Carbon black (CB) and carbon nanotube (CNT) co-doped polyimide (PI) modified glassy carbon electrode (CB-CNT/PI/GCE) was first prepared for the simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The CB-CNT/PI/GCE exhibited persistent electrochemical behavior and excellent catalytic activities. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used for the simultaneous detection of AA, DA, and UA in their ternary mixture. The peak separations between AA and DA, and DA and UA, are up to 166 mV and 148 mV, respectively. The CB-CNT/PI/GCE exhibited high sensitivity to DA and UA, with the detection limit of 1.9 µM and 3 µM, respectively. In addition, the CB-CNT/PI/GCE showed sufficient selectivity and long-term stability, and was applicable to detect AA, DA, and UA in human urine sample. Dopamine (DA) is an important catecholamine neurotransmitter for message transfer in the mammalian central nervous system . It play2 [On account of the comprehensive consideration of in vivo environment, it is necessary to develop a simultaneous, rapid, and accurate analytical method for the determination of the ternary mixture of AA, DA, and UA. Electrochemical techniques are the most used method for the quantitative determination of AA, DA, and UA with excellent accuracy. Numerous materials such as carbon nanotube ,12, grap2  metal an2 ,18,19, c2 ,21, boro2 , silica 2 , and naf2  have bee2 . Typical2 . The higIn this study, we suggest a method to fabricate a thin PI composite film on glassy carbon electrode (GCE). The PI was doped with the hybrid material of carbon black (CB) and carbon nanotube (CNT). By taking advantages of high electronic conductivity of CB and CNT, the CB-CNT/PI composite exhibits excellent responses to the simultaneous detection of AA, DA, and UA. Owing to the desirable structure and stability of PI, the present CB-CNT/PI/GCE exhibits good selectivity and long-term stability. N,N′-dimethylacetamide (DMAc), Pd/C (10%), hydrazine hydrate, acetone, nitric acid, ethanol, acetic anhydride, pyridine, K2HPO4, KH2PO4, uric acid (UA), ascorbic acid (AA) and dopamine (DA), urea, glucose, fructose, maltose, hydrogen peroxide (H2O2), potassium chloride (KCl), ironic chloride (FeCl3), ammonium chloride (NH4Cl), sodium sulphate (Na2SO4), sodium chloride (NaCl), and citric acid were purchased from Sinopharm Chemical Reagent Co., Ltd., Beijing, China. DMAc was dried using anhydrous molecular sieves before using. Carbon black (CB) was obtained from CABOT Corporation and used as received. Carbon nanotube (CNT) was obtained from Showa Denko Co. Ltd., Tokyo, Japan. Glassy carbon electrode  was obtained from Shanghai Chen Hua Instrument Co. Ltd., Shanghai, China. A 0.1 M phosphate buffer solution  was used to prepare electrolyte. Doubly distilled water was used throughout the experiments. All other chemicals were of analytical grade and were used without further purification.The reagent 4,4′-(Hexafluoroisopropylidene) diphthalic dianhydride (6FDA) was purchased from ChinaTech  Chemical Co. Ltd., which was recrystallized from acetic anhydride, and vacuum dried before using. The reagent 9,9′-bis(4-hydroxy phenyl) fluorene (BHF) was purchased from Sinosteel Anshan Research Institute of Thermo-energy Co. Ltd., Anshan, China. −1, the average scanning frequency of the instrument was 16 times. The synthesized monomer and polyimide were analyzed by proton nuclear magnetic resonance (1H-NMR) spectra on a Bruker Advance-AV 500 MHz instrument  in deuterated dimethyl sulfoxide. The morphologies of the modified electrode surfaces were observed using the field emission scanning electron microscope . Electrochemical measurements were performed with a CHI 750D workstation . A conventional three electrode system with PI modified GCE as working electrode, a 1.0 mm diameter of Pt wire as counter electrode, and Ag/AgCl (sat. KCl) as reference electrode was employed in this study. All measurements were performed in air at room temperature of approximately 20 °C.The X-ray diffraction (XRD) pattern was taken from 5° to 60°  with Cu-Kα radiation (λ = 1.541 Å) by an X’ Pert Powder X-ray diffractometer . Fourier transform infrared spectrometry (FT-IR) spectra were measured using a Nicolet IS 10  equipped in the range of 500–4000 cm1H-NMR spectrum of BNHOPF , 7.97 , 7.52 , 7.45 , 7.38 , 7.23 , 7.14–7.07 , 6.52 .The BHF (1 mol) was reacted with 5-fluoro-2-nitrophenol (2 mol) by nucleophilic aromatic substitution in the presence of potassium carbonate (2 mol) and DMAc under 12 h of heating. The mixture was poured into distilled water for cooling, the yellow solid compound was filtered and dried. . The detailed reaction procedure is shown in the first step of 1H-NMR spectrum of BAHOPF , 7.91 , 7.49–7.36 , 7.31 , 7.03 , 6.75 , 6.55 , 6.34 , 6.26 ).Thirty milliliters of ethanol, 0.01 mol (BNHOPF), and 0.25 g Pd/C were mixed in a three-necked flask. Twenty milliliters of hydrazine hydrate were dropped into the three-necked flask with a constant pressure funnel within 1 h. The reaction was fulfilled after heating for 12 h. The white crystals were obtained after recrystallization . The reaction procedure is shown in the second step of 2 atmosphere to obtain a viscous poly (amic acid) solution. Finally, the solution was poured into methanol to obtain a white precipitate, which was collected by filtration and vacuum dried at 150 °C for 12 h.The preparation process of polyimide is shown in PI (30 mg) was diluted in DMAc solvent (0.57 g) and sonicated for 30 min to make a homogeneous solution. The CB solution was prepared by putting 15 mg of CB into 0.5 mL DMAc and sonicated for 30 min. The CNT solution was prepared by putting 15 mg of CNT into 0.5 mL DMAc and sonicated for 30 min. Then the CB solution was mixed with CNT solution (v:v = 1:1) and sonicated for 30 min. The PI solution was mixed with the CB-CNT mixture and sonicated for 30 min which donated as CB-CNT/PI solution. The GCE surface was polished successively with 1.0, 0.3, 0.05 µm α-alumina slurries to make a shiny surface. The cleaned GCE was rinsed and sonicated with distilled water and ethanol to remove any adhering alumina. Then the CB-CNT/PI solution (10 µL) was dropped onto the GCE surface for 12–24 h to prepare the sensor. The fabrication of CB-CNT/PI/GCE and the detection of the substances with the electrochemical detection process is shown in −1 and 1330 cm−1) and the appearance of characteristic absorption peaks of amine groups (3382 cm−1 and 3318 cm−1) are good evidence for the successful reduction reaction.The structures of BNHOPF and BAHOPF were confirmed by FT-IR. −1, 1714 cm−1, 1383 cm−1, and 722 cm−1 correspond to asymmetric and symmetric imide ring stretching vibrations of C=O, C–N vibration, and C=O bending vibration, respectively. The broad peak around 3400 cm−1 corresponds to the asymmetric stretching of O–H bond. [FT-IR spectra of PI was investigated . The pea–H bond.  FurthermThe SEM technique was applied to study the morphology of the CB-CNT/PI/GCE . The PI-The primary purpose of the present study is simultaneous detection of AA, DA, and UA. Usually, the voltammetric peaks for the oxidation of AA, DA, and UA are overlapped and hard to distinguish. To check the usefulness of the doping of CB and CNT within the PI, we compared the cyclic voltammetry (CV) of ternary mixture of AA, DA, and UA by using various electrodes. In general, differential pulse voltammetry (DPV) has been widely utilized for the simultaneous detection of AA, DA, and UA, due to high sensitivity and selectivity ,29,30. FDA: 3–300 µM), the linear regression equation is IDA = 51.84CDA − 0.2474, with a correlation coefficient of R2 = 0.9993. In a high concentration range of DA (CDA: 300–2000 µM), the linear regression yields a relationship of IDA = 16.14CDA + 0.3667, with a correlation coefficient of R2 = 0.9987. Similar results were observed for UA  and IUA= 1.9586CUA + 2.1075 . The observed two slopes could be explained by monolayer adsorption followed by multilayer adsorption [Next, we tried individual determination of each substance in the ternary mixture with DPV. d for UA C,F, and d for UA F. The lisorption , or exissorption . As showsorption ,33,34,35AA= 0.9504CAA − 0.0146, with a correlation coefficient of R2 = 0.998 (n = 3). The detection limit of AA was found to be 75 μM. DA = 0.9709CDA + 0.0244, a correlation coefficient of R2 = 0.9893 (n = 3). The detection limit of DA was found to be 60 μM. Similarly, UA = 3.2261CUA + 0.2878, a correlation coefficient of R2 = 0.9963 (n = 3). The detection limit of UA was found to be 8.8 μM. The insets in The steady-state amperometric responses of the CB-CNT/PI/GCE to AA, DA, and UA are presented in 3, NH4Cl, NaCl, KCl, glucose, fructose, maltose, citric acid, urea, H2O2, and Na2SO4 were added into the mixture of 0.3 mM AA, 0.3 mM DA, and 0.3 mM UA, no obvious interference can be observed  of FeClobserved , indicatThe practical application for determination of AA, UA, and DA in human urine samples was carried out to check the performance of the present sensor. A fresh urine sample was prepared by 50-fold dilution of urine with PBS (pH 7). Amperometry technique was used in an electrochemical cell containing 400 μL of urine sample and 20 mL of PBS. Different concentration of AA, DA, and UA was added into the sample. The CB and CNT-co-doped PI was modified on the glassy carbon electrode, and as-prepared electrode (CB-CNT/PI/GCE) was successfully utilized for the simultaneous detection of AA, UA, and DA. The CB-CNT/PI/GCE exhibited high electrocatalytic activities towards the oxidation of AA, DA, and UA, and sufficient peak separation between AA, DA, and UA was observed with CV and DPV. The CB-CNT/PI/GCE showed sufficient selectivity and stability, and was applicable to the analysis in a human urine sample. The large capacity of PI and high surface area of CB and CNT make it a promising candidate for electrocatalytic applications and biomedical applications.


