Cerebral stroke is the leading cause of death and permanent disability among elderly people. In both humans and animals, cerebral ischemia damages the nerve cells in vulnerable regions of the brain, viz., hippocampus, cerebral cortex, cerebellum, and hypothalamus. The present study was conducted to evaluate the therapeutic efficacy of nanoencapsulated quercetin (QC) in combating ischemia-reperfusion-induced neuronal damage in young and aged Swiss Albino rats. Cerebral ischemia was induced by occlusion of the common carotid arteries of both young and aged rats followed by reperfusion. Nanoencapsulated quercetin (2.7 mg/kg b wt) was administered to both groups of animals via oral gavage two hours prior to ischemic insults as well as post-operation till day 3. Cerebral ischemia and 30 min consecutive reperfusion caused a substantial increase in lipid peroxidation, decreased antioxidant enzyme activities and tissue osmolality in different brain regions of both groups of animals. It also decreased mitochondrial membrane microviscosity and increased reactive oxygen species (ROS) generation in different brain regions of young and aged rats. Among the brain regions studied, the hippocampus appeared to be the worst affected region showing increased upregulation of iNOS and caspase-3 activity with decreased neuronal count in the CA1 and CA3 subfields of both young and aged rats. Furthermore, three days of continuous reperfusion after ischemia caused massive damage to neuronal cells. However, it was observed that oral treatment of nanoencapsulated quercetin (2.7 mg/kg b wt) resulted in downregulation of iNOS and caspase-3 activities and improved neuronal count in the hippocampal subfields even 3 days after reperfusion. Moreover, the nanoformulation imparted a significant level of protection in the antioxidant status in different brain regions, thus contributing to a better understanding of the given pathophysiological processes causing ischemic neuronal damage. Cerebral ischemic diseases are the third most common cause of death and one of the leading causes of senile dementia in the world. The reduction in glucose and oxygen transport to the brain during cerebral ischemic conditions causes bioenergetic failure which leads to oxidative stress, inflammation, blood-brain-barrier (BBB) dysfunction, and eventually leads to cell death Some of the brain regions viz. cerebrum, cerebellum, hypothalamus and hippocampus are more vulnerable to ischemia than others. Severe damage to the hippocampus results in difficulties in forming new memories (ante retrograde amnesia), and often affects memories formed before the damage (retrograde amnesia) It is necessary to introduce exogenous antioxidants as drugs as well as free radical scavengers to counter cerebral ischemia and reperfusion induced oxidative attack, especially in aged individuals. At present, there are hardly any efficient neuroprotective agents that may be used for treating ischemic stroke, especially in the elderly. Quercetin (QC) is an important bioflavonoid polyphenolic antioxidant and it can be found in fairly large amounts in fruits, vegetable oils, red wine, and tea Long circulating polymeric nanoparticles made of polylactide-co-glycolide (PLGA) are well accepted as effective drug carriers because of various technological advantages viz., long shelf life, high carrier capacity and feasibility of various routes of administration including oral route The aim of our present study is to ascertain whether oral treatment with PLGA nanoparticulated quercetin exert any neuroprotective effect against cerebral-ischemia reperfusion evoked oxidative damage in the neuronal cells in different brain regions, especially the hippocampus, of young and aged rats.Polylactide-co-glycolide  didodecyldimethylammonium bromide (DMAB) and quercetin were purchased from Sigma – Aldrich . Ethyl acetate (AR grade) was purchased from Rankem Fine Chemicals . Chloroform and methanol were purchased from E. Merck. All other reagents were of analytical grade.A modified emulsion – diffusion – evaporation method The Transmission Electron Microscopy observations were performed with TECNAI G2 BIOTWIN system at a magnification of 9.9X. Initially 10 μl of the nanoencapsulated quercetin suspension was retrieved and placed upon 300 mesh carbon coated copper grids. The grids containing the samples were dried extensively under lamp. The grids were then stained with 2% Phosphotungstic acid solution followed by vigorous washing with thin flow of MilliQ water. The stained grids were then dried again, inserted in the sample receiver and allowed to wait until the machine created a vacuum before obtaining images. The images of the samples were transferred to the computer screen of the instrument and the particle diameter was measured using the TEM Software program at different magnifications Male Sprague Dawley rats of two age groups, young (2 months) and aged (20 months) weighing 150–170 g and 415–440 g respectively, were used. The animals were kept in a temperature and humidity controlled housing with 12 hrs light and dark cycles. They were acclimatized for 3–5 days to the new environment before use and allowed free access to food and water. Rats from each category (2 months old and 20 months old) were subdivided into nine groups, each group consisting of 6 animals. Two groups of animals were used for saline treatment . Three experimental groups of rats (both young and aged) were respectively treated with free quercetin (2.7 mg/kg b wt), empty PLGA nanoparticles (2.7 mg/kg b wt) and PLGA nanoparticulated quercetin (2.7 mg/kg b wt) via oral gavage, two hours prior to ischemic induction. Rest of the four experimental groups of rats (both young and aged) were treated with free quercetin (2.7 mg/kg b wt) and nanoparticulated quercetin (2.7 mg/kg b wt) till day 1 and day 3 post-operation respectively via oral gavage. All the rats used in this study received proper care in compliance with the Institutional Animal Ethics Committee (IAEC), Indian Institute of Chemical Biology, India, Registration No. 147/99/CPC SEA and the Committee specifically approved this study.Rats from both young and aged groups  were anesthetized by a single intraperitoneal injection of Urethan (35 mg/kg) and were exposed to ischemia by bilateral clamping of the common carotid arteries. After 30 min. of ischemic insult, animals were further subdivided into three groups (both young and aged) subjected to reperfusion (by withdrawing clamps) intervals of 30 min, 24 h and 72 h respectively. The animals that underwent reperfusion for 24 h and 72 h were given appropriate postoperative care till day 3. After the respective reperfusion intervals, all the animals were killed by decapitation The brain tissues of all the other animals were isolated immediately and rinsed with ice-cold saline. Different brain regions viz. hippocampus, hypothalamus, cerebral cortex and cerebellum were isolated. Small portions of all these brain regions were also kept for the analysis of edema development. The brain regions were then homogenized in phosphate buffer saline with Teflon coated homogenizer for different biochemical assays.2DCFDA (5-(and-6)- chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate acetyl ester) (2 μM) for 15 min at 30°C in dark, and fluorescence was monitored. H2DCFDA, an uncharged, cell permeable fluorescent probe readily diffuses into cells and gets hydrolyzed by intracellular esterases to yield H2DCF, which is trapped inside the cell. Then it is oxidized from the non-fluorescent form to a highly fluorescent compound dichlorofluorescein primarily by hydroxyl radical (•OH), hydrogen peroxide (H2O2) or other low-molecular-weight peroxides produced in the cells. Thus the fluorescence intensity is proportional to the amount to the ROS produced by the cells. Fluorescence was measured through a spectrofluorometer  by using 499 nm as excitation and 520 nm as emission wavelengths. The data were normalized to normal values, and the normal was expressed as a value of 100%.Intracellular ROS level of different brain regions were measured Lipid peroxidation in different brain regions of experimental rats was determined by measuring the amount of conjugated diene. The brain tissue homogenates were extracted in a chloroform-methanol mixture , and the extracted lipid solvent mixture was evaporated to dryness under nitrogen atmosphere at 25°C and dissolved in cyclohexane. The lipid content in cyclohexane was assayed at 234 nm, and the results were expressed as μmol lipohydroperoxide/mg protein by using a εm of The intracellular GSH content in different brain regions was assayed at the end of the specified time. Samples of the brain tissues were removed from each animal, rinsed in ice-cold saline and blotted carefully. The samples were placed in a previously weighed ice-cold glass homogenizer containing phosphate buffer (pH 7). The weight of the added sample was determined. After homogenization, an equal volume of 10% metaphosphoric acid was added and mixed by vortexing. The mixture was allowed to stand for 5 min at room temperature. After centrifugation for 5 min, the supernatant was collected carefully without disturbing the precipitate. The GSH contents of the neutralized supernatant were assayed using Ellman's Reagent  according to the method described by Griffith (1980) 2O2Remaining portions of the brain regions were homogenized in 0.25 M sucrose with Teflon coated homogenizer and centrifuged at 10,000 g for 15 min. A portion of supernatant was further centrifuged at 1,05,000 g for 60 min. The supernatant from the second centrifugation contains the cytosolic fraction of brain cells The entry of plasma water into swollen brain of young and aged rats has been calculated by the resultant change in cerebral tissue osmolality by the induction of cerebral ischemia – reperfusion. Comparative studies in brain tissue osmolality have been performed among normal, ischemia-reperfused and other groups of ischemia-reperfused rats pre-treated either with free QC (2.7 mg/kg b.w.) or mannosylated liposome entrapped QC by the following the equation Where ΔII represents the resultant change in tissue osmolality, n denotes the osmols in an initial weight of water W0 (g) of brain tissue; DW, an additional amount of water (g) enters into brain tissue and RT =  {1.83–107(mmHg) cm3/mol} indicates the product of gas constant and absolute temperature.The hippocampal regions were dissected out from both groups of animals, were homogenized with a Dounce homogenizer in lysis buffer containing 100mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxicholate, 0.1% sodium dodecylsulphate (SDS), 5 mM EDTA, 1mM phenyl-methylsulfonyl fluoride, 5 μg/ml protease inhibitor cocktail at 4°C. The homogenates were centrifuged at 15,000× g for 15 min. The supernatants were used to determine the total protein content II and II are the fluorescence intensities parallel and perpendicular to the direction of the excited light. The microviscosity parameters [(r0/r) − 1]−1 were calculated in each case using 0.362 as the maximal limiting fluorescence anisotropy for DPH The fluorescence depolarization, associated with the hydrophobic fluorescence probe diphenyl hexatriene (DPH), was used to monitor the changes in the fluidity of the lipid matrix accompanying the gel to liquid crystalline phase transition. The mitochondrial membrane fraction was incubated at 37°C by the addition of DPH dissolved in tetrahydrofuran . The excitation and emission maxima were 365 and 430 nm, respectively. The fluorescence anisotropy was calculated by using the equation:Rats were anaesthetized and after they underwent ischemia-reperfusion, they were sacrificed. A predetermined region of injured hippocampus was dissected, weighed and stored at −70°C for batch analysis. The tissue was then homogenized in cell Lysis buffer and the homogenate was centrifuged first at 6000 rpm for 10 min and the supernatant was again subjected to centrifugation at 1,05000 rpm for 1 hr. The supernatant was collected and used as the cytosolic preparation for further analysis of Caspase-3 activity using Caspase-3 Assay kit from Sigma-Aldrich. 