasing evidences suggesting that some EDCs can act as obesogens and interfere with body’s natural weight control mechanisms by disrupting adipose tissue biology, endocrine hormone systems, or central hypothalamic-pituitary-adrenal axis Sex hormones are critical to the regulation of body composition, fuel homeostasis, and reproduction in humans However, epidemiological studies that investigate the associations of postnatal phthalate exposures with obesity or body size measurements are scarce. To our knowledge, there were four epidemiological studies for this, including two cross-sectional studies and one longitudinal study involved in children, another cross-sectional one in men Children and adolescents are in rapid growth and development and are more susceptible to external disturbance than adults. Moreover, it has been reported that children are extensively exposed to phthalates and the exposure levels are higher compared with adults The study was approved by the Ethical Review Board of the School of Public Health of Fudan University. Guardians provided written informed consent before the study.The study design and study population has been described in detail in a previous report of the association of urine bisphenol a (BPA) with BMI Body weight (kg) and height (cm) were measured using the same type of apparatus and followed the standard procedures recommended by Cameron For a better comparability of urine phthalate metabolites among individuals, first morning urine was collected and participants themselves carried out the urine collection. In order to minimize the contamination of phthalates during urine sampling and storing, urine samples were collected in glass centrifuge tubes, which was rinsed by acetone and baked at 350°C for 2 hours, and the participants were told to avoid a contact of urine with plastic products in the process of urine collection. The collected urine samples were transported to lab as soon as possible and stored in dark at −20°C until analysis. All the urine samplings were collected between January 3 and 6, 2012.Fourteen phthalate metabolites studied here included MEHP, mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), MBP, monoisobutyl phthalate (MiBP), MEP, mono[(2-carboxymethyl) hexyl] phthalate (MCMHP), mono(4-hydroxybutyl) phthalate (MHBP), monomethyl phthalate (MMP), monocyclohexyl phthalate (MCHP), MBzP, monoisononyl phthalate (MiNP), and monooctyl phthalate (MOP). MEHP is the primary metabolite of DEHP, and MEHHP, MECPP, MCMHP, and MEOHP are secondary metabolites of DEHP. MBP and MHBP are primary and secondary metabolites of DBP, respectively. MiBP is derived from DiBP, MMP from DMP, MEP from DEP, MCHP from DCHP, MBzP from BBzP, MiNP from DiNP, and MOP from DOP.Fourteen urine phthalate metabolites (free and conjugated) were analyzed using a method modified from Guo et al. All urine analyses were carried out in random and information on BMI subgroup was blinded to analytical technicians. To minimize a potential systematic drift caused by analytical process, all the analyses of urine samples were performed in a short time span  and by the same analytical team. The recoveries of phthalate metabolites of spiked urine samples used for quality control in the process of whole urine analyses varied between 70.3% and 124%, and their standard deviations varied between 9.2% and 19.1%. The detailed analytical method was supplied in Supporting Information File S1 (Tables S2 and S3 in Supporting Information File S1).For measurements below the limit of detection , a default value of LOD divided by the square root of 2 was assigned, which is a method of handling nondetectable values that produces reasonably nonbiased means and standard deviations c = P × [(1.024–1)/(SG –1)], where Pc is the specific gravity-corrected phthalate metabolite concentration (ng/mL), P is the experimental phthalate metabolite concentration (ng/mL), and SG is the specific gravity of the urine sample Due to the glucuronidation of phthalate metabolites in the liver and its elimination by active tubular secretion, creatinine correction may not be appropriate for urine phthalate metabolite concentration For descriptive statistics, arithmetic mean and standard deviation (SD) of BMI and WC, and geometric mean (GM), 95% confidence interval (CI), and median of SG-uncorrected urine phthalate metabolite concentrations were calculated for all subjects or by sex, age, and BMI subgroups  . BecauseMultiple linear regression analysis was also performed to study the association of SG-corrected urine phthalate metabolite concentrations with BMI and WC values  and 5. LIn adjusted model 2, in order to avoid the collinearity derived from strong correlations among daughter metabolites of one parent phthalates in linear regression model, only one daughter metabolite was included, whereas all others not. In this model, sum of DEHP was used to represent for five metabolites of DEHP, and sum of DBP was used to represent for MBP and MHBP. When one of the phthalate metabolites from DEHP or DBP was included in this model, sum of DBP or sum of DEHP was not added as covariable into the model. In addition, sum of high MWP, sum of low MWP, and sum of all were not used as independent covariables in all these models. The detail information about covariable for specific metabolites modelling was provided in As shown in In multiple linear regression analysis, similar results were obtained before and after sampling weights were accounted for, and weighted estimates were presented. The detail sampling weights of each selected school was provided in Table S5 in Supporting Information File S1. As shown in On the whole, urine phthalate metabolite concentrations in this study were comparable to those from previous studies conducted in other countries (Table S8 in Supporting Information File S1), although the profiles of urinary phthalate metabolites demonstrated some differences in terms of quantity and species of metabolites. For example, MBzP was detected only in 38.6% of subjects in this study, but detected in almost 100% in the studies of U.S., German, and Spanish children As a result of an extensive use of phthalate-related products, human can be exposed to phthalates through almost all possible pathways, such as ingestion, inhalation, dermal contact, and placenta transmission In the present study we found nine urine phthalate metabolites and five sums were positively associated with BMI or WC in Chinese school children when adjusted for individual SG, age, and sex (in adjusted model 1), but most of these significant associations disappeared and some even reversed when further adjusted for other urine phthalate metabolites (in adjusted model 2). Only MEHP and MEP remained significant. Close correlations among phthalate metabolite measurements may be one reason for these association changes (Table S4 in Supporting Information File S1), and these changes may mean other possibilities. One possibility was that some phthalate metabolites might act as confounders. When these metabolites were included as covariables, they corrected the associations of metabolites of interest with BMI or WC. Another possibility was that some phthalates might have modifying effects on BMI or WC Given the cross-sectional nature of this study, the possibility of reverse causation cannot be ruled out. It is possible that increased overall caloric intake, different diet composition, and longer daily indoor time for obese individuals might increase the risk of phthalate exposures, compared to normal weight ones An underlying assumption in this study and other similar studies is that one single measurement of spot urine could represent a long-term exposure of corresponding participant to no-persistent environmental chemicals In our studies, we observed some age- or sex-related differences in the associations of phthalate metabolites with BMI or WC. MEHHP and MEOHP were found to be significantly associated with BMI only in the 8–11 year age group, not in all the subjects. These age- or sex-related differences were also reported in some previous studies There were some epidemiologic studies supporting the associations of postnatal phthalate exposures and body size measures in children. Using data from the National Health and Nutrition Examination Survey (NHANES) 1999–2002, Hatch et al. found that BMI increased with urinary MEP quartiles and inversely related to MEHP quartiles in girls 12–19 years of age, but no metabolite quartiles were significantly associated with BMI in the 6–11 year age group There are some plausible explanations for these discrepancies. Firstly, creatinine correction for urine phthalate metabolite concentrations can weaken or even reverse the association due to the positive associations of urine phthalate metabolite concentrations and BMI with urinary creatinine In this study we found nine urinary phthalate metabolites and three sums were positively associated with BMI or WC in Chinese school children, and it was more evident for MEHP and MEP. Some associations showed sex and/or age related difference. Given the cross-sectional nature of this study and lack of some important obesity-related variables, the associations discovered here need to be further investigated.Supporting Information File S1Table S1 BMI-based criteria by age and sex for screening of overweight and obese school children proposed by Working Group on Obesity in China (WGOC) in 2005. Table S2 Settings for multiple reaction monitoring (MRM) mode. Table S3 Results of quality assurance/quality control. Table S4 Pearson correlation coefficients among phthalate metabolites uncorrected by specific gravity. Table S5 Calculation of sampling weights of each selected school (sampling weights = number of source population/number of participants). Table S6 Association of urinary phthalate metabolite concentrations corrected by specific gravity with body mass index by age groups after adjustment for age in years and sex in 259 school children of Shanghai, China in 2011–2012. Table S7 Association of urinary phthalate metabolite concentrations corrected by specific gravity with body mass index by sex after adjustment for age in 259 school children of Shanghai, China in 2011–2012. Table S8 Comparison of urinary phthalate metabolite concentrations   between this study and other several populations.(DOC)Click here for additional data file.


