The vesicular neurotransmitter transporters (VNTs) are small proteins responsible for packing synaptic vesicles with neurotransmitters thereby determining the amount of neurotransmitter released per vesicle through fusion in both neurons and glial cells. Each transporter subtype was classically seen as a specific neuronal marker of the respective nerve cells containing that particular neurotransmitter or structurally related neurotransmitters. More recently, however, it has become apparent that common neurotransmitters can also act as co-transmitters, adding complexity to neurotransmitter release and suggesting intriguing roles for VNTs therein. We will first describe the current knowledge on vesicular glutamate transporters (VGLUT1/2/3), the vesicular excitatory amino acid transporter (VEAT), the vesicular nucleotide transporter (VNUT), vesicular monoamine transporters (VMAT1/2), the vesicular acetylcholine transporter (VAChT) and the vesicular γ-aminobutyric acid (GABA) transporter (VGAT) in the brain. We will focus on evidence regarding transgenic mice with disruptions in VNTs in different models of seizures and epilepsy. We will also describe the known alterations and reorganizations in the expression levels of these VNTs in rodent models for temporal lobe epilepsy (TLE) and in human tissue resected for epilepsy surgery. Finally, we will discuss perspectives on opportunities and challenges for VNTs as targets for possible future epilepsy therapies. Epilepsy is one of the most common acquired chronic neurologic disorders, affecting approximately 1% of the human population and displaying an annual incidence of 50,000–100,000 persons , caused by abnormal synchronized, high frequency neuronal discharges  or potassium (K+) channels, (2) alterations in γ-aminobutyric acid (GABA)ergic inhibition via actions on GABAA receptors or on GABA synthesis, reuptake or degradation, (3) decreased synaptic excitation via actions on ionotropic glutamate receptors and (4) modulation of neurotransmitter release via presynaptic mechanisms, with most relevant action on glutamate release  modulation of voltage-dependent sodium, calcium , the vesicular excitatory amino acid transporter (VEAT), and the vesicular nucleotide transporter (VNUT)  for serotonin (5-HT), dopamine (DA), noradrenaline (NE) and histamine and the vesicular acetylcholine transporter (VAChT)   Figure . The rat Reimer, . The SLCGasnier, .H+) generated by the vacuolar H+-ATPase. Despite the utmost important function of VNTs, the main regulation of this vesicular transport remains unknown. Two mechanisms have been suggested to modulate vesicular transport: (1) influencing the membrane potential (Δψ) by cation and anion fluxes and (2) a direct interaction between the heterotrimeric G-protein, Gα o2 and VNTs  probably creates a lumen-positive Δψ and consequently increases the ΔμH+ for ACh and 5-HT vesicular accumulation , heterozygous (+/−) and wild-type (+/+) mice, identified SV2A as the binding target for LEV. In addition, these transgenic mice were phenotyped in kindling and distinct acute seizure models, unveiling a decreased seizure threshold and accelerated kindling development of the SV2A+/− mice compared to the SV2A+/+ mice. SV2A−/− mice, on the other hand, exhibit early severe seizures and die within 2–3 weeks after birth , the binding site of levetiracetam (LEV). These data prove that vesicular proteins are accessible and propose a promising role for proteins, involved in vesicle exocytosis, as novel targets for the development of AEDs. Unfortunately, only few studies have followed this concept and brivarecetam is the only new AED in the pipeline targeting a vesicular protein. As outlined for SV2A, results from transgenic mice, distinct acute and chronic rodent epilepsy models and resected human epileptic tissue are crucial to constitute a clear and complete conception of new, potential AED targets. Consequently, in this review we will use the same outline structure to describe the known literature on the different VNTs in epilepsy. Unveiling and understanding the structure of VNTs is of utmost importance to deduce their functional domains and their physicochemical properties in order to develop compounds that can interact with these transporters. Therefore, we will also shortly describe and show the known VNT structure models and the most active and used modulators of their activity. In conclusion, we will point out the remaining challenges for research on VNTs as possible targets for future epilepsy therapies.SLC17 gene family consists of nine members . The MFS is the largest group of secondary active transporters, regulating the transport of a wide variety of substrates, such as inorganic ions, sugars, amino acids, and xenobiotics, across cellular and intracellular membranes   was reduced in punctate structures of one or several layers of the peritumoral neocortex of patients with epilepsy that was secondary to low-grade tumors, a common cause of epilepsy in which the epileptogenic region presents a loss of neurons and excitatory synapses. This decrease was correlated with gliosis, neuronal loss, and a decrease in the number of asymmetrical synapses  levels have been shown in the VGLUT1+/− mice . Both disorders are marked by accumulation of sialic acid in lysosomes  neurotransmission could contribute to the severe neurological defects of Salla disease  decreased KCl-triggered release of ATP, confirming the involvement of this transporter in vesicular storage and subsequent exocytosis of ATP -IR was decreased in the striatum, hippocampus, thalamus, frontal cortex, and cerebellum, while VMAT-IR was increased in the striatum. This significant increase of VMAT2 is suggested to be a compensation for deficient dopaminergic/noradrenergic innervation, as a consequence of decreased TH , but not VMAT2 mRNA levels are transiently elevated in locus coeruleus (LC) neurons. Noradrenergic LC efferents innervate most forebrain areas and are activated postictally in epilepsy kindling models. Moreover, LC stimulation has been demonstrated to exert seizure-suppressing effects in several epilepsy animal models. Consequently, it is suggested that the increase of TH and NET, responsible for synthesis and reuptake of NE, respectively, could be an adaptive mechanism to restore intracellular NE levels and enable LC neurons to counteract hyperexcitability , which is highly innervated by cholinergic terminals from the basal forebrain  and nicotinergic ACh receptors (nAChRs). Pilocarpine, a non-selective mAChR agonist, is a widely used chemoconvulsant in both actue and chronic animal epilepsy models  and VAChT are decreased following acute stress , responsible for the vesicular accumulation of electrically neutral substrates, GABA, and glycine  act selectively through the GABA-ergic system. The former inhibits neuronal and glial uptake of GABA (including GAT-1) whilst the latter increases the synaptic concentration of GABA by inhibition of GABA-aminotransferase. Other new anticonvulsants are gabapentin and its structure-related pregabalin that are GABA analogues. They easily cross the blood-brain barrier and increase brain synaptic GABA, but their effects are complex and have not been fully characterized 18.5 and birth. They exhibit a hunched posture and are completely immobile and stiff, most likely symptoms of overexcitation  mice  vs. wiltype brain tissue, is necessary.Currently commercial antibodies cannot be used for immunolocalization of sialin and from previous immunohistochemical studies  mice clearly exhibit seizures, this transporter, whether or not crucial for vesicular import of aspartate, still might be a novel AED target. Subsequently, the currently available tools should be used to further investigate this role. The seizure threshold of sialin(+/−) can be compared to their wildtype littermates in different animal models for epilepsy and moreover, the expression levels of VEAT in human epileptic tissue should be determined.However, since sialinThe lack of tools is a major issue in order to unravel the possible involvement of VNUT in epilepsy and epileptogenesis. Although ADK overexpression and adenosine deficiency are considered to be pathological hallmarks in distinct rodent models and in human tissue resected from TLE patients  promoter. These VMAT2The precise role VAChT plays in epilepsy still needs to be unraveled. The sparse data from human epileptic tissue and homozygous VAChT knock-down mice, suggest that inhibition of this VNT might lead to protection against seizures. However, cholinergic neurotransmission in the hippocampus is linked to cognitive functions, such as learning and memory. Hippocampal LTP is negatively affected in homozygous VAChT knock-down mice, which is of great importance in learning and memory processes  controllers should be considered as hot topic therapeutic interventions to regulate VNTs expression levels.SCL18A2 hypermethylation was observed in ~90% of all cases, subsequently epigenetic silencing of VMAT2 was defined as a novel adverse predictor of biochemical recurrence after radical prostatectomy (Sorensen et al., Epigenetics is defined as information that is heritable during cell division other than the DNA sequence itself, including DNA methylation or histone tail modifications, which can produce lasting alterations in chromatin structure and gene expression (Kobow and Blumcke, miRNA on the other hand is a group of small non-coding RNA able to control post-transcriptional gene expression by fine-tuning protein production, through sequence-specific binding within the 3′ untranslated region of mRNA transcripts (Jimenez-Mateos and Henshall, Epilepsy remains a hard-to-treat disorder for millions of patients. There is an urgent need of non-invasive antiepileptogenic or disease-modifying treatments. VNTs are appealing new intracellular targets for future AEDs. Nevertheless, LEV and brivaracetam are, respectively, hitherto the only approved AED and the only AED currently in the pipeline targeting a vesicular protein (Kaminski et al., 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.