100 μg of cytosolic protein was diluted with 50 μl of cell lysis buffer. 50 μl of 2× Reaction buffer containing 10 mM DTT was added to each sample. Each sample was assayed with and without addition of 5 μl of the caspase-3- like tetrapeptide substrate DEVDp- nitroanilide. Samples were incubated at 37°C for 1 hr. Cleavage of the chromophore from the substrate was detected spectrophotometrically at a wavelength of 405 nm v – Volume of sample in ml,d – Dilution factor,t – Reaction time in minutes.The left and right hippocampal regions of both normal and experimental groups of young and aged rats were dissected out after their sacrifice and fixed by immersion in formalin solution. The tissue block was trimmed until it was 1cm thick and was then processed into paraffin wax. One hundred serial 5 μm sections were cut perpendicular to the long axis of the hippocampus. Every tenth section was taken, mounted onto glass slides and stained with cresyl violet r. A difference was considered significant when p was less than 0.05.Mean and SEM were calculated for all data. Significant difference between mean were evaluated by analysis of variances. Linear correlations between two data were calculated by means of Pearson's correlation coefficient The nanocapsules obtained were in the size range 20–50 nm. The TEM and AFM images  revealedLipid peroxidation is a major marker for stress induced membrane damage. The mean ± S.E. values of conjugated diene that are generated in different brain regions of young and aged rats by the induction of cerebral ischemia and reperfusion is summarized in Age related alteration in different antioxidant enzymes viz. superoxide dismutase (SOD) and catalase in different brain regions by the induction of cerebral ischemia and reperfusion is shown in Ischemic brain edema results from both cell swelling (cytotoxic edema) and increased blood vessel permeability (vasogenic edema). A change in brain tissue osmolality is the index of cerebral edema. Cerebral tissue osmolality is the index of cerebral edema. Cerebral tissue osmolality decreased with development of cerebral edema in different brain regions of both young and aged rats after the induction of ischemia and reperfusion . TreatmeIn ischemic brain tissue, ROS generation is accelerated by cytosolic pro-oxidant enzymes and failure to adequately replenish antioxidants. These overproduced ROS cause macromolecular damage and activation of various pathways. In this study, the generated ROS were assessed with the membrane-permeable fluorescent probe 2′,7′-dichlorodihydrofluorescien diacetate (H2DCFDA). The fluidity of mitochondrial membranes is especially crucial for the production of energy. In the present study, cerebral ischemic insult resulted in reduction in the mitochondrial membrane microviscosity in different brain regions of young and aged rats , the worAfter studying the effects of ischemia and reperfusion in four different vital brain regions of both age groups of animals, we observed that the hippocampus was the most affected amongst all other brain regions. In view of this, we conducted our further studies on hippocampal region of both young and aged rats.Inducible nitric oxide synthase (iNOS) is an enzyme that produces toxic levels of nitric oxide (NO) and is expressed in a number of brain pathologies, including cerebral ischemia. In our present study we observed that ischemic insults followed by respective intervals of reperfusion for 30 min, 24 h and 72 h resulted in the upregulation of iNOS protein expression in the hippocampus of experimental rats as observed from western blot analysis . Free quCaspase-3, an executioner caspase, is believed to play a vital role in apoptosis in a wide variety of cells. Activated caspase-3 cleaves proteins that are important in maintaining neuronal process integrity. Ischemic insult followed by reperfusion intervals of 30 min, 24 h and 72 h resulted in an increase in caspase-3 activity in the hippocampal region of both young and aged rats . Free quThe hippocampal formation is the most frequently examined region in studies of the mechanism of ischemic cell death and selective neuronal vulnerability. A brief period of ischemic insults is known to cause cell death in hippocampal CA1 and CA3 pyramidal neurons. Analysis of the data from the control young and aged rat hippocampal brain region  revealedCellular damage arising from the oxidative stress (OS) has been implicated in neuronal degeneration associated with normal ageing in-vivo. The size and size distribution of nanoparticles are important in determining their stability for drug release and cellular uptake efficiency The primary advantage of NP carrier technology is that NPs mask the BBB limiting characteristics of the therapeutic drug molecule. Furthermore, this system may also slow drug release in the brain, decreasing peripheral toxicity Lipid peroxidation is vulnerable to reactive oxygen radicals (ROS) induced by ischemia-reperfusion, which cause oxidative damage to brain bio membrane, lipids, proteins and DNA leading to brain dysfunctions and nerve cell death The antioxidant enzyme capacity of the brain tissue affected by ischemia-reperfusion is particularly important for the primary endogenous defence against the free radical induced injury Brain cells require a constant supply of energy substrates to maintain ionic equilibrium across neural membranes Reactive oxygen species have been implicated in the pathophysiology of many neurologic disorders and brain dysfunctions Several studies have shown that oxidative stress might lead to several alterations in membranes, including changes in the biophysical properties such as asymmetry and fluidity Ischemic insults result in delayed neuronal death in selectively vulnerable brain regions such as the hippocampal subfields Excessive release of nitric oxide (NO) has been implicated in pathophysiology of neurodegeneration in ischemic stroke Upregulation of iNOS expression leads to excessive nitric oxide (NO) production. This excess NO reacts with superoxide to form peroxynitrite, a powerful radical that induces neuronal death after cerebral ischemia Based on the results presented herein, we propose that oral treatment with nanoencapsulated quercetin might play a protective role against oxidative damage by preventing the loss of pyramidal neurons from the hippocampal CA1 and CA3 subfields in ischemia reperfusion induced young and aged rats. However, shorter durations of reperfusion produce significantly less damage than longer durations and early treatment with nanodrugs increase the chances of survival owing to reduced neuronal damage. This approach of delivering a non-toxic herb origin antioxidant, quercetin, to brain offers the potential clinical application in human neurodegenerative diseases in future.


Recently we employed phylogenetics to predict that the cellular interpretation of TGF-β signals is modulated by monoubiquitylation cycles affecting the Smad4 signal transducer/tumor suppressor. This prediction was subsequently validated by experiments in flies, frogs and mammalian cells. Here we apply a phylogenetic approach to the Hippo pathway and predict that two of its signal transducers, Salvador and Merlin/Nf2  are regulated by monoubiquitylation. This regulatory mechanism does not lead to protein degradation but instead serves as a highly efficient “off/on” switch when the protein is subsequently deubiquitylated. Overall, our study shows that the creative application of phylogenetics can predict new roles for pathway components and new mechanisms for regulating intercellular signaling pathways. Phylogenetic analyses of amino acid conservation in developmental signaling pathways have traditionally focused on pathway origins Here we report a phylogenetic analysis of nine families that participate in the Hippo pathway and compare the results to the TGF-β (five families) and Wnt (ten families) pathways The Hippo serine-threonine kinase, assisted by its co-factor Salvador, then phosphorylates Warts and its co-factor Mats. Salvador and Mats are WW/Sarah and Phoecin domain adapter proteins, respectively. Phosphorylated Warts then phosphorylates Yorkie, a WW domain transcription co-factor. Phosphorylated Yorkie is recognized and sequestered in the cytoplasm by 14-3-3 proteins. In the absence of phosphorylation Yorkie continually translocates to the nucleus, binds the transcription factor Scalloped and influences gene expression that leads to increases in cell proliferation and decreased apoptosis DrosophilaPreviously, a phylogenetic study of the Yorkie/Yap/Taz family and its binding partners in the Scalloped/Tead family revealed evidence of co-evolution Three lines of phylogenetic data converge to support predictions that two non-enzymatic Hippo pathway signal transducers, Salvador and Merlin/Nf2 are strong candidates for regulation by monoubiquitylation. Merlin/Nf2 is a well-known tumor suppressor and there is suggestive experimental evidence in the literature consistent with our prediction. From a larger perspective, our study demonstrates that the creative exploitation of amino acid sequence conservation via phylogenetics can predict new roles for pathway components and new regulatory