While the functional role of CRF1 in hormonal and behavioral adaptation to stress is well-known, the physiological significance of CRF2 remains to be fully appreciated. Accumulating evidence has indicated that CRF2 and its selective ligands including urocortin 3 (Ucn 3) are important molecular mediators in regulating energy balance. Ucn 3 is the latest addition of the CRF family of peptides and is highly selective for CRF2. Recent studies have shown that central Ucn 3 is important in a number of homeostatic functions including suppression of feeding, regulation of blood glucose levels, and thermoregulation, thus reinforcing the functional role of central CRF2 in metabolic regulation. The brain loci that mediate the central effects of Ucn 3 remain to be fully determined. Anatomical and functional evidence has suggested that the ventromedial hypothalamus (VMH), where CRF2 is prominently expressed, appears to be instrumental in mediating the effects of Ucn 3 on energy balance, permitting Ucn 3-mediated modulation of feeding and glycemic control. Thus, the Ucn 3-VMH CRF2 system is an important neural pathway in the regulation of energy homeostasis and potentially plays a critical role in energy adaptation in response to metabolic perturbations and stress to maintain energy balance.The vital role of the corticotropin-releasing factor (CRF) peptide family in the brain in coordinating response to stress has been extensively documented. The effects of CRF are mediated by two G-protein-coupled receptors, type 1 and type 2 CRF receptors (CRF Corticotropin-releasing factor (CRF) was discovered in 1981 by Vale and colleagues at the Salk Institute . Its exiIn addition to CRF, additional members of the CRF peptide family including urocortins (Ucns) 1, 2, and 3 have been identified in mammals, including humans . AccumulAdditionally, the Ucns have been shown to be involved in various physiological regulations including energy balance, cardiovascular function, and behavioral modulation. Ucn 1, 2, and 3 all suppress feeding , and defUrocortin 3 is the latest addition of the CRF family of peptides, initially identified in the brains of humans and rodents . Sequenc1 and CRF2), and amino acid sequence analysis has shown that the two receptors share approximately 70% homology , phospholipase C, protein kinase B, and ion channels have also been shown to couple to CRFRs  is mainly to sequester CRF to reduce its HPA axis stimulation. Levels of CRFBP are elevated in pregnancy to dampen the negative effect of stress responses on the developing fetus . In addi as well . This sp as well .1 and CRF2 have distinct distributions in the brain. CRF1 has a wide distribution throughout the brain with high density in the medial septal area, amygdala, and cerebellum , CRF2(b), and CRF2(c). In murine brains, CRF2(a) is predominantly expressed in the hypothalamus, lateral septum (LS), and dorsal raphe (2(b) is found predominantly in the periphery, including in skeletal muscle, the gastrointestinal tract, and the heart (2(c) is found only in the human brain he heart . CRF2(c)an brain .1 has been closely associated with the stress response, including the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary and behavioral adaptation to stressors  mice have more active metabolism and lose heat faster than wildtype (WT) mice (2 KO mice have higher brown adipose tissue (BAT) temperature and, when given a choice between room temperature and warm areas, prefer warmer areas more than WT mice  activity to promote lipolysis (2 (discussed below), in most cases, results in increased SNS activity. This apparent discrepancy may result from a number of possibilities including compensatory mechanisms due to total body KO of the receptor as compared to acute, local stimulation of the receptor in the brain. Obviously more studies are needed to further elucidate this issue.In addition to feeding, CRFWT) mice , suggest WT mice . The KO  WT mice , indicatipolysis . This hy2 is not essential for the effects of leptin on energy balance, including feeding and body weight regulation. Again this discrepancy may be due to the nature of global CRF2 KO, which potentially results in functional compensation such as exaggerated CRF1 activity in these mice  have higher body mass-adjusted food intake than WT and are heavier than WT controls due to increased lean body mass  have an increased respiratory exchange ratio and increased motor activity in their home cages , when injected into the lateral ventricles of rats . Tempera by CRF2 . Noramin by CRF2 . This inptor EP3 . It has ptor EP3 . Furtherptor EP3  suggestsThe expression of Ucn 3 in the brain has been determined in a number of stress paradigms and metabolic challenges. It was found that restraint stress rapidly elevates Ucn 3 gene expression in the MeA and that the elevated Ucn 3 mRNA levels return to basal levels 4 h after the stress . RestraiFigure 1; The expression of Ucn 3 has also been examined in genetically obese rodent models. Food deprivation increases Ucn 3 mRNA expression in the dorsal part of the medial amygdala (MeD) in obese Fa/Fa Zuker rats and has no effect on Ucn 3 expression in the rPFH . In cont2 in the brain has provided important insight into possible areas that mediate the effects of CRF2 in energy balance. The VMH has received significant attention due to its abundant expression of CRF2 and well-known role in regulating energy homeostasis, feeding, and blood glucose levels.The anatomical distribution of CRFThe VMH is parceled on cytoarchitectonic grounds into three major divisions: dorsomedial (VMHdm), central, and the ventrolateral (VMHvl) parts . The VMHThe VMH has long been considered a critical brain area in the regulation of energy homeostasis. Lesion of the VMH results in hyperphagia, hyperinsulinemia, reduction of SNS activity, increase of fat mass, and reduction of energy expenditure that ultimately leads to storage of excess of energy and obesity . Convers2 expression  with less colocalization of these two materials in the VMHvl . Taken together, it is possible that the functional role of CRF2 in the VMH in glucose homeostasis is context dependent. When blood glucose is low as a result of