Despite employing diverse molecular mechanisms, many different cell signaling systems avoid losing information by transmitting it in a linear manner. Related research article Nunns H, Goentoro L. 2018. Signaling pathways as linear transmitters. eLife7:e33617. doi: 10.7554/eLife.33617Much as explorers might use compasses and altimeters to establish where they are and radios to communicate with one another, the cells building a multicellular organism sense chemical gradients to determine their position and send and receive chemical signals to communicate. This sensing and communication works best if signals get transmitted into the cell’s interior without any loss of information . In the Now, of course, we know that signaling is far more complex, with different pathways operating via different molecular components and biochemical mechanisms. Modeling and experimentation have shown how such complex signaling mechanisms can carry out sophisticated signal processing. For example, protein kinase cascades can convert graded signals to ultrasensitive ones , feedbacThis enthusiasm to discover how signaling pathways performed signal processing had the effect of helping to obscure decades of pre-existing work which showed that many signaling systems act as linear transmitters e.g. . HoweverThese pathways have disparate topologies and molecular mechanisms. Nunns and Goentoro found that established mathematical models of the three pathways can be simplified to demonstrate a linear relationship between the input and output. They also performed experiments to confirm this linearity for the Wnt and EGF-ERK pathways. Finally, they showed experimentally that perturbations predicted to break linearity  did exactly that.The idea that ligand-bound receptors directly produced the cellular response endured for decades. However, once the complexity of key signaling pathways was realized, it became apparent that maintaining linearity in multistep pathways constituted a significant biochemical problem. Even simple binding reactions have nonlinear responses, and cascades of them result in increasingly sensitive responses . So far,The first mechanism corrects an ultrasensitive response with negative feedback . This coThe second mechanism involves the modulation of an unsaturated or unsaturable cycle . SignaliThe third mechanism is a push-pull mechanism in which the active form of a signaling species stimulates downstream activity, while the nominally inactive form reduces downstream activity . ModelinThis work prompts two immediate thoughts. The first is to wonder why so many quantitative biologists neglected to study linear input-output signaling for so long. By the 1970s, pharmacologists routinely regarded the equivalence of percent ligand occupancy and percent of maximum downstream response as a criterion that a given molecule was likely to be the receptor for that ligand . By the The second thought is to admire the established models of the three pathways studied by Nunns and Goentoro. These models were developed by different research groups, none of which set out to test linear signal transmission. The fact that the models nevertheless exhibit linearity, a behavior they were not built to reproduce, suggests that they represent the key molecules and interactions correctly.This work also suggests topics for future research. The finding that these very different signaling pathways produce signaling without distortion is consistent with the idea that the consequences of such mechanisms were strongly selected for before the emergence of multicellular life. It would be interesting to build engineered systems that enable the careful study of the selective advantages that linear signaling mechanisms confer. Such systems might build on work which showed the linearity of downstream responses controlled by engineered autoregulating repressors  that rep


The role of Hedgehog (Hh) signaling in vascular biology has first been highlighted in embryos by Pepicelli et al. in 1998 and Rowitch et al. in 1999. Since then, the proangiogenic role of the Hh ligands has been confirmed in adults, especially under pathologic conditions. More recently, the Hh signaling has been proposed to improve vascular integrity especially at the blood–brain barrier (BBB). However, molecular and cellular mechanisms underlying the role of the Hh signaling in vascular biology remain poorly understood and conflicting results have been reported. As a matter of fact, in several settings, it is currently not clear whether Hh ligands promote vessel integrity and quiescence or destabilize vessels to promote angiogenesis. The present review relates the current knowledge regarding the role of the Hh signaling in vasculature development, maturation and maintenance, discusses the underlying proposed mechanisms and highlights controversial data which may serve as a guideline for future research. Most importantly, fully understanding such mechanisms is critical for the development of safe and efficient therapies to target the Hh signaling in both cancer and cardiovascular/cerebrovascular diseases. The Hedgehog (Hh) family of morphogens, which includes Sonic Hedgehog (Shh), Indian hedgehog (Ihh), and Desert Hedgehog (Dhh), was identified nearly four decades ago in Drosophila as critical regulators of cell fate determination during embryogenesis .Shh is the most widely expressed and studied. It has been implicated in the development of multiple organs including the central nervous system (CNS), lungs, foregut, heart, and limbs  by partiThe role of the Hh signaling in vascular biology has first been highlighted in embryos by Pepicelli et al. in 1998 . Indeed,Shh is synthetized as a preprotein of which the signal sequence is first cleaved to produce a full-length unmodified form. An autocatalytic reaction removes then the carboxy-terminal domain and attaches a cholesterol moiety to the newly exposed carboxy-terminus. Shh is further modified by Hedgehog acyltransferase (Hhat), which catalyzes the addition of a palmitate to the amino-terminus  Figure . Ihh andSecretion and solubility of cholesterol-modified Hh ligands depend on the transmembrane protein Disp1 (dispatched RND transporter family member 1) and the cell surface protein Scube2   25]. Bo. Bo25]. The interaction of the Hh proteins with their specific receptor Patched-1 (Ptch1) de-represses the transmembrane protein Smoothened (Smo), which activates downstream pathways, including the Hh canonical pathway leading to the activation of Gli transcription factors and so-called Hh noncanonical pathways, which are independent of Smo and/or Gli  26]..26].Activation of the Hh canonical pathway promotes cell survival and proliferation through the regulation of Bcl2, N-myc and CyclinD1 while the noncanonical signaling has been involved in cytoskeleton changes and cell migration . Hh-induHh binding to Ptch1 is regulated by several coreceptors. Among these, Cell adhesion molecule-related/downregulated by oncogenes (Cdon), Brother of Cdon (Boc) and Growth arrest specific 1 (Gas1) are suggested to promote Hh ligand interaction with Ptch1 while Hedgehog interacting protein (Hhip) inhibits it  Figure A.Dzip1/Iguana recapitulates most features of Shh deficiency [Beside, Hh signaling activity has been shown to depend on the primary cilium. Indeed, several Hh signaling pathways elements including Smo, Sufu, Kif7, Gli2, and Gli3 have been located at the primary cilium and disruption of genes encoding for cilia proteins such as intraflagellar transport proteins (Ift), talpid3, and ficiency .KO embryos fail to form yolk sac blood vessels [KO embryos do not form any blood vessels, IhhKO embryos do form ones which fail to undergo vascular remodeling, i.e., ramification into large and small branches and pericyte coverage [Hh signaling has been shown to orchestrate angiogenesis in the yolk sac as Smo vessels . Ihh, se vessels . Neverthcoverage ,31. Thiscoverage ,32, whilAt the cellular level, it is still not clear which cell types respond to Ihh signals: in vitro, endothelial cells (EC) themselves are suggested to respond to Ihh since C166 cells, a mouse yolk sac EC line, respond to Shh recombinant protein by overexpressing Gli1 and Ptch1 and migrating more. In these cells, Shh also increases expression of neuropilin 1 (Nrp1), Kruppel like factor 4 (Klf4), jagged canonical Notch ligand 1 (Jag1), and collagen type IV alpha 1 chain (Col4a1), major factors implicated in EC biology .KO and SmoKO mouse embryos especially in the distal part of the lungs [KO embryos, but appropriate to the decrease in airway branching. In the same study, Vegfa expression is reportedly not different from that of control lung [KO embryos likely because of possible compensatory effects from the other Hh ligands. On the contrary, vascular stabilization is defective because of angiopoietin 1 (Angpt1) downregulation [Shh-deficient mouse lungs have first been reported as poorly vascularized by Pepicelli et al. in 1998 [he lungs ,35. Notarol lung  and earlgulation .ECKO lungs [At the cellular level, the lung capillary network development does not depend on a direct effect of Shh on EC since it is normal in SmoKO lungs . In contKO lungs   has defects in the ciliary basal body protein Dzip1, causing improper cilia formation and disruption of Hh signaling [Artery and vein identity was reported to be established normally in ignaling . Hh signaling is involved in the vascular development of other organs notably the retina, brain, skeleton, and heart.The retina: Hh signaling is implicated in retinal angiogenesis, as cyclopamine inhibits retinal angiogenesis , and is The brain: Gli2 deficient embryos have been reported to lack the entire brain basilar artery  and Shh,The skeleton: Ihh promotes skeletal blood vessel 3D organization and stability  and Shh The heart: Shh is necessary for FGF9-induced Vegfa, Vegfb, Vegfc, and Angpt2 expression in cardiomyocytes and perivascular cells  and the Somite: In avian embryos, inhibition of Smo and Shh compromise intersomitic vessel growth , while aFinally, ectopic Shh overexpression in the dorsal neural tube was shown to induce spinal cord hypervascularization , while eEven though Hh signaling appears to promote vascular development in the yolk sac, lungs, bones, heart, retina, and ovaries, organ specificity exists and contradicting observations have been made: for example, superficial vessel development in the zebrafish eye is increased due to excessive vessel sprouting in Smo deficient fish , while, In addition to its role in embryo vascularization, the Hh signaling has also been identified as regulating postnatal angiogenesis, especially in the setting of ischemia  and cancThe Hh signaling has been shown to be reactivated in ischemic conditions especially in the hindlimb muscle. Shh ,62, Gli1Reactivation of Hh signaling is suggested to be impaired in the setting of aging since Gli1 expression in the ischemic muscle is diminished in aged mice ,64. Dhh It still remains unclear which Hh ligand(s) regulate ischemia-induced angiogenesis. Indeed, angiogenesis has been shown to be transiently accelerated in Shh iKO mice  suggestiECKO