mechanisms influencing developmental signaling pathways.Phylogenetic analyses of amino acid alignments have successfully improved our understanding of TGF-β pathway regulation. The underlying logic of the phylogenetic approach is to exploit evolutionary conservation as a guide to amino acids involved in the post-translational regulation of protein activity. As both mono- and polyubiquitylation occur solely on lysine (with very rare exceptions) the identification of potential monoubiquitylation sites requires consulting available biochemical data to eliminate lysines conserved for structural reasons. Monoubiquitylation predictions for a specific lysine are then based on: 1) universal conservation in a broad range of species, 2) occurrence within an highly conserved context and 3) that is not essential for protein structure In this study we also required that the conserved context include an upstream hydrophobic residue (at positions -1 or -2) as we noted for the two monoubiquitylated lysines (K519 and K507) in human Smad4 The nine highly divergent species employed in this study and their Linnaean classifications are shown in In the Hippo family, human Mst1 contains thirty-six lysines . EighteeHuman Salvador1 contains eighteen lysines  of whichC. elegans as sma-4 K538, the polyubiquitylated K19 in human ß-catenin is not conserved in C. elegans wrm-1 Differences in lysine conservation between Salvador and the polyubiquitylated Armadillo/ß-catenin family are consistent with the prediction of Salvador K345 monoubiquitylation. Human ß-catenin contains twenty-six lysines of which three (11.5%) are universally conserved. These lysines are structural components of the interaction surface that binds the transcription factor TCF In the Warts family, human Lats1 contains sixty-four lysines  of whichIn the Yorkie family, human Yap1 has fourteen lysines  of whichIn the Yorkie family, human Yap1 has fourteen lysines  of whichIn the Kibra family, human Wwc1 has seventy-four lysines  of whichIn the Merlin family, human Nf2 has sixty lysines  with sevIn summary, the lysine conservation data most strongly predicts the monoubiquitylation of Salvador and Merlin/Nf2. For Salvador, this is based on the presence of only one conserved lysine that is not required structurally and that is present in a conserved context containing a hydrophobic residue at -1 or -2 in all species. For Merlin/Nf2, where suggestive experimental data exists in the literature, this is based on the presence of a conserved lysine that is not required structurally and that is present in a conserved context containing a hydrophobic residue at -1 in all species.We then sought to identify additional parallels between Hippo pathway families and monoubiquitylated members of the Co-Smad subfamily (Smad4 in vertebrates and Medea in flies). A survey showed that vertebrate species in all Hippo families contain a variable number of family members.In the Hippo/Mst family  there arM. musculus Frmd1) but fish have lost two copies. In the Merlin/Nf2 family . Alternatively, many TGF-β and Wnt families in fish contain four or five copies reflecting both rounds of genome duplication. For example, Dishevelled is present in five copies and Smad2 is present in four copies There is only one protein family in each of the TGF-β and Wnt pathways that is reduced to one copy in mammals and both of these proteins have pleiotropic functions. Smad4 belongs to the Co-Smad subfamily because it forms transcriptional complexes with many partners  and thus acts as a signal transducer for multiple TGF-β pathways. β-catenin family members play roles in Wnt signaling and in adherens junctions where they modulate the turnover of membrane-bound cadherins The logic that copy number reduction serves to prevent pathway crosstalk for Smad4 seems apt for Salvador. The sole copy of Salvador in vertebrates is thought to function as a co-factor for both Hippo/Mst kinases The logic that copy number reduction serves to prevent pathway crosstalk for Smad4 also seems apt for Merlin/Nf2. The sole copy of Merlin/Nf2 is thought to function primarily via protein-protein interactions that regulate the function of its many partners including Expanded/Frmd and Kibra/Wwc family members Unlike copy number and lysine conservation data that have small variable ranges, amino acid substitution rates in animals can vary up to 67,000-fold, For example, histoneH4 shows a rate of 0.001 substitutions per residue per billion years  while ATP-cone domain proteins show a rate of 66.6 substitutions a priori reason to expect a bias in substitution rates for ligands versus signal transducers whereas there is a well documented bias towards increasing copy number in ligands First we calculated overall rates  for six When examining interacting proteins with similar roles in the TGF-β and Wnt pathways we frequently saw comparable substitutions rates. Concordant rates suggest the proteins are under similar selective constraints. Examples of interacting proteins with concordant substitution rates are: 1) the TGF-β receptor complex components , 2) Wnt receptor complex components  and 3) Wnt transcription factor complexes . Alternatively interacting proteins in which each plays a distinct role frequently showed discordant rates (differing by more than 2-fold). An example in the TGF-β pathway is the Smad multimeric complex composed of Co-Smads regulated by monoubiquitylation (0.595) and receptor activated Smads regulated by phosphorylation (0.258). An example in the Wnt pathway is the multimeric complex composed of Shaggy/Gsk3 kinases (0.333) with the adapter proteins APC (1.221) and Axin (1.583).In the Hippo pathway, we found examples of concordance such as: Warts/Lats kinase (0.298) and its co-factor Mats/Mob (0.151) as well as Kibra/Wwc (0.802) and Expanded/Frmd (0.997). Discordance in substitution rates between interacting proteins is evident for Hippo/Mst (0.156) and Salvador  as well as Merlin/Nf2 (0.498) and Expanded/Frmd (0.997) These findings are not expected from Salvador’s perceived role as a passive co-factor for the Hippo kinase or from the perceived roles of Merlin/Nf2 and Expanded/Frmd as simple adapter proteins. In summary, comparisons with discordant pairs in the TGF-β and Wnt pathways suggest that Salvador and Merlin/Nf2 have undiscovered roles distinct from their partners , within the Hippo pathway or beyond.Overall, this study builds upon successful phylogenetics-based predictions for the regulation of Smad signal transducers, including Smad4, in the TGF-β pathway. Comparison of phylogenetic data for nine Hippo families to results from studies of the TGF-β and Wnt pathways led us to predict new roles for Salvador and Merlin/Nf2 and to suggest that these may involve pathway regulation via monoubiquitylation. If validated, and suggestive experimental evidence is already visible in the literature for Merlin/Nf2, these predictions will reinforce the view that the creative exploitation of amino acid conservation via phylogenetics can illuminate new regulatory mechanisms affecting developmental pathways.All members of the Hippo/Mats, Salvador, Warts/Lats, Mats/Mob, Yorkie/Yap/Taz, Scalloped/Tead, Kibra/Wwc, Expanded/Frmd and Merlin/Nf2 families for nine fully sequenced organisms were retrieved and analyzed as described Alignments for each gene family were created in MAFFT. In one case an alignment was annotated manually (in the Expanded/Frmd family an excessive tail on Expanded was removed). See Figure S1Bayesian trees of Hippo pathway proteins. Bayesian trees are displayed in the same format as the Maximum Likelihood trees in the main text with explicit posterior probabilities shown at the nodes. Branches with posterior probabilities <0.90 are considered weak and colored in blue. Branch lengths denote the number of amino acid changes per site. Organisms are abbreviated as in the Maximum Likelihood trees in the main text. A) Hippo/Mst Bayesian tree, B) Salvador Bayesian tree, C) Warts/Lats Bayesian tree, D) Mats/Mob Bayesian tree, E) Yorkie/Yap/Wwtr Bayesian tree, F) Scalloped/Tead Bayesian tree, G) Kibra/Wwc Bayesian tree, H) Expanded/Frmd Bayesian tree and I) Merlin/Nf2 Bayesian tree.(PDF)Click here for additional data file.Table S1Accession numbers.(PDF)Click here for additional data file.Table S2Alignment lengths. The total lengths of protein alignments used to create trees are shown below. Bayesian trees were made from MUSCLE alignments, while MAFFT alignments were used to create Maximum Likelihood trees. Expanded alignments were corrected by hand since the presence of numerous large D. melanogaster insertions and deletions resulted in many gaps, making it difficult to create trees. Original alignment lengths for Ex are shown in parentheses. Alignments are available upon request.(PDF)Click here for additional data file.


Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies. Human clinical studies consistently link greater blood leukocyte count to increased risk of atherothrombotic disease events, such as myocardial infarction (MI) and stroke 12/13Cathepsin G is one of multiple plausible molecular mediators of the association between leukocytes and thrombotic vascular occlusion We hypothesized that neutrophil cathepsin G is a physiologic platelet activator and key molecular mediator of vascular thrombus formation in vivo. We tested these hypotheses in experiments using both human and mouse blood and three separate mouse models of vascular thrombus formation in vivo. The aim of these studies was to identify a molecular mechanism that directly links neutrophils to arterial thrombosis and to determine the relevance of cathepsin G as a potential target for anti-thrombotic therapies.Healthy male and female volunteers  were enrolled to examine human platelet aggregation in vitro. Volunteers were eligible for study participation if they were free of chronic and acute medical illness and had not taken aspirin or nonsteroidal anti-inflammatory drugs in the past 10 days. This study was approved by the Johns Hopkins Institutional Review Board and all participants signed written informed consent.3 neutrophils/µl) with the freshly-prepared PRP (1.7×105 platelets/µl) and maximum platelet aggregation was determined after stimulating samples for 5 min at 37°C with thrombin receptor activating peptide-6  in a dual channel lumi-aggregometer . In some experiments, PAR4 agonist peptide  was also added to stimulate platelet aggregation. The effect of neutrophil count on platelet aggregation was assessed by varying the number of neutrophils present in PRP through the pathophysiologic range (0–20×103 neutrophils/µl).Platelet aggregation was assessed in platelet rich plasma (PRP) by