hyperinsulinemia, CRF2 in the VMH negatively regulates the CRR response. On the other hand, under normoglycemia, CRF2 induces acute hyperglycemia to facilitate fuel mobilization perhaps in response to stress. We have also found that CRF2-positive neurons in the VMH are sensitive to glucose, as high glucose inhibits and low glucose stimulates the neuronal activity (In addition to appetite suppression, activation of CRFe levels . This ise levels . On the nephrine , indicatactivity .2 KO mice display altered HPA hormonal secretion, and central Ucn 3 injection facilitates stress-induced ACTH secretion (2 in the VMH fails to modulate HPA hormone secretion (2-positive brain loci that are important for modulation of the HPA axis remain to be determined.Type 2 CRF receptor has been shown to modulate the HPA axis. For example, CRFecretion . On the ecretion , indicat2 in the VMH, VMH-specific CRF2 knockdown mice were generated by injection of a lentiviral vector expressing CRF2 small hairpin RNA (shRNA; 2 shRNA displayed significantly reduced CRF2 mRNA levels and gain more weight, mostly in white fat, than control mice. Furthermore, similar to Ucn 3 null mice, mice with reduced CRFR2 in the VMH exhibited elevated basal food intake and ate more than the control mice after overnight fasting. This result indicates that CRF2 in the VMH serves as a brake to facilitate the cessation of feeding. This study suggests that CRF2 in the VMH plays a critical role in mediating the effect of central Ucn 3 in energy balance.To probe the physiological role of endogenous CRFA shRNA; . Mice in2 in the VMH display reduced lipolysis and increased adiposity in white fat (2 knockdown in the VMH has no major impact in heat production or uncoupling protein 1 expression in BAT, suggesting that CRF2 in the VMH does not significantly modulate thermogenesis in BAT. This result appears to disagree with earlier reports that VMH is involved in regulating thermogenesis in BAT (2-positive neurons are a subpopulation of cells in the VMH that regulate SNS outflow to white fat without significant functional impact on BAT. Heterogeneity of VMH neurons has been reported as overlapping but distinct subpopulation of neurons in the VMHvl that are critical in regulating fighting and mating (In addition to elevated feeding, mice with decreased expression of CRFhite fat . This ishite fat . Interess in BAT . Anatomis in BAT , 2006. Id mating .2 in regulating blood glucose levels, mice with reduced CRF2 expression in the VMH show improved glucose homeostasis compared to control mice (2 knocked down in the VMH exhibit an exaggerated rebound in blood glucose levels compared to control mice after the initial hypoglycemic response to insulin challenge. This result agrees with the study by 2 is a critical molecular mediator in VMH regulation of glucose homeostasis.Consistent with the function of CRFrol mice . Moreove2 neurons regulate output functions and to describe an anatomical link between these neurons and the SNS, it is necessary to determine their axonal projections to identify downstream targets in the brain. A number of anterograde tracing studies utilizing different tracers have been performed to evaluate the projections of VMH neurons. Generally, it was found that VMH neurons project extensively to neighboring hypothalamic nuclei including the anterior and paraventricular nucleus, BNST, and periaqueductal gray (PAG; To understand how VMH CRFray PAG; . These a2-positive cells in the VMH show similar axonal projections to these brainstem areas (Figure 3; 2, can potentially modulate SNS activity by direct projections to brainstem autonomic centers.Recently, using a conditional viral tracing approach, we have found that VMH neurons project to a number of important brainstem autonomic centers including the parabrachial nucleus, C1 catecholaminergic cell group in the rostral ventrolateral medulla, and the nucleus of solitary tract . Moreove2 receptors are involved in the regulation of energy homeostasis. Recent studies using various genetic mouse models and molecular tools have further ascertained the critical role of CRF2 and its selective ligands, including Ucn 3, in feeding, blood glucose regulation, SNS output, and peripheral metabolism. Moreover, CRF2 in the VMH mediates most, if not all, of the effects of central Ucn 3 on energy homeostasis.The function of CRF peptides and their receptors in coordinating hormonal, neuronal, and behavioral responses to stress is well recognized. Pharmacological studies have determined that the CRF2 and its selective ligands in the brain will be aided by brain region-specific transgenic animal models permitting manipulation of ligand or receptor expression. Study of such models will provide insight into the specific roles of CRF2 in modulating metabolic functions.It is clear that conflicting results have been observed between whole body and region- or tissue-specific KO or overexpression mouse models. Furthermore, several studies have demonstrated that anatomical or even functional heterogeneity exists within a seemingly single Ucn 3 cell population in the hypothalamus. Thus, a more detailed understanding of the physiological function of CRF2 and Ucn 3 are involved in this regulation, as the expression of the receptor and ligands are closely regulated under these challenges. It is thus conceivable that dysregulated CRF2 or ligand function potentially cause or exacerbate metabolic perturbations. This hypothesis can be easily tested with the above mentioned rodent models to determine the functional role of the CRF2 system in metabolic diseases. Moreover, a better understanding of the molecular mechanisms by which various stressors or metabolic signals regulate the expression and/or function of CRF2 and its ligand will provide significant insight into the potential role of the CRF family and its receptors in the pathophysiology of metabolic disorders including obesity and diabetes.Regulation of energy balance under diverse challenges including stress, starvation, or high-fat diet requires numerous adaptive mechanisms in both central and peripheral tissues. It is now clear that central CRFThe 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.


