Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

Pires-Oliveira M, Maragno AL, Parreiras-E-Silva LT, Chiavegatti T, Gomes MD, Godinho RO. Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo. J Appl Physiol 108: 266-273, 2010. First published November 19, 2009; doi:10.1152/japplphysiol.00490.2009.-Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. in the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. in addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, BrazilUniv São Paulo, Fac Med Ribeirao Preto, Dept Biochem & Immunol, Ribeirao Preto, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, BrazilFAPESP: 05/59006-1FAPESP: 2006/58629-8Web of Scienc

ISG15 Modulates Development of the Erythroid Lineage

Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15-/- bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation

Study of the role of the ubiquitin-like protein ISG15 in erythroid development

L’érythropoïèse constitue un processus continu et ordonné au cours duquel des progéniteursérythroides commis prolifèrent et se différencient en globules rouges. Au cours d’un criblage visant à identifierdes gènes dont l’expression est régulée au cours de la différenciation terminale, nous avons identifié ISG15comme un gène dont l’expression est induite dans les stades tardifs de la différenciation érythroïde. ISG15appartient à la famille des protéines apparentées à l’ubiquitine et est conjuguée directement à des résidus Lysinede protéines cibles. La séquence des évènements moléculaires qui président à la modification de résidus Lysinedes protéines par ISG15 (ISGylation) est très semblable à ceux mis en oeuvre dans l’ubiquitination des protéines.Nous avons montré que l’expression d’ISG15 et des enzymes liées au processus d’ISGylation Ube1L, UbcM8 etHerc6 est induite au cours de l’érythropoièse in vivo et aussi dans des érythroblastes en culture induits à sedifférencier en réponse à l’Epo. Grâce à ce système de culture in vitro, nous avons montré que l’induction de cesgènes est majoritairement indépendante de la voie de signalisation IFN, et partiellement dépendante de la voie designalisation Epo. La comparaison de l’érythropoïèse dans des souris contrôles et des souris dans lesquelles legène ISG15 a été inactivé (ISG15-/-), montre une diminution du nombre de progéniteurs BFU-E et CFU-E dansla moelle osseuse avec une augmentation des BFU-E/CFU-E dans la rate des animaux ISG15-/-. Nous avonsaussi montré que les érythroblastes ISG15-/- sont inhibés dans leur différenciation terminale, à la fois in vivo et invitro. A l’inverse, la surexpression d’ISG15 dans les érythroblastes se révèle faciliter la différenciationérythroide. Au niveau moléculaire, nous avons montré que des acteurs importants de la différenciation érythroidepeuvent être ISGylés, comme par exemple STAT5, Globine, PLCγ et ERK2. En ce qui concerne plusprécisément STAT5, nous avons montré que son ISGylation est induite au cours de la différenciation et avonscherché à identifier les conséquences de son ISGylation. Dans le contexte d’une protéine de fusion, STAT5ISGylée se lie mieux à son site de liaison à l’ADN, une propriété associée avec une régulation positive de TfR etBCL-XL, deux gènes précédemment décrits comme étant régulés par STAT5 dans les érythroblastes. L’ensemblede ces résultats établit un nouveau rôle pour ISG15 au cours de la différenciation érythroide, en plus de sesfonctions anti-virales bien caractériséesErythropoiesis is an orderly continuous process during which committed erythroid progenitorcells proliferate and differentiate into mature red blood cells. In a search for genes that are deregulated duringthis differentiation process, we have identified ISG15 as being induced during the late stages of erythroiddifferentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins bythe enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we haveshown that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6are induced during erythroid differentiation. Moreover, using in vitro differentiating erythroblasts, we haveshown that the induction of these genes is mostly independent of IFN signaling, while it is partially dependent onEpo signaling in these cells. Our analysis of the ISG15 deficient mice have shown a decreased number of BFUE/CFU-E in the bone marrow, associated with an increased number of these cells in the spleen of these animals,a phenotype reminiscent of stress erythropoiesis. While ISG15-/- bone marrow and spleen-derived erythroblastsshowed a less differentiated phenotype both in vivo and in vitro, over-expression of ISG15 in erythroblasts wasfound to facilitate erythroid differentiation. At the molecular level, we have shown that important effectors oferythroid differentiation can be ISGylated, including STAT5, Globin, PLCγ and ERK2. Attempt to identify theconsequences of ISGylation has only been performed for STAT5. When studied in the context of a fusionprotein, ISGylated STAT5 was found endowed with a higher capacity to bind DNA, a property associated withup-regulation of TfR and Bcl-XL, two genes previously described as being regulated by STAT5 during erythroiddifferentiation. This establishes a new role for ISG15, in addition to its well-characterized anti-viral functions,during erythroid differentiation