mice [ECKO does not alter ischemia-induced angiogenesis, while angiogenesis is impaired in mice in which Gli3 expression is disrupted in myoblasts [The Hh signaling to ECs does not participates in ischemia-induced angiogenesis since angiogenesis occurs normally in SmoCKO mice ,70. On tCKO mice ,63. Hh lyoblasts . Moreoveyoblasts ,72. Shh,yoblasts , suggestyoblasts .The proangiogenic properties of the Hh ligands have been confirmed in other ischemic tissues including the heart ,74, braiOnce again, the role of the endogenous Hh signaling in the heart is not clear, while Lavine KJ et al. reported that Hh-blocking antibodies administration decreases capillary density and cardiomyocyte survival in the setting of myocardial infarction , it has Hyperactivation of the Hh signaling observed in tumors is suggested to promote tumor angiogenesis. In detail, the Hh signaling blockade with Smo antagonists, including GDC-0449 and Cyclopamine, reduces the vascular density of Hh-producing colon cancer xenografts  and oralHyperactivation of Hh signaling in cancer cells themselves  has been shown to increase proangiogenic factor expression including vegfa ,89,90, mApart from being expressed by cancer cells, Hh ligands are also expressed by ECs (Shh and Ihh) ,96,97, eWhile most studies agree in reporting that hyperactivated Hh signaling in tumors may promote tumor angiogenesis, a few studies have shown opposite results: first, Smo inhibition using IPI-926 has been reported to increase tumor vessel density in pancreatic ductal adenocarcinoma . Shh-defHh signaling has been involved in atherosclerosis plaque angiogenesis. Insulin resistance adipocyte-derived exosomes (IRADEs) carry Shh, which promotes plaque vulnerability partially by inducing vasavasorum angiogenesis. This is associated with increased Gli1 and Vegfa expression levels . In addiKO embryos and cyclopamine-administered adult mice display brain vascular leakage [ECKO mice  demonstrating that ECs are the cells mediating Hh regulation of BBB integrity. Activation of the Hh signaling in ECs decreases BBB permeability and increases trans-endothelial electrical resistance of brain ECs by promoting expression of both tight , Occludin, F11 receptor (Jam-A), (tight junction protein 1) ZO-1) [The critical role of Hh signaling in maintaining BBB integrity has first been highlighted in 2011 by Prat’s laboratory . This st leakage . Interes1) ZO-1) ,107, and1) ZO-1)  junction1) ZO-1) ,108, and1) ZO-1)  expressi1) ZO-1)   integrity since Smoammation . Schwannammation ,121. As ammation ,122.Moreover, the increased BNB permeability associated with diabetic neuropathy has been shown to result from Dhh downregulation . In the ECKO mice display spontaneous vascular leakage and exacerbated LPS-induced neutrophil recruitment in the lung. Notably, Dhh is a downstream target of Klf2 which promotes interaction of the Cdh5 with its partner the β-catenin and decreases Icam-1 and vascular cell adhesion molecule 1 (Vcam-1) expression; nevertheless Dhh effects are more likely independent on Gli transcription factors since neither Gli1 nor Gli2 expression is modulated in Dhh KO ECs [We recently reported that blood vessel integrity depends on Hh signaling not only within the central and peripheral nervous system, but also in other organs including the heart and the lung. Endothelial adherens junction integrity and immune quiescence depends on Dhh expression by ECs themselves. Indeed, Dhhh KO ECs . Interesh KO ECs ,124.Hh regulation of blood vessel survival, especially in the heart, is also proposed to depend on the Hh signaling activity in cardiomyocytes and on the resulting overexpression of Vegfa, Vegfb, Vegfc, Angpt1, and Angpt2 .Finally, in the retina of Akika diabetic mice, the deceased expression of Shh has been associated with decreased Angpt1 expression, increased capillary permeability, and pericyte loss .Apart from regulating endothelial intercellular junctions and endothelial immune quiescence, Hh ligands may also regulate vasoactive properties of ECs since administration of microparticles carrying Shh have been shown to promote both nitric oxide synthase 3 (NOS3) expression and NOS3 phosphorylation ,126. MorECKO and DhhECKO mice are reported to display abnormal vessel permeability and several studies report expression of Hh ligands by ECs themselves: for instance, Shh is expressed by dental pulp and liver sinusoidal ECs [While Hh regulation of angiogenesis is mediated via Hh-induced upregulation of Vegfa in fibroblast-like cells, Hh regulation of blood vessel integrity seems to be cell-autonomous. Indeed, both Smoidal ECs ,129 and idal ECs . Finallyidal ECs ,96,97.Nevertheless, signaling pathways mediating Hh-regulation of intercellular junctions or VCAM-1 expression are unknown. For instance, the Hh canonical signaling involvement (i.e. Gli dependant transcription) remains controversial while several study have reported Gli1 overexpression in ECs upon Shh treatment ,33,108, hi409 mutantift81 [The primary cilium is suggested to be necessary for BBB integrity since zebrafishes deficient for cilia biogenesis have increased risk of developmental intracranial hemorrhage which can be rescued either by activation of the Hh pathway (PKA dominant negative or Sufu KD) or by the endothelial-specific re-expression of intraflagellar transport genes . However9 mutant .Finally, overexpression of Gli3, a negative regulator of Hh canonical signaling, in cultured ECs promotes Cxcl1, Cxcl2, Cxcl8, and TYMP expression. However, it is suggested to act independently from the Hh canonical pathway .Shh was suggested to promote muscularization and maturation of blood vessels in 2004, in a study showing that Shh gene therapy promotes the formation of enlarged and more muscularized vessels in diabetic nerves . Such obfo10aigu mutants is not due to an EC structural defect but to a pericyte defect [Interestingly, both igu mutant and cyclopamine-treated zebrafish embryos display decreased mesenchymal Angpt1 expression and hemorrhages located in multiple tissues throughout the body including the somites and pharyngeal area and the head. Tight junctions between ECs display normal morphology and size in mutant embryos. On the contrary, perivascular mural cells fail to make contact with ECs. These results suggest that hemorrhage in e defect . Neverthe defect .Muscularization of new blood vessels is more likely the consequence of a direct response of mural cells (SMCs or pericytes) to Hh signaling stimulation, since both SMCs and pericytes express Shh, Ptch1, Smo and Gli1 ,138,139.Differentiation of Sca1+ progenitor cells into SMCs has been shown to be mediated through Ptch1 and Gli2 . NotablyFinally, Shh increases the expression of factors promoting vessels stabilization such as Angpt1, platelet derived growth factor, BB dimer (Pdgf-BB), and transforming growth factor beta 1 (Tgfβ) ,153.SIX homeobox 1 (Six) or EYA transcriptional coactivator and phosphatase 1 (Eya1) deficient embryos display an increased Shh expression especially because it fails to decrease after E16.5; these mutant embryos have severe vascular defects in the SMC compartment leading to the major vessel rupture and to hemorrhage. This is prevented when Shh expression is decreased  ,155. MorBesides participating in blood vessel maturation, Hh signaling, especially Shh and Scube2, has been shown to be overexpressed in injured arteries especially during intimal formation after carotid artery ligation ,156,157 Altogether, these data highlight the wide actions of the Hh signaling in vascular biology. First, the Hh signaling is essential for angioblast assembly into vascular tubes, i.e., vasculogenesis. This has been observed in the yolk sac  and duriWhile cellular and molecular mechanisms underlying the vascular effects of the Hh signaling remain elusive, some consensuses appear. Both Hh-driven vasculogenesis and angiogenesis more likely involve activation of mesenchymal-type cells. Vasculogenesis depends on the Hh-induced BMP4 overexpression in these third cells while Hh-induced angiogenesis is mediated by Vegfa upregulation. On the contrary Hh-induced endothelial barrier tightness seems to depend on the Hh signaling activation in ECs themselves.However some mechanisms remain to be elucidated. Indeed, differentiation of angioblast is also suggested to depend on EC-activation by Hh ligands directly  and Hh-iMoreover, while Vegfa appears to be the main effector of Hh-induced angiogenesis, investigation of the regulation of Vegfa expression by the Hh signaling has been poorly investigated and limited to cancer cells. In breast cancer cells, Vegfa mRNA expression is shown to be specifically activated by a truncated form of Gli1 (splicing variant), while full-length Gli1 seems unable to bind Vegfa promoter and to activate its transcription . WhetherNevertheless, even if most studies agree in reporting that Hh signaling is proangiogenic, several conflicting data exist. Indeed, the endogenous Hh signaling has first been shown to promote ischemia-induced angiogenesis and systemic administration of Hh-blocking antibodies to decrease Vegfa mRNA levels and ischemic limb reperfusion . HoweverTherefore, further investigations are required to fully understand the role of the endogenous Hh signaling in the ischemic hindlimb. For instance, it would be interesting to compare the Hh signaling activity in mice administered with Hh-blocking antibodies and in mice deficient for Shh using reporter mice so that we could quantify the Hh signaling activity and Vegfa expression in different cell types. Indeed, while Pola et al. study involves the activation of the Hh signaling in fibroblast, studies conducted by Renault et al. investigated the role of the Hh signaling in myocytes ,69. It iOverall, the main reasons why mechanisms underlying the Hh action on the vasculature remain poorly understood are (1) lack of reliable, specific, and sensitive antibodies that could be used to detect expression of the Hh ligands and receptors so that we can identify Hh producing cells and Hh responding cells properly and (2) the limited amount of studies that have used tissue specific conditional KO mice. Moreover, the most widely used anti-Shh antibodies do also recognize Ihh and Dhh; this is the case of the rabbit anti-Shh antibodies , goat anti-Shh antibodies , and mouse anti-Shh antibodies ). In fact, vascular effects induced by Hh ligands have been mainly attributed to Shh, while it may not be the case. For instance, Shh has been suggested to be the one promoting BBB tightness both in healthy and pathologic conditions , while aThe proangiogenic potential of the Hh signaling agonists is of growing interest in the treatment of ischemic diseases especially myocardial infarction and peripheral artery diseases as pointed out in several reviews ,20,21,22Moreover, in the light of more recent studies, Hh signaling agonists may be beneficial in the setting of diseases in which endothelium integrity is compromised notably cardiovascular diseases like diabetic microangiopathies and cerebrovascular disorders, such as multiple sclerosis and HIV-induced encephalopathy.