impedance aggregometry in the presence and absence of neutrophils and a specific cathepsin G inhibitor, cathepsin G inhibitor I 3/µl) were reconstituted with freshly prepared PRP (1.7×105 platelets/µl) and maximum aggregation in response to TRAP determined by impedance aggregometry as described above.HL60 cells , an immortalized myelocytic cell line, were cultured in RPMI 1640/20% fetal calf serum  and 1.5% DMSO to induce neutrophil differentiation 5 platelets/µl), before and after stimulating platelet aggregation with 15 µM TRAP. Knock-down of cathepsin G expression in HL60 cells was also confirmed by Western blotting. Briefly, PRP supernatant or HL-60 cells were treated with lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 2 mM EDTA, 0.1% sodium dodecyl sulfate (SDS) and inhibitor cocktail . Proteins were boiled for 5 minutes and proteins separated (10 µg/lane) by 10% SDS-polyacrylamide gel electrophoresis. Nitrocellulose membranes  were blocked with 10% nonfat dry milk/0.1% Tween-20 and incubated overnight at 4°C with mouse anti-human cathepsin G  or rabbit polyclonal β-actin IgG antibody , followed by HRP-conjugated goat anti-mouse IgG antibody  or goat anti-rabbit IgG-HRP antibody . Proteins were visualized by enhanced chemiluminescence .Western blotting was used to quantify cathepsin G protein in human PRP, in the presence and absence of neutrophils  were used to examine platelet-mediated thrombus formation in vivo using three distinct models: tail bleeding time, mesenteric arteriolar thrombus formation, and collagen-coated filament occlusion of the middle cerebral artery (MCA). Cathepsin G deficient (CatG−/−) mice that were backcrossed for 10 generations on a C57BL/6 background were the generous gift of Dr. Timothy Ley . Strain characterization of backcrossed mice  identified >98% of chromosomal markers belonging to this strain. Mouse studies were approved by the Johns Hopkins University Animal Care and Use Committee .The distal 5 mm of tail was amputated from anesthetized mice (ketamine 100 mg/kg IM injection) and the tail immersed in 37°C phosphate-buffered-saline. Time to visible cessation of bleeding was recorded in wildtype (WT) mice injected intravenously with vehicle , cathepsin G inhibitor I , or aspirin , and in CatG−/− mice.Blood neutrophil counts in vivo were increased by treating mice with mouse recombinant granulocyte colony stimulating factor  (1 µg subcutaneous injection×3 consecutive days) and decreased by treatment with cyclophosphamide (Sigma) . Complete blood counts were determined by automated cell counter  and leukocyte differential verified by manual cell counting. Cell counts and bleeding times, as described above, were determined on the day of the third GCSF  injection. Blood monocyte counts were manipulated in vivo by intaperitoneal injection of a monocyte depleting IgG antibody against CCR2, MC21 , or isotype control antibody, IgG2b  Neutrophil-platelet conjugate formation was assessed by flow cytometry in WT and CatG−/− mouse blood in vivo and in vitro as previously described with modification 8 platelets) into an anesthetized (ketamine [100 mg/kg]/xylazine [10 mg/kg] IM injection) recipient mouse. In another set of experiments, native platelets were labeled in vivo by intravenous injection of X488 , a fluorescently labeled IgG derivative against mouse GPIbβ that does not alter platelet adhesion or aggregation 3 (15%)-soaked piece of Number 2 Whatman paper onto the arteriolar surface for 90 sec. Fluorescent images of platelet accumulation onto the injured vascular surface were captured with a digital camera  and analyzed with imaging software . Time to vascular occlusion  by platelet thrombus was determined for WT and CatG−/− mice in the presence and absence of cathepsin G inhibitor I  or aspirin .Ferric chloride-induced mesenteric arteriolar thrombosis was assessed by intravital fluorescence microscopy as previously described with modification Transient filament occlusion of the MCA was performed as previously described with modification Data are mean ± SD unless otherwise indicated. Data were analyzed by t-test or analysis of variance (ANOVA) with Bonferroni correction for multiple post-hoc comparisons, as appropriate. Correlations between leukocyte counts and bleeding times were determined by parametric (ln[count]) and nonparametric (raw count) methods with similar results. All significance testing was two-tailed and results considered significant at an alpha of 0.05.Reconstitution of human neutrophils with autologous PRP increased agonist-induced platelet aggregation by ∼50% fig. 1A.We hypothesized that cathepsin G inhibitor I antagonized the pro-aggregatory effect of neutrophils by blocking the effect of neutrophil cathepsin G on platelet PAR4. To test this hypothesis we repeated aggregation experiments in the presence of a maximal dose of PAR4 agonist peptide, which exerts its platelet activating effect downstream of the enzymatic activity of cathepsin G. PAR4 peptide did not significantly increase platelet aggregation over TRAP alone in the absence of neutrophils , suggest3/µl. We performed Western blotting experiments on plasma from aggregation samples to confirm release of cathepsin G from neutrophils. These experiments demonstrated that the increase in platelet aggregability related to neutrophils was associated with an increase in the plasma concentration of cathepsin G in the presence of TRAP-activated, but not unactivated, platelets  or bind fibrinogen  in response to collagen stimulation in vitro among the four treatment conditions , consistent with the effects of GCSF and cyclophosphamide being specific to neutrophil count and not to an alteration in platelet function. Furthermore, because monocytes share multiple molecular pathways with neutrophils and have been reported to contribute to thrombus formation We compared the effect of cathepsin G inhibitor I on bleeding time to the effect of aspirin. Dose-response studies demonstrated that aspirin was maximally effective at prolonging tail bleeding time at an intravenous dose set to approximate a blood concentration of 500 µM fig. 4A,We next examined the effect of cathepsin G on platelet thrombus formation under arterial flow conditions in mesenteric microvessels. As previously described Comparing CatG−/− to WT mice provided results similar to those obtained with pharmacologic inhibition of cathepsin G- i.e., time to thrombus formation was prolonged in knockout mice (The impact of cathepsin G on CBF and ischemic brain injury was examined using a previously described mouse stroke model These experiments demonstrate the physiologic importance of neutrophils to platelet activation and vascular thrombus formation in vivo and its mechanistic dependence on cathepsin G. The ability of neutrophil cathepsin G to promote platelet aggregation and vascular occlusion was demonstrated in human PRP in vitro, after tissue trauma in the mouse in vivo, under arterial flow conditions in mouse microvessels, and in a vascular occlusive stroke model. Specificity of effect was demonstrated using both a pharmacologic inhibitor of cathepsin G, which provided effective inhibition at submicromolar concentrations and in a dose-dependent manner, and in cathepsin G deficient mice. These translational research experiments provide a direct causal mechanism to help explain the strong association between neutrophil count and arterial thrombotic morbidity previously described for patients with atherosclerosis There was a striking dose-response relation between neutrophil count and both platelet activation in vitro and bleeding time in vivo. In human PRP, this dose-response relation was observed at neutrophil counts within the range clinically encountered under physiologic and pathologic conditions. The pro-aggregatory effect of neutrophils was markedly reduced in the absence of cathepsin G activity, whether induced pharmacologically or by expression knockdown. The effect of neutrophil cathepsin G on platelet aggregation appeared to be mediated in part by cleavage of PAR4 because inhibition of cathepsin G activity failed to diminish the pro-aggregatory effect of neutrophils when PAR4 agonist peptide was used to activate platelets. Our data suggest that cathepsin G acts as a physiologic platelet activator in the blood milieu whose availability is regulated, in part, by the number of accessible neutrophils. However, the impact of neutrophils on platelet aggregation may involve mechanisms in addition to the action of cathepsin G on PAR4 because abrogation of the pro-aggregatory effects of neutrophils appeared incomplete at high neutrophil counts and in knockdown experiments with HL60 cells. Additional mechanisms through which neutrophils may promote platelet aggregation include release of prostaglandin metabolites The ability of neutrophils to form mixed aggregates with platelets is well recognized In vivo, the number of blood neutrophils appeared to modulate the speed with which hemostasis could be achieved after traumatic injury in the mouse. Bleeding time was highly and inversely correlated with the neutrophil count but showed no association with lymphocyte, monocyte, or platelet counts. These data should not be construed to mean that these other cell types have no influence on hemostasis; platelets are well recognized to play a key role in primary hemostasis, and monocytes have been reported to influence clot formation through formation of tissue factor bearing microparticles Neutrophils have long been recognized to possess multiple mechanisms with potential to both promote The discovery of a cathepsin-G-dependent relationship between neutrophil count and thrombus formation in vivo has important clinical implications. Given that acute stress is accompanied by demargination of neutrophils and a rapid rise in blood neutrophil count, it might be reasonable to postulate that neutrophilia offers a protective mechanism to promote hemostasis under conditions of traumatic injury. While potentially protective, a pro-thrombotic effect from neutrophil cathepsin G might increase risk of pathologic thromboses in individuals with pre-existing pro-thrombotic states as has been described for patients with atherosclerosis In sum, our data indicate that neutrophil cathepsin G is a physiologic regulator of platelet activation and thrombus formation in vivo. Our experiments suggest that cathepsin G is a potential target for novel anti-thrombotic therapies, but like all such therapies, inhibition of cathepsin may increase bleeding risk as well. Additional experiments are needed to determine the potential benefits and risks of cathepsin G inhibitors in animal models of vascular occlusive diseases.