Shh, Lmx1b, and Nurr1, but not in the levels of Otx2 and Pitx3, in saline-pretreated rats. METH pretreatment followed by METH challenges also decreased Nurr1 but increased Otx2 and Pitx3 expression in the midbrain. These findings suggest that, in adult animals, toxic doses of METH can differentially influence the expression of transcription factors involved in the developmental regulation of dopamine neurons. The combined increases in Otx2 and Pitx3 expression after METH preconditioning might represent, in part, some of the mechanisms that served to protect against METH-induced striatal dopamine depletion observed after METH preconditioning.Methamphetamine (METH) is an addictive and neurotoxic psychostimulant widely abused in the USA and throughout the world. When administered in large doses, METH can cause depletion of striatal dopamine terminals, with preservation of midbrain dopaminergic neurons. Because alterations in the expression of transcription factors that regulate the development of dopaminergic neurons might be involved in protecting these neurons after toxic insults, we tested the possibility that their expression might be affected by toxic doses of METH in the adult brain. Male Sprague-Dawley rats pretreated with saline or increasing doses of METH were challenged with toxic doses of the drug and euthanized two weeks later. Animals that received toxic METH challenges showed decreases in dopamine levels and reductions in tyrosine hydroxylase protein concentration in the striatum. METH pretreatment protected against loss of striatal dopamine and tyrosine hydroxylase. In contrast, METH challenges caused decreases in dopamine transporters in both saline- and METH-pretreated animals. Interestingly, METH challenges elicited increases in dopamine transporter mRNA levels in the midbrain in the presence but not in the absence of METH pretreatment. Moreover, toxic METH doses caused decreases in the expression of the dopamine developmental factors,  Methamphetamine (METH) is a psychostimulant that is abused throughout the world. Acute administration of the drug causes behavioral changes that are secondary to activation of dopaminergic systems located in various brain regions In preclinical studies, injections of moderate-to-large doses of METH cause depletion of DA as well as loss of DAT and TH in the striatum of rodents and non-human primates Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the National Institute on Drug Abuse, Intramural Research Program. The research was conducted under Animal Study Protocol #09-CNRB-25.Male Sprague-Dawley rats  weighing approximately 350–400 g were habituated for one week prior to drug treatment. Animals were housed in polyethylene cages containing hardwood bedding in a temperature-controlled room with a 12 hour light∶dark cycle and free access to food and water. All animal procedures were performed according to the National Institutes of Health d,l-METH hydrochloride for two weeks as described in After habituation, rats were injected with saline or with progressively higher doses of Rats were weighed three times per week during the pretreatment period and both challenge days to ensure proper dosing. Tympanic temperatures were taken 30 minutes prior to the first injection and 30 minutes after every other injection on the second challenge day. The animals were euthanized 14 days following the METH challenge by decapitation. Their brains were then quickly removed, dorsal striata and midbrain regions were dissected over ice, snap frozen on dry ice, and stored at −80°C until used in biochemical experiments.4 and centrifuged at 14000× g for 15 min. DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels were analyzed in the striatal extracts using HPLC with electrochemical detector as described previously For monoamine analysis, the dorsal striata of 5–8 mice per group were homogenized in 0.01 M HClOTotal RNA extracted from a midbrain region that encompasses the ventral tegmental area and substantia nigra of the rat was used to analyze the expression of genes of interest by qRT-PCR as previously described c Protein assay . 20 µg of total protein were electrophoresed on 10% SDS-polyacrylamide gels and then transferred to Hybond-P™ membrane . The membranes were blocked and then immunolabeled with antibodies against DAT (1∶1000), TH (1∶10000), Pitx-3 (1∶1000)  and Nurr1  at 4°C overnight. Immune complexes were detected with HRP-labeled second antibody and ECL+ chemiluminescence reagents . To confirm equal protein loading, blots were stripped and reprobed with anti-α-tubulin antibody  for 2 hours at room temperature. Signal intensity was measured using densitometric analysis  and quantified using Carestream Molecular Imaging Software .Western blot analyses were performed as previously published http://www.systat.com). Statistical analysis of HPLC, qRT-PCR and Western blot data was performed using one-way ANOVA for repeated measures followed by Fisher's protected least significant difference (PLSD) . The null hypothesis was rejected at p<0.05.All data are presented as means ± SEM. Statistical analysis of the experimental data on body temperature and weights was performed using two-way ANOVA for repeated measures followed by a pairwise multiple comparison procedure (Tukey's test) to identify differences between the groups or sessions, respectively  experienced significant reductions in Wnt1 expression regardless of pretreatment  and substantia nigra pars compacta (SNpc), play important roles in the control of motor and psychomotor behaviors Dat mRNA levels in the midbrain Dat mRNA in remaining SNpc dopaminergic neurons was decreased whereas the level of Th mRNA was not affected in these animals The substantial loss of DA markers observed in the present study following injections of toxic METH doses is consistent with the observations of other investigators who used similar doses of the drug Otx2 mRNA only in the animals pre-exposed to lower doses of METH. These observations suggest that treatment with low non-toxic doses of the drug might have altered the transcription of Otx2 to such an extent that challenges with large METH doses were able to increase Otx2 mRNA levels in midbrain DA neurons in METH-preconditioned rats but not in saline-pretreated animals. In contrast to Otx2 mRNA expression which was not affected by METH in saline-pretreated animals, Shh transcript levels were markedly reduced in these rats. SHH is thought to be responsible for ventral fate determination during development Shh after 8 days, but not after 11 days, of gestation causes loss of DA populations Shh transcript after METH challenges in the two saline-pretreated groups, which show decreased dopaminergic markers, in contrast to normal Shh mRNA levels in the METH-preconditioned rats that exhibit normal levels of DA and TH protein, suggest that SHH may play a partial role in maintaining DA homeostasis in the striatum of METH pretreated rats. Although much remains to be done to elucidate the role of SHH by itself or in combination with OTX2 expression in maintaining DA neurons in the adult brain, it is unlikely that either one of them is involved in the METH-induced changes in DAT expression since the drug caused significant decreases in DAT expression in both saline- and METH-pretreated rats, albeit to a lesser extent in the latter than in the former group.The differential METH-induced changes in TH, DAT and DA levels could also be secondary, in part, to perturbations in transcription factors that influence the expression of these DA markers. Among these transcription factors, OTX2, WNT1, and SHH are involved in regulating the early development of DA neurons Nurr1 mRNA and protein levels in both saline- and METH-pretreated rats. NURR1 is a member of the nuclear receptor superfamily of transcription factors Nurr1 in the adult brain suggests that it might play additional roles in the nervous system Nurr1 mRNA and protein levels in midbrain after METH challenges both in saline- or METH-pretreated animals are disconcerting because they might render DA neurons more vulnerable to cell death. However, preclinical toxic doses of METH or amphetamine that cause decreases in TH, DAT, and DA levels in the striatum are not associated with DA cell loss in SNpc Click here for additional data file.