Aging is a complex process characterized by a gradual decline in organ functional reserves, which eventually reduces the ability to maintain homeostasis. An exquisite feature of elderly subjects, which constitute a growing proportion of the world population, is the high prevalence of cardiovascular disorders, which negatively affect both the quality of life and the life expectancy. It is widely acknowledged that physical activity represents one of the foremost interventions capable in reducing the health burden of cardiovascular disease. Interestingly, the benefits of moderate-intensity physical activity have been established both in young and elderly subjects. Herein we provide a systematic and updated appraisal of the literature exploring the pathophysiological mechanisms evoked by physical activity in the elderly, focusing on the functional role of the β adrenergic system. Aging is a multifaceted process characterized by a gradual decline in organ functional reserves , a prolongation of isovolumetric relaxation time, a modification of the dynamic longitudinal wall relaxation, and diastolic suction . All these differences have been reported across a wide range of physiological filling pressures , heart failure (HF), and atrial fibrillation (AF) increases dramatically with age , performed twice weekly for 10 years confers a sustained improvement in quality of life compared with non-trained patients  sensitivity and density has been shown in the cardiac muscle and has been mainly attributed to down-regulation and impaired coupling of βAR to adenylate cyclase Lakatta, . The ageYoung individuals are more responsive than elderly subjects to isoproterenol-induced increases in blood flow in the brachial artery (van Brummelen et al., The age-linked decline in adrenergic responsiveness impairs also vasodilatation, increasing thereby total peripheral resistances (Santulli and Iaccarino, 2AR mechanism (Maisel et al., 2AR-selective ICI 118,551 but not by the β1AR-selective bisoprolol (Van Tits et al., The βAR system is activated in lymphocytes during prolonged aerobic physical exercise both in healthy subjects and in HF patients Maki, . The num2AR has been indicated as a mechanism underpinning the increased vasodilator response to isoproterenol after exercise (Gaballa et al., 2AR receptor selective antagonist ICI 118,551 is able to significantly decrease coronary blood flow velocity and increase late diastolic coronary resistance during a running session (DiCarlo et al., The effects of exercise on cardiovascular catecholamine responsiveness have been extensively studied, pointing out the functional role of βAR. An increased responsiveness to isoproterenol has been demonstrated in the myocardium of trained rodents, when compared with sedentary controls (Takeda et al., Other recent studies have shown in animal models that physical exercise ameliorates sensitivity of βAR when the vasodilator response mediated by such receptors has been previously reduced by the aging process. In particular, a 6-week training program of 5 days/week swimming exercise improved vasodilator response to the non-selective βAR agonist isoproterenol, in coronary arteries, compared with the sedentary group (Santulli and Iaccarino, Collectively, the aforementioned studies show the beneficial effects of physical training on vascular sensitivity in the aging process. However, βAR responsiveness to exercise is not homogeneous, depending on several factors, including the region of vascular bed to be studied. Indeed, regions of different diameters in the same artery might respond differently to physical exercise. Another noteworthy variable is the type of artery studied: resistance and conductance vessels show indeed different responses.Aging has become one of the most critical issues for industrialized nations, because population average age, and thus the incidence of age-associated disorders, has markedly risen to create a major burden as patients draw heavily on the need for continuing medical treatment and hospital and other community services. In the present review, we examined the relationship between physical training and aging, focusing on the functional role of βARs. The amelioration of βAR responsiveness, obtained through means of regular physical training, contributes to the clinical improvement in cardiovascular health reported in elderly subjects.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.


This review aims to provide a snapshot of the actual state of knowledge on genetic variants of nuclear receptors (NR) involved in regulating important aspects of liver metabolism. It recapitulates recent evidence for the application of NR in genetic diagnosis of monogenic (“Mendelian”) liver disease and their use in clinical diagnosis. Genetic analysis of multifactorial liver diseases such as viral hepatitis or fatty liver disease identifies key players in disease predisposition and progression. Evidence from these analyses points towards a role of NR polymorphisms in common diseases, linking regulatory networks to complex and variable phenotypes. The new insights into NR variants also offer perspectives and cautionary advice for their use as handles towards diagnosis and treatment. Systematically, genetic analysis with regard to disease onset and progression can be separated into pre- and post-hoc examination of monogenic or polygenic diseases. Monogenic (“Mendelian”) diseases are caused by a single gene defect and follow relatively straightforward inheritance patterns. The most prominent of these disorders are rather rare, often severe, and characterized by early onset. Genetic testing for monogenic liver disease in symptomatic patients is based on known disease-associated gene variants, thereby confirming the genetic etiology and sometimes allowing prediction of disease progression . In contrast, polygenic diseases such as fatty liver disease and gallstones result from combinations of multiple gene variants and environmental factors, all of which play a role in disease initiation and progression . The assNuclear receptors (NRs) are a subclass of regulatory molecules that orchestrate gene transcription in response to the presence or absence of specific ligands. Due to these functional requirements, they are characterized by the presence of a ligand-binding and a DNA-binding domain. NRs represent a central point of interaction between environment and gene regulation. They are the “hinge” connecting endogenous and environmental stimuli, that is, ligands, with the cells' transcriptional response . This position makes them a prime target for medical intervention by agonistic or antagonistic binding of synthetic compounds. However, the regulatory orchestra of molecules conducted by NR is highly complex and abounds with redundancy and crosstalk, hence any impact might be potentially difficult to predict as well as disappointingly diffuse. Even identical variations in a single NR can result in a wide variety of phenotypes due to genetic differences in the cofactors involved and higher order networks based on mutual regulatory interaction .NR2A1)  allele at position −1 of the FXR-coding sequence.Quantitative trait locus mapping in inbred mice identified the s et al.  describe = 0.02) . The assl et al.  used an l et al.  went on l et al.   is a fatty acid-activated member of the PPAR subfamily of NR. These receptors play important roles in lipid and glucose metabolism. Members of the family have been implicated in obesity-related metabolic diseases such as hyperlipidemia, insulin resistance, and coronary artery disease. Like FXR, PPARs form heterodimers with RXR, and these heterodimers regulate the transcription of various genes in liver. Polymorphisms in ipidemia –40  and the level of circulating leptin in obesity. Obese subjects carrying at least one [T] allele displayed higher plasma leptin levels than homozygous carriers of the common allele. The [T] allele was also associated with lower BMI at a given leptin level, indicating a complex interaction between PPAR-gamma and leptin signaling. These findings could be confirmed and extended by a study in 96 Chinese patients with nonalcoholic fatty liver disease (NAFLD), which reported an association of this variant with adiponectin levels and the development of NAFLD [PPARG c.C161T and other polymorphisms are associated with the levels of tumor necrosis factor (TNF)-alpha, leptin, and adiponectin in NAFLD. When patients in Germany  [n = 202) [Meirhaeghe et al.  describeof NAFLD . Zhou etof NAFLD  replicatn = 363)  and Italn = 202)  were anaα/PPARGC1). The synthetic agonist GW4064 enhances expression of PGC1α and thus mitochondrial function, however enhanced expression of FXR increases PPARGC1 RNA not directly, but via protection from repression by the atypical corepressor SHP [Bile salts are intimately entwined with lipid metabolism, and besides their well-known role in dietary lipid absorption and cholesterol homeostasis, evidence is accumulating that the body uses blood levels of bile salts as sensor for metabolic processes . Hence,ssor SHP .PXR locus in 188 patients with NAFLD showed an association of two variants (rs7643645 and rs2461823) with several phenotypes of the disease, among others ALT levels [The pregnane X receptor (PXR/NR1I2) is known to be involved in the regulation of hepatic detoxification processes. Using PXR knockout and humanized mouse models, PXR was found to influence drug × drug interactions, hepatic steatosis, and the homeostasis of vitamin D, bile salts, and steroid hormones , 54. InvNr5a2 in mice results in compromised intestinal lipid absorption as well as defective embryogenesis and cholesterol homeostasis [In vitro methylation of the SHP promoter reversibly decreased transactivation and LRH-1 binding; overexpression of SHP inhibited HCC foci formation, arrested HCC tumor growth in xenografted nude mice, and increased the sensitivity of HCC cells to apoptotic stimuli [Liver receptor homologue 1  is an orphan member of the NR superfamily, that is, it has no known endogenous ligand. LRH-1 is involved in the regulation of genes that participate in steroid, bile salt, and cholesterol homeostasis . Knockoueostasis , 57. Appeostasis . In mous stimuli .ABCB11 might be associated with sustained virological response [In vitro experiments using HCV replicon-harboring cells have shown that the impact of bile salts on HCV replication might be through the action of FXR rather than a direct effect of bile salts themselves [FXR variants implicated in ICP [The replication of hepatitis C virus (HCV) is linked to lipid droplets, and a combined genomic/metabolomic analysis of HCV-infected HUH-7.5 cells by RNA sequencing, microarray, and proteomics revealed profound changes in, among others, PPAR signaling and PXR/RXR activation . Viral rresponse  and progresponse . In vitremselves . FXR antemselves . Hence, d in ICP . We spec Differential regulation of the pre-C and pregenomic promoters of HBV by members of the NR superfamily  has been known for some time . RamièreABCB11 are known to be associated with cholestasis induced by oral contraceptives and other drugs [While diet-induced metabolic overload, alcohol and HCV are the most common insults to the liver, drug-induced injury (DILI) is gaining in prominence due to increasing age and multimorbidity of the general population. The likelihood of medication-induced liver damage increases substantially beyond the threshold of 50 mg per day cumulative ingestion . The inver drugs , as revier drugs .PXR [CAR [ The role of gene polymorphisms in predisposition towards DILI has been reviewed comprehensively , 73. SufPXR  and CAR PXR [CAR , 75, 76 NR1H3 gene encoding LXR-alpha, which affects HDL cholesterol level with an effect size of only 4% but shows a risk allele frequency of 42%, the study also detected some higher impact rare alleles. A variation in the gene for the androgen receptor AR (NR3C4) exerted a strong influence on LDL cholesterol levels, with a considerable effect size of 30% set aside a risk allele frequency of only 2% [Identifying and quantifying genes associated with metabolic traits is one of the prime challenges when devising means to deal with the epidemics of diabetes and metabolic syndrome. This information might be used as cost-efficient leverage to identify and motivate at-risk individuals towards lifestyle changes, but to date there is no evidence for clinical impact . A long  only 2%   was identified as a new contributory factor for HDL cholesterol levels, with an effect size of 19% [ A GWAS with more than 2.5 million SNPs in 19,840 individuals, with replication in up to 20,632 individuals, identified more than 30 loci that had an impact on serum LDL cholesterol, HDL cholesterol, or triglyceride levels. A rare variant  in e of 19% .in silico. Phenotypes examined included various metabolic traits such as fat mass at different diets, cholesterol, and triglyceride levels under normal and atherogenic diet. Ten of the QTL regions identified in this study contained candidate genes that had previously been characterized and shown to cause metabolic phenotypes in agreement with the trait used for mapping, serving as a proof of principle for the application of this methodology.Technical progress, particularly the combination of increased speed and decreased cost of genotyping and expression quantification, is ushering in systematic approaches to metabolic traits. Once sufficiently large numbers of individuals from any population  have been genotyped for markers covering the entire genome, statistics can be used to calculate the likelihood of correlation between the inheritance of genotypes and the expression values of quantifiable phenotypes, including the abundance of transcripts assayed by expression arrays. This methodology, denoted as “expression quantitative trait loci (eQTL) mapping” can be used to unravel gene regulation by association and identify regulatory networks involving multiple loci. To extend the information from these large datasets, Schadt et al.  proposed A note of caution is obviously due when transferring data and results from mouse models into the human genomic context. Transcriptional regulation, particularly organ-specific mechanisms and binding sites, has diverged significantly between man and mouse, probably more so than NR function . Neverthtransregulatory effects by eQTL might help to pinpoint functional mechanisms for these disease-associated variants. eQTL data enables us to identify a mechanism by which a SNP controls expression of a remote locus, hence causing predisposition to disease by allelic variation across long genomic distances [The identification and functional characterization of regulatory SNPs have encouraged the use of eQTL data for the interpretation of GWAS results. These genome-wide scans using anonymous SNP markers usually detect associations of disease with polymorphic markers, often in regions without known candidate genes . For an istances .Technical advances increase our knowledge regarding the biochemical and gene-regulatory mechanisms underlying metabolic diseases. Transgenic animals inform us about gene function, eQTL studies in human samples and model systems provide us with information about genetic loci that are associated with the inheritance of multiple metabolic parameters, and GWAS in genetically well-characterized cohorts yield candidate genes for metabolic disturbances at ever increasing resolution and depth. Considering all this amassed knowledge, our understanding of disease pathobiology is improving constantly. Considering the reality of how little lifestyle modification is achieved by risk information, as illustrated by antismoking campaigns, it remains a challenging task to employ this knowledge to combat the epidemics of the metabolic syndrome and its associated burden of disease.


