The role of LEKTI in fate determination of keratinocyte stem cells

SPINK5 (serine protease inhibitor Kazal-type 5) encodes the putative proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor). In skin, LEKTI expression is restricted to the stratum granulosum of the epidermis and the inner root sheath of hair follicles. Mutations that create premature termination codons in SPINK5 have been reported as the cause of Netherton syndrome (NS), a human autosomal recessive disorder characterized by congenital ichthyosis with defective cornification, a specific hair shaft defect known as trichorrexis invaginata or 'bamboo hair', and severe atopic manifestations, including atopic dermatitis and hayfever. Althought recombinant human LEKTI inhibits a battery of serine proteases including plasmin, trypsin, subtilisin A, cathepsin G, and elastase, the precise role of LEKTI in the physiopathology of NS remains unclear. Spink5−/− mice display a NS-like phenotype. Surprisingly, a psoriasis-like hyperplasia, basement membrane breakdown followed by evagination of spindle-shaped epidermal cells into the dermal compartment, and the presence of numerous sweat gland-like structures were also observed when the skin of Spink5−/− newborn mice, which die at birth, was transplanted onto the back of nude mice. Collectively, these observations suggest that LEKTI may play a role on cell proliferation and stem cell fate. Our current work aims at elucidating the mechanisms by which LEKTI impact these biological processes. Using keratinocyte stem cells obtained from NS patients, we have identified LEKTI as a regulator node in several signaling pathways involved in stem cell behavior.

Follicular Peripheral T-cell Lymphoma Expands the Spectrum of Classical Hodgkin Lymphoma Mimics.

Epstein-Barr virus (EBV)-infected B cells with Reed-Sternberg-like cell (RS) features may occur in peripheral T-cell lymphomas (PTCLs), especially in angioimmunoblastic T-cell lymphoma. Here, we report 5 patients presenting with lymphadenopathy whose first biopsies demonstrated nodular lymphoid proliferations containing scattered CD30, CD15, EBV Hodgkin and Reed-Sternberg-like cells, which led to an initial diagnosis of lymphocyte-rich classical Hodgkin lymphoma. However, the uncommon clinical features and/or the occurrence of relapse as PTCL prompted review of the biopsies with expanded immunohistologic and molecular studies and revision of the diagnoses to follicular variant of PTCL (F-PTCL). All cases had atypical small to medium-sized CD3 T cells that expressed CD10 (4/5) and the follicular helper T-cell (TFH) antigens BCL6, PD1, CXCL13, and ICOS. All demonstrated clonal T cells with a similar pattern in multiple samples from 4 patients. In 2 cases, flow cytometry demonstrated circulating lymphocytes with an abnormal sCD3, CD4, ICOS immunophenotype. Two patients had a skin rash at presentation, and 1 had B symptoms. Two of the 4 patients treated with polychemotherapy are alive at 3 and 6 years after first diagnosis. These cases highlight how some F-PTCLs may closely mimic lymphocyte-rich classical Hodgkin lymphoma requiring careful assessment of the T cells before rendering the latter diagnosis. The functional properties of TFH cells might lead to the presence of EBV-positive B blasts with RS-like features in TFH-derived PTCL such as angioimmunoblastic T-cell lymphoma and F-PTCL.

Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions.