Based on these findings, we propose a hypothesis of normal and malignant transformation in the breast, which centers on OCT4 and its associated gene network. Although minimal expression of these embryonic genes can be seen in the breast in its resting state throughout life, a controlled program of upregulation of this gene network may be a potential regulator of the normal remodeling of the breast toward a milk-secretory organ during pregnancy and lactation. Deregulation of this gene network either within or outside pregnancy and lactation may lead to aberrant breast cell proliferation and malignant transformation, suggesting a role of these genes in both normal lactation and breast oncogenesis.Breast cancers with lactating features, some of which are associated with pregnancy and lactation, are often poorly differentiated, lack estrogen receptor, progesterone receptor, and HER2 expression and have high mortality. Very little is known about the molecular mechanisms that drive uncontrolled cell proliferation in these tumors and confer lactating features. We have recently reported expression of OCT4 and associated embryonic stem cell self-renewal genes in the normal lactating breast and breastmilk stem cells (hBSCs). This prompted us to examine OCT4 expression in breast cancers with lactating features and compare it with that observed during normal lactation, using rare specimens of human lactating breast. In accordance with previous literature, the normal resting breast  showed minimal OCT4 nuclear expression (0.9%). However, this increased in the normal lactating breast (11.4%), with further increase in lactating adenomas, lactating carcinomas, and pregnancy-associated breast cancer (30.7–48.3%). OCT4 was expressed in the epithelium and at lower levels in the stroma, and was co-localized with NANOG. Comparison of normal non-tumorigenic hBSCs with OCT4-overexpressing tumorigenic breast cell lines (OTBCs) demonstrated upregulation of OCT4, SOX2, and NANOG in both systems, but OTBCs expressed OCT4 at significantly higher levels than SOX2 and NANOG. Similar to hBSCs, OTBCs displayed multi-lineage differentiation potential, including the ability to differentiate into functional lactocytes synthesizing milk proteins both  Breast cancer associated with pregnancy and lactation  is the most frequent malignancy in pregnant women, affecting 1:3000 pregnancies in patients between 32 and 38 years old  that is found silenced in the vast majority of somatic cells. It has been established that the normal resting breast  as well as cell lines derived from it have very little expression of OCT4 and associated embryonic TFs  or available OCT4-overexpressing breast cancer cell line models  possessing tumor-initiating features. Although expression levels between ESC genes were similar in both hBSCs and hESCs, OTBCs had imbalanced ESC gene expression, with OCT4 levels being significantly higher than those of SOX2 and NANOG. However, similar to hBSCs and hESCs, OTBCs were able to differentiate both in vitro and in vivo into cells from all three germ layers, suggesting multi-lineage differentiation potential. Importantly, they were also able to differentiate into functional lactocytes that synthesized milk proteins. These findings highlight OCT4 with its associated embryonic TFs as potential key regulators of normal self-renewal and differentiation in the breast during pregnancy and lactation. Further, they suggest that deregulation of OCT4 expression in the breast may result in malignant transformation of breast cells, acquisition of tumorigenic properties, and lactating differentiation potential.To address this, we quantified OCT4 expression and subcellular localization by immunohistochemical (IHC) staining in rare specimens of normal human breast during the resting (non-lactating) and lactating stages, and in breast cancers with lactating features. Although minimal OCT4 expression was observed in the normal resting breast, OCT4 was upregulated in the normal lactating breast, with a further increase in expression in breast tumors with lactating features. The majority of OCT4N = 2) and lactating (N = 6) breast tissues were obtained from the tissue archive of the School of Anatomy, Physiology, and Human Biology, The University of Western Australia. Human breast tumor tissues were obtained from the Tissue Procurement Core Facility of The University of North Carolina Lineberger Comprehensive Cancer Center in accordance with approved institutional review board (IRB) protocols. Tumor tissues were examined by a pathologist and were identified as lactating breast adenomas (N = 7), lactating breast carcinomas , and PABC (N = 1). The tumor tissues were assigned lactating features by the pathologist based on the presence of lipid droplets characteristic of lactation. This was confirmed by immunofluorescence (IF) staining for milk proteins both in the tumors and in the normal lactating breast specimens . Slides were deparaffinized in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Heat induced epitope retrieval was performed at 100°C for 30 min in Bond-Epitope Retrieval solution 1 pH 6.0 (AR9961) and blocked with Dako Serum-Free Ready-To-Use Protein Block for 10 min (X0909). Slides were incubated with anti-OCT4 antibody , and manual annotations. A first separation using Tissue Studio Composer was followed by inspection of the annotation results and manual adjustment of annotation using Manual Region of Interest Selection for regions where the automated approach did not yield appropriate annotation. The cellular analysis was identical for the squares where Composer was used and those that were hand annotated. For each square, expression in epithelial versus stromal cells was quantified, including information on positiveness (either expression or no expression), subcellular localization , and intensity of positive expression . This was done via a cell scoring system that grouped each cytoplasm into an intensity group  and each nucleus into an intensity group . Then, the two numbers were added together to give a unique score based on the resulting sum . OCT4 expression was quantified in two to five 0.9-μmImmunofluorescence staining was done as described in Hassiotou et al. . AntibodN = 31) provided informed written consent and expressed mature breastmilk (5–200 ml) on one or more occasions under aseptic conditions using a Medela Symphony pump . Freshly expressed breastmilk was diluted with equal volume of sterile PBS . After centrifugation at 805 × g for 20 min at 20°C, the cell pellet was separated from the fat and liquid part skim milk, washed three times in PBS, and was then resuspended in 7% Fetal Bovine Serum  in PBS (blocking buffer). Total breastmilk cell content and viability were measured using a Neubauer hemocytometer by Trypan Blue exclusion.The study protocol was approved by the Human Research Ethics Office of The University of Western Australia. Breastmilk was sourced from healthy breastfeeding women located in Perth, WA, Australia. All participants (Ex vivo flow activated cell sorting (FACS) analysis of OCT4 and NANOG expression in freshly isolated breastmilk cells was done as described in Hassiotou et al.  and in breast tumors that display lactating features and/or are associated with pregnancy and lactation Figure A. The laP = 0.011) and lactating breast (P = 0.007) (Table P = 0.043) and lactating breast (P = 0.07)  and lactating breast tissues , with the highest expression observed in lactating adenomas (Table P = 0.061). OCT4 was co-localized with NANOG in the majority of positive cells , given that cell lines isolated from tumors that have high OCT4 expression are not commercially available. Breastmilk cells were characterized s Figure A, which s Figure A. In sphs Figure A. RT-PCRs Figure B. Express Figure B . Some of these tumors may be associated with pregnancy and lactation, although further research is needed to underpin the molecular characteristics of these tumors. Comparison of normal hBSCs with tumorigenic OCT4-tranduced cells showed differential expression of ESC TFs in the two systems. We propose that the levels and balance of expression of ESC TFs in breast cells distinguish between a normal and a CSC , often concentrated in the basal layer. This suggests that OCT4 confers a tumorigenic character when expressed at levels higher than the normal range. In support of this, forced ectopic expression of OCT4 in resting breast cells produced cells with high proliferation capacity that resembled the claudin-low subtype, and which formed primary tumors and metastases in nude/SCID mice (Beltran et al., In agreement with the association of these TFs with lactation, tumors with lactating features showed upregulation of OCT4, but with much higher expression than the normal lactating epithelium (Taken together, these results suggest that the distinguishing factor between normal MaSCs and breast CSCs may be the balance between and expression levels of TFs, which control breast cell self-renewal. These findings are integrated in Figure +/NANOG+ cells in the mammary stroma both in the normal lactating and the tumor tissues examined (Figures In addition to self-renewal, OCT4, SOX2, and NANOG control pluripotency in hESCs and iPSCs (Takahashi et al.,  Figures  and 4 fuTo our knowledge, this is the first study that directly compares ESC TF expression and subcellular localization between normal lactation and breast cancer, enabling a first examination of expression differences in these rare specimens. A limitation of the current data is the number of samples available for IHC analysis. However, normal human lactating breast tissue specimens are extremely rare, which is why the normal human lactating breast has been scarcely studied previously. Similarly, although breast tumor tissues with lactating features may not be as rare as previously thought, pathologists do not routinely look for and designate lactating features in tumor samples, rendering the acquisition of such samples problematic. Further studies are needed to confirm and expand the current findings, also including normal breast samples during early, middle and late pregnancy, to improve understanding of ESC TF expression during the normal pregnancy/lactation cycle.An important outcome of our study was the demonstration of ESC TF expression changes in different developmental stages of the normal adult breast. Previous focus on the resting breast, where MaSCs are scarce (Tiede and Kang, OCT4 and its associated embryonic TFs emerge as potential regulators of self-renewal and potency in early-stage MaSCs, which are scarce in the resting breast, but are activated to undergo a controlled program of proliferation and differentiation toward milk-secretory cells during pregnancy and lactation. In addition to their role during normal lactation, these genes may control breast oncogenesis by reverting normal mammary cells to a CSC self-renewing phenotype when expressed aberrantly. It is proposed that disruption of this controlled program of gene expression during pregnancy and lactation, failure to appropriately silence these genes during postpartum involution, and/or their aberrant imbalanced upregulation in the resting breast can be at the origin and progression of aggressive breast cancers, which may display lactating features. Future work will utilize the cellular hierarchy of breastmilk as a model to elucidate genetic events that transform cells along the mammary developmental continuum to give rise to different subtypes of breast cancer.Foteini Hassiotou: conception and design, financial support, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; Anna R. Hepworth: statistical analysis and final approval of manuscript; Adriana S. Beltran: collection of data and final approval of manuscript; Michelle M. Mathews: provision of study material, collection of data, and final approval of manuscript; Alison M. Stuebe: provision of study material and final approval of manuscript; Peter E. Hartmann: financial support, data interpretation, and final approval of manuscript; Luis Filgueira: data interpretation and final approval of manuscript; Pilar Blancafort: conception and design, financial support, data interpretation, and final approval of manuscript.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.