Phase contrast cardiovascular magnetic resonance (CMR) is able to measure all three directional components of the velocities of blood flow relative to the three spatial dimensions and the time course of the heart cycle. In this article, methods used for the acquisition, visualization, and quantification of such datasets are reviewed and illustrated.Currently, the acquisition of 3D cine (4D) phase contrast velocity data, synchronized relative to both cardiac and respiratory movements takes about ten minutes or more, even when using parallel imaging and optimized pulse sequence design. The large resulting datasets need appropriate post processing for the visualization of multidirectional flow, for example as vector fields, pathlines or streamlines, or for retrospective volumetric quantification.Multidirectional velocity acquisitions have provided 3D visualization of large scale flow features of the healthy heart and great vessels, and have shown altered patterns of flow in abnormal chambers and vessels. Clinically relevant examples include retrograde streams in atheromatous descending aortas as potential thrombo-embolic pathways in patients with cryptogenic stroke and marked variations of flow visualized in common aortic pathologies. Compared to standard clinical tools, 4D velocity mapping offers the potential for retrospective quantification of flow and other hemodynamic parameters.Multidirectional, 3D cine velocity acquisitions are contributing to the understanding of normal and pathologically altered blood flow features. Although more rapid and user-friendly strategies for acquisition and analysis may be needed before 4D velocity acquisitions come to be adopted in routine clinical CMR, their capacity to measure multidirectional flows throughout a study volume has contributed novel insights into cardiovascular fluid dynamics in health and disease. Phase contrast (PC) cardiovascular magnetic resonance (CMR) can measure, non-invasively, all three directional components of the velocities of blood flow relative to all four spatio-temporal dimensions of the heart and great vessels. The underlying principles have been known and applied over several decades -6. The mAlternatively, Doppler ultrasound can be employed to assess regional blood flow velocities. The technique is widely and routinely used in numerous applications and cardiovascular pathologies and has a number of advantages compared to MRI including widespread availability, ease of use, and no contraindications in case of pacemakers or metallic implants. However, Doppler ultrasound is also limited by its inter-observer variability and by detecting only the component of velocity directed to or from the transducer.Computed tomography provides relatively rapid 3D scans with excellent spatial resolution that can show the intravascular distribution of contrast agent at a given moment, but without being able to measure the velocities of blood flow.This review paper deals principally with the methods and applications of the more comprehensive, 3 dimensional, 3 directionally encoded, time resolved (cine) velocity acquisition,17, whicThe concise naming of such acquisitions has varied between groups. While '4D' has come to be widely used and recognized, having the advantage of brevity, use of '3D cine' is arguably more correct and consistent with the use of '2D cine' to describe routine velocity mapping  of all required (N) phase encoding steps are collected (k-space segmentation). The procedure has to be repeated for a total of N/nk heart beats to acquire the full data set. Gating can be accomplished using either prospective or retrospective gating. In prospective gating full 3D datasets are acquired at defined time points from the ECG trigger. In retrospective gating, full 3D datasets are retrospectively assembled by interpolation at percentages of the cardiac cycle. Prospective gating will be sufficient for studying systolic or early diastolic events, but differences in the length of involved heart cycles will prevent measurement at late diastolic phases. Using retrospective gating, full coverage of the cardiac cycle can be obtained.A typical measurement strategy is schematically illustrated in figure x, vy, vz). To minimize artifacts in phase difference images related to subject motion, interleaved velocity encoding is often performed, for which the different flow encodes are kept as close together as possible in time as shown in figure Res = 4 TR.Four acquisitions are necessary to complete the velocity encoding for each cine time frame. Successive acquisitions have to be performed for reference scan and three velocity sensitized acquisitions to calculate the three-directional blood flow velocity  currently allows the acquisition of 4D velocity data with a spatial and temporal resolution on the order of 2 mmhe heart ,20,43,44It is well known that phase contrast CMR can be subject to phase offset errors associated with eddy currents, Maxwell terms, and gradient field imperfections -47. ThesAdditional problems, such as velocity aliasing should be corrected in order to improve the data quality before further data processing or visualization. Appropriate correction and pre-processing strategies should thus be applied to ensure accurate flow measurement as described in more detail in references -48.The exact correction approaches may need to be adapted to the specific CMR system, imaging protocol and cardiovascular region of interest. A systematic validation strategy, incorporating both in-vitro and in-vivo validation methods, is essential to assess whether the applied correction is effective for the chosen application ,49.Three directional phase contrast data can not only be used for flow velocity analysis but also to gain information on vascular geometry by calculating a 3D phase contrast angiogram (3D PC MRA), information in the acquisition being used to identify vascular boundaries without the need for additional measurements -53. AlgoAlthough the derived 3D PC-MRA does not provide as detailed a depiction of vascular boundaries as contrast enhanced MRA, it can contribute to the presentation of flow results by displaying the 3D vascular form relative to which flows can be traced and orientated, as illustrated for the aorta in Figures In the heart, the boundaries move considerably through the cardiac cycle, which makes 3D PC-MRA data less useful. Combining the flow visualization with separately acquired balanced steady-state free precession data, often part of the standard protocol, can improve orientation and presentation considerably, as demonstrated in Figures The comprehensive nature of the 4D velocity data  requires data analysis strategies to deliver clinically relevant visualization and quantification. A typical CMR exam of the thoracic aorta comprises about 4000-8000 raw magnitude and velocity component images, depending on the spatial and temporal resolution and coverage. Efficient visualization and quantification strategies are needed to convert the wealth of information contained into meaningful images and measurements. Interactive methods of analysis offer versatility for the exploration of novel or alternative approaches, whereas standardized and automated methods can be more user-friendly, less operator dependent, and so more suitable for clinical use or larger patient studies.For the analysis and visualization of multidirectional blood flow in a 3D volume, several visualization tools, including 2D vector-fields, 3D streamlines and 3D pathlines  -14,61. MAn advantage of 4D velocity acquisition relative to traditional 2D velocity mapping is its suitability for retrospective quantitative analysis at any anatomical location within the acquired volume. An example of a strategy for retrospective quantification is illustrated in figure Advantages of 4D relative to the more conventional acquisitions of several, specifically located 2D velocity maps include simpler planning and retrospective choice of locations and types of analysis. Volumetric flow may be calculated in regions not previously known to be relevant, identified retrospectively and interactively. An example of such an approach is the measurement of flow through all four heart valves, using retrospective tracking of the valves . 4D acquDirect Flow: Blood that enters the LV during diastole and leaves the LV in the following systole. Retained Inflow: Blood that enters the LV during diastole but does not leave during systole in the analyzed heart beat. Delayed Ejection Flow: Blood that starts and resides inside the LV during diastole, but then leaves with the following systole. Residual Volume: Blood that stays in the LV for more than two cardiac cycles. This information can be used to color-encode blood regions based on their rates of passage, giving an informative type of visualization  ,55,82-84In the healthy thoracic aorta, characteristic flow patterns such as helical flow and mild early diastolic retrograde flow in the ascending aorta have been identified. Normal large scale aortic flow features include the following: During systole, left ventricular outflow initially generates flow that tends to be skewed towards the shorter, inner curvature. The peak velocity stream tends to migrate to the outer curvature, and later in systole, to curve postero-laterally, back towards the inner curvature in a right-handed helix in the ascending aorta and arch. Flow velocities are highest in the ascending aorta  and slightly lower in the arch, where flow enters the brachiocephalic branches. In the proximal descending aorta, velocities increase where streamlines tend to separate from the inner curvature, converging slightly towards the outer wall  and pulse wave velocity (PWV) in the healthy aorta. 4D velocity CMR was used to derive the normal distribution of vectorial WSS in the entire thoracic aorta. Marked regional variations of WSS found in these studies may help explain why atherosclerotic lesions tend to develop and progress at specific locations in the aorta such as the inner aortic curvature and supra-aortic branches . The feaResidual Volume, related to the high end systolic volume of the cavity, other flow components show changes such as a decrease in the Direct Flow [A number of studies have investigated abnormal intracardiac flow. Considerable changes in the presence, position and extent of vortices, as well as relative size of the flow components have been found in patients with dilated cardiomyopathy ,76,90. Tect Flow ,76.Multidirectional velocity acquisitions can be used for assessments of valve abnormalities before surgery and during follow-up . It has A number of studies have described blood flow in patients with common vascular pathologies such as aortic atheroma, aneurysms, dissection, coarctation and graft repair, identifying regions of complex and potentially pathological flow ,57,58,84A study using 4D velocity acquisition provided novel evidence for a potential source of otherwise cryptogenic embolic stroke. Pathline visualization showed a high incidence of retrograde flow connecting complex atheromatous plaques in the proximal descending aorta with inflow to the carotid arteries. It suggested that descending aortic atheroma, even if located several centimeters distal to the left common carotid artery, should be regarded as a potential source of embolic stroke .Other studies showed that relatively subtle geometric changes of the ascending aorta, for example age related ectasia or moderate aortic valve stenosis, resulted in marked alteration of flow patterns in and downstream of the affected region ,94. In pFindings in a patient with aortic valve stenosis and dilated ascending aorta are illustrated in figure Such studies can help to identify regions of abnormal turbulence and shear rates. From the literature it is known that unfavorable shear forces near the vessel wall can change endothelial function and create areas at risk for vascular remodeling . Flow abInitial studies in larger cohorts of patients with aortic atherosclerosis have confirmed the expected increase of global pulse wave velocity (i.e. stiffening of the aorta) related to age and presence of atherosclerosis . Wall shThe more intense, turbulent instabilities associated with jet flow beyond a stenosis have been investigated, as outlined above, by turbulent intensity analysis ,73. HereStudies in patients with aortic coarctation demonstrated that 4D velocity acquisition can help evaluate collateral blood flow as a potential measure of hemodynamic significance. Additionally, 3D visualization showed distorted flow patterns in the descending aorta after coarctation repair such as marked helical and vortical flow in regions of post-stenotic dilation .3) and temporal (40-50 ms) resolution.By exploiting the trade-off that can be made between volume coverage and scan time, 3D flow maps covering the entire heart and surrounding large