Anthropogenic global changes in biodiversity are generally portrayed in terms of massive native species losses or invasions caused by recent human disturbance. Yet these biodiversity changes and others caused directly by human populations and their use of land tend to co-occur as long-term biodiversity change processes in the Anthropocene. Here we explore contemporary anthropogenic global patterns in vascular plant species richness at regional landscape scales by combining spatially explicit models and estimates for native species loss together with gains in exotics caused by species invasions and the introduction of agricultural domesticates and ornamental exotic plants. The patterns thus derived confirm that while native losses are likely significant across at least half of Earth's ice-free land, model predictions indicate that plant species richness has increased overall in most regional landscapes, mostly because species invasions tend to exceed native losses. While global observing systems and models that integrate anthropogenic species loss, introduction and invasion at regional landscape scales remain at an early stage of development, integrating predictions from existing models within a single assessment confirms their vast global extent and significance while revealing novel patterns and their potential drivers. Effective global stewardship of plant biodiversity in the Anthropocene will require integrated frameworks for observing, modeling and forecasting the different forms of anthropogenic biodiversity change processes at regional landscape scales, towards conserving biodiversity within the novel plant communities created and sustained by human systems. Human populations and their use of land have transformed more than three quarters of the terrestrial biosphere into anthropogenic biomes  results when humans interact with native patterns of species richness. Within a given area, ASR can be quantified as the sum of native species richness (N), anthropogenic loss of native species (ASL) and anthropogenic species increase (ASI):Anthropogenic species richness (ASL within a given area is commonly predicted as a function of N and the area of native habitat lost to agriculture and settlements (HL) by applying classic species-area relationships , agricultural domesticates , and exotic ornamental species (OS) present in an area. Estimating ASR based on these very basic assumptions oversimplifies and even omits some key processes by which humans alter plant species richness, including global climate change ASR, ASL, and ASI that summarize the state of current knowledge of anthropogenic global patterns of plant species richness and may serve as hypotheses against which more comprehensive data and models may be tested in the future. While these estimates must therefore be considered preliminary, we present these with the aim of stimulating global change and biodiversity science as well as the conservation community to embrace a more comprehensive and long-term view of the novel anthropogenic patterns of biodiversity sustained by human systems in the Anthropocene ips SAR; . ASI witThe use of global models and empirical estimation procedures  enabled N, or both , either by causing at least 5% of their native species to be lost, or by introducing exotic species at levels equivalent to 5% or more of  or both . Model rglobally . Yet thiN, with exotics exceeding 10% of native richness across more than a quarter of the biosphere, causing a median global exotic increase of 7% [3–18%] relative to N  to those observed by Stohlgren et al. Most regional landscapes (89% [31–100%] of ice-free land) have likely experienced substantial increases in exotic species, by ≥5% of ive to N . AnthropN or more; ASR; N; ASR can be predicted by global variation in N  of exotic species invasions , or crop introductions (CS: 0.03), based on standardized coefficients from multiple regression .Though it appears that the vast majority of the terrestrial biosphere has gained or lost a substantial number of species , models ion in N . Indeed,N, have created artificial relationships between loss and gain, balancing out their relative effects . Yet an even simpler explanation is random chance. Substantial gains occurred across 89% [31–100%] of the terrestrial biosphere and substantial losses across 51% [14–64%] /N; R2 = 0.47 vs. 0.32; data not shown). Taken together, these results indicate that human population density, which drives land use intensification, might ultimately be an even better indicator of anthropogenic ecological change than land use or habitat loss Assessing land use and population as global drivers of anthropogenic ecological succession is a challenge because of their complex interrelationships. Land use drives habitat loss and varies with human population density  and all ere used . HabitatWhatever the mechanism, by enriching plant communities with exotic species and thinning native species locally and globally, humans are causing a vast biotic homogenization of plant communities across the terrestrial biosphere ASR≤N; N; Today, few native plant communities remain undisturbed and without exotic companions . Though ASR/N ∼132%; ASL/N = 14%; ASR/N; HL = 66%; As observed globally, anthropogenic species richness usually followed native richnesspatterns across biomes and biogeographic realms . TemperaHL = 40%) and densely populated (median population = 29 persons km−2) than the terrestrial biosphere as a whole  and much of the anthropogenic biosphere . Nevertheless, eight hotspots appeared to have lost more than 10% of native species from their regional landscapes - substantially more than the median for the anthropogenic biosphere (median = 8%). While no hotspot gained more non-natives than the temperate broadleaf forest biome (41% of N), six hotspots gained more than 20% non-natives. As with the rest of the biosphere, anthropogenic species increase usually balanced native species loss, and only four hotspots showed a substantial net decline in species richness. Exceptionally large non-native gains were observed in two hotspots  as a result of extremely high levels of invasion in Japan, and high invasions plus high ornamentals in California. In terms of total species gains and losses, the most anthropogenically altered hotspot overall was Japan (median = 42%), and the wildest was the East Melanesian Islands, with species loss near zero and the lowest anthropogenic species increase relative to native levels as well . Still, 18 of 32 hotspots had a greater total species loss + gain than the global median for the anthropogenic biosphere (18%), confirming that the most biodiverse regions on Earth also tend to be among the most challenged by anthropogenic transformation.Biodiversity hotspots . This de2, respectively HL 73% and 65%) and native species losses (16 and 21% respectively) observed across anthromes, biomes, realms and hotspots  and overall human alteration ((ASI+ASL)/N; ASI; N ∼1000 species/cell), while leaving the most species rich and species poor regions less intensively used for agriculture and less densely populated  were estimated from Monfreda et al. OS) from urban area and published counts of urban exotic domestic plant species . Uncertainties in model predictions for ASL, IS, OS and the estimates derived from them  were characterized using upper and lower error bounds derived from a worst case sensitivity analysis Click here for additional data file.Appendix S2Global statistics(XLS)Click here for additional data file.Appendix S3Maps and spatial data.(PDF)Click here for additional data file.


Poly-(ADP-ribose) polymerase (PARP) activity was assessed by poly-ADP-ribose immunostaining. Increased PAR staining in ovaries and circulating leukocytes from DHT rats showed enhanced DNA damage, which was reduced by concomitant vitamin D3 treatment. Surprisingly, PAR staining was reduced in both the endothelium and vascular smooth muscle cells of the aorta rings from hyperandrogenic rats. Thus in the early phase of PCOS, vascular tone is already shifted towards vasoconstriction, characterized by reduced vasorelaxation and vascular dysfunction is concomitant with altered PARP activity. Based on our findings, PARP inhibitors might have a future perspective in restoring metabolic disorders in PCOS.Polycystic ovary syndrome (PCOS) is a complex endocrine disorder characterized by hyperandrogenism and insulin resistance, both of which have been connected to atherosclerosis. Indeed, an increased risk of clinical manifestations of arterial vascular diseases has been described in PCOS. On the other hand endothelial dysfunction can be detected early on, before atherosclerosis develops. Thus we assumed that vascular dysfunction is also related directly to the hormonal imbalance rather than to its metabolic consequences. To detect early functional changes, we applied a novel rodent model of PCOS: rats were either sham operated or hyperandrogenism was achieved by implanting subcutaneous pellets of dihydrotestosterone (DHT). After ten weeks, myograph measurements were performed on isolated aortic rings. Previously we described an increased contractility to norepinephrine (NE). Here we found a reduced immediate relaxation to estradiol treatment in pre-contracted aortic rings from hyperandrogenic rats. Although the administration of vitamin D Polycystic ovary syndrome (PCOS) is the most common endocrine disorder, affecting 4–11% of women. The lifelong disease often remains hidden because of its multidisciplinarity. The main problem is infertility but the disease influences the whole body in many different ways Indeed, haemodynamic changes of internal carotid artery Estrogens have a multiple cardiovascular protective effect. These include both a rapid and a long-term vasorelaxing effect, the latter of which depends on gene expression and employs different signalling pathways not as in short term effects Vitamin D was effective in PCOS: a daily intake of vitamin D reduced obesity in PCOS patients Poly-(ADP-ribose) polymerase-1 (PARP-1) is a ubiquitously expressed nuclear enzyme, which has a central role in responses to cellular injury This signalling pathway has been implicated in both experimental models and in human cardiovascular diseases 3 has been shown to inhibit PARP by Mabley et al., it was demonstrated that UV irradiation-mediated PARP activation in human keratinocytes can be inhibited by treatment with vitamin D, 7-dehydrocholesterol or 1alpha,25-dihydroxyvitamin D3Besides the available pharmacological inhibitors, there is cumulating amount of evidence for endogenous modulators, too. Recently the active forms of vitamin DThe same group examined the downstream mediators of NO/PARP activation to investigate possible mediators of ischemic sexual dimorphism. Apoptosis inducing factor translocation and PAR formation did not mediate ischemic injury in the female mice brain; although equivalent activation of cell death pathway occurred in both sexes after ischemia, detrimental effects were only present in males The aim of this study was to show altered regulation of vascular tone in PCOS applying a novel rodent model. We wanted to detect the possible beneficial effect of chronic vitamin D administration and acute estradiol treatment. Also we aimed to look at altered DHT induced PAR response in different organs and sought to identify a correlation between the changes in vascular reactivity and PAR responses.Ethics approval The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and was approved by Semmelweis University Animal Care Commission .3. No medical or surgical complications were observed throughout the whole period. Conventional rat chow and tap water were provided ad libitum. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and was approved by Semmelweis University Animal Care Commission  conforming to the Institutional Animal Care and Use Committee (IACUC). Thirty adolescent, 21–28 day-old, female Wistar rats, weighing 100–140 g were purchased from Semmelweis University Animal Colony . The animals were randomized into 3 treatment groups: control, DHT and DHT+vitamin D3  every week, which is a slight modification of the protocol previously described by Przybylski et al. To induce experimental PCOS, twenty animals received 70-day-long continuous-release subcutaneous pellets of 7.5 mg dihydro-testosterone  underneath the back skin, releasing a daily dose of 83 μg DHT as described by Manneras et al. 2, 1.17 MgSO4, 20 NaHCO3, 1.18 KH2PO4, 0.027 EDTA, and 11 glucose . Hyperkalaemic solution was prepared from normal KR. It was supplemented with KCl to achieve 124 mM potassium level. Temperature of the solution was kept at 37°C, and it was aerated with a 5% CO2/95% O2 mix . Norepinephrine and 17- estradiol were obtained from Sigma-Aldrich. Drugs were freshly prepared in nKR solution on the day of the experiment.Normal Krebs-Ringer (nKR) solution was composed of (in mM): 119 NaCl, 4.7 KCl, 2.5 CaClAfter 70 days of treatment, rats were anesthetized. For anesthesia we used penthobarbital  45 mg/kg intraperitoneally. Animals were perfused transcardially with 10 ml heparinized (10 IU/ml) nKR solution. The aorta of each animal was then removed. The distal part of the thoracic aorta (TA) was isolated and four rings were prepared and placed into a vessel chamber filled with nKR solution aerated with 95% O2/5% CO2 mix. Thoracic aorta segments of 3 mm length from each experimental group were mounted on stainless steel vessel holders (200 µm in diameter) of a conventional myograph setup . The organ chambers of the myographs were filled with 8 ml of nKR solution. The bath was warmed to 37°C, and the resting tension of TA rings was adjusted to 15 mN, according to previous studies + to elicit a reference contraction. Twenty minutes later, precontraction was induced by norepinephrine (5×10−8 M) and for estradiol-mediated vasorelaxation cumulative dose-response measurements were used (10−5–10−4 M). Norepinephrine contraction was expressed as a percentage of K+-precontraction. Aortic relaxations were tested after a stable plateau of contraction had been reached. Relaxant responses were expressed as a percentage of the precontraction produced by norepinephrine (percentage =  (tension after norepinephrine-tension after estradiol)/(tension after norepinephrine-baseline tension) * 100). The isometric tension recording of the thoracic aorta segments was made with the MP100 system, and recorded data were analyzed with AcqKnowledge 3.7.3 software . Vasoactive substances were dissolved in nKR. All concentrations are expressed as the final concentration in the organ bath.Segments were exposed to 124 mM KOvaries and aorta rings of the animals were collected and freshly fixed for histological examinations. Tissue samples were immersion fixed in 4% buffered formaldehyde, hematoxylin-eosin staining was prepared and specimens were examined using a Zeiss AxioImager. A1 microscope coupled with Zeiss AxioCAm MRc5 CCD camera for evaluation Pannoramic viewer software was used; 3DHISTECH Ltd., Budapest, Hungary.Leucocytes were isolated from venous blood samples using Histopaque-1083 . Methanol-fixed smears were prepared from the cell suspension. Samples of ovaries and aortas were fixed with formalin, embedded in paraffin and 5 μm thick sections were prepared.After deparaffinization and antigen retrieval , samples were incubated with a mouse monoclonal anti-poly-ADP-ribose (PAR) antibody overnight at 4°C . Secondary labelling was achieved using biotinylated anti-mouse horse antibody  . Horseradish peroxidase-conjugated avidin  and nickel-enhanced diaminobenzidine  was used (Vector Laboratories). Tissue sections and smears were counterstained with nuclear fast red.To evaluate PAR-staining, the following semiquantitative score system was applied by a blinded observer as described previously Parametric variables were analyzed by applying ANOVA. As a post hoc test, Newman-Keuls` test was applied. P<0.05 was uniformly accepted as a significant difference. Data were presented as mean ± SEM.3 (To examine the vascular dysfunction in the early phase of polycystic ovary syndrome (PCOS) we first treated rats with dihydrotestosterone (DHT) for 70 days as described by Manneras et al. 3 . Histolo3 .−8 M) for approximately 15 minutes and then were treated with estradiol . Estradiol readily induced vasorelaxation in the precontracted aortas. Surprisingly this effect was significantly weaker in the DHT group compared to the control group. Vitamin D3 did not restore the vascular responsiveness to estradiol, namely E2 induced a similar relaxation in the DHT+vitamin D3 as in the DHT alone group. . Thus short-term estrogen treatment is less potent as a vasorelaxing agent in large arteries from hyperandrogenic animals. In the rat aortas treated by dihydrotestoterone, the estradiol dependent vasorelaxation was substantially reduced (p<0.01) compared to the control group, and vitamin D3 treatment could not correct it  and showed similar contractility in all treatment groups. Since the relative hypoestrogenic state in PCOS is considered to contribute to the vascular dysfunction we asked whether acute estrogen treatment can restore normal vascular tone. To explore this, aorta rings were precontracted with norepinephrine . On the contrary, in the vascular endothelium and smooth muscle cells the PAR staining in the DHT group was significantly weaker (3 p<0.05).PARP activity is a marker of cellular DNA damage, and it was also shown to be controlled by estrogens 3 therapy on it in a recent rodent model of PCOS. Parallelly, we were detecting the DHT-induced PAR response, PAR poly(adenosinphosphate-ribose) accumulation and the effect of vitamin D3 therapy on it in different organs to assess the activation and inhibition of PARP enzyme in our PCOS simulating clinical circumstances.Our experiment has been the first to study the relaxing capacity of estradiol on the vessel wall under a hyperandrogenic milieu and the effect of vitamin DIn women with PCOS significant pharmacological reactivity changes develop as well as mechanical damages of the large blood vessels Estrogen has a variety of effects on vasculature including the regulation of vascular tone. The vasodilatory effects of estrogen can be endothelium dependent and independent, and they can work by genomic and nongenomic mode of action 2+ channels and/or activating voltage – operated Ca2+ and K+ channels Testosterone also has a moderate vasorelaxant effect acutely, especially by the modulation of vascular smooth muscle ion channel function, particularly the inactivation of L- type voltage operated CaIn our study we found an impaired vasorelaxant effect of estrogen. Athough it was still working, the vasorelaxant effect was substantially reduced in hyperandorgenic milieu caused by the PCOS . New's [3 therapy reduced it significantly.  polymerase compared to controls and DHT treated animals.As it was detected in many clinical conditions but not in policystic ovary syndrome, we studied the PAR activation in a rodent model of PCOS. In PCOS the cardiovascular risk is elevated as unfavourable hormonal – hyperandrogenic, metabolic, prediabetic and inflammatory alterations are present from a younger age till menopause Our results were coherent with earlier studies in hypertension and diabetes mellitus 3 treatment did not have any significant influence on this effect.To sum up, the activation of the poly(ADP-ribose) polymerase was shown in many different clinical and pathological settings , vascular diseases  ischaemic diseases , and in chronic heart failure 3 on them was not significant. This result might give the clue to one of the key-mechanisms of early cardiovascular damage in PCOS.The DHT treatment caused alterations in relaxant capacity. Estrogen in hyperandrogenic milieu could not work as well as in the control female rat aortic rings and the influence of vitamin D3 did not influence it significantly. Similarities of PARP expression and estrogen dependent relaxation may raise the possibility of causal connections, which require further investigation.In PCOS, caused by DHT treatment, deleterious effects were exerted partly via PARP activity (ovaries and leukocytes), and these could be reversed by vitamin D. But in aortic walls, both in endothelium or vascular smooth muscle, the PARP activity was significantly reduced by DHT treatment and vitamin D


Schistocerca gregaria) show an extreme form of phenotypic plasticity and can transform between a cryptic solitarious phase and a swarming gregarious phase. The two phases differ extensively in behavior, morphology and physiology but very little is known about the molecular basis of these differences. We used our recently generated Expressed Sequence Tag (EST) database derived from S. gregaria central nervous system (CNS) to design oligonucleotide microarrays and compare the expression of thousands of genes in the CNS of long-term gregarious and solitarious adult desert locusts. This identified 214 differentially expressed genes, of which 40% have been annotated to date. These include genes encoding proteins that are associated with CNS development and modeling, sensory perception, stress response and resistance, and fundamental cellular processes. Our microarray analysis has identified genes whose altered expression may enable locusts of either phase to deal with the different challenges they face. Genes for heat shock proteins and proteins which confer protection from infection were upregulated in gregarious locusts, which may allow them to respond to acute physiological challenges. By contrast the longer-lived solitarious locusts appear to be more strongly protected from the slowly accumulating effects of ageing by an upregulation of genes related to anti-oxidant systems, detoxification and anabolic renewal. Gregarious locusts also had a greater abundance of transcripts for proteins involved in sensory processing and in nervous system development and plasticity. Gregarious locusts live in a more complex sensory environment than solitarious locusts and may require a greater turnover of proteins involved in sensory transduction, and possibly greater neuronal plasticity.Desert locusts ( Schistocerca gregaria) are notable for their extreme form of phenotypic plasticity. Depending upon changes in population density, their genome can generate two markedly different phenotypes, which were thought to be different species when first discovered Desert locusts  S. gregaria EST sequences contained in our database S. gregaria transcript sequences in GenBank. We also included in the microarray design all available L. migratoria EST sequences that have no apparent ortholog in the S. gregaria set. In this study we have used these microarrays to carry out a transcriptome-wide analysis of molecular differences between the CNS of adult solitarious and gregarious desert locusts, comparing the expression of thousands of genes.Very little is currently known about the molecular and mechanistic basis of phase polyphenism of the desert locust. Because of the primacy of behavior and therefore the CNS in desert locust phase transition Schistocerca cDNAs and the remaining 48 (22%) by probes from Locusta. Some 40% of the differentially expressed genes have been annotated to date , Neurological system process (GO:0050877), Response to stress (GO:0006950), Generation of precursor metabolites and energy (GO:0006091) and Cellular macromolecule biosynthetic process (GO:0034645). In addition, differential transcript levels were also observed for genes encoding pacifastins, hexamerins, three cytochrome P450 proteins and DNA methyltransferase 2.In total, 214 genes out of a total of 20,755 represented on the microarrays were differentially expressed between solitarious and gregarious locusts , 100 of which were more strongly expressed in the gregarious phase and 114 in the solitarious phase. Of the differentially expressed transcripts, 166 (78%) were identified by probe spots representing  to date , allowin to date . By combMulticellular organismal development are likely to be involved in nervous system development and/or remodeling. Our previous findings Drosophila melanogaster, Cullin-3 indirectly controls dendritic actin rearrangements via degradation of its target, the actin-cross-linking Kelch protein Several of the differentially expressed genes classified under D. melanogaster Mi-2 chromatin remodeler. Both are known transcriptional regulators of sensory nerve cell differentiation in D. melanogasterD. melanogaster homolog of the human Nova-1 and Nova-2 proteins Chromatin-remodeling is an important epigenetic mechanism that influences development through control of transcription The pronounced phase differences in the expression of genes normally implicated in nervous system development  are unexpected in adult animals. Although these genes have not yet been functionally characterized in locusts, our data suggest that the nervous system of gregarious locusts may show more ongoing structural remodeling than that of solitarious locusts. The key mechanisms of neuroplasticity include the alteration of existing connections and the establishment of new ones. Enhanced plasticity in the nervous system may allow gregarious locusts to adapt more easily or rapidly to new environments