Few years after HTLV-1 identification and isolation in humans, STLV-1, its simian counterpart, was discovered. It then became clear that STLV-1 is present almost in all simian species. Subsequent molecular epidemiology studies demonstrated that, apart from HTLV-1 subtype A, all human subtypes have a simian homolog. As HTLV-1, STLV-1 is the etiological agent of ATL, while no case of TSP/HAM has been described. Given its similarities with HTLV-1, STLV-1 represents a unique tool used for performing clinical studies, vaccine studies as well as basic science. The first human oncogenic retrovirus was discovered in the USA, in a T cell line obtained from blood cells of a patient suffering from a disease then called “cutaneous T-cell lymphoma” , 2. Few Few years later, Tropical Spastic Paraparesis/HTLV-1 associated myelopathy (TSP/HAM), a severe neuromyelopathy, was also identified as another disease caused by HTLV-1 . Thus, AMacaca fuscata) were co-cultured with chronically and productively infected T-cells from the MT-2 cells, an HTLV-1-transformed cell line. This allowed the authors to obtain a simian cell line persistently infected by HTLV-1, thus suggesting that Japanese monkeys might be susceptible to HTLV-1 natural infection [In 1982, lymphocytes from a Japanese monkey . They are divided in two subfamilies, ntinents .Papio anubis, Papio hamadryas, Papio papio and Papio cynocephalus) originating from South Africa and Ethiopia [To determine which simian species carry STLV-1, seroepidemiological studies were performed using kits that had been previously developed for the detection of anti-HTLV-1 human antibodies, as well as by PCR  had the same genomic organization i.e. LTR-gag-pol-env-pX-LTR [In 1984, Watanabe et al.  demonstrv-pX-LTR , 20. Seqv-pX-LTR .Mandrillus sphinx, Cercopithecus cephus, C. agilis, C. pogonias, G. agilis and C. nictitans share close relationships with HTLV-1D and -F from Cameroon and Gabon [Env region clusters with STLV-1 isolated from two baboon species, Papio ursinus and Papio cynocephalus [Phylogenetic studies revealed that HTLV-1 subtype B is very closely related to STLV-1 strains infecting chimpanzees (98% identity), Allen’s swamp monkeys (around 96% identity) and gorillas from Zaïre, Central African Republic and Cameroon , 53–55. nd Gabon , 56–58. cephalus . No dataMacaca tonkeana (living in Indonesia) and Macaca arctoides  [Macaca tonkeana virus is related to the most divergent HTLV-1 subtype C that is present in Melanesia and Australia. Molecular clock data inferred STLV-1 introduction around 156,000 to 269,000 years ago on the Asian continent [Most divergent STLV-1 strains were described in Asian d China) –62. Macaontinent . These rChlorocebus pygerythrus sample. Therefore, it should now be possible to use this technique to determine STLV-1 virus evolution over time using available Egyptian or Asian NHP mummies.Finally, Calvignac et al.  demonstrPrevalence of HTLV-1 may reach 1 to 40% in adults depending on age, sex and geographic location . It is wAs it is the case for HTLV-1-infected individuals, most STLV-1-infected monkeys remain lifelong asymptomatic hosts . For somPapio anubis, Gorillas and AGM [Papio anubis suffering from ATL [On the contrary, a number of ATLL-like diseases sharing clinical and pathological features with human ATLL were reported in NHPs , 73–79.  and AGM , 81, 82.from ATL . In thatMandrillus sphinx and Papio anubis) have been used for performing molecular studies [Given the high degree of sequence similarities between STLV-1 and HTLV-1 genomes and the fact that both viruses cause ATL, STLV-1 infected NHPs  expression. Simian SBZ and Tax amino-acid sequences are highly similar to human HBZ and Tax , thanks to a Tax effect on STAT5a. This might explain why Tax is more potent in these cells, and why HTLV-1 induces ATL in the periphery .Depending upon STLV-1 strain, SBZ protein sequence is highly similar or contain insertions and deletion compared to HBZ  or an HBZ LL/AA mutant  was linked to a temporary decrease of STLV-1 PVL .pol mutant viruses still able to infect animals remains unexplained.A limited number of reports described STLV-1 inter-simian species transmission , 93, 94 Saimiri sciureus, i.e. non-human primates that are not naturally infected with STLV-1 [Saimiri sciureus, the virus also replicates through clonal expansion after having used reverse transcription (RT) at the initial stages [Finally, given the high degree of similarity between HTLV-1 and STLV-1 genomes and the abundance of molecular tools available in the HTLV-1 field, some laboratories decided to use the HTLV-1 molecular clone or HTLV-1 infected cells to perform studies in non-human primates . TInterestingly, the authors also demonstrated that the results obtained in rhesus macaques were different from those obtained in rabbits infected with the same viral clones, thus reinforcing the fact that NHPs are the more relevant system for studying HTLV-1 pathogenesis.In addition to STLV-1, other retroviruses, i.e. Simian Immunodeficiency Virus (SIV) and Simian Foamy Virus (SFV) infect NHPs. Cases of natural coinfection have been reported both in humans and in NHPs: HTLV-1/HIV-1, HTLV-1/HFV, STLV-1/SFV or STLV-1/SIV-1 , 107–115Natural STLV-1/SIV-1 co-infection induces the development of a neoplastic disease in sooty mangabey  and of aPapio anubis, was recently shown to be much higher compared to SFV mono-infected animals [Finally, blood SFV proviral load from STLV-1/SFV naturally co-infected  animals . These rAltogether, these data demonstrate that STLV-1 is a useful tool to understand mechanisms of HTLV-1 transmission and ATL pathogenesis. PTLV-1 mono-infected as well as SIV co-infected animals could also be used to develop possible new anti-HTLV-1 clinical approaches and to modify anti-HIV treatment.


Direct reactions with NO are likely to produce new signaling molecules such as peroxynitrite and nitrosothiols, and it is probable that chemical competitions will exist which will determine the ultimate end result of signaling responses. How NO is generated in plants cells and how NO fits into this complex cellular environment needs to be understood.Nitric oxide (NO) is an immensely important signaling molecule in animals and plants. It is involved in plant reproduction, development, key physiological responses such as stomatal closure, and cell death. One of the controversies of NO metabolism in plants is the identification of enzymatic sources. Although there is little doubt that nitrate reductase (NR) is involved, the identification of a nitric oxide synthase (NOS)-like enzyme remains elusive, and it is becoming increasingly clear that such a protein does not exist in higher plants, even though homologues have been found in algae. Downstream from its production, NO can have several potential actions, but none of these will be in isolation from other reactive signaling molecules which have similar chemistry to NO. Therefore, NO metabolism will take place in an environment containing reactive oxygen species (ROS), hydrogen sulfide (H Since nitric oxide (NO) was mooted to be an important signaling molecule in animals in 1987 ,3,4, the2·-) and hydrogen peroxide (H2O2), along with hydrogen sulfide (H2S), works as part of a suite of relatively reactive small molecules in cells which help to control the cell’s activity and the function of proteins. NO has been implicated in seed germination  ID: EAZ02807.1. This had the following match:Using the NOS signature from the rat nNOS sequence (NM_052799 XM_346438) in Blastp at NCBI had the highest score hit of hypothetical protein OsI_24933 [        Query 1   RCVGRIQW 8        RC G IQW        Sbjct 224  RCTGKIQW 231      Arabidopsis thaliana hypothetical protein  revealed nothing in plants of significance, but it did pull out NOS-like sequences from a range of other organisms, including Staphylococcus and insects. Therefore, there is little evidence of this short NOS signature sequence being in either Arabidopsis or Oryza databases, at least to date.NOS is likely to interact with other peptides, and this would be a way to identify important functional regions. The calmodulin-interacting regions (CaM) from the rat sequence revealed nothing of note. When the three human NOS sequences were aligned, these CaM motifs were not represented in all NOS peptides, and therefore it could be argued that they are not essential, and not finding them does not rule out the presence of a plant NOS. Others have looked for other interacting regions as well ,38; for It can be concluded so far, in that case, that there is no significant evidence from the sequence searching of a NOS-like protein in two plant sequences for which major genome sequencing projects have been undertaken ,44.Homo sapiens NOS interacting protein isoform (NP_057037) was revealed in both the Arabidopsis and Oryza data proteins, which have already been annotated as NOS-interacting proteins . Such proteins may be used as lures to find interacting partners in plant extracts, some of which may have NOS-like activity.NOS in other species is not a stand-alone protein, but has interacting partners. NOS-interacting proteins can be found in the literature , such asOverall, the bioinformatic searching carried out here, although by no means exhaustive, showed no clear evidence of a NOS-like protein in plants, although elements such as a reductase do clearly exist. These data are not contrary to those found and reported by others .·. However, with the loss or gain of an electron, other forms are nitroxyl (NO−) and nitrosonium (NO+) ions [2S. If cadmium ion stress in plants is taken as an example, the cellular response includes the generation of NO and ROS [2S [When the chemistry of nitric oxide is discussed, it is often assumed that this involves the radical form: NOO+) ions . It is i and ROS , as well ROS [2S , all preS-nitrosation (often referred to as S-nitrosylation) is the modification of the –SH group to –SNO [S-persulfidation by H2S, glutathionylation by GSH, or reaction with another thiol to create a disulfide , and soS-nitrosation, the enzyme translocates to the nucleus, thus abandoning its role in glycolysis to take up a new role in the control of gene expression [S-nitrosated, and cytosolic GAPDH can interact with nuclear DNA, specifically to a partial gene sequence of NADP-dependent malate dehydrogenase [2O2 [S-persulfidated by H2S [The protein modification by NO is, however, an important signaling process. Many proteins in plants have been identified as being nitrosated , with a pression . In planrogenase . Howeverase [2O2  and in ad by H2S , with thd by H2S , and it d by H2S ,58.S-nitrosation also has a role in mediating the interplay between NO and other reactive signaling mechanisms, such as those involving ROS. For example, key enzymes which generate ROS, such as NAPDH oxidase, can be modified by NO. It has been reported that RBOHD is S-nitrosated at Cys890 which inactivates the enzyme and thus reduces its ROS-generating activity [activity . TherefoS-nitrosation, NO interaction will lead to conformational changes in the protein and commensurate changes in activity, either increased or decreased. Some of these modifications may have the result of altering other signaling pathways mediated by other reactive signals; for example, tyrosine nitration can alter superoxide dismutase (SOD) activity and hence ROS signaling [The second important modification of proteins brought about by NO is tyrosine nitration , and agaignaling .−)  . This hamolecule , perhaps2O2, with the latter being important in signaling. In a similar manner, NO can alter catalase activity [2O2, perhaps prolonging ROS-mediated signaling.The generation of NO will also be into an environment rich in antioxidants. NO may affect the activity of enzymatic antioxidants, as mentioned above, where NO, through a peroxynitrite-mediated mechanism, altered SOD activity , and henactivity , thus lo· will be prolonged, and so this will enhance NO·-mediated signaling.A large part of the antioxidant capacity of the cell is due to the presence of low molecular-weight antioxidants. There are a range of small low molecular-weight thiols in cells , but oneImportantly, NO and glutathione can react together to produce GSNO. This potentially has the capacity to reduce GSH/GSSG levels in cells, and hence potentially alter the intracellular redox environment, especially if the reaction is compartmentalized. The reaction will also remove NO from directly partaking in further signaling. However, GSNO has important roles as well. GSNO can act as a donor and therefore a reservoir of NO, and it has been suggested that GSNO can mediate some NO effects , having 2S). H2S has recently been found to be an important signaling molecule in both animals and plants [2S can react directly with NO to produce nitrosothiol  [2 may alter antioxidant levels in cells [2 and some nitrogen compounds [2 has been shown to have effects in plants [2 gas effects [2 signaling will be revealed in the future.Lastly, it has recently been suggested that signaling in animals and plants may involve hydrogen gas (H2) . The prein cells  and so iompounds ,79. Certn plants ,81 and hn plants . NO has  effects ,84 and nNO is a key signaling molecule in plants, being important in plant reproduction , developIf there is an oxidase-type enzyme in higher plants it would need to obtain its electrons from somewhere; most likely a reductase, as seen with P450. As can be seen in 2S, and so NO will not work in isolation. The reaction of NO with ROS or H2S will lower the bioavailability of NO, but also produce new signaling molecules, such as peroxynitrite [S-nitrosation, but this may not be possible if other reactive compounds such as H2S or ROS have already modified the relevant thiol. Therefore, the downstream actions of NO cannot always be assumed.The role of NO is also complex and not fully understood. NO is made in plants cells in response to the same cues that initiate the generation of ROS and Hynitrite  and nitrynitrite , which wynitrite . NO willynitrite . Further2S do not interfere, or is there a competition between all these signals, keeping each other in check, as already been mooted [In conclusion, two major barriers exist to the progression of NO research in plants. Firstly, the controversy surrounding the presence of NO-generating enzymes needs to be resolved. Here, is it suggested that the term NOS-like is dropped to avoid continual confusion by drawing parallels with the mammalian system, as clearly the homology does not exist. The term nitric oxide-generating (NOG) would be more accurate. Secondly, the way NO is interwoven into the signaling of other important reactive chemicals needs to be understood. Is NO metabolism compartmentalized in such a way that ROS, GSH or Hn mooted ? Until s