vessels can be acquired in a single acquisition. However, currently available techniques still require scan times of about 20-40 minutes for adequate spatial  are promising since redundancies in two spatial encoding and the temporal dimensions can be utilized to speed up data acquisition ,104. It New methods based on the combination of phase contrast MRI and fast sampling strategies, e.g. echo-planar imaging and radial imaging with 3D PC-VIPR, have been reported and are promising for further reduction in total scan time and/or increased spatial or temporal resolution ,87. A nuImaging at even higher magnetic field strength (7T) may thus be promising for achieving high spatial resolution and assessing small vascular structures  which are difficult to assess with currently existing 4D velocity implementations. One of the ultimate goals, the acquisition of time-resolved 3D hemodynamics in the coronary arteries, has to date not been accomplished. Due to their low diameter, complex structure, and particular substantial motion during the cardiac cycle, the assessment of coronary 4D velocities is expected to be challenging even at ultra-high fields. We speculate that a combination of spatio-temporal image acceleration, improved and reliable respiration synchronization, high field, and possibly even intra vascular contrast agents may be necessary.The time needed for acquisition also means that, while relatively large scale cyclically repeating flows can be visualized, smaller scale flow instabilities are not. In the heart and large vessels, in some regions more than others, blood flow tends to fragment and mix through fluctuating eddy formation, particularly in regions of flow separation and high shear. These are inevitable given in the velocities, viscosity, dimensions, diameter-changes and direction-changes of normal as well as abnormal cardiovascular flow fields. Flow instabilities may be glimpsed in vivo as local variations of blood signal in breath-hold or real-time CMR cine images, particularly when flow in a dilated cavity is unusually slow. However, CMR lacks the spatio-temporal resolution to show all instabilities. They are generally too small and rapidly passing to be resolved, particularly by prolonged 4D velocity acquisitions. This has implications for the interpretation of some attempted measurements, notably those of wall shear stress. Powerful small scale velocity fluctuations, as present in disturbed or turbulent blood flow, can however be displayed from a 4D PC-MRI acquisition by quantification of the intravoxel standard deviation .In addition to long acquisition times, another drawback of 4D velocity CMR is related to the complex and often time-consuming post acquisition data analysis. More automated methods for flow visualization and retrospective quantification would thus be helpful for applications within a clinical workflow. New software tools and algorithms need to be developed, for example to define standardized analysis planes in routinely acquired 4D velocity data. Similarly, for all aorta or cardiac studies, standard flow images might be generated automatically to expedite interpretation of the data sets.The spatial resolution of 4D acquisitions is likely to be suboptimal for small vessel regions and jet flows, and this affects e.g. the accuracy of vascular segmentation by 3D phase contrast angiography from the same acquisition. The moving boundaries of the heart cavities and atrio-ventricular valves are relatively hard to determine by the approach. Precisely registered cine acquisitions could help, although this may depend on the use of a corresponding respiratory position.Comprehensive 4D velocity CMR imaging, visualization strategies, and quantification of cardiovascular hemodynamics are contributing to the understanding of cardiac and vascular pathologies, but inherent limitations of the technique should be born in mind. Due to the time currently needed to acquire and analyse the datasets, they have yet to become accepted in routine clinical CMR practice as alternatives or adjuncts to targeted 2D breath hold velocity measurements of flow.The authors declare that they have no competing interests.MM was responsible for studies and presented data regarding aortic and whole heart 4D velocity mapping and quantitative analysis of flow wall parameters, TE was responsible for studies and presented data regarding cardiac and aortic 4D velocity mapping and quantitative analysis of flow and turbulent kinetic energy, MM and TE drafted the manuscript, PK participated in critical revision of the manuscript and review of the current state and limitations of 4D MR velocity mapping. All authors participated in the literature review and read and approved the final manuscript.Time-resolved velocity vector field and planar color coding in a plane longitudinally transecting the thoracic aorta in a normal volunteer.Click here for fileTime-resolved 3D pathlines in a normal thoracic aorta illustrating the temporal evolution of blood flow during the cardiac cycle.Click here for fileTime-resolved 3D pathline visualization of blood flow in the heart and the great vessels. Pathlines are emitted from planes located at the mitral valve (red-yellow) and the tricuspid valve (blue-turquoise) at early and late diastolic ventricular inflow. A separately acquired balanced steady-state free precession three-chamber image provides anatomical orientation.Click here for fileRotating view of flow characterization in the heart of a healthy volunteer at late diastole. Vortex cores are shown as white isosurfaces. In addition, short streamlines have been emitted from these isosurfaces.  vortex rings can be seen below the mitral valve and the tricuspid valve.Click here for fileTime-resolved 3D pathlines in the aorta of a patient with and ascending aortic aneurysm illustrating the dynamics of disturbed blood flow during the cardiac cycle.Click here for fileTime-resolved pathline visualization of all blood flow involved in one cardiac cycle in the left ventricle (LV) of a healthy, 61 year old male. The pathlines are color coded according to their behavior over the cardiac cycle in 4 different components: Direct Flow (green) enters the LV during diastole and leaves the LV during systole in the analyzed heart beat, Retained Inflow (yellow) enters the LV during diastole but does not leave during systole in the analyzed heart beat, Delayed Ejection Flow (blue) starts and resides inside the LV during diastole and leaves during systole, Residual Volume (red) resides in the LV during more than two cardiac cycles[ac cycles.Click here for fileTime-resolved volume rendering of turbulence intensity  in a patient with an aortic coarctation located distal to the left subclavian artery. The elevated values of turbulent kinetic energy in the ascending aorta were caused by a minimally obstructive subvalvular membrane, exemplifying the sensitivity of this measure to minor abnormalities in blood flow. A semi-transparent three-dimensional isosurface rendering of CE-MRA data is shown for orientation.Click here for fileTime-resolved volume rendering visualization of turbulence intensity  in the left atrium of a patient with severe mitral regurgitation.Click here for file


Genome-wide association studies (GWAS) have identified genetic variants associated with numerous cardiovascular and metabolic diseases. Newly identified polymorphisms associated with myocardial infarction, dyslipidemia, hypertension, diabetes, and insulin resistance suggest novel mechanistic pathways that underlie these and other complex diseases. Working out the connections between the polymorphisms identified in GWAS and their biological mechanisms has been especially challenging given the number of non-coding variants identified thus far. In this review, we discuss the utility of expression quantitative trait locus (eQTL) databases in the study of non-coding variants with respect to cardiovascular and metabolic phenotypes. Recent successes in using eQTL data to link variants with functional candidate genes will be reviewed, and the shortcomings of this approach will be outlined. Finally, we discuss the emerging next generation of eQTL studies that take advantage of the ability to generate induced pluripotent stem cell lines from population cohorts. NOD2 (nucleotide-binding oligomerization domain containing-2) mutations in inflammatory bowel disease  of common cardiovascular disease have successfully identified many new genetic loci that explain the heritability of myocardial infarction, dyslipidemia, hypertension, and diabetes  mapping. Simply put, it is the combination of mRNA expression and genotype data to determine which variants correlate with transcript levels of genes. In principle, eQTLs can be mapped for known SNPs in any tissue type of interest. When the regulatory effect of a polymorphism is on a nearby gene the relationship is termed Application of eQTL data to animal models and large human databases has established the heritability of gene expression  and transcript levels of three genes  in liver, a targeted eQTL analysis was performed in a replication cohort of 62 human liver samples. Minor allele homozygotes displayed more than 12-fold higher SORT1 expression than major allele homozygotes. This differential gene expression by genotype was not apparent in the analysis of the adipose samples or in previously reported data from lymphocytes  cholesterol and the hepatic expression of the SORT1 to cholesterol biology, the same approach is being utilized to uncover more genes of biological significance. Besides the SORT1 locus, GWAS for blood cholesterol levels have also uncovered at least 94 other associated loci , 38 SNP-to-gene eQTLs in liver, 28 in omental fat, and 19 in subcutaneous fat were identified. Some lead SNPs were shown to be remote from their associated gene transcripts. For example, rs9987289 [associated with LDL-C and high-density lipoprotein (HDL)-C] correlated with a twofold change in liver expression of PPP1R3B, yet is 174 kb away from the gene. Similarly, rs2972146 correlated with IRS1 expression in omental fat, despite being located 495 kb away from the gene. The eQTL data for these two loci provide another layer of insight into their biological significance.With the successful use of eQTL data to link PPP1R3B, and the TTC39B gene, each of which lies in or in proximity to a locus associated with HDL cholesterol  as well as impaired endothelial function measured by flow-mediated vasodilation, but not with traditional risk factors, LDL cholesterol, or triglycerides.One exciting prospect of GWAS is uncovering novel pathways and mechanistic insights into disease pathogenesis. With coronary artery disease (CAD), eQTL analyses in novel cell types are being undertaken to link genetic associations with pathways in inflammation and thrombosis. Monocytes represent one such cell type; its relative accessibility (from blood samples) and ease of use in eQTL studies make it an attractive cell type in which to link genotype and expression data, though it represents only one of the full spectrum of cell types that need to be studied to comprehensively find novel mechanisms for cardiovascular disease. One study linked two SNPs on chromosome 10q23.31 with increased CAD risk and increased PDGFD gene (Coronary Artery Disease (C4D) Genetics Consortium, A second CAD-associated SNP did not show association with transcript expression in liver, whole blood, or monocytes, but instead was strongly associated with expression in aortic media, aortic adventitia, and mammary artery tissue , the response to radiation, and even response to drugs, hormones, and peptides  as a renewable source of patient-specific cell lines. Recent advances in nuclear reprogramming technology allow for the transformation of terminally differentiated adult cells into induced iPSCs that are phenotypically indistinguishable from embryonic stem cells , which is a unique, community-based cohort in which three generations of individuals have been extensively phenotyped and genotyped. Although limited eQTL analyses have been performed within the FHS (Levy et al., Gene expression analyses can yield important information about genetic architecture and can point to mechanisms that link genetics and cardiovascular disease. The application of eQTL analyses has already forged connections between GWAS data and mechanistic pathways. Technical and conceptual limitations, however, limit the ability to systemize this approach to more polymorphisms for more diseases. The advent of more comprehensive biobanks, high-throughput RNA sequencing, and collections of iPSC lines holds much promise for the field. Ultimately, the ability to explain the heritability of common diseases such as cardiovascular and metabolic disorders should be greatly facilitated by future eQTL efforts.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.