and deal with the sensory complexity of a swarm. This is consistent with the substantially larger brains of gregarious locusts compared to solitarious locusts. Although the primary sensory neuropils in the brain are relatively smaller in gregarious locusts, the higher integration centers are up to 50% larger than in solitarious animals 2+-activated K+ channel Slowpoke. In D. melanogaster, Slowpoke is involved in habituation A transcript that was more abundant in the solitarious CNS encodes a homolog of the CaTranscripts encoding the long wavelength-sensitive opsin, a typical rhodopsin visual pigment protein S. gregaria chemosensory proteins (CSPs) CSP1 and CSP3 L. migratoria. RNA interference against CSP3 in the gregarious phase of L. migratoria reduced aggregation behavior and preference for pheromones L. migratoria does not contain appreciable amounts of CSP3 transcripts S. gregaria may point to a role for CSPs beyond that of peripheral olfaction in this species.Transcripts encoding the Tribolium castaneum genome Our data suggest that gregarious and solitarious locusts differ in some aspects of acute stress tolerance. Gregarious locusts had higher transcript levels for a thaumatin-like protein. Thaumatins were originally described as plant stress proteins where they have glucanase activity and function in a defense mechanism against pathogens L. migratoriaGregarious animals also had higher levels of transcripts encoding heat-shock proteins (HSPs). An elevation of HSP-encoding transcripts has previously been shown in gregarious Solitarious locusts in contrast to gregarious locusts seem to invest more in oxidative stress resistance; that is, resistance to the chronic accumulation of deleterious effects that occur over a lifetime. Organisms are protected from reactive oxygen species (ROS) by antioxidant systems. Ageing is associated with an accumulation of cellular waste and the detrimental effects of ROS and other free radicals Generation of precursor metabolites and energy), ribophorin and eight ribosomal proteins (GO: Cellular macromolecule biosynthetic process). This suggests that solitarious locusts, with their long lives and their enhanced protection from oxidative stress also invest more strongly in ongoing anabolic renewal than do gregarious locusts. By contrast gregarious locusts with their enhanced HSP production may instead preferentially protect already existing proteins.Genes encoding homologs of proteins involved in basic cellular processes, such as protein synthesis and cellular respiration, showed higher expression in solitarious locusts. These included genes encoding electron-transferring flavoprotein dehydrogenase, mitochondrial ATP synthase coupling factor 6  are differentially expressed in the two phases S. gregaria. These encoded gene products are arylphorin-like and closely resemble L. migratoria JHPH proteins. Arylphorins are members of the hexamerin family of proteins, which were thought simply to provide haemolymph stores of amino acids In Solitarious desert locusts had higher levels of DNA methyltransferase 2 (Dnmt2)-encoding transcripts. This agrees with our previous observation of reduced Dnmt2 transcript levels following crowding of solitarious hoppers Two pacifastin-like peptide precursors were differentially expressed. Pacifastins are a family of serine peptidase inhibitors found in arthropods 2(FC) = 5.11; FC: fold change). Also present in the S. gregaria EST database is a highly similar sequence  (2(FC) = 3.18). We could not determine the biological role of these transcripts based on sequence comparisons. It is probable that these transcripts do not code for proteins. Only very short ORFs (ranging in length from 3 to 7 codons) could be detected in the different reading frames and it is unclear whether these are actually protein coding or merely contain motifs consistent with a protein coding transcript. A large part of eukaryotic genomes is transcribed into non-coding RNA species (ncRNA), whilst the protein-encoding genes only make up a small portion of the genome. The biological role of only a few classes of ncRNA molecules have been determined, but many more remain to be identified and/or functionally characterized Of the 214 genes that were differentially expressed in the microarray study, 128 have not yet been annotated . Using Rtig4392) . The micS. gregaria and L. migratoria. The pattern of differential gene expression in S. gregaria shows several similarities with that of L. migratoria. For example expression of transcripts for CSP3 and HSPs is stronger in gregarious locusts of both species. By contrast transcripts for hexamerins (high in gregarious L. migratoria heads and solitarious hind legs), cytochrome P450 proteins and alcohol dehydrogenases showed opposite patterns of expression in the two species. S. gregaria and L. migratoria belong to different sub-families of the Acrididae and have presumably evolved density dependent polyphenism independently. It remains to be seen just how strongly the convergent phase change processes are underpinned by similar changes in gene expression.The current microarray study revealed 214 genes that are differentially expressed between solitarious and gregarious desert locusts. Independent support for our data is provided by several previously published studies in both The radically different lifestyles of solitarious and gregarious locusts mean that they face very different challenges. For example, solitarious locusts rely on crypsis to avoid predation, whereas gregarious locusts in a swarm have a greatly reduced individual predation risk. Life in a swarm has its own risks however: there is increased competition for food and a higher risk of disease, and there is a need to move continually to find new resources. The challenge of extracting meaningful sensory information from the constantly moving throng of other locusts in a swarm is also considerable. The risks inherent to the solitarious lifestyle arise from their very low population densities, where for example, finding a mate or suitable oviposition sites may be difficult and require prolonged searching. Our microarray analysis has identified genes whose altered expression may enable locusts of either phase to deal with the different challenges they face. For example, genes for proteins such as HSPs that confer protection from acute physiological challenges are upregulated in gregarious locusts, as are genes encoding proteins that confer protection from infection. By contrast solitarious locusts, which require a long adult life, are more strongly protected from the slowly accumulating effects of ageing by an upregulation of genes related to anti-oxidant systems, detoxification and anabolic renewal. Gregarious locusts had a greater abundance of transcripts for proteins involved in sensory processing. Solitarious locusts have larger eyes and antennae than gregarious locusts, but the latter experience a more complex sensory environment that may require a greater turnover of proteins involved in sensory transduction, and possibly greater neuronal plasticity.S. gregaria colony in Leuven originated from the Aquazoo in Düsseldorf , locusts originally from Nigeria. Gregarious locusts were reared under crowed conditions in acrylic plastic cages (40×43×85 cm) under a 13 hour light/11 hour dark photoperiod at 32°C. They were fed daily with fresh cabbage and oat flakes ad libitum. Solitarious locusts were reared individually for 30 successive generations in plastic boxes (15×8×7 cm), and received the same food as the crowd-reared animals. The solitarious breeding room had identical temperature and light conditions as the crowd-rearing room. For more detail on the solitarious culture, see Hoste et al. (2002) et al. (2010) The 2; 0.746 g KCl; 0.407 g MgCl2; 0.336 g NaHCO3; 30.807 g sucrose; 1.892 g trehalose; pH 7.2). The samples were collected in 2 ml tubes with 1.4 mm MagNa Lyser Green Beads (Roche), immediately frozen in liquid nitrogen and stored at −80°C. The samples were homogenized using a MagNA Lyser instrument (Roche)  and total RNA was extracted from the homogenates . A DNase treatment  was also performed to eliminate any genomic DNA contamination. The quantity and integrity of the RNA was measured spectrophotometrically on an Agilent 2100 Bioanalyzer (Agilent Technologies).Sampling was done separately for males and females. Each condition  was represented by three independent biological samples, which each contained pooled tissues from five adult locusts aged between 2 and 10 days after final eclosion. Head ganglia  and thoracic ganglia were dissected, cleaned and rinsed in Ringer solution , plus all L. migratoria EST sequences S. gregaria set (blastn hits producing E<1E-10 were considered orthologs). Probes were designed according to the following eArray criteria: (i) probe length: 60 nucleotides, (ii) two probes per target sequence (best probe methodology) and (iii) probe orientation: sense. Other parameters were set at default settings. In total 20,755 unique locust transcript sequences  were represented on the arrays. Each sequence was represented by two probes, allowing for the design of the arrays in 4x44K slide format (Agilent Technologies), which also includes control features. The platform information has been deposited in the NCBI Gene Expression Omnibus (GEO) database, http://www.ncbi.nlm.nih.gov/geo) and is accessible under the accession number GPL11172.The locust microarrays were designed by means of the eArray system (Agilent Technologies). They contained probes representing all available i.e. eight hybridizations per run were performed in a balanced and even design  The current microarray study aimed at identifying genes differentially expressed in the solitarious and gregarious desert locust CNS. In order to rule out sex-dependent biases in the identification of phase-specific transcriptional differences, our sampling and hybridization design were strictly balanced across the two sexes. To that purpose, the experiment consisted of two independent hybridization runs, one per sex. Each one of them involved six samples (three solitarious and three gregarious), and an n+2 A-optimal design was chosen (n = 6 samples per run), Samples were labeled using the Quick Amp Labeling Kit , which generates fluorescent cRNA starting from total RNA or poly(dA) RNA. Each sample was labeled with Cy5 (red) and with Cy3 (green). For each sample, 2 µg total RNA was reverse transcribed by using a poly(dT) primer coupled to an antisense T7 promoter. The reverse transcriptase also catalyzes synthesis of the second cDNA strand (which contains the sense T7 promoter sequence), providing the template for synthesis and linear amplification of cRNA strands. During the transcription step Cy3- or Cy5-labeled dCTPs were incorporated in newly synthesized cRNA strands. Prior to cDNA synthesis, Spike A Mix (Agilent) was added to each sample to be labeled with Cy3 and Spike B Mix to each sample to be labeled with Cy5. These Spike-In mixes test for linearity, sensitivity and accuracy of the microarray workflow. The labeled samples were purified . The cRNA yield and relative amount of incorporated labeled dCTPs were determined on a NanoDrop ND-1000 UV-VIS Spectrophotometer (Thermo Scientific).Hybridizations were performed using the Gene Expression Hybridization Kit (Agilent) following the manufacturer's instructions for 17 hours at 65°C. Next, the microarrays were washed twice in GE Wash Buffer 1 (Agilent) for 1 minute each and finally in GE Wash Buffer 2 at 37°C for 1 minute.limmahttp://www.ncbi.nlm.nih.gov/geo) and are accessible under the GEO series accession number GSE31237.Microarrays were scanned using a Genepix Personal 4100A confocal scanner (Axon Instruments) at a resolution of 5 µm and excitation wavelengths of 635 nm and 532 nm. The PMT voltages for each wavelength were adjusted to obtain an overall green/red ratio as close as possible to 1. Spot identification and quantification of the fluorescent signal was carried out using GenePix Pro 6.0 software (Axon Instruments). The hybridization designs for both sexes were considered identical repeats and only phase was treated as a factor. The data obtained from all 16 hybridizations were pooled and used in a single statistical analysis -adjusted, with a cut-off at FDR  = 10%.(DOC)Click here for additional data file.Table S2Non-annotated differentially expressed genes. ID refers to the EST or GenBank ID; log2FC is the log2-transformed fold-change in expression in gregarious over solitarious CNS; p-values are false discovery rate (FDR)-adjusted, with a cut-off at FDR  = 10%.(DOC)Click here for additional data file.