A new culture model was developed to study the role of proliferation and apoptosis in the etiology of keloids. Fibroblasts were isolated from the superficial, central, and basal regions of six different keloid lesions by using Dulbecco's Modified Eagle Medium containing 10% fetal calf serum as a culture medium. The growth behavior of each fibroblast fraction was examined in short-term and long-term cultures, and the percentage of apoptotic cells was assessed by in situ end labeling of fragmented DNA. The fibroblasts obtained from the superficial and basal regions of keloid tissue showed population doubling times and saturation densities that were similar to those of age-matched normal fibroblasts. In contrast, the fibroblasts from the center of the keloid lesions showed significantly reduced doubling times (25.9 +/- 6.3 hours versus 43.5 +/- 6.3 hours for normal fibroblasts) and reached higher cell densities. In long-term culture, central keloid fibroblasts formed a stratified three-dimensional structure, contracted the self-produced extracellular matrix, and gave rise to nodular cell aggregates, mimicking the formation of keloid tissue. Apoptotic cells were detected in both normal and keloid-derived fibroblasts, but their numbers were twofold higher in normal cells compared with all keloid fibroblasts. To examine whether apoptosis mediates the therapeutic effect of ionizing radiation on keloids, the cells were exposed to gamma rays at a dose of 8 Gy. Under these conditions, a twofold increase in the population of apoptotic cells was detected. These results indicate that the balance between proliferation and apoptosis is impaired in keloid fibroblasts, which could be responsible for the formation of keloid tumors. The results also suggest that keloids contain at least two different fibroblast fractions that vary in growth behavior and extracellular matrix metabolism.

Investigating mineralocorticoid hypertension.

About 3% of our hypertensive patients have high blood pressure induced by corticosteroids. Muscle weakness, tiredness, polyuria and polydipsia may indicate hypokalaemia. Hypokalaemic hypertension in the presence of a low plasma renin activity is the typical finding of corticosteroid hypertension. The most frequent cause of corticosteroid hypertension is primary aldosteronism (Conn's syndrome) due to an adrenal adenoma or bilateral hyperplasia of the adrenal glands. The plasma concentration of aldosterone and the ratio between plasma aldosterone and renin concentrations are high, and the kaliuresis exceeds 30 mmol/24 h in the presence of hypokalaemia. Adrenal carcinomas are rare and very malignant. The localization of an adrenal tumour is made by computer tomography (CT-scan) or nuclear magnetic resonance imaging and by measurement of the aldosterone/cortisol concentrations in the adrenal venous blood. Adenomas are removed under laparoscopy, and adrenal hyperplasias are treated with spironolactone (50-400 mg daily) or amiloride (5-30 mg daily). In rare cases (<1%), excessive stimulation of the mineralocorticoid receptor is due to cortisol (apparent mineralocorticoid excess, Cushing's disease, liquorice, or hereditary deficiency of 11beta-hydroxysteroid dehydrogenase) or to a chimeric gene coding for 11beta-hydroxylase (CYP11B1/CYP11B2). In these rare cases, the synthesis of aldosterone is under the control of the adrenocorticotrophic hormone, so treatment with glucocorticoids (dexamethasone 0.25-1.0 mg daily) is therefore possible (glucocorticoid-remediable aldosteronism). Excessive deoxycorticosterone (DOC) causes the same symptoms and signs as hyperaldosteronism. Excessive DOC is found in patients with adrenal tumours that secrete DOC, in those with hereditary or acquired disorders with dysfunctioning glucocorticoid receptors, or in those with congenital hyperplasia of the adrenal glands (deficiency of 17alpha-hydroxylase or 11beta-hydroxylase). Liddle's syndrome is a constitutive hyperactivity of the transepithelial transport of sodium, which under normal conditions is controlled by the mineralocorticoid receptor. Plasma renin and aldosterone concentrations are suppressed and the plasma potassium concentration may be normal. In contrast, plasma aldosterone and renin concentrations are increased in patients with hypokalaemic hypertension which represents secondary aldosteronism. The increased aldosterone is the consequence of stimulated renin activity due to renal or renovascular or other disorders, antihypertensive drugs or other medications. In conclusion, a work-up for corticosteroid-induced hypertension is indicated in patients with hypokalaemic hypertension and in those with severe hypertension even in the absence of hypokalaemia, and in hypertensive patients with a family history of cardiovascular diseases.

Overexpression of a mutant form of TGFBI/BIGH3 induces retinal degeneration in transgenic mice.

PURPOSE: Despite ubiquitous expression of the