A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.Previous reports have described  Polygalacturonases (PGs) are enzymes directed toward the degradation of D-galacturonic acid moieties of pectic substances. Several PG genes have been isolated from filamentous fungi, due to their importance in host-pathogen cross-talks and their industrial relevance, for example, in food and textile processing , a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several different growth conditions, even in the presence of non-inducing sugars such as sucrose, as opposed to pgg1, expressed only in pectin-containing medium, after 76 h of growth  mycelia grown for 24 h on minimal medium containing pectin as sole carbon source (pgg2 gene clone) with forward primer 5' TGAGGAATGAATGAATGAATG 3' and reverse primer 5' GGCCATTCTAGACTAGGTGG 3'. The restriction generated products of 85 bp, 80 bp and 170 bp. The 170 bp fragment was purified from the agarose gel, using the Wizard SV Gel and PCR Clean-Up System , and radiolabeled with [γ-32P]dATP (32P]dATP using polynucleotide kinase (Promega). For competition assays, a 25- or 50-fold molar excess of the unlabeled oligonucleotide was added to the binding reaction.Nuclear extracts were prepared from 32P]dATP . LabeledWhen the 170 bp DNA fragment was used as probe, a band shift was observed, independently of the extract concentration employed in the binding reactions, which provides evidence that proteins in the extract recognized the CCAAT element, since it was the most probable cis-element present in the 170 bp fragment . The expP. griseoroseum mycelia that may be important for the expression in vivo. Since pectin and sucrose activate pgg2 gene transcription in the initial 24 h of in vitro growth, it is reasonable to postulate that the CCAAT sequence may be involved in PG gene expression in other carbon sources as well. Sucrose is not a normal constituent of the pectin molecule, so gene activation may follow a different regulatory pathway not exclusive to the pectinolytic system, but possibly a general one controlled by a CCAAT element. We believe that this element regulates pgg2 expression in pectin as well, but is not involved in the expression of the pgg1 gene that is induced solely by pectin in 76 h of fungus growth (pgg1 promoter (results not shown). CCAAT boxes have been implicated in the modulation of transcript levels in eukaryotes, and their functionality has also been reported for some genes that code for other cell wall degrading enzymes, such as cellulase and xylanase, and enzymes of biotechnological importance like amylase and penicillin (Aspergillus nidulans and Neurospora crassa and resemble the Hap complex of Saccharomyces cerevisiae (Penicillium.Taken together, these results show that in the polygalacturonase gene studied the sequence CCAAT is responsible for the binding of protein complexes from induced in vivo and gene expression level. Since the polygalacturonases are the most extensively studied enzymes in the pectinase family, we believe that new insights into the detailed mechanisms of expression of other polygalacturonase genes will be reported in the near future.Further studies are needed to determine the relationship between the binding of transcriptional regulators


In recent decades, several biomarkers have been investigated as predictors of breast cancer risk, development, prognosis and treatment efficacy.The detection of biomarkers strongly associated with breast carcinogenesis has an enormous potential, especially for selecting subjects at high risk of developing breast cancer who could benefit from chemopreventive treatments.Although the number of potential biomarkers continues to increase, a unique biomarker for breast cancer risk prediction has not been identified and it is probable that a panel of biomarkers will prove optimal. Further studies are needed to validate breast cancer biomarkers evaluation for individual risk assessment.This review summarizes the main biomarkers, which are important at different stages of breast carcinogenesis with updates from the recent literature. Efficacy in breast cancer early diagnosis, treatment and prevention is a big challenge considering that breast cancer is the most common diagnosed cancer in women worldwide. Biomarkers have a critical role both in monitoring cancer progression and assisting the identification of high-risk subjects. The general characteristics of a biomarker should include high reproducibility and detectability, easy collection from patients by minimally invasive techniques and high correlation with the disease with different expression in healthy versus affected subjects.An ideal biomarker for monitoring cancer treatment should be strongly correlated with cancer growth and should be an index for drug efficacy. A good candidate biomarker for cancer prevention would be a marker directly correlated with risk and with molecular mechanisms of carcinogenesis in pre-cancerous tissues, should be differentially expressed in average versus high-risk populations and should predict response to chemopreventive agents. Several tumour markers have shown evidence of clinical usefulness and have been recommended for use in practice in breast cancer patients: CA 15-3, CA 27.29, carcinoembryonic antigen (CEA), oestrogen and progesterone receptors , human epidermal growth factor receptor 2 (HER-2), urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) and certain multiparametre gene expression assays  [In the last decade, much effort has been focused on discovering biomarkers that can be used to predict breast cancer risk. Among the established risk biomarkers, deleterious germline mutations in BRCA1/BRCA2/TP53 genes have been demonstrated as strong predictors of breast cancer development ,7, whereOther potential biomarkers that have been investigated for an association with breast cancer include hormonal and nuclear receptors, membrane receptors and signal transduction factors, anti-inflammatory and antioxidant factors, apoptosis and angiogenesis factors, proliferation markers and antigens, epigenetic modulation factors, cellular inducible enzymes, cell cycle regulators, oncogenes and tumour suppressor genes ,14.In this review, we will discuss the main biomarkers which have been reported to have a role in breast cancer early development and progression.Oestrogen and progesterone are essential hormones for breast growth, and their receptors (ER and PR) are established predictive factors for breast cancer treatment efficacy and disease outcome. ERs are transcription factors, which mediate the action of oestrogens, whereas the PR gene is regulated by oestrogen, and so the PR could be a marker of oestrogen action in breast cancer .Previous studies have shown that ER and PR levels are associated with a favourable breast cancer prognosis and are highly predictive of benefit from endocrine treatment in both the adjuvant and metastatic settings –18. Two In normal breast, PRA and PRB are co-expressed at similar levels on luminal epithelial cells, suggesting that both proteins are required to mediate physiologically relevant progesterone signalling, and an imbalance in the native ratio of the two isoforms may lead to alterations in PR signalling ,24. PrevAndrogen receptors (AR) are expressed by the majority of human breast carcinomas, at a frequency comparable to or higher than that reported for ER and PR ,28. AR sAll these findings highlight the role of ER, PR and AR in promoting breast cancer and consequently their potential as drug targets in both preventive and therapeutic settings.Other members of the steroid/thyroid hormone receptor super family include the nuclear retinoid receptors. Receptor selective retinoids inhibit the growth of both normal and cancerous human breast cells predominantly through induction of a G1 cell cycle block . There aThe RARs and RXRs are thought to mediate the effects of retinoids on cell growth, differentiation and apoptosis and, therefore, to have a role in mammary carcinogenesis suppression –43. RARaThe PPARs are also members of the nuclear receptor super family, which includes steroid, retinoid and thyroid hormone receptors ,48. Threin vitro through the activation of PPARg [Previous studies suggested that PPARg signalling may exacerbate mammary gland tumour development in mice  and PPARof PPARg . Furtherof PPARg .All these results document important roles for retinoid receptors and PPARs in oncogenesis, thereby identifying new targets for cancer therapy and prevention. Further evidence supporting the importance of the retinoid pathway in breast cancer development has been provided by a large randomised trial with the synthetic retinoid fenretinide that has shown a reduced risk of recurrence after primary surgery in pre-menopausal women .3, the biologically active form of vitamin D, that modulates differentiation, cell cycle and apoptosis of stromal and epithelial cells derived from mammary gland and breast cancers in vitro [The vitamin D receptor is a nuclear receptor present on over 80% of human breast cancers and mediates the action of 1,25-dihydroxy-vitamin Din vitro –60. A viin vitro .in situ and infiltrative carcinomas compared to benign breast diseases or normal breast tissue [3 has been correlated with increased breast cancer risk and metastasis [The expression patterns of the heterodimers formed by VDR and RARs or RXRs have been studied by immunohistochemistry in benign and malignant human breast tissues. Higher levels of all receptor types were found in both t tissue ,63. Prevt tissue ,64. An it tissue –67 and atastasis ,69.Several epidemiologic, mechanistic and experimental data support the chemopreventive potential of vitamin D and its analogues, although hypercalcemic side effects represent a major obstacle to their clinical development. A proposed strategy for using a non-calcemic dose in the chemopreventive setting is to administer vitamin D or its analogues in combinations with other agents, such as retinoids or anti-oestrogenic compounds .et al found a significantly higher expression of EGFR in ductal cells of needle aspirates from women at high risk for breast cancer development than in low-risk control subjects [Tyrosine kinase signalling has been demonstrated to have an important role in the genesis and progression of human breast cancer as well as in all the steps of the normal mammary gland development . Receptosubjects , suggestThe HER2 gene is amplified and over-expressed in about 25% of breast cancers, conferring a more aggressive biology . Over-exIt has been reported that tamoxifen treatment up-regulates HER2 by 40% in breast cancer cells expressing both HER2 and ER , and thaThe other two members of the HER family, HER3 and HER4, are of particular interest because of their ability to interact directly with HER2. A recent study presented striking evidence that alterations in HER3 exist in breast cancer  and anotin vivo bone metastatic capacity of human breast cancer cells [Two other membrane receptors demonstrated to have a role in breast carcinogenesis are the transforming growth factor beta (TGF-b) receptor and the insulin-like growth