Study of the role of the ubiquitin-like protein ISG15 in erythroid development

L érythropoïèse constitue un processus continu et ordonné au cours duquel des progéniteursérythroides commis prolifèrent et se différencient en globules rouges. Au cours d un criblage visant à identifierdes gènes dont l expression est régulée au cours de la différenciation terminale, nous avons identifié ISG15comme un gène dont l expression est induite dans les stades tardifs de la différenciation érythroïde. ISG15appartient à la famille des protéines apparentées à l ubiquitine et est conjuguée directement à des résidus Lysinede protéines cibles. La séquence des évènements moléculaires qui président à la modification de résidus Lysinedes protéines par ISG15 (ISGylation) est très semblable à ceux mis en oeuvre dans l ubiquitination des protéines.Nous avons montré que l expression d ISG15 et des enzymes liées au processus d ISGylation Ube1L, UbcM8 etHerc6 est induite au cours de l érythropoièse in vivo et aussi dans des érythroblastes en culture induits à sedifférencier en réponse à l Epo. Grâce à ce système de culture in vitro, nous avons montré que l induction de cesgènes est majoritairement indépendante de la voie de signalisation IFN, et partiellement dépendante de la voie designalisation Epo. La comparaison de l érythropoïèse dans des souris contrôles et des souris dans lesquelles legène ISG15 a été inactivé (ISG15-/-), montre une diminution du nombre de progéniteurs BFU-E et CFU-E dansla moelle osseuse avec une augmentation des BFU-E/CFU-E dans la rate des animaux ISG15-/-. Nous avonsaussi montré que les érythroblastes ISG15-/- sont inhibés dans leur différenciation terminale, à la fois in vivo et invitro. A l inverse, la surexpression d ISG15 dans les érythroblastes se révèle faciliter la différenciationérythroide. Au niveau moléculaire, nous avons montré que des acteurs importants de la différenciation érythroidepeuvent être ISGylés, comme par exemple STAT5, Globine, PLCg et ERK2. En ce qui concerne plusprécisément STAT5, nous avons montré que son ISGylation est induite au cours de la différenciation et avonscherché à identifier les conséquences de son ISGylation. Dans le contexte d une protéine de fusion, STAT5ISGylée se lie mieux à son site de liaison à l ADN, une propriété associée avec une régulation positive de TfR etBCL-XL, deux gènes précédemment décrits comme étant régulés par STAT5 dans les érythroblastes. L ensemblede ces résultats établit un nouveau rôle pour ISG15 au cours de la différenciation érythroide, en plus de sesfonctions anti-virales bien caractériséesErythropoiesis is an orderly continuous process during which committed erythroid progenitorcells proliferate and differentiate into mature red blood cells. In a search for genes that are deregulated duringthis differentiation process, we have identified ISG15 as being induced during the late stages of erythroiddifferentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins bythe enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we haveshown that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6are induced during erythroid differentiation. Moreover, using in vitro differentiating erythroblasts, we haveshown that the induction of these genes is mostly independent of IFN signaling, while it is partially dependent onEpo signaling in these cells. Our analysis of the ISG15 deficient mice have shown a decreased number of BFUE/CFU-E in the bone marrow, associated with an increased number of these cells in the spleen of these animals,a phenotype reminiscent of stress erythropoiesis. While ISG15-/- bone marrow and spleen-derived erythroblastsshowed a less differentiated phenotype both in vivo and in vitro, over-expression of ISG15 in erythroblasts wasfound to facilitate erythroid differentiation. At the molecular level, we have shown that important effectors oferythroid differentiation can be ISGylated, including STAT5, Globin, PLCg and ERK2. Attempt to identify theconsequences of ISGylation has only been performed for STAT5. When studied in the context of a fusionprotein, ISGylated STAT5 was found endowed with a higher capacity to bind DNA, a property associated withup-regulation of TfR and Bcl-XL, two genes previously described as being regulated by STAT5 during erythroiddifferentiation. This establishes a new role for ISG15, in addition to its well-characterized anti-viral functions,during erythroid differentiation.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

Pires-Oliveira M, Maragno AL, Parreiras-E-Silva LT, Chiavegatti T, Gomes MD, Godinho RO. Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo. J Appl Physiol 108: 266-273, 2010. First published November 19, 2009; doi:10.1152/japplphysiol.00490.2009.-Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[05/59006-1]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)FAPESP[2006/58629-8

FBXO25-associated nuclear domains: A novel subnuclear structure

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [FAPESP][03/08055-7]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [06/58140-9](FAEPA) Fundacao de Apoio ao Ensino, Pesquisa e Assistenci

<i>ISG15</i> deficiency impairs <i>in vitro</i>-induced erythroid differentiation.