Many different receptor systems are regulated by endomembrane traffic, including signals from receptors activated by extracellular cues such as receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), morphogen receptors, and immune receptors. Notably, with few exceptions, each receptor system undergoes internalization from the cell surface upon activation, followed by receptor-specific control of intracellular membrane traffic through complex intracellular endosomal compartments. The canonical view of regulation of receptor signaling by endocytosis involves downregulation of receptors by their sequestration from the extracellular milieu rich in ligands or degradation of receptor-ligand complexes following traffic to the lysosome. However, it has become apparent that a wide variety of receptors and their downstream signals exhibit actions unique to specific endomembrane compartments , a phenomenon which may require endosomal recruitment of receptor and signaling intermediates. In addition, the potential for pharmacological intervention of putative endosomal signals activated by PAR1 and other GPCRs that trigger p38 MAPK activation for treatment of patients with intracranial hemorrhage and other injuries is highlighted.The role of ubiquitinylation in promoting the lysosomal degradation of signaling receptors has been well-documented. Focusing on GPCRs, Lin and Liu reported that mTOR in skeletal muscle is recruited to and activated at macropinosomes. Mechanical stretch and osmotic shock induced activation of phospholipase D2, but not D1, and stimulated phosphatidic acid production on macropinosomes, leading to mTOR signaling. The surprising finding that mTOR can be activated in macropinosomes, in addition to lysosomes, underscores the complexity of the spatiotemporal dynamics of mTOR signaling that has implication in development and disease progression.mTOR (mechanistic target of rapamycin) signaling that regulates cell growth and proliferation has been classically associated with the lysosomes. Under mechanical stimulation, Inapanathan and Botelho examine how lysosomes serve as signaling hubs, functioning as platforms for recruitment and regulation of important signaling proteins such as AMP-activated protein kinase (AMPK), mTORC complex 1 (mTORC1), and glycogen synthase kinase 3ß (GSK3ß). This review examines how the highly regulated localization of each of these signals to the surface of the lysosome allows integration of cues such as from cell metabolism, extracellular mitogens, and immune signals into a coordinated cellular response. The review further discusses how these lysosome-derived signals in turn control autophagy, lysosome adaptation, cell proliferation and survival and immune cell function and many other outcomes, and how these observations support the view of lysosomes as a major signaling hub of the endocytic system.Lysosomes serve the critical function of effecting degradation of macromolecules within cells; however, these organelles are becoming well-appreciated as having far more complex functions that impact many aspects of cellular physiology. Onnis and Baldari reviewed a body of literature that points to trafficking pathways that deliver endosomal T-cell antigen receptor (TCRs), kinases, and adapter proteins to the IS in order to sustain signaling. TCRs can be internalized by both clathrin-dependent or clathrin-independent pathways, in a ligand-dependent or independent manner. Although classically known for signal termination, TCR endocytosis is followed by distinct polarized recycling pathways that contributes to IS assembly. Thus, paradoxically, TCR endocytosis is important for both signal termination and sustained signaling. The exocytic trafficking at the IS is also believed to be involved in intercellular communication to modulate antigen presenting cell function.The interplay between plasma membrane organization and vesicular trafficking and signaling is nicely illustrated in the context of immunological synapse (IS), a specialized contact area between a T cell and an antigen presenting cell. Sugiyama et al., we examine the compartmentalization of PI3K and Akt activation, regulation and signaling to downstream substrates on various membrane compartments including the plasma membrane, endosomes, lysosomes, and mitochondria, as well as within nanoscale domains within compartments such as the plasma membrane. A particular focus is the recruitment of PI3K-Akt signals and regulators to the plasma membrane and endocytic vesicles derived therefrom, as this may suggest a mechanism for spatiotemporal coordination of positive and negative regulators of this signaling axis. Furthermore, this review examines how the compartmentalization of these key signals at various scales within the endocytic network impacts cellular processes such as invadopodia formation, metabolism, and lysosomal function.The endocytic system is intimately linked with signaling from RTKs, in particular for signals involving phosphatidylinositol-3-kinase (PI3K) and Akt. In DeNies et al., we investigated the relationship between clathrin-mediated receptor internalization and signaling for a chemokine receptor CXCR4. As expected, CXCR4 internalization is significantly reduced when clathrin is knocked down. Interestingly, we reported an increase in chemokine CXCL12-dependent ERK1/2 signaling but a decrease in cell migration under clathrin-knock down condition. However, disrupting lipid raft and CME simultaneously abrogated ERK1/2 signaling. An antibody sensitive to CXCR4 post-translational modification (PTM) revealed a decrease in CXCR4 PTM upon clathrin knock down, despite an unchanged total CXCR4 protein and mRNA levels. The study highlights the growing understanding that modulation of endocytosis affects not only receptor signaling and internalization but also receptor PTM.The interplay between receptor internalization and signaling is expected to take place during directed cell migration process such chemotaxis. In Altogether, these studies and topical reviews underline the importance and regulation of signal compartmentalization at different cellular locales. Important unanswered questions remain, such as exactly how intracellular pools of receptors communicate with cell surface activated receptors and extracellular ligands. Could internal receptors be post-translationally modified in the absence of ligand stimulation? If so, what might be the physiological relevance of such signal compartmentalization? What are the unique physiochemical properties of specific endocytic compartments that endow these with unique signaling factors, and what is the specific repertoire of signaling factors in each endocytic compartment? Systems biology of receptor trafficking and signaling approaches will be important to unravel the mystery of spatial compartmentalization of receptor signaling. Finally, how receptor crosstalk plays a role in signaling compartmentalization, and how alterations of endocytic traffic can contribute to specific diseases by driving changes in receptor signaling will also be important future research directions.CA and AL both conceived and wrote the editorial.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Growing evidence suggests that synaptic signaling is compromised in the aging brain and in Alzheimer’s disease (AD), contributing to synaptic decline. Wnt signaling is a prominent pathway at the synapse and is required for synaptic plasticity and maintenance in the adult brain. In this review, we summarize the current knowledge on deregulation of Wnt signaling in the context of aging and AD. Emerging studies suggest that enhancing Wnt signaling could boost synaptic function during aging, and ameliorate synaptic pathology in AD. Although further research is needed to determine the precise contribution of deficient Wnt signaling to AD pathogenesis, targeting Wnt signaling components may provide novel therapeutic avenues for synapse protection or restoration in the brain. Wnt signaling was originally discovered as a tumorigenic pathway in the early 1980s. Almost four decades later, this prominent pathway has been implicated in numerous cellular processes from early embryonic development to tissue homeostasis. This signaling cascade is also linked to a variety of diseases, from cancer to bone and cardiovascular diseases. In the nervous system, Wnt signaling plays a vital role from early patterning of the nervous system to higher functions including synaptic plasticity and memory in the adult brain. In this review, we will focus on new studies indicating a deregulation of the Wnt signaling pathway in the aging brain and Alzheimer’s disease (AD).Alzheimer’s disease is a devastating neurodegenerative disorder that accounts for two thirds of all dementia cases, and is characterized by progressive cognitive impairment and memory loss leading to difficulties in the performance of daily tasks . The AD 2+ pathways, which lead to changes in gene expression and/or cytoskeleton reorganization. The most extensively studied cascade is the canonical Wnt pathway that controls the expression of Wnt target genes by stabilizing cytoplasmic ß-catenin and by inducing the reorganization of the cellular cytoskeleton  activation, release of Ca2+ from intracellular stores and activation of CaMKII and PKC, resulting in transcriptional changes and actin remodeling , and Wnt/Caskeleton . In abseskeleton . Upon biskeleton . The Wntskeleton . This siskeleton . The thimodeling .In vivo studies show that canonical Wnt signaling is required for the early phase of LTP and also sufficient to enhance it  is upregulated in the aged human brain . A possible explanation is that the studies have been conducted in different areas of the brain, where Wnt components are differentially expressed under basal conditions. For example, most of rodent studies analyzed the hippocampus, whereas human studies have been performed in the prefrontal cortex. Nonetheless, the data presented showed that Wnt signaling is reduced in the aging brain, possibly contributing to the observed age-related synaptic decline . Thus, bDeregulation of Wnt signaling may also contribute to synapse vulnerability in the context of AD. Indeed, reduced Wnt signaling has a direct impact on synapses . In addiDkk1 expression is rapidly elevated by Aß in hippocampal neurons, resulting in reduced canonical Wnt signaling and concomitant synapse loss  and knock-out of LRP6 leads to increased Aß production in an AD mouse model . This isechanism . In addiechanism . Second,echanism , a key pechanism . CollectAPOE, TREM2, and Clusterin) are linked to aberrant Wnt signaling. For example, ApoE4, a major genetic risk factor for late-onset AD, inhibits canonical Wnt signaling in cell lines  mouse model (Pten gene, however, the ablation of one ß-catenin allele decreases the incidence and delays the appearance of the T-ALL (Given the role of Wnts in cancer, the challenge is how to develop treatments to boost Wnt signaling in neurodegenerative diseases without promoting cancer. Mutations in several Wnt signaling pathway components have been linked to colorectal cancer, in particular loss of function mutations in APC leading to high levels of active ß-catenin . Also, tse model . In thishe T-ALL . These she T-ALL . Therefoin vivo role has not been demonstrated. Moreover, we still lack a comprehensive view of the expression and activity of other Wnt ligands in the context of brain aging and AD. Further studies on key Wnt components such as Wnt secreted factors and Wnt receptors could provide more mechanistic insight into how deregulation of Wnt signaling contributes to cognitive decline during normal aging and in AD. Targeting components of the Wnt signaling pathway could open therapeutic avenues for boosting synaptic resilience and enhancing cognition in elderly people and people living with AD.Compelling evidence supports the notion that Wnt signaling is deregulated in the aging brain and in AD . NotablyAll authors contributed the same in the process of reading the literature and writing the manuscript. Figures were prepared by EP.