factor (IGF) receptors. TGF-b signalling pathway is implicated in cell senescence, regulation of cell growth and differentiation . TGF-b her cells –102.Another recent study reported that TGF-b is a potential mediator of retinoic acid and tamoxifen-induced apoptosis in breast cancer cell lines, suggesting that the TGF-b pathway may have a role also in the breast chemoprevention setting .The IGFs are peptide hormones involved in regulation of cell proliferation, differentiation and apoptosis . Severalet al investigated the possible correlation between celecoxib administration and prostaglandin concentration in serum and nipple aspirate fluid (NAF), reporting a reduction of prostaglandin levels in NAF of post-menopausal high-risk women and a reduction in both NAF and plasma prostaglandin levels in women with newly diagnosed breast cancer [Cyclooxygenase-2 (COX-2) is an important inflammatory mediator, responsible for the conversion of arachidonic acid into prostaglandins. In the last decade, several studies reported a correlation between COX-2 expression and breast cancer. Elevated COX-2 protein levels have been detected in approximately 40% of invasive breast cancers and COX-2 over-expression has been demonstrated to correlate with large tumour size, high grade, high proliferation, hormone receptor negative status and over-expression of HER-2 –117. Thet cancer . FurtherMatrix metalloproteinases (MMPs) are a family of proteases involved in the regulation of the cell microenvironment, matrix turnover, growth factor bioavailability and several aspects of immunity and inflammation. Other classes of proteases, which interact with MMPs are a disintegrin and metalloproteinases (ADAMs), and tissue inhibitor of metalloproteinases (TIMPs) ,126. Epiin situ and invasive carcinoma [C-reactive protein (CRP) is an acute-phase protein, synthesized in the liver, which is considered a marker of inflammation . CRP serarcinoma .et al suggested that the OPN-c splice variant could be a selective and prognostic marker for human breast cancer, as OPN-c mRNA was identified in 80% of breast carcinomas [et al demonstrated a significant positive association of OPN-b and OPN-c expression with adverse pathological and clinical outcomes [Osteopontin (OPN) is a phosphoglycoprotein involved in a variety of physiopathologic processes such as bone remodelling, angiogenesis, inflammatory response, cell growth and tissue differentiation and wound healing . OPN posrcinomas . Patani outcomes .In a cohort of women with newly diagnosed metastatic breast cancer, high-baseline OPN plasma levels or OPN increase > 250 ng/ml at any time during follow-up were found to be associated with poor survival  and highThe apoptosis-signalling pathway has an important role in cancer onset and progression. Many apoptotic proteins have been studied for an association with breast cancer pathogenesis. The inhibitors of apoptotic proteins are a more recently described family of proteins that function as endogenous inhibitors of caspases . SurviviTP53 is a tumour suppressor gene that encodes the p53 protein, which is activated in response to several forms of cellular stress and mediates many cellular processes, including cell cycle arrest, apoptosis, senescence and differentiation ,168. SevP53 has been shown to be a possible target of the immune system. Antibodies against p53 have been detected in different types of malignant disease, with the highest positivity rates observed for breast cancer . There aCyclin D1 is a key factor involved in the regulation of the G1-S phase transition during the cell cycle. Cyclin D1 over-expression has been observed in DCIS, suggesting a possible role in breast cancer development –178. Prein vitro data suggested that retinoids reduce cyclin D1 expression in human breast cancer cell lines [It has been shown that cyclin D1 can activate oestrogen receptor transcriptional activity in the absence of estradiol, and that this activity can be inhibited by 4-hydroxytamoxifen . Furtherll lines ,183. Thell lines .Vascular endothelial growth factor (VEGF) is a key enhancer factor of angiogenesis, the process of new blood vessels formation, involved in cancer development and progression. Several studies have investigated the association between VEGF expression in cancer tissues and plasma with breast cancer. High levels of VEGF expression in breast cancer tissues have been associated with poor prognosis and decreased overall survival ,186, andSeveral studies have been conducted to investigate the possible use of proliferation markers as breast cancer prognostic indicators. Various methods are available for the measurement of proliferation rates in tumours, including mitotic counts, estimation of the fraction of cells in S-phase of the cell cycle and proliferation-associated antigens . A previProliferating cell nuclear antigen (PCNA), a nuclear protein involved in DNA repair processes, has been investigated, but appear to correlate poorly with Ki67 and mitotic count, suggesting a more limited use in assessing proliferation .The biomarkers that have been discussed in this review are involved at different stages of the breast carcinogenesis multi-step process. Each of the described biomarkers may be a target for drug treatment or prevention. Notwithstanding, we should take into account that the absolute benefit of a treatment varies greatly between individuals. Biomarkers may also be helpful for identification of high-risk subjects who will benefit from a given treatment. However, the currently available data do not support the implementation of a cancer risk model based on biomarkers evaluation. Further studies and prospective clinical randomised trials are necessary to better define whether and which biomarkers could be used in clinical practice for better treatment selection and patient follow-up.


Before birth, glucocorticoids retard growth, although the extent to which this is mediated by changes in insulin signalling pathways in the skeletal muscle of the fetus is unknown. The current study determined the effects of endogenous and synthetic glucocorticoid exposure on insulin signalling proteins in skeletal muscle of fetal sheep during late gestation. Experimental manipulation of fetal plasma glucocorticoid concentration was achieved by fetal cortisol infusion and maternal dexamethasone treatment. Cortisol infusion significantly increased muscle protein levels of Akt2 and phosphorylated Akt at Ser473, and decreased protein levels of phosphorylated forms of mTOR at Ser2448 and S6K at Thr389. Muscle GLUT4 protein expression was significantly higher in fetuses whose mothers were treated with dexamethasone compared to those treated with saline. There were no significant effects of glucocorticoid exposure on muscle protein abundance of IR-β, IGF-1R, PKCζ, Akt1, calpastatin or muscle glycogen content. The present study demonstrated that components of the insulin signalling pathway in skeletal muscle of the ovine fetus are influenced differentially by naturally occurring and synthetic glucocorticoids. These findings may provide a mechanism by which elevated concentrations of endogenous glucocorticoids retard fetal growth. In all species studied to date, there is an increase in the circulating glucocorticoid concentration in the fetus near term The insulin receptor (IR) and insulin-like growth factor type 1 receptor (IGF-1R) are members of the family of receptor tyrosine kinases. When activated by insulin or IGFs, these kinases phosphorylate insulin receptor substrates (IRS) which, in turn, lead to the activation of a number of cascades, including the phosphophatidylinositol 3-kinase (PI3K) signalling pathway. This pathway can induce Akt phosphorylation which induces tissue-specific metabolic effects, such as translocation of the glucose transporter-4 (GLUT4) and consequently stimulation of glucose uptake in skeletal muscle and adipose tissue, activation of the peroxisome proliferator activated receptors involved in fat metabolism and storage, and stimulation of glycogen synthase activity and glycogen deposition in skeletal muscle and liver in utero was hypothesised to decrease the abundance of proteins associated with growth, such as mTOR and S6K, and increase the proteins associated with glucose transport, such GLUT4. These changes may be responsible for the decline in fetal growth towards term and the preparation for greater dependence on insulin-stimulated glucose disposal after birth, respectively. Experimental manipulations of fetal plasma concentrations of glucocorticoid were achieved by fetal cortisol infusion and maternal dexamethasone treatment. These two experimental models enabled the impact of glucocorticoid exposure on insulin signalling proteins to be assessed.In fetal life, insulin has both glucoregulatory and anabolic actions from at least mid-gestation and developmental changes in the expression of insulin receptors and intracellular signalling proteins have been observed in fetal tissues, including skeletal muscle −1; 18% protein and 10 MJ kg−1; Sheep Nuts #6; H&G Beart, Kings Lynn, UK) with free access to hay, water and a salt-lick block. Overall, the pregnant ewes consumed between 8–11 MJ day-1 of metabolizable energy. Food, but not water, was withheld for 18–24 h before surgery. All surgical and experimental procedures were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 and were approved by the University of Cambridge animal ethics committee.Twenty-one Welsh Mountain sheep fetuses of known gestational age were used in this study; all were singleton fetuses . The ewes were housed in individual pens and fed concentrates (200 g day2-N2O), catheters were implanted into the femoral artery and vein of eleven fetuses at 115–117 days of gestation using surgical methods described previously (term 145±2 days) −1day−1 in 3.0 ml 0.9% saline; EF-Cortelan, GlaxoSmithKline, Brentford, Middlesex, UK; n = 5) or the saline vehicle  for five days beginning at 125–126 days of gestation. The dose of cortisol was chosen to mimic the plasma concentrations normally observed in the immediate prepartum period in fetal sheep Under halothane anesthesia  in 2 ml saline or the saline vehicle  at 24 hour intervals from 125 days of gestation. Tissues were collected 10 hours after the final maternal injection. The dexamethasone treatment regime resembles that recommended for use in human clinical practice −1 sodium pentobarbitone i.v.). At delivery, a 10 ml blood sample was taken by venepuncture of the umbilical artery, and a number of tissues were collected from the fetus after the administration of a lethal dose of barbiturate (200 mg kg−1 sodium pentobarbitone). Skeletal muscle tissue samples taken from the hind limb (biceps femoris) were immediately frozen in liquid nitrogen and stored at −80°C until analysis. All blood samples obtained at tissue collection were immediately placed into EDTA-containing tubes and centrifuged for 5 min at 1000 x g and 4°C. The plasma aliquots were stored at –20°C until analysis.All fetuses were delivered by Caesarean section under general anaesthesia of the ewe .Muscle protein levels were quantified by Western