<p>(<b>A</b>) Erythroblasts from the BM of either wild-type or <i>ISG15^-/-</i> mice were maintained under proliferation conditions (SCF, Epo, Dex) and switched to Epo alone to induce differentiation. Cumulative cell number of three independent wild-type (black symbols) and <i>ISG15^-/-</i> erythroblasts cultures (open symbols) are shown both under proliferation and differentiation conditions. Cells were counted with an electronic counter (CASY Scharfe). (<b>B</b>) Quantitative determination of hemoglobin contents of differentiating WT and <i>ISG15^-/-</i> erythroblasts 24, 48 and 72 hours after differentiation induction. Normalized values (hemoglobin level per 10⁶ live cells) are shown. (<b>C</b>) Cytocentrifugation analyses followed by Benzidine-May-Grunwald staining of cells maintained either under proliferation conditions (day 0) or induced to differentiate in response to Epo. Differentiating cells are stained in brown by Benzidine (black arrow), immature eryhroblasts stain in blue. Note the presence of a significant proportion of immature cells in <i>ISG15^-/-</i> culture (open arrow) as compared to WT culture. Representative fields are shown. (<b>D</b>) Quantification of cells of increasing maturity 24 h, 48 h and 72 h after the cells had been induced to differentitate. Cells (≥200) were counted per slide and mean values ±s.d. calculated from at least three independent experiments.</p

<i>ISG15</i> expression during erythroid differentiation is independent of IFN signaling and partially dependent upon Epo signaling.

<p>(<b>A</b>) semi-quantitative RT-PCR analyses of <i>ISG15, Ube1L, UbcM8, Herc6, Irf7, Bcl-x_L</i> and <i>ß-Major-Globin</i> mRNA expression in WT or <i>IFNAR^-/-</i> differentiating primary erythroblasts. Bone marrow erythroblasts of each genotype were maintained in proliferation conditions (SCF, Epo and Dex) for one week and next induced to differentiate in response to Epo alone for three days. Cells were collected every 24 hours as indicated and RNA extracted. The experiment was normalized to <i>ß-Actin</i> mRNA expression. (<b>B</b>) Statistical analysis of the induction of the expression of <i>ß-Maj-Globin, Bcl-X_L</i>, <i>ISG15</i>, <i>Ube1L</i>, <i>UbcM8</i> and <i>Herc6</i> mRNA in differentiating WT and IFNAR^-/- erythroblasts. Quantification was performed as described in Mat & Met. Note the unchanged expression of a second housekeeping gene <i>HPRT</i>. au = arbitrary unit. (<b>C</b>) Whole cell protein extracts were prepared from WT or <i>IFNAR^-/-</i> erythroblasts maintained as in A and analyzed on a 10% acrylamide gel for <i>ISG15</i> expression using an anti-ISG15 antibody. Anti-ß-Actin and anti-GAPDH were used as loading controls. (<b>D</b>) Statistical analysis of the induction of ISG15 during erythroid differentiation as normalized to ß-Actin. Quantification was performed as described in Mat & Met. au = arbitrary unit. (<b>E and F</b>) A p53^-/- erythroid cell line expressing exogenous hBcl-X_L was switched from proliferation conditions (Epo, SCF, Dex) to differentiating medium in the presence or absence of Epo. Cells were collected every 24 hours as indicated and analyzed for their ability to (e) survive as measured by propidium iodide staining in flow cytometry analyses; (f) differentiate as shown by analysis of their morphology after benzidine/May-Grunwald staining. Note the significant induction of cell death in control cells maintained in absence of Epo; in contrast, hBCL-xL erythroblasts are strongly protected from apoptosis. (<b>G</b>) Cells were lyzed according to cell number and volume. ISG15 expression was analyzed on a 15% acrylamide gel using anti-ISG15 antibody, activation of the EpoR/STAT5 signaling pathway was monitored using anti-P-STAT5 antibody, differentiation was monitored using anti-Globin antibody and loading control was performed using anti-SAM68 antibody. (<b>H</b>) Mock and mscv-puro-STAT5^S710F transduced p53^-/- erythroid cell line maintained under proliferation conditions were lyzed and analyzed for ISG15 expression using anti-ISG15 antibody on a 10% acrylamide gel (Top panel). P-STAT5 was detected at a higher level in mscv-puro-STAT5^S710F transduced cells while only a modest increase in the total amount of STAT5 can be noted. Anti-β -Actin was used as a loading control.</p

<i>ISG15</i> expression and protein ISGylation are induced during <i>in vivo</i> erythroid differentiation.