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Seriphium plumosum L. in South African semi-arid grassland. Grassland community had a significant effect on S. plumosum density, canopy size, and on components of soil fertility (P< 0.05). Slope position had a significant effect on S. plumosum density and canopy size (P< 0.05). Seriphium plumosum density was significantly lower (23 plants/50 m2±2.39 (SE)) in CDG than in RHG (40 plants/50 m2±4.19). By contrast, S. plumosum canopy size was significantly higher (1.39 m2±0.11) at CDG than at the RHG (1.06 m2±0.08). The interaction of grassland community and slope position had a significant effect on S. plumosum density and soil sodium content (P< 0.05). Bottom slope regions in RHG (59.00 plants/50 m2±8.62) had higher S. plumosum density than bottom slope regions (19.75 plants/50 m2±3.01) in CDG. Soil sodium content was similar in CDG slopes and higher than in RHG slopes. Seriphium plumosum density was positively related to components of soil fertility; P , K , Na , Ca , and SOC content . However, Seriphium plumosum canopy size did not show any relationship with components of soil fertility. This study revealed that S. plumosum density in grassland communities increased with increasing soil texture and fertility.Most studies suggest that multiple factors are responsible for woody plant encroachment, but are inconclusive on its causes. Woody plant encroachment is modified by local patterns of disturbance, topography, soil texture and fertility, and their relative importance and interaction strength varies among locations. We used grassland communities, Carletonville Dolomite Grassland (CDG), Rand Highveld Grassland (RHG) and abiotic factors (Soil Organic Carbon (SOC), Total nitrogen (TN), Phosphorus (P), Potassium (K), Sodium (Na), Calcium (Ca), Magnesium (Mg) and pH) to investigate habitat preferences of  Woody plant encroachment is an increase in density, cover and biomass of trees or shrubs, especially in the arid and semi-arid grassland and savanna biomes . Causes Woody plant encroachment has global and local drivers. The global drivers includes climate change and increased atmospheric carbon dioxide . The priSoil properties  and topography interact to influence the pattern of woody plant abundance and distribution . The extS. plumosum encroachment on soil fertility. This is because woody or shrubby plant encroachment can also create “islands of fertility”, by increasing nutrient availability below the shrubs, thus providing habitat for species that are sensitive to low nutrient availability [S. plumosum encroachment affects soil fertility, yet important to understand its general role on ecosystems.The study also aims to clarify the effect of lability . Nutrienlability . In Soutlability . Thus, wlability . Althouglability , thus enlability . HoweverGenerally, the outcomes of woody plant encroachment in semi-arid grassland and savanna communities are not universal . There iSeriphium plumosum L., an indigenous, unpalatable shrub in the Asteraceae family, has increased in abundance within its historic range in the fynbos biome and expanded its geographic range in to the grassland biome [Seriphium plumosum encroachment has converted extensive grazing areas into less productive rangelands [S. plumosum encroachment. Consequently, solutions that integrate knowledge of the ecology of S. plumosum species, such as soil—water relationships in rangelands communities, will probably improve our ability to control its encroachment in grasslands.In South Africa, nd biome . Seriphingelands , throughngelands , and redS. plumosum density, cover and soil fertility , Phosphorus (P), Potassium (K), Sodium (Na), Calcium (Ca), Magnesium (Mg) and Soil Organic Carbon (SOC) and pH). 2) The type of association found between S. plumosum densities, cover, and soil fertility. We predicted that 1) Seriphium plumosum density, size and soil fertility will be high at bottom slopes due to run-on of nutrients and rain water. 2) Seriphium plumosum density will vary between grassland communities, because of local differences in environmental factors and disturbance regimes, among others.This study is aimed at determining; 1) the effect of grassland communities, slope position  and their interactions on S. plumosum coexisted with a diversity of herbs, many of which also belong to Asteraceae family. Seriphium plumosum occurs in predominantly disturbed and overgrazed areas [Seriphium plumosum.The study was conducted on Kaalfontien and Schietfontein private owned farms situated in Gauteng, South Africa. The farm owners gave us permission to do field work on their farms and there was no specific permission required for each locations, since both farms were private owned and the study did not involve any endangered or protected species. These farms are located in Carletonville Dolomite Grassland (Gh15) and Rand Highveld Grassland (Gm 11) veld types, respectively . Kaalfoned areas , 44 of ted areas , Madagased areas . Both faGenerally, the sites on Carletonville Dolomite Grassland (CDG) are higher (mean 1614.28 m ± 0.78 (SE)) in altitude compared to the Rand Highveld Grassland (RHG) sites (1457.24 m ± 0.85). Similarly, soils in the CDG had higher silt (8.66% ± 0.96) and clay (28% ± 0.29) than silt (2.66% ± 0.96) and clay (11% ± 0.20) in RHG. Rain in both veld types where the farms are located falls almost exclusively in summer (October- April), with means of 593 mm and 654 mm per annum for CDG and RHG, respectively . HoweverAristida congesta, Brachiaria serrata, Eragrostis chloromelas and Alloteropsis semialata. The soils are mostly from the Dolerite and chert of the Malmani Subgroup, which support mostly shallow Mispah and Glenrosa soil forms [Themeda, Eragrostis, Heteropogon and Elionurus. The soils are from the Quartzite ridges of the Witwatersrand Super group, which support soils of various quality such as Glenrosa and Mispah, along rocky ridges [Carletonville Dolomite Grassland has a complex mosaic pattern of grasses such as il forms . The RHGy ridges .A factorial design consisting of two grassland communities (CDG and RHG) x three slope positions  with twelve replicates. The sampling sites at each grassland community had six replicates and two subplots at each of the three slope positions  on the north-facing slopes. The two subplots at each slope position were chosen randomly from four previously established subplots. There were 10 m buffer zones between subplots and slope positions. The distance (measured from the center of each subplot) between the two subplots used in this study ranged between 35 m (i.e. for adjacent subplots) and 75 m (i.e. for nonadjacent subplots) apart within slope positions and 35 m (i.e. for adjacent subplots) and 130 m (i.e. nonadjacent subplots) between slope positions .S. plumosum density and canopy size, and components of soil fertility: Soil organic carbon (SOC), Total Nitrogen (TN), Phosphorus (P), Potassium (K), Sodium (Na), Calcium (Ca), Magnesium (Mg) and pH.There were 50 m buffer zones between the replicates at each of the grassland communities. The centers of the replicates were approximately 150 m apart from each other. The variables measured included Seriphium plumosum density, canopy size and soil samples were collected from March to June 2015 at both veld type-sites. Seriphium plumosum density was determined by counting all individuals rooted in a 50 m2 (25 m x 2 m) belt-transect, running downhill, at the center of each 625 m2 (i.e. 25 m x 25 m) subplots. Density was expressed as mean individual plants per unit area (50 m2). Canopy size was determined by measuring horizontal diameter (to the nearest 2 cm) of the long and short axis of the canopy of individuals whose canopy intercepted the 25 m line transect placed down slope in each of the subplots and slope positions. Canopy size was calculated using an ellipse function (C = abπ / 4.0), where a represents the long axis and b represents the short axis of the canopy [2). Since S. plumosum requires water and nutrients to germinate, recruit and survive, but reported to dislike fertile soils [S. plumosum plants in each belt transect. The soils were analyzed for eight components of fertility using Ambic I extraction followed by AA spectroscopy. The sub-sample of soils from each of the two subplots at each slope position in each replicate were pooled together and analysed for texture (silt and clay) at the Agricultural Research Council-Institute for Soil, Climate and Water (ARC-ISCW) soil analyses accredited laboratory.e canopy  and exprle soils , the soiSeriphium plumosum density, canopy size and soil fertility at two grassland communities and three slope positions was analysed as a completely randomised design with a 2 × 3 factorial analysis of variance (ANOVA) using GLM procedures. Data for S. plumosum density, size and soil fertility for each slope position were averaged before the analysis. Data met the assumption of ANOVA concerning homogeneity of variance before being analysed for the effect of grassland community, slope position and their interaction on S. plumosum density, canopy size, and components of soil fertility using SAS [S. plumosum density, cover, and components of soil fertility were compared using the Turkey’s HSD test, and the differences declared significant at P < 0.05. Using linear regression analyses in SAS [S. plumosum density, individual size and the eight components of soil fertility.sing SAS . When ths in SAS , we explS. plumosum density and canopy size are presented in table below  than in the RHG (40 plants/50 m2 ± 4.19). In general, Seriphium plumosum density at bottom slopes (39 plants/50 m2 ± 6.06) was significantly higher than at the top slopes (25 plants/50 m2 ± 2.88). Seriphium plumosum density at the mid slopes (31 plants/50 m2 ± 3.61) was not significantly different from the bottom and top slopes. Grassland community x slope position had a significant interaction effect on S. plumosum density (P < 0. 0016) (0. 0016) .2 ± 8.62) had significantly higher S. plumosum density than in the mid slopes (36 plants/50 m2 ± 4.88) and top slopes (25 plants/50 m2 ± 3.94) as well as all slope positions in the CDG. Canopy size of S. plumosum was larger in CDG (1.39 m2 ± 0.11) than in RHG (1.06 m2 ± 0.09). In general, S. plumosum individual canopy size were bigger at the top slopes (1.42 m2 ± 0.13) than at the bottom slopes (0.99 m2 ± 0.12), while canopy size at the mid slopes (1.29 m2 ± 0.11) was similar to both top and bottom slopes.Bottom slope regions in RHG (59.00 plants/50 mGrassland community differed significantly in soil organic carbon (P < 0.0001), total nitrogen (P < 0.0001), phosphorus (P < 0.0001), potassium (P < 0.0001), sodium (P < 0.0001), calcium (P < 0.0001) and magnesium (P < 0.0001) content. However, these differences were not observed for soil pH content .All the components of soil fertility were significantly higher in CDG than in RHG, except pH that was similar at both grassland communities .No effect of slope position in any soil fertility components was found (P = 0.1006). The interaction of grassland community and slope position had significant effect on soil sodium content (P = 0.0181). Soil sodium content was the same at all slope positions in Carletonville Dolomite Grassland and significantly higher than similar slopes in Rand Highveld Grassland .S. plumosum increased with soil fertility. The effect was weak but significant (P < 0.05). Canopy size did not show any significant relationship with the components of soil fertility , need further investigation. The fact that, as woody plants encroach, grass productivity declines, causing soil erosion and reducing the amount of soil nutrients [Seriphium plumosum density and components of soil fertility, and S. plumosum relative abundance in bottom slope may suggests the sensitivity of S. plumosum to low soil nutrient availability. Although this finding confirm our predictions that, S. plumosum encroachment would be positively correlated with some components of soil fertility, which would facilitate its germination and recruitment, other research have found the opposite. Consequently, it is suggested in this study that the effect of woody plant encroachment are not universal [Interestingly, components of soil fertility  were positively correlated with  reached  and/or w reached . It is a reached . The fin reached  that S. utrients  may applutrients , 34, dueutrients . This crutrients . The posniversal .S. plumosum encroachment of mostly, fine textured and fertile soils. Previous research found that, S. plumosum occurs mainly in coarse-textured, nutrient poor soils, often with relatively low sodium content. These results and that of other, confirm the similarity on the occurrence of S. plumosum on coarse textured soils, but the opposite on the soil nutrient status. The fact that in this study, S. plumosum density has shown a weak positive correlation with components of soil fertility and negative correlation reported in the other study, suggests that controlled research on manipulation of soil nutrient resources, especially soil organic carbon (SOC), Phosphorus (P) Potassium (K), and Sodium (Na). This could contribute to understanding how S. plumosum responds to soil fertility. Furthermore, an interaction of abiotic factors at grassland communities found in this study, suggests that S. plumosum encroachment is influenced by a complex interaction of multiple abiotic factors. Consequently, a better understanding of the complex interaction of these abiotic factors on S. plumosum encroachment is needed to aid in its control and management on South African grassland communities.This study shows S. plumosum encroachment on South African semi-arid grassland communities is also lacking. Consequently, more research to understand the relationship between changes in abiotic factors and the extent, rate and times of S. plumosum encroachment is needed to aid in the development of management and control methods for its encroachment in South African semi-arid grassland communities.The relationship between the changes in abiotic factors and the extent, rate and the times of S1 Fig(JPG)Click here for additional data file.S1 Appendix(RTF)Click here for additional data file.