blotting using methods described previously The glycogen content of skeletal muscle was determined by a biochemical assay as described previously P<0.05.Values are presented as mean values ± SEM. Data were analysed using Student’s t-test. Ratios of muscle protein expression were arcsine transformed prior to statistical analysis, and are presented as a fold change from the mean values observed in the respective control group. For all comparisons, statistical significance was accepted when Plasma cortisol concentration was significantly higher in the cortisol-infused fetuses than in the saline-infused fetuses p<0.05; . Fetal cAt delivery, plasma cortisol concentration was significantly lower in fetuses whose mothers were injected with dexamethasone compared to those whose mothers were treated with saline p<0.05; . Exposurin utero. In agreement with the hypothesis of the study, fetal infusion of cortisol led to downregulation of the phosphorylated levels of the anabolic proteins, mTOR and S6K, while maternal dexamethasone treatment increased GLUT4 expression in fetal skeletal muscle.The results demonstrate that components of the insulin signalling pathways relating to both growth and glucoregulation in skeletal muscle of the ovine fetus are sensitive to glucocorticoid exposure in utero. In fetal sheep, the decline in growth rate seen near term is known to be glucocorticoid-dependent, since it can be prevented by removal of the fetal adrenal gland and induced prematurely by five days of cortisol infusion in vivoThese findings contribute to the understanding of the regulation of growth by glucocorticoids in vivoin vitroin utero remains unclear.In the present study, reductions in muscle p-mTOR and p-S6K in the cortisol-infused fetuses were associated with increased Akt2 and p-Akt protein content. This finding was unexpected given that the PI3K/Akt pathway is a key activator of mTOR phosphorylation in adult tissues in vivoin utero remains unknown.Muscle GLUT4 protein expression in the ovine fetus was increased by maternal dexamethasone treatment. This observation confirms that previously reported in fetuses of pregnant ewes treated with either single or repeated doses of dexamethasone at an earlier gestational age (106–107 days) Cortisol and dexamethasone exerted different effects on muscle insulin signalling proteins in the fetuses of the two experimental models of intrauterine glucocorticoid exposure. There are several possible explanations for these observations. First, the experimental models differ in the duration and level of glucocorticoid exposure and timing of tissue collection. In the dexamethasone-treated animals, dexamethasone concentrations in the fetal circulation are significantly elevated 1–2 h post-injection and, although still detectable, fall thereafter at tissue and blood collection at 10 hours after the second maternal injection Second, natural and synthetic glucocorticoids differ in their properties of action. The synthetic glucocorticoids have longer half-lives and are more potent than the endogenous glucocorticoids, and, unlike the endogenous glucocorticoids that act on both glucocorticoid and mineralocorticoid receptors, synthetic glucocorticoids only bind and activate glucocorticoid receptors. The relative effects of activation of mineralocorticoid receptors by cortisol on insulin signalling pathways in fetal tissues, however, are unknown. The fall in plasma cortisol seen in the dexamethasone-exposed fetuses is in accordance with the negative feedback effects of synthetic glucocorticoids on endogenous glucocorticoid production and has been reported previously in the fetuses of pregnant ewes treated with dexamethasone in utero may be due to concomitant changes in other circulating and/or local hormones and growth factors. In both of these fetal sheep models, increases in plasma triiodothyronine and leptin have been demonstrated in response to fetal cortisol infusion and maternal dexamethasone treatment in uteroThird, some of the effects of glucocorticoid exposure on insulin signalling protein expression in skeletal muscle in utero, multiple doses lead to impaired body size at birth while a single dose often has little effect on growth Suppression of mTOR and S6K in skeletal muscle of cortisol-treated sheep fetuses may also be due, at least in part, to local changes in IGF synthesis. Previous studies have shown that cortisol infusion for five days in fetal sheep downregulates both IGFI and IGFII mRNA abundance in skeletal muscle In summary, the current study demonstrated that endogenous and synthetic glucocorticoids alter components of the insulin signalling pathways in skeletal muscle of the ovine fetus in different ways, with potentially different consequences for the regulation of muscle growth in the fetus and insulin sensitivity in postnatal life. An infusion of cortisol to the fetus for five days reduced phosphorylated levels of mTOR and S6K which, in turn, are likely to contribute to the mechanisms of glucocorticoid-induced growth retardation. In contrast, a single course of maternal dexamethasone treatment had little effect on the insulin signalling molecules measured in fetal skeletal muscle, apart from an increase in GLUT4 expression which may be a homeostatic response to the fetal hyperglycaemia seen previously after dexamethasone exposure


Factors affecting transcriptional elongation have been characterized extensively in in vitro, single cell (yeast) and cell culture systems; however, data from the context of multicellular organisms has been relatively scarce. While studies in homogeneous cell populations have been highly informative about the underlying molecular mechanisms and prevalence of polymerase pausing, they do not reveal the biological impact of perturbing this regulation in an animal. The core components regulating pausing are expressed in all animal cells and are recruited to the majority of genes, however, disrupting their function often results in discrete phenotypic effects. Mutations in genes encoding key regulators of transcriptional pausing have been recovered from several genetic screens for specific phenotypes or interactions with specific factors in mice, zebrafish and flies. Analysis of these mutations has revealed that control of transcriptional pausing is critical for a diverse range of biological pathways essential for animal development and survival. Cells within a mature animal differ dramatically in their size, shape, function, longevity and ability to keep dividing even though, with few exceptions, every cell contains the same set of genes. The great diversity of cells found in animals is a consequence of different cell types expressing different profiles of genes during cell fate determination and differentiation. Failure of gene regulation usually has catastrophic effects on the developing embryo, and in adult life leads to disease, including cancer.All gene expression is controlled at the level of RNA polymerase recruitment and successful formation of the pre-initiation complex (PIC). Obviously, if RNA polymerase cannot bind to the transcription start site of a gene, then no RNA can be transcribed. However, transcription in eukaryotes may be regulated at several additional levels, including elongation, processing, termination and export from the nucleus.Drosophila. In the absence of heat shock, transcriptionally engaged RNA polymerase II (RNAP II) accumulates just downstream of the transcriptional start sites (TSSs) of Hsp genes and is associated with short 20–60 nucleotide long nascent RNAs Hsp genes It had been established for over 30 years that transcription elongation may be a rate-limiting step in gene expression, but it is only in the past five years or so that the prevalence and importance of elongation control has been recognized  and interacts with RNA polymerases I, II and III Spt5 (foggym806) was recovered from a genetic screen for mutations affecting neuronal development in zebrafish foggym806 fish look relatively normal but have neuronal defects that ultimately prove lethal, including a deficit of dopamine-containing neurons and corresponding excess of serotonin-containing neurons foggym806 is a missense mutation that leads to an amino acid substitution (V1012D) in the very C-terminal region of Spt5, which is conserved only amongst higher eukaryotes. Subsequently, null alleles of Spt5 were characterized in zebrafish that had originally been generated in a large-scale screen for mutations affecting embryogenesis Spt5 null alleles have additional phenotypes with respect to foggym806 homozygotes, including reduced pigmentation, short tails, small ears and pericardial oedema The first mutant allele of Spt5 coupled to time-course microarray analysis of early zebrafish embryos indicated that Spt5 plays an erythroid-specific role in early embryogenesis through the induction of gata1 gene expression There is a maternal component to Spt5 expression in zebrafish, thus homozygous null animals do contain some residual wild-type protein. However, the phenotypes observed in these embryos as the maternal component diminishes are highly specific and reproducible, indicating that the expression of a specific subset of genes during development is more sensitive to a reduction in Spt5 availability than others. Subsequently, expression profiling of over 10,000 protein coding genes in zebrafish embryos 24 hours postfertilization identified that only 5% of genes were differentially expressed between null mutants and their wild-type siblings Spt5 (fh20) was isolated from a genetic screen to identify genes that control the posterior migration of facial branchiomotor neurons in the zebrafish hindbrain fh20 allele causes a splicing defect, leading to a mix of correctly and incorrectly spliced mRNA and a hypomorphic phenotype. Spt5 null facial branchiomotor neurons survive to at least five days postfertilization while failing to migrate posteriorly in a wild-type host A further zebrafish allele of hsp70 in the absence of heat shock in vivo The N-terminal region of Spt5 (NSpt5: lacking the repeats phosphorylated by P-TEFb) acts in a dominant manner to disrupt development when expressed in zebrafish embryos Drosophila Spt5W049 allele was recovered from a genetic screen for maternal factors that affect anterior-posterior patterning during embryogenesis Spt5W049 leads to defects in segmental patterning of the embryo. The effects of Spt5W049 are gene specific, since expression of the gap genes is normal while expression of two of the three primary pair-rule genes, even-skipped (eve) and runt (run), is aberrant. Furthermore, enhancer-reporter constructs reproducing specific stripes of eve expression are affected differentially by Spt5W049: expression driven by the stripe 2 enhancer is weak but broadened, while expression of stripe 3 appears wild-type. These results indicate that Spt5 is sensitive to the different combinations of trans-acting factors that drive expression of stripe 2 and stripe 3.The Spt5W049 missense mutation maps to the same region of Spt5 as the foggym806 mutation in zebrafish, implicating this domain of Spt5 in interactions with