Conservation of orthologous regulatory gene expression domains, especially along the neuroectodermal anterior-posterior axis, in animals as disparate as flies and vertebrates suggests that common patterning mechanisms have been conserved since the base of Bilateria. The homology of axial patterning is far less clear for the many marine animals that undergo a radical transformation in body plan during metamorphosis. The embryos of these animals are microscopic, feeding within the plankton until they metamorphose into their adult forms.Patiria miniata, a sea star with an indirectly developing planktonic bipinnaria larva. We find that the animal-vegetal axis of this very simple embryo is surprisingly well patterned. Furthermore, the patterning that we observe throughout the ectoderm generally corresponds to that of "head/anterior brain" patterning known for hemichordates and vertebrates, which share a common ancestor with the sea star. While we suggest here that aspects of head/anterior brain patterning are generally conserved, we show that another suite of genes involved in retinal determination is absent from the ectoderm of these echinoderms and instead operates within the mesoderm.We describe here the localization of 14 transcription factors within the ectoderm during early embryogenesis in Our findings therefore extend, for the first time, evidence of a conserved axial pattering to echinoderm embryos exhibiting maximal indirect development. The dissociation of head/anterior brain patterning from "retinal specification" in echinoderm blastulae might reflect modular changes to a developmental gene regulatory network within the ectoderm that facilitates the evolution of these microscopic larvae. The astonishing diversity of animal forms, coupled with the complex life histories typical of many marine invertebrates, presents numerous challenges in inferring the ancestral character of members of the closely related phyla collectively known as the deuterostomes. Modern molecular phylogenies place four phyla within the monophyletic deuterostomes: Echinodermata and Hemichordata comprise a distinct clade called the Ambulacraria -3 that iWithin the Ambulacraria, the free-swimming, bilaterally symmetric larvae of echinoderms, especially the bipinnaria larva of sea stars and the auricularia larva of sea cucumbers, share many similarities with the tornaria larva of indirectly developing hemichordates. These microscopic larvae have an apical concentration of serotonergic neurons  and one Saccoglossus kowalevskii [Not all Ambulacrarians develop through a larval stage, however, and recent comparisons of regulatory gene expression have revealed that orthologs of many genes and signaling molecules involved in vertebrate neural patterning are expressed in spatially restricted domains along the anterior-posterior (AP) axis of the direct developing vermiform hemichordate juvenile alevskii ,14, a spalevskii ,16) theralevskii . In addialevskii , suggestPatiria miniata (previously Asterina miniata), which forms a typical bipinnaria larva. We show that these genes are expressed in diffuse concentric ectodermal domains that pattern the early embryonic axis. Furthermore, we observe in the sea star a general correspondence of domains of orthologous gene expression to those found along the AP and dorsal-ventral (DV) axis of direct developing deuterostomes. In addition, we detect expression of retinal determining gene orthologs in the mesoderm of echinoderm larvae, but not within the ectoderm of gastrulating embryos. We discuss the role of this implied modularity of regulatory patterning during evolution.Here we examine the expression of regulatory gene orthologs that have known or suspected roles in patterning the axial neuroectoderm of many protostome and deuterostome embryos within the indirectly developing sea star, P. miniata orthologs of regulatory genes that have known or suspected roles in patterning the axial neuroectoderm of many protostome and deuterostome embryos were isolated using a candidate gene approach. Recombinants for the following seven genes that encode homeodomain proteins were obtained: retinal homeobox (rx), optix-like homeobox 3 (six3), gastrulation and brain-specific homeobox (gbx), lim domain homeobox 2 (lhx2) and paired box homeobox 6 (pax6), as well as members of the Nkx gene family, nk2.1 and nk1. We also identified partial sequences of eyes absent (eya), the ets family gene pea3, and two C2H2 zinc-finger genes, zic and krupple-like factor 13 (klf13). The following four winged-helix forkhead box genes were isolated: foxq2, foxj1, foxd and foxg. A complete list of orthologs, sequence lengths, and orthology of gene sequences is provided in Additional file vegetal plate [Sea star late blastulae have a morphologically distinct animal-vegetal (AV) axis that is first readily observed when elongation of cells at the vegetal pole results in a noticeable thickening of epithelium termed the al plate . During al plate . At thiszic, foxq2, rx and nk2.1, are found only in the animal-most ectoderm  of foxg and lhx2, a gene localized to the aboral ectoderm, further highlights the oral side restriction of foxg transcripts in the gastrula . At prenk2.1 and foxd are expressed in part of the preoral ciliary band directly above the mouth , they are further subdivided into pre- and postoral regions on the basis of the localization of foxd, nk2.1, nk1 and gbx.The expression patterns at this later stage also show that the regulatory state of the early larval ciliary bands is heterogeneous, for example, P. miniata, does not appear to have a morphologically distinct animal pole domain at this stage. Transcripts of foxq2, pax6 and pea3  using the ciliary band marker, foxg, and the animal pole domain gene, pax6 -most ectoderm.Comparisons of the regulatory gene expression patterns that we observed in these indirectly developing sea star embryos with those known in directly developing bilaterians illuminate additional surprising patterns of conservation. We observe a general mapping of gene expression patterns along body axes  is involved in the formation of motor neurons in the ventral telencephalon [Saccoglossus nk2.1 orthologs also show a ventral bias in expression [foxg plays a role in ventral forebrain development, while lhx2 specifies dorsal telencephalic fates [foxg and nk2.1 is restricted to the oral  ectoderm, while lhx2 orthologs are expressed within the aboral  ectoderm  inherited from a common ancestor. Conservation, however, is not maintained for orthologs of genes expressed within regions posterior to the midbrain-hindbrain boundary in chordates and hemichordates as rudiment .pax6, six3 and rx play known roles in pattering and specifying anterior vertebrate sensory systems, most notably