<p>(<b>A</b>) Sorting procedure of the different erythroblasts populations from bone marrow cells of WT Bl6/J mice. (<b>B</b>) RT-PCR analyses of <i>ISG15, UbcM8, Herc6, Ube1L, Bcl-X_L</i> and <i>ß-Major-Globin</i> mRNA in sorted Pro-erythroblasts (Pro-E: large cells, CD71^high, Ter119^med), basophilic erythroblasts (EryA: large cells, CD71^high, Ter119^high), polychromatic (EryB: small cells, CD71^med, ter119^high) and orthochromatic erythroblasts/reticulocytes (EryC: small cells, CD71^low, ter119^high) as described in Mat & Met. Experiments were normalized to 18S Ribosomal RNA expression. (<b>C</b>) Statistical analysis of the induction of the expression of <i>ß-Maj-Globin, Bcl-X_L, ISG15</i>, <i>Ube1L, UbcM8</i> and <i>Herc6 m</i>RNA. Quantification was performed as described in Mat & Met. Note the two-fold decreased expression of a second housekeeping gene, ß-actin during differentiation. au = arbitrary unit. (<b>D</b>) Western blot analyses of whole cell extracts of the indicated erythroid subpopulations using anti-ISG15 (top panels) antibody. Middle panels show GAPDH levels as loading control. Bottom panels show globin accumulation as detected with Ponceau staining of the membranes. Whole cell extracts were prepared from sorted wild-type bone marrow cells as in A or from RBC of WT and <i>ISG15^-/-</i> mice. (<b>E</b>) Statistical analysis of the induction of ISG15 at the protein level during <i>in vivo</i> erythroid differentiation normalized to ß-Actin. Quantification was performed as described in Mat & Met. au = arbitrary unit.</p

Identification of several ISGylation substrates in erythroid cells and increased ubiquitination in <i>ISG15^-/-</i> RBCs.

<p>(<b>A</b>) Proliferating erythroblasts were left either untreated (1), or stimulated with IFNß (100 U/ml) for 24 h (3), or induced to differentiate for 72 h (lane 4 to 8). Cells were lyzed according to size and number and ISG15/ISGylation level was compared by western blotting to the level of ISG15/ISGylation present in proliferating Flag-ISG15-expressing p53^-/- erythroid cell line. The arrow points to ISG15 band (<b>B</b>) Quantification of ISG15 level in independent western blot experiments. (<b>C</b>) Proliferating control and Flag-ISG15-expressing p53^-/- erythroid cell line were lysed and ISGylated proteins searched after a Flag immunoprecipitation. Anti-Flag detect ISG15-Flag and ISGylated proteins (arrow heads) in the crude lysates (dashed line) and are found enriched after the immunoprecipitation (plain line bar). Stars point to light and heavy chains of immunoglobulin. For the detection of ISGylated Globins, cells were induced to differentiate for 72 hours in order to induce globin expression. ISGylated proteins are indicated by arrowheads. Normal unmodified molecular weight of the proteins are: ERK2 (42kDa), PLCγ (150kDa), STAT5 (90kDa), Globin (13kDa). For ERK2 and STAT5 detection, cell lysates were run on a 10% acrylamide gel, for ISG15 and Globins detection on a 15% acrylamide gel and for PLCγ on a 8% gel. (<b>D</b>) Proliferating or 48 hours-differentiating control and STAT5-Flag-expressing p53^-/- cell line were lysed and analyzed for exogenous STAT5 expression either using a Flag (upper left panel) or a STAT5 antibody (upper right panel). Note the modest increase in the total amount of STAT5 induced by the expression of STAT5-Flag. ISGylated STAT5 was searched after a Flag immunoprecipitation followed by either a Flag (bottom left panel) or an ISG15 western blot analyses (bottom right panel). Extracts were run on a 7% acrylamide gel. (<b>C</b>) Western blot analysis of RBC extracts from WT and <i>ISG15^-/-</i> mice using a 15% acylamide gel. ISG15 expression and ISGylation were analysed using anti-ISG15 antibody (top panel), ubiquitination was monitored using anti-ubi antibody (intermediate panel) and anti-β-Actin was used as a loading control.</p