contextual factors that regulate its activity during development. Assays performed in nuclear extracts demonstrated that both the Foggym806 and W049 protein variants have a diminished ability to inhibit transcription prior to the phosphorylation events of the P-TEFb checkpoint eve expression in the early embryo; stripes of eve pair-rule protein are broadened or fused in late, cellularizing Spt5W049 embryos when the pattern should be fully resolved into seven distinct stripes. Thus, the inhibitory activity of Spt5 on transcription prior to the P-TEFb checkpoint has a role in repression of eve in the early embryo. In wild-type embryos, a subset of the cells that repress eve expression during the seven stripe stage re-activate eve expression around 30 minutes later during gastrulation to form interstripes. Thus, the repressive mechanism involving Spt5 is rapidly reversible.Remarkably, the Drosophila Spt5 were recovered in a genetic screen for factors that modify Presenilin-dependent Notch phenotypes Notch phenotypes, indicating that Spt5 is required to activate gene expression in response to Notch signalling. Animals homozygous for the Spt5W049 missense mutation eve locus and activation of heat shock gene expression, thus Spt5 clearly has both positive and negative effects on transcription in vivo dependent on context.Two null alleles of Drosophila Spt5 have been recovered from a genetic screen for factors mediating dosage compensation Drosophila, males (XY) make additional transcripts from their single X chromosome, to match the amount transcribed from females (XX). The increased transcription in males is dependent on the MSL complex, which contains at least five different proteins and two non-coding RNAs. Rather than localizing to the promoter or TSS, the MSL complex is found across the gene bodies of active genes Spt5 genetically interacts with genes encoding components of the MSL complex and Spt5 protein physically interacts with MSL1. Thus, it seems likely that in the case of dosage compensation, Spt5's role is to promote active elongation across the gene body rather than in establishing the P-TEFb checkpoint Very recently, further alleles of Caenorhabditis elegans. NELF is recruited to a large subset of genes Drosophila S2 culture cells leads to both up and down regulation of target genes The NELF complex, which is made up of four subunits , is found only in higher metazoans; it is not found in yeasts or the nematode WHSC1 and WHSC2 genes WHSC2 encodes NELF-A. The clinical presentation of WHS is complex and its severity correlates with the size of the chromosomal deletion. Clinical features include a distinctive craniofacial phenotype, growth and mental retardation, seizures and cardiac abnormalities. The precise contribution of WHSC2/NELF-A gene deletions to this syndrome are not yet understood due to the variability of the associated chromosomal aberrations that remove numerous genes, and the lack of good animal models Mutations in the human gene encoding the NELF-A subunit have been implicated in the pathogenesis of Wolf–Hirschhorn syndrome (WHS). WHS is caused by deletions of the distal part of the short arm of chromosome 4 that remove the NELF-B mRNA is associated with metastatic breast cancer NELF-B was recovered in a yeast 2-hybrid screen for factors that interact with breast cancer susceptibility gene BRCA1 and is sometimes referred to as COBRA1 (co-factor for BRCA1) NELF-B have an inner cell mass deficiency and die at the time of implantation Mice homozygous for a mutation in Drosophila development NELF-A cease to develop before the cellular blastoderm stage and display abnormal nuclear morphology. The remaining embryos gastrulate normally, however, they generally stop developing during germband retraction and exhibit head defects and incomplete dorsal closure. Embryos derived from maternal germ line clones of a hypomorphic allele of NELF-E show similar, but less severe developmental defects eve, fushi tarazu (ftz), and sloppy paired 1 (slp1) at the cellular blastoderm stage is apparently normal in both the NELF maternal germ line clone embryos, although the expression of some reporter transgenes derived from these genes is greatly diminished NELF also plays a critical role during early Drosophila contain paused polymerases and are expressed at basal levels in the absence of pathogens CecA1, DptB and AttA] do not contain paused polymerases but do become rapidly and highly activated. Stimulus-sensing genes, which must always be expressed at some low level, are more likely to contain paused polymerases than effector proteins, which need only be expressed in response to an immune challenge There is a reduction in activation of immune response pathways in larvae depleted of NELF by RNAi, supporting a role for NELF in gene activation Drosophila salivary glands reduces the level of paused polymerase found on Hsp70Hsp transcription to decrease down to basal levels after the heat shock stimulus has ceased. The presence of NELF somehow facilitates the dissociation of heat shock factor from target genes NELF localizes to the heat shock genes when they are not induced, where it collaborates with DSIF to establish pausing. Depletion of NELF-E in The immune and heat shock responses demonstrate that promoter proximal pausing is not an absolute requirement for rapid activation of transcription. Moreover, promoter proximal pausing often appears to suppress expression of inducible genes to basal levels when there is no stimulus present.Drosophila. It is not possible to make embryos from germ line clones of null alleles of Spt5 or Spt4 as complete loss of either of these factors is lethal to cells in Drosophila (B.H.J. unpublished result), whereas the NELF-A null clones do survive for a time eve gene whereas Spt5W049 embryos lose repression of eve in interstripe regions Although it is well established that DSIF and NELF act together, the biological consequences of perturbing DSIF and NELF activity are distinct from each other in P-TEFb plays a critical role in the activation of all transcription and the regulation of its activity can be a rate-limiting step in metazoan gene expression The 7SK small non-coding RNA (snRNA) inhibits RNAP II transcription by binding P-TEFb and recruiting an RNA binding protein HEXIM  to block Cdk9 activity HEXIM1 gene (referred to as CLP-1 in some publications) generally die before birth and have heart defects Mice homozygous for a targeted knockout of the Drosophila contains just one member of the HEXIM family, which, along with the 7SK snRNA, is ubiquitously expressed during development GAL4>UAS-RNAi system Drosophila development, there are noticeable differences in levels in different cell types. For example, LARP7 is expressed strongly in the ommatidial clusters in the eye imaginal disc and a cluster of cells in the presumptive notum of the wing disc Proteomic analysis of human proteins associated with mouse Brd4 revealed that Brd4 interacts with P-TEFb Drosophila orthologue of Brd4 is encoded by female sterile (1) homeotic . Fsh is a Trithorax group protein, whose main action is to maintain gene expression. Binding of Fsh to promoter regions is predictive of transcriptional activity in Drosophila S2 cells fs(1)h function results in segmental abnormalities including homeotic transformations in the progeny of mutant mothers The c-myc gene is one of the most highly amplified oncogenes isolated from human cancer The c-Myc protein contains a transactivation domain that interacts directly with the Cyclin T subunit of P-TEFb to stimulate transcription by releasing paused polymerases A small fraction of P-TEFb is present in SECs where the Cdk9 kinase is highly active MLL gene to other proteins are often recovered from acute and aggressive myeloid and lymphoblastic leukaemias. Five integral components of SECs  are frequent translocation partners to MLL Key components of SECs have been identified as frequent translocation partners of the mixed lineage leukaemia (MLL) protein in leukaemia Inactivation of the mouse AFF1/AF4 gene by homologous recombination led to growth defects and revealed that AFF1/AF4 is critical for normal lymphocyte development, but not for other cell types in the haematopoietic system lilliputian (lilli) gene encodes the single AFF-like protein found in Drosophila, so is the best fly equivalent to AFF1/AF4. Alleles of lilli were first isolated in a screen for factors that suppress activated Raf in eye development (referred to as Su(Raf)2A; lilli plays multiple roles during Drosophila development.The Drosophila contains a single member of the ELL family of proteins, which is encoded by the Su(Tpl) gene Su(Tpl) lead to embryonic segmentation defects and genetically interact with the Ras signalling and Notch signalling pathways Mutations recovered in genes encoding elongation factors have revealed their involvement in a diverse range of biological pathways including the pathogenesis of numerous cancers, embryonic patterning, haematopoiesis, and also neuronal development, migration and degeneration. However, we are only beginning to appreciate the contribution that promoter proximal pausing and control of P-TEFb activity has during animal development and adult life.The core components regulating elongation are expressed in all cells and recruited to numerous genes; however, disruption of their function in vivo leads to distinctive defects revealing that promoter proximal pausing must be influenced by contextual transcription factors. A number of proteins have been shown to promote P-TEFb recruitment to stimulate transcription at specific target genes including heat shock factor, Tat and c-Myc Promoter proximal pausing is not an absolute requirement for either rapid or high induction of gene expression, but appears to be a common feature at genes that are normally expressed at some basal level, but which have the capacity to be rapidly induced by changes in cellular environment. Expression of such genes requires very precise control as too little expression may render the cells unable to respond to incoming signals, and too much may trigger expression of downstream effectors in the absence of the appropriate signal.Rigorous control of cell division is essential in multicellular organisms during development to generate functional tissues and organs and in adult life to prevent tumours. Excessive stimulation of P-TEFb activity often leads to increased cell proliferation; mutations that increase c-Myc activity or fuse the MLL transcription factor to SEC components have been isolated from numerous cancers. Perhaps the tight control of the P-TEFb checkpoint observed in higher animals has evolved as an additional barrier to deregulated cell proliferation.Animal studies confirm that correct regulation of promoter proximal pausing is critical for development and health in adult life. Analysis of gene expression changes in ES cells and other cell culture systems may hint at the consequences of disrupting the pausing checkpoint in vivo, but to truly understand the biological relevance and role of pausing in vivo, more genetic studies in whole model organisms are necessary.