Gramd1b knockdown caused morphological changes of the cells, characterized by the formation of membrane ruffling and protrusions, implicating its role in cell migration. Consistently, GRAMD1B inhibition significantly enhanced cell migration, with an increase in the levels of the Rho family of GTPases. We also found that Gramd1b knockdown-mediated pro-migratory phenotype is associated with JAK2/STAT3 and Akt activation, and that JAK2 or Akt inhibition efficiently suppresses the phenotype. Interestingly, AG490 dose-dependently increased p-Akt levels, and our epistasis analysis suggested that the effect of JAK/STAT inhibition on p-Akt is via the regulation of GRAMD1B expression. Taken together, our results suggest that GRAMD1B is a key signaling molecule that functions to inhibit cell migration in breast cancer by negating both JAK/STAT and Akt signaling, providing the foundation for its development as a novel biomarker in breast cancer.Dysregulated JAK/STAT signaling has been implicated in breast cancer metastasis, which is associated with high relapse risks. However, mechanisms underlying JAK/STAT signaling-mediated breast tumorigenesis are poorly understood. Here, we showed that GRAMD1B expression is upregulated on IL-6 but downregulated upon treatment with the JAK2 inhibitor AG490 in the breast cancer MDA-MB-231 cells. Notably,  Remarkably, breast cancer-associated mortality has been attributed to metastases of the cancer to secondary distant sites, rather than to the primary tumor2. 10–15% of breast cancer patients show symptoms of metastasis within 3 years of initial detection of tumor, while others may show symptoms after 10 years or more3. This heterogeneity in metastatic rates of breast tumors further adds to the complexity of the disease and makes prognosis, as well as the development of treatment strategies, difficult.Breast cancer is a clinically heterogeneous disease and has been ranked as the most common malignancy in women worldwide, with incidence rates particularly high in developed countries and relative mortality rates greatest in less developed countries4. The JAK/STAT cascade, a cytokine and growth factor signaling pathway5, has been well established in breast tumorigenesis6. Constitutively-activated STAT3 has been detected in approximately 50–60% of all breast cancers7  where it contributes to several hallmarks of cancer, including proliferation, angiogenesis and metastasis6. In particular, the autocrine/paracrine IL-6/JAK/STAT3 feed-forward loop has been implicated as a key player of tumor progression and metastasis8. Immunohistochemical analyses with human breast tumor samples further revealed an increased level of IL-6 at the leading edge of invasive breast tumors, with its level positively correlated with advanced stage, confirming a pivotal role of IL-6 signaling in breast tumor metastasis in vivo8.Metastatic breast tumors are the more chemoresistant forms of breast cancer and are hence associated with poor survival10. Mutations in genes of this signaling pathway have been reported in more than 70% of breast tumors9 where they were found to promote tumor growth and progression, and contribute to chemoresistance12. Furthermore, PI3K/Akt signaling was shown to promote the expression of Twist, a master transcription factor of epithelial-mesenchymal transition (EMT), leading to enhanced TGF-β receptor signaling which in turn functions to maintain hyperactivated PI3K/Akt signaling, cooperatively driving breast tumor metastasis14. More recently, cPLA2α was also reported to facilitate TGF-β-induced EMT in breast cancer through the PI3K/Akt signaling cascade15, suggesting the pivotal role of PI3K/Akt signaling in breast cancer cell migration. Interestingly, several studies have also reported a cross-talk between PI3K/Akt and JAK/STAT signaling in metastatic breast tumors16. However, there is a gap in knowledge of the exact molecular mechanisms by which these signaling cascades interact with each other to facilitate breast cancer metastasis.Another signaling pathway commonly dysregulated in breast cancer is the PI3K/Akt signaling cascadeDrosophila to identify additional signaling components of the JAK/STAT pathway17. The Drosophila ortholog of GRAMD1B (GRAM domain-containing protein 1B) was identified as a putative component of the signaling cascade17. GRAMD1B contains a GRAM domain that is known to function as a protein-binding or lipid-binding intracellular signaling domain19. More recently, GRAMD1B has been implicated in human malignancies. Specifically, it was reported to play a role in chemoresistance of ovarian cancer patients, such that GRAMD1B inhibition led to an anti-tumor effect20. Furthermore, a genome-wide association study in chronic lymphocytic leukemia patients revealed that single nucleotide polymorphism in Gramd1b is associated with increased risk of disease in a European population21. In gastric cancer, GRAMD1B regulates cell survival by upregulating expression of the anti-apoptotic molecule Bcl-xL22. In this study, JAK/STAT signaling was found to positively regulate GRAMD1B expression in the breast cancer MDA-MB-231 cells. Knockdown of Gramd1b resulted in distinct morphological changes of the cells, accompanied by increased rates of cell migration. Intriguingly, p-JAK2 and p-Akt levels were drastically induced upon GRAMD1B inhibition, but treatment with AG490 or MK-2206 almost completely suppressed the pro-migratory phenotypes induced by Gramd1b knockdown. Lastly, our epistasis analysis suggested a central role of GRAMD1B in the linkage between JAK/STAT and Akt signaling.A genome-wide RNAi screen was conducted in 24. Since the Drosophila ortholog of GRAMD1B was initially identified as a signaling component of the Drosophila JAK/STAT pathway17, we determined whether GRAMD1B expression is also modulated by JAK/STAT signaling in the breast cancer MDA-MB-231 cells. Interestingly, we observed an increase in the expression of GRAMD1B of 49 kDa (UniProtKB Q3KR37-3) on IL-6 stimulation  accompanied by membrane ruffling (dotted line)  and confirmed a change in morphology of MDA-MB-231 cells, with formation of membrane protrusions  Fig. , hallmarGramd1b knockdown cells almost entirely covered the wound by 36 hours, only ~40% of the wound had been covered by control cells , which binds to ubiquitin that functions to mark endocytosed receptors for lysosomal degradation46. In the JAK/STAT signaling pathway, the ligand-receptor internalization and trafficking to the early endosome were reported to be associated with the signaling intensity47, suggesting that GRAMD1B may function to promote ligand-receptor decay, causing the de-activation of JAK/STAT signaling.The JAK/STAT signaling pathway is one of many control pathways that promotes cell motility by regulating actin dynamics and activating key metastasis-promoting genesion Fig. . Notablyion Fig. , suggestogy Fig.  and migrogy Fig. , as wellown Fig. . Notably49. For example, Ca2+-dependent Akt activation was implicated in TRPV4-mediated breast cancer cell migration and metastasis50, and Twist-Akt2 signaling axis was shown to be essential in promoting the invasive ability and survival of breast cancer cells13. Furthermore, Akt has been found to be required for the formation of membrane ruffles and lamellipodia through interaction with actin filaments and co-localization with the ARP2/3 complex52. Notably, our data suggested that GRAMD1B acts downstream to PI3K, but upstream/parallel to Akt. The spatial localization of PI3K and its products at the leading edge of motile cells was shown to be crucial for effective cell migration. Specifically, PI3K and its interacting PH domain-containing protein binding partners such as Akt have been reported to translocate to the plasma membrane in response to a chemoattractant stimulus54. At the leading edge, the membrane translocated Akt interacts with the PI3K products PIP3 and PIP2, triggering well-coordinated cell movement53. Since GRAM domain has been implicated in membrane-coupled lipid/protein-binding, it is conceivable that GRAMD1B may play a role in translocation of Akt and/or de-stabilization of the interaction between Akt and the PI3K products at the leading edge of motile cells. In support of this, in another GRAM domain-containing protein MTMR2, the GRAM domain has been recognized to play a role in PIP2 and PI(5)P substrate recognition55, thereby highlighting the possible function of GRAM domain in phosphoinositide recognition.Another signaling cascade often implicated in breast tumorigenesis is the PI3K/Akt signaling pathway, where it is considered a potential therapeutic target due to its role in tumor initiation and progression16. Specifically, co-inhibition of the PI3K/mTOR and JAK2 signaling cascades was found to synergistically reduce breast tumor growth and metastasis, as well as improve overall survival in vivo16. Therefore, we undertook epistasis analysis to further elucidate the regulatory hierarchy between JAK/STAT and PI3K/Akt signaling cascades on loss of GRAMD1B activity. Interestingly, we detected a dose-dependent increase in p-Akt levels on JAK/STAT inhibition by AG490 , containing 10% FBS (Gibco) and 1% Penicillin-Streptomycin (Gibco). IL-6  was used to activate JAK/STAT signaling at working concentrations of 20–60 ng/ml for 6 hours. Cells were treated with the JAK2 inhibitor AG490 (Sigma-Aldrich) and the Akt inhibitor MK-2206  for 24 hours. The Rho inhibitor Rhosin hydrochloride (Tocris Bioscience), Rac1 inhibitor NSC23766 (Santa Cruz Biotechnology) and Cdc42 inhibitor ML141 (Santa Cruz Biotechnology) were reconstituted in DMSO. Small interfering RNA (siRNA) targeting Gramd1b  was designed and synthesized by Singapore Advanced Biologics Pte. Ltd. . si-Gramd1b-2  was used to rule out off-target effects of siRNA for Gramd1b. Non-targeting siRNA (Ambion) was used as a negative control. Cells were transfected with siRNA using the transfection reagent Lipofectamine 3000  as per the manufacturer’s instructions.The human MDA-MB-231 breast cancer cell line  was maintained in RPMI-1640 medium  was used to extract total RNA, which was subsequently converted to cDNA using the Revert Aid First Strand cDNA Synthesis Kit . The samples were loaded in triplicates in a 96-well plate for each sample set, and quantitative PCR was carried out using the FAST SYBR green cocktail  in the HT7900 FAST Realtime PCR system . Sequence of the primers (IDT technologies) used for this study are listed in Supplementary Table TM Mammalian Protein Extraction Reagent  containing HaltTM Protease Inhibitor cocktail and EDTA . Protein concentration was determined by the Microtiter Bio-Rad Protein Assay solution . 