the eyes [pax6, the six gene family members and eya operate in a similar gene network for retinal determination in both vertebrates and Drosophila .Having established that orthologs of many regulatory genes involved in anterior neural specification are also expressed within the anterior ectoderm of sea star embryos, we sought to determine if orthologs of transcription factors involved within the retinal determination network are also expressed within echinoderm embryos.pax6 ortholog is expressed within the animal pole domain  in 1-week-old larvae  was performed as described previously [eviously ,38. Partescribed . Whole meviously .WMISH was performed essentially as described by Hinman et al. [et al. [in situ riboprobe in a reaction catalyzed by horseradish peroxidase, allowing for fluorescence detection of DIG- and DNP-labeled riboprobes.n et al. , with mo [et al. . In brieThe authors declare that they have no competing interests.WMISH and FISH. CKJ cloned sea urchin orthologs and performed WMISH. MLM cloned sea star orthologs and performed WMISH and FISH. All authors read and approved the final manuscript.VFH and KAY conceived of the study, participated in its design and drafted the manuscript. KAY cloned sea star orthologs and performed Table 1. List of sea star, P. miniata, orthologs and orthology of gene sequences.Click here for file


Type 2 diabetes (T2D) is predicted to affect one in three Americans by the year 2050 and is a leading cause of blindness, end stage renal failure and loss of limb with associated costs exceeding $180 billion/year. Insulin resistance in liver and skeletal muscle are major factors in the pathogenesis of T2D, with a strong relationship between ectopic lipid content in liver and skeletal muscle with insulin resistance and T2D in both humans and rodent models of NAFLD and T2D .50/ED50 of 13.Evidence in support of the key role of ectopic lipid in the pathogenesis of hepatic insulin resistance and T2D in humans comes from studies where we  and others  have shown that modest weight loss (~8 kg) resulted in large (~80%) reductions in liver triglyceride content, which were associated with normalization of fasting plasma glucose concentrations, hepatic glucose production and hepatic insulin sensitivity. Thus we hypothesized that reversal of non-alcoholic fatty liver disease (NAFLD), by promoting mild mitochondrial uncoupling would reverse insulin resistance and diabetes in rat models of NAFLD and T2D. Previous studies by our group had found that treating high fat fed rats with 2,4-dinitrophenol (DNP), a well established mitochondrial protonophore, would lead to mitochondrial energy uncoupling leading to reductions in liver triacylglycerol (TAG) and DAG content, decreased PKCε activation and decreased hepatic insulin resistance . DNP uncouples mitochondrial glucose and fat oxidation from ATP production by shuttling protons across the mitochondrial membrane resulting in increased mitochondrial energy consumption, but can be fatally toxic due to its on-target side effect of hyperthermia. We hypothesized that we could markedly increase the therapeutic window of DNP by targeting it to the liver. We therefore generated several derivatives of DNP, which would be preferentially metabolized by the cytochrome P-450 system in the liver to the active protonophore, DNP, and screened them in isolated hepatocytes for their ability to promote increased oxygen consumption. From this screen we identified DNP-methyl ether (DNPME), which raised oxygen consumption rates with similar potencies to DNP. As predicted, the ratio of toxic to effective dose was 50-fold greater for DNPME than DNP, without any evidence of hyperthermia or renal or hepatic toxicity in rats treated for 6 weeks with DNPME. This therapeutic index also compares favorably with other drugs that are in common use such as acetaminophen, which has a LDGiven the markedly increased therapeutic window for DNPME versus DNP we then examined whether DNPME would reverse insulin resistance, hepatic steatosis and diabetes in the rat. Despite identical body weights at the time of study, DNPME-treated rats had lower fasting plasma glucose, insulin, and triglyceride concentrations; improved glucose tolerance; and increased hepatic and peripheral insulin sensitivity. These improvements in liver and muscle insulin responsiveness were associated with reductions in liver and muscle TAG and DAG content as well as decreases in PKC activity in liver and skeletal muscle. In contrast there were no changes in tissue ceramide content or inflammatory markers with DNPME treatment, disassociating these factors from reversal of insulin resistance and diabetes in these animals.Given the surprising effect of DNPME to increase peripheral insulin sensitivity we examined the effects of DNPME on hepatic VLDL production and found that DNPME treatment resulted in a ~50% reduction in hepatic VLDL export, consistent with DNPME induced reductions in plasma triglyceride concentrations. In addition we observed that DNPME treatment caused a 60% increase in hepatic tricarboxylic acid (TCA) cycle flux whereas there were no observable effects of DNPME treatment on relative rates of mitochondrial fatty acid/TCA cycle flux in heart, skeletal muscle, brain, or kidney. Taken together these data imply that DNPME treatment promotes increased hepatic mitochondrial oxidation rates resulting in lower hepatic TAG and DAG content and reduced VLDL production resulting in decreased plasma triglyceride concentrations and decreased muscle TAG and DAG content .Because DNPME treatment promoted regression of NAFLD and insulin resistance in high fat fed rat models of NAFLD, we next examined whether DNPME would also be effective in two rat models of T2D. DNPME treatment led to a reduction in fasting plasma glucose, insulin, and triglyceride concentrations, improved glucose tolerance and reduced hepatic and peripheral muscle lipid content without any indication of hepatic or renal toxicity in both rat models, highlighting its potential for use as a therapeutic agent in patients with T2D.In summary these data demonstrate that by promoting a relatively small increase in hepatic mitochondrial uncoupling, DNPME can safely reverse hepatic steatosis, hypertriglyceridemia and insulin resistance in a rat model of NAFLD without inducing hyperthermia or associated systemic toxicities. In addition we also found that DNPME reduces fasting plasma glucose and insulin concentrations and improves glucose tolerance in two rat models of T2D. Taken together these data demonstrate the potential feasibility of disassociating the toxicity of DNP from its efficacy by altering the pharmacokinetics of DNP metabolism to treat the related epidemics of NAFLD, metabolic syndrome and type 2 diabetes.