Atrial fibrillation (AF) is the most common heart rhythm disorder in the Western world and a common cause of hospitalization and death. Pharmacologic and non-pharmacologic therapies have met with limited success, in part due to an incomplete understanding of the underlying mechanisms for AF. AF is traditionally characterized by spatiotemporally disorganized electrical activation and, although initiating triggers for AF are described, it is unclear whether AF is sustained by spatially meandering continuous excitation (re-entrant waves), localized electrical sources within the atria, or some other mechanism. This has limited therapeutic options for this condition. Here we show that human AF is predominantly caused by a small number (1.8±0.9) of localized re-entrant waves or repetitive focal beats, that remain stable with limited spatial migration over prolonged periods of time. Radiofrequency ablation that selectively targeted the sites of these sources was able to immediately terminate fibrillation and eliminate the arrhythmia with high success. Our results show that human AF, despite apparent spatiotemporal disorganization, is often perpetuated by a few spatially-constrained and temporally conserved sources whose targeted ablation can eliminate this complex rhythm disorder. Heart rhythm disorders are defined by abnormal spatial or temporal patterns of electrical propagation. Fibrillation, the most prevalent human rhythm disorder, is characterized by extremely rapid, temporally irregular and spatially disorganized electrical activity with potentially disastrous consequences. In the ventricles, fibrillation causes an abrupt loss of cardiac output (sudden death), and is the leading cause of death world-wide Several mechanisms have been proposed for the maintenance and recurrence of AF, including electrical remodeling a priori as sustaining mechanisms for human AF In some animal models, reentrant spiral waves (rotors) may act as sources for fibrillation To define the predominant mechanisms underlying the perpetuation of AF in humans, we developed an approach to map human AF during minimally invasive procedures, combining high temporospatial resolution mapping of both atria  with patWe studied 80 subjects with a broad range of AF phenotypes including paroxysmal AF (defined as episodes that self-limit within 7 days), persistent AF (episodes that terminate only with cardioversion) or longstanding persistent AF (continuous AF for over 1 year) As a validation for our approach, we first computed activation patterns in patients during normal sinus rhythm using baskets in both the left and right atrium. This approach led to no adverse events in our experience. When applied to AF, our computational mapping technique revealed localized and stable sources in 96% of patients. This is in contrast to recent human studies in which localized sources for AF were rarely or never observed Repetitive focal beats in AF, defined in this study as activation radiating from a source region, were revealed in 29% of patients . AlthougStudy patients often demonstrated more than one coexisting electrical rotor or repetitive focal beat , for an 2 and 2.1±1.8 cm2, respectively. Notably, source locations were conserved between maps acquired 115±57 minutes apart throughout mapping  or >30% of the area of a 4.6 cm diameter atrium The spatially constrained and temporally conserved nature of a small number of rotors and focal beat sources in human AF presented an opportunity to establish their causal role via targeted elimination. We ablated the rotational center or origin of localized sources during ongoing AF by directly applying radiofrequency energy in 26 patients (of the population of n = 80), of whom 19 had persistent AF. Of 16 of 26 patients who exhibited 1 or 2 coexisting sources, ablation targeted to the limited migration loci of rotational centers or focal origins, with no other ablation, terminated AF in 3.9±3.8 minutes of total ablation to sinus rhythm n = 13,  and 4, oAblation of rotors or focal beats was successful independent of the type or location of sources. For example, targeted ablation at the rotational center of a left atrial rotor for less than 1 minute converted AF to sinus rhythm , while tTen of the 26 patients had 3 or more organized AF sources, although our protocol permitted ablation at only 2 regions. Localized ablation at 2 sites slowed AF  by 15±12% in these patients after 6.3±4.3 minutes of targeted ablation. These results show that ablation of all identified localized sources can terminate fibrillation, regardless of the location of the source and whether it is a rotor or a focal beat.After performing targeted ablation, we also performed standard-of-care ablation per our approved protocol  defines the practical resolution required for mapping.Clinically, while conventional ablation can isolate initiating mechanisms for AF in the pulmonary veins, other initiating mechanisms must remain to explain its suboptimal success Our results demonstrate that cardiac fibrillation in human atria, despite its substantial spatiotemporal variability, may be directly caused by very few stable electrical rotors or repetitive focal beats, that are spatially constrained and temporally conserved. Accordingly, limited and rapid ablation was able to terminate fibrillation for long-term elimination. These results were observed in patients with a wide range of AF phenotypes. The demonstration of localized perpetuating sources for human AF using this approach may enable the development of a number of targeted interventions in addition to ablation, including pacing, pharmacologic, gene or regenerative therapies.The study was approved by the joint Ethics committee of the University of California and Veterans Affairs Medical Centers, San Diego, and written informed consent was obtained from each subject. The authors' responsible joint institutional review board approved the study.We studied 80 consecutive patients with drug-resistant AF referred for ablation , all of Electrophysiology study was performed after discontinuing anti-arrhythmic medications for >5 half-lives (>60 days after amiodarone). Catheters were advanced to the heart from peripheral veins. After trans-septal puncture, 64-pole basket catheters  were advanced to the left atrium  and the right atrium (n = 54 patients) . CatheteElectrode locations were verified within atrial geometry by fluoroscopy and clinical mapping  for analysis.We computed spatial activity in AF using 2 approaches. First, we determined AF activation from potentials (electrograms) at each electrode to construct isochronal maps. Second, we performed phase analysis to fibrillation, as first described by Gray et al. followed by current standard-of-care ablation In phase II, targeted ablation was performed directly at AF rotor and focal beat locations to demonstrate their mechanistic role in perpetuating AF in each patient. This is quite distinct to prior mapping studies of AF that generally did not use such interventions to establish whether mapped features were causal or simply associative. Targeted ablation at rotors and focal beats was performed prior to any other intervention. Ablation was performed using an irrigated catheter  at 25–35 W or, in patients with heart failure, a non-irrigated catheter  at 40–50 W, target 52°C. In n = 2 patients with prior conventional ablation in whom targeted ablation terminated AF to sinus rhythm, the pulmonary veins were still isolated and no other ablation was performed. In all other patients, targeted ablation was To establish that identified AF sources caused the clinical AF phenotype, and not just AF seen acutely in the laboratory, we rigorously followed patients for recurrent AF after targeted elimination of sources. Anti-arrhythmic medications were discontinued after a 3 month blanking period t-test. Contingency tables were analyzed using the Fisher exact or the Chi-tests when appropriate. A p-value of <0.05 was considered statistically significant. Statistics were calculated using SPSS 19 .Continuous data are represented as mean ± standard deviation (SD). The t-test was used to compare variables between 2 groups. Paired continuous variables were compared using linear regression and the paired Table S1Patient Characteristics.(DOCX)Click here for additional data file.Table S2Characteristics of Human AF Sources.(DOCX)Click here for additional data file.Figure S1Phase map of Human Left Atrial Activation During AF. The phase map was computed using the Hilbert Transform (TIF)Click here for additional data file.Figure S2Directionality Analysis of a Rotor During Human Atrial Fibrillation.A. shows propagation emanating from the left atrial rotor to the remaining atrium, in the same patient shown in B. Recurrence of predominant direction, shown as the correlation at each site of the direction over consecutive cycles, showing high recurrence (repeatability) in the annulus adjacent to the rotational center (warm colors) with markedly reduced correlation in a surrounding annulus of tissue (cool colors) with some recovery of repeatability at distant sites.(TIF)Click here for additional data file.Figure S3Temporal Conservation of a Left atrial rotor in human AF for 237 days.A. Isochronal map of a left atrial rotor obtained prior to conventional ablation that passed outside this source, and did not target it. Atrial fibrillation failed to terminate during ablation, and recurred after the procedure. B. Isochronal map of a left atrial rotor at the same location obtained at repeat electrophysiology study 237 days later. Targeted ablation at this source eliminated AF.(TIF)Click here for additional data file.Figure S4Additional Examples of Brief Targeted Ablation at Stable Sources for Human Atrial Fibrillation.A. Isochronal map of a left atrial focal beat source that lay outside traditional ablation lesion locations. Localized ablation at this site  terminated AF directly to sinus rhythm in <5 minutes. B. Isochronal map of a counterclockwise left atrial rotor on the posterior left atrium near (but not within) the left pulmonary vein antra, where localized ablation (red dots in right panel) terminated AF to sinus rhythm within 3 minutes. Both patients are free of AF on implanted monitors.(TIF)Click here for additional data file.Figure S5Kaplan-Meier curves for freedom from AF, detected using rigorous monitoring including implanted continuous ECG recordings.(TIF)Click here for additional data file.


Sir,In their recent article, The novelty of the association of decreased Dicer expression with EMT and mesenchymal phenotype is questionable considering our previous published work. Using 21 breast cancer cell lines, two cellular models of human and mouse mammary cancer progression, and human cohort data, we had shown previously a significant downregulation of Dicer expression when cells exhibit mesenchymal phenotypes (Dicer-Like1 expression in Arabidopsis (As levels of the full-length protein were decreased with mesenchymal phenotypes, we wondered about the deregulated miRNAs targeting the Dicer 3′UTR during EMT, as it was already published that this region contains multiple different miRNA target sites. Dicer mRNA presents several let-7 target sequences, both in the coding region and in its 3′UTR , differebidopsis .In addition to regulation via its long 3′UTR, mammalian Dicer mRNA appears as multiple spliced isoforms exhibiting 5′ and 3′ end truncations. We previously described 14 putative variants in human Dicer mRNA . We alsoIn contrast to our results, in vivo where Dicer exists as multiple mRNA isoforms and can be targeted by multiple miRNAs? Is there a competition between the different miRNAs in binding their targets as some miRNA consensus sequences overlap? Is there a competition between miRNA binding and the HuR RNA binding protein also present in 3′UTR of Dicer?Many questions remain unaddressed with regard to Dicer mRNA regulation. What is the relevance of the Dicer and miR-103/107 expression relationship In summary, both our results (