30 μg of proteins were loaded onto SDS-PAGE gels, and standard western blot analysis was performed. The following antibodies were used: GRAMD1B , Rac1/RhoA/Cdc42 , p-STAT3 (Tyr705) , STAT3 , p-JAK2 (Tyr1007/1008) , JAK2 , p-Akt (Ser473) , Akt , p-PI3K p85 (Tyr458)/p55 (Tyr199) , PI3K p85  and β-actin .Protein lysates were extracted using M-PERFollowing 72 hours of transfection with siRNA, cells were fixed using 4% paraformaldehyde, and then permeabilized with 0.1% Triton X-100. For F-actin cytoskeleton staining, the cells were incubated with TRITC-conjugated Phalloidin  at 1:500 dilution at room temperature for 1 hour. The slides were imaged under the Confocal Laser Scanning Microscope (Olympus Fluoview FV 1000).Following 48 hours of transfection with siRNA, cells were treated with AG490 at 50 μm or MK-2206 at 10 μm for an additional 24 hours. Following the treatment, cells were re-suspended in serum free RPMI-1640 medium and seeded into polycarbonate membrane transwell inserts . RPMI-1640 medium containing 10% FBS was used as a chemoattractant, and cells were incubated at 37 °C for 18 hours to allow migration. Following incubation, the migrated cells were stained using 0.5% crystal violet and visualized using the Nikon SMZ1500 microscope. The average number of migrated cells per insert was calculated by imaging five different fields.A linear scratch or wound was made across the confluent monolayer of the 72 hours post transfected cells using a fine 10 μl pipette tip. Three random fields were marked out, and images were subsequently taken at 12 hour intervals to monitor cell migration. The average gap width and average number of migrated cells across the three marked fields were then measured and calculated at each time point.P < 0.05.The GraphPad prism 6 software  was used to carry out statistical analysis. For comparing means between two groups, a two-tailed student T-test was used. A one-way ANOVA was used for tests involving more than two groups. For wound healing assay, the two-way ANOVA statistical test was adopted. Data is represented as means ± SEM, and results are considered statistically significant if Supplementary Information


Calcium signaling is emerging as a key pathway controlling cellular senescence, a stable cell proliferation arrest playing a fundamental role in pathophysiological conditions, such as embryonic development, wound healing, cancer, and aging. However, how calcium signaling is regulated is still only partially understood. The inositol 1, 4, 5‐trisphosphate receptor type 2 (ITPR2), an endoplasmic reticulum calcium release channel, was recently shown to critically contribute to the implementation of senescence, but how ITPR2 expression is controlled is unclear. To gain insights into the regulation of ITPR2 expression, we performed an siRNA screen targeting 160 transcription factors and epigenetic regulators. Interestingly, we discovered that the retinoid X receptor alpha (RXRA), which belongs to the nuclear receptor family, represses ITPR2 expression and regulates calcium signaling though ITPR2 and the mitochondrial calcium uniporter (MCU). Knockdown of RXRA induces the production of reactive oxygen species (ROS) and DNA damage via the ITPR2‐MCU calcium signaling axis and consequently triggers cellular senescence by activating p53, whereas RXRA overexpression decreases DNA damage accumulation and then delays replicative senescence. Altogether, our work sheds light on a novel mechanism controlling calcium signaling and cellular senescence and provides new insights into the role of nuclear receptors. Senescent cells are mainly characterized by a stable cell cycle arrest and the appearance of features such as senescence‐associated‐β‐galactosidase activity  and senescence‐associated secretory phenotype  superfamily. NRs mainly function as transcriptional regulators, controlling organismal development, homeostasis, and metabolism  in three different strains of primary human lung fibroblasts  and observed the most significant and reproducible upregulation of ITPR2 expression by knocking down the gene encoding the nuclear receptor RXRA  ChIP‐seq database and observed three peaks of RXRA on intron 2 of ITPR2 in one dataset  co‐expression database and found out that RXRA is among the genes which expression is the most significantly inversely correlated with the one of s Figure f. Convers Figure g. These 2.2ITPR2 expression, regulates calcium fluxes. Using the cytosolic calcium indicator Fluoforte and calcium imaging, we observed that RXRA knockdown by a siRNA pool or two individual siRNAs increased histamine‐induced calcium release from the ER  calcium release channel, we next investigated whether RXRA, by repressing R Figure a and thaMCU expression was not significantly regulated by RXRA , an antioxidant  until they became mycoplasma‐free before performing experiments. Experiments were performed in IMR90 at p14–p16, in WI38 at p21–24, and in MRC5 at p21–p25. NAC, Trolox, and 9‐cis‐retinoic acid were purchased from Sigma‐Aldrich and used at 1 µM, 50 µM, and 500 nM, respectively. BAPTA was purchased from Santa Cruz Biotechnology and used at 1 µM.IMR90, WI38, and MRC5 human fetal lung fibroblasts (ATCC), GP‐293 retroviral, or 293 T lentiviral packaging cells (Clontech) were cultured in Dulbecco's modified Eagle's medium  containing GlutaMax and supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Life Technologies). Cells were maintained at 37°C under a 5% CO4.2pLNCX2‐mito‐GCaMP2 , ON‐TARGETplus siRNA SMARTpools targeting the genes of interest with four different siRNAs and ON‐TARGETplus individual siRNAs were used . Sequences of siRNAs comprised in the pools and of individual siRNAs are listed in Supporting Information Tables 4.4ITPR2 was calculated for each siRNA pool according to its level in cells transfected with the control nontargeting siRNA pool.For the screen, a library of 160 siRNA pools each targeting an epigenetic regulator or a transcription factor was used. The full list of the 160 genes targeted by the library is displayed in Supporting Information Table 4.5The Maxima first‐strand cDNA synthesis kit (Life Technologies) was used to synthesize cDNA from total RNA, according to manufacturer’s instructions. cDNAs obtained by this reverse transcription (RT) were used as templates for quantitative PCR (qPCR). The qPCR mixture also contained TaqMan mix (Roche), 100 µM of a Universal Probe Library probe (Roche), and 200 nM of primers (Sigma‐Aldrich). Primer sequences are listed in Supporting Information Table 4.6Cells were directly lysed in Laemmli buffer. Cell lysates were resolved by SDS‐PAGE electrophoresis and transferred to nitrocellulose membranes (Bio‐Rad). Membranes were then blocked with TBST‐Milk 5% for 1 hr and incubated with primary antibodies overnight at 4°C. Primary antibodies and dilutions used are listed in Supporting Information Table 4.7Chromatin immunoprecipitation (ChIP) was performed with the Millipore ChIP Assay Kit (17‐295). MRC5 cells were crosslinked with 1% formaldehyde for 10 min at 37°C. Chromatin was prepared according to the Millipore protocol and sonicated to an average size of 300–500 bp using a Diagenode Bioruptor. Chromatin fragments were immunoprecipitated at 4°C overnight with RXRA antibody or normal rabbit IgG used as negative control , live cells were treated either with 5 µM Fluoforte  or with 10 µM Rhod‐2  for 1 hr at 37 °C in Hank’s balanced salt solution (HBSS) with calcium, magnesium, and no phenol red . Cells were then washed and incubated in HBSS with no calcium, no magnesium, and no phenol red  for calcium imaging. Fluorescence was recorded every 1.5 s using Zeiss LSM 780 confocal microscope. Histamine  was injected after 1 min of measurement at a final concentration of 100 nM. Average changes in fluorescence intensities in multiple regions of interest (ROI) were calculated. Results are shown as (F1‐F0)/F0, where F0 is the mean of the intensities from 10 to 50 s. Alternatively, cells stably expressing mitoGCaMP2, a genetically encoded mitochondrially targeted calcium indicator, were transfected with siRNA in glass‐bottom dishes. Six days later, fluorescence (Ex 488 nm/Em 500–570 nm) was monitored in live cells using Zeiss LSM 780 confocal microscope. Fluorescence intensity was quantified using the ImageJ software.4.10Six days after siRNA transfection in glass‐bottom dishes (Thermo Scientific), cells stably expressing roGFP2‐ORP1 were washed and incubated in HBSS with calcium, magnesium, and no phenol red  for ROS detection. Fluorescence (Ex 405 and 488 nm/Em 500–554 nm) was monitored in live cells using Zeiss LSM 780 confocal microscope. The ImageJ software was used to quantify fluorescence intensity, and the ratio between 405 nm (oxidized state) and 488 nm (reduced state) was plotted, as previously described , cells were fixed in ice‐cold methanol for 10 min at −20°C and blocked in PBS‐Tween 0.05% containing 20% FBS (PBST‐FBS). Incubation with primary antibodies in PBST‐FBS was performed overnight at 4°C. Primary antibodies and dilutions used are listed in Supporting Information Table 4.12SD as errors bars, and Student’s t test was used to determine the p‐value. *p < 0.05; **p < 0.01; ***p < 0.001.Graphs are presented with None declared.D.B. and N.M. conceived and supervised the project. D.B., N.M., and X.M. designed the experiments. X.M., M.F., M.W., C.R., and N.M. performed experiments. X.M., M.W., O.K., P.A.D., N.M., and D.B. performed data analysis. D.B., N.M., and X.M. wrote the manuscript. Click here for additional data file. Click here for additional data file.