It is widely accepted that specific human papillomavirus (HPV) types are the central etiologic agent of cervical carcinogenesis. However, a number of infected women do not develop invasive lesions, suggesting that other environmental and host factors may play decisive roles in the persistence of HPV infection and further malignant conversion of cervical epithelium. Although many previous reports have focused on HPV and environmental factors, the role of host susceptibility to cervical carcinogenesis is largely unknown. Here, we review the findings of genetic association studies in cervical carcinogenesis with special reference to polymorphisms of glutathione-S-transferase (GST) isoforms, p53 codon 72, murine double-minute 2 homolog (MDM2) gene promoter 309, and FAS gene promoter -670 together with HPV types including our recent research results. Cervical cancer is the second most common cancer in women worldwide, and is both a preventable and a curable disease especially if identified at an early stage. It is widely accepted that specific human papillomavirus (HPV) types are the central etiologic agent of cervical carcinogenesis. Other environmental and host factors also play decisive roles in the persistence of HPV infection and further malignant conversion of cervical epithelium . AlthougP < .05 was accepted as statistically significant.A large number of previous studies have suggested the possible correlation between genetic polymorphisms of cancer susceptibility genes and the higher risk of human malignant tumors , 3. GeneThe genes of glutathione-S-transferase (GST) family encode enzymes that appear to be critical in cellular protection against the cytotoxic effects. GSTs play an important role in conjugating glutathione to the products of endogenous lipid peroxidation and inactivating organic hydroperoxides via selenium-independent glutathione peroxidase activity, thus protecting the cell from the deleterious effects of oxidative stress . GST isoWe conducted GST genotype analysis together with HPV typing in a total of 198 cervical smear samples obtained from the patients who received cervical cancer screening. They consist of 54 normal, 102 low-grade squamous intraepithelial lesion (LSIL), and 42 high-grade SIL (HSIL). The protocol of this study was approved by our institutional review board, and all samples were obtained from Japanese women with informed consent. The exfoliated cervical cells were disrupted with lysis buffer, and genomic DNA was extracted with phenol-chloroform and precipitated with ethanol using standard techniques. The GSTM1 and GSTT1 genetic polymorphisms were evaluated using multiplex PCR techniques according to the method reported by Chen et al.  with somPrevious epidemiological studies of GST and cervical neoplasia found no significant differences in the frequency of GSTM1 or GSTT1 in women with SIL or cancer compared to controls with normal cervical pathology , 9, 10. p53 is a tumor suppressor gene involved in multiple pathways including apoptosis, cellular transcriptional control, and cell cycle regulation , 15. A lWe conducted genotype analysis of p53 codon 72 together with HPV typing in a total of 198 cervical smear samples obtained from the patients who received cervical cancer screening. They consist of 54 normal, 102 LSIL, and 42 HSIL, as described above. PCR restriction fragment length polymorphism (RFLP) analysis of codon 72 of the p53 gene, which was modified from a technique described by Ara et al. , was conAs shown in Our present results revealed that the differences in the polymorphic frequency of p53 Arg, Arg/Pro, and Pro genotypes between SILs and controls were statistically not significant. Moreover, neither Arg nor Pro allele affected the increased risk of SILs with or without high-risk HPVs compared to controls. Some previous studies have reported no correlation between germline polymorphisms of the p53 codon 72 and increased risk of cervical cancer –21. The Murine double-minute 2 homolog (MDM2) is the key negative regulator of p53, and dysfunction of these genes may be associated with an increased rate of accumulation of genetic errors, thereby enhancing the progression of the disease. A single nucleotide polymorphism (SNP) in the MDM2 gene promoter, SNP309 (a T to G change at nucleotide 309 in the first intron), increases the affinity of the promoter for the transcription activator Sp1, resulting in higher level of MDM2 mRNA and MDM2 protein and a subsequent attenuation of the p53 pathway . SNP309 We conducted genotype analysis of MDM2-SNP309 together with HPV typing in a total of 195 cervical smear samples obtained from patients with consent who received cervical cancer screening. They consist of 52 normal, 102 LSIL, and 41 HSIL. Eight human cervical squamous carcinoma cell lines  were also used for genotype analysis of MDM2-SNP309 together with HPV typing. All cell lines were originating from Japanese women, as previously described , were peRecently, Meissner Rde et al.  tested tOur present results using exfoliated cervical cell samples demonstrated that there was an increased OR for TG + GG genotype in HSIL cases compared to controls among the patients with high-risk HPV. We observed that HPV types 16 and 18, the most prevalent and aggressive types worldwide, are predominant in cases with TG + GG genotype and G allele. Moreover, 7 of 8 human cervical squamous carcinoma cell lines that possess high-risk HPV except YUMOTO also showed TG or GG genotype. These observations suggest that MDM2-SNP309 and high-risk HPV infection may be cooperatively associated with cervical carcinogenesis. It would be of interest to further evaluate whether MDM2-SNP309 has the potential to be used in conjunction with HPV-DNA testing and cervical cytology for the management of SIL patients.Apoptosis is a physiological process that regulates normal homeostasis, and alterations of apoptosis-related genes are likely to contribute to the pathogenesis of autoimmune diseases  and maliWe conducted genotype analysis of Fas gene promoter -670 together with HPV typing in a total of 279 cervical smear samples obtained from the patients with consent who received cervical cancer screening. They consist of 63 normal, 167 LSIL, and 49 HSIL. Eight human cervical squamous carcinoma cell lines  were also used for genotype analysis of this SNP together with HPV typing, as described above. PCR-RFLP analysis of the Fas gene promoter -670, modified from a technique described by Lee et al. , was conPolymorphisms in the promoter region or 5′ flanking region of genes can lead to different levels of gene expression and have been also implicated in a number of diseases. Recently, Lai et al.  conducte Previous studies , 43 haveHere, we review the findings of genetic association studies in cervical carcinogenesis with special reference to polymorphisms of GST isoforms, p53 codon 72, MDM2-SNP309, and FAS gene promoter -670 together with HPV types including our recent research results. Our studies using exfoliated cervical cell samples or human cervical squamous carcinoma cell lines have demonstrated that the GSTT1 null genotype, the TG/GG genotype of MDM2-SNP309, and the GA/GG genotype or G allele of Fas promoter -670 are closely associated with cervical carcinogenesis together with high-risk HPV infection. It would be of interest to further evaluate whether these polymorphisms could be used as a disease marker for the natural history of cervical neoplasms in a setting of longitudinal cohort study and for the determination of appropriate screening interval in patients with or without high-risk HPV. HPV are the etiologic agents of cervical and other epithelial cancers. Persistence of infections by high-risk HPV types is the single greatest risk factor for malignant progression. Vaccination against HPV types 16 and 18 has commenced or will soon commence in a number of countries. Our studies have demonstrated that the frequency of G allele increased from LSIL to HSIL and that there was an increased OR for G allele in HSIL cases with high-risk HPV types including 52 and 58. It is known that geographically different oncogenic HPV types 52 and 58 are more prevalent than 16 and 18 in East Asia. HPV prevalence should be considered and treated individually regarding the strategy that best suits the HPV types in a given geographical area. It is likely that novel strategies in combination with vaccination against HPV types 16 and 18 are required in those countries where other types of HPV may be more prevalent. Moreover, further studies on the differential gene expression profiles between normal cervical keratinocytes and cervical cancer cell lines may provide the better understanding for the effect of these polymorphisms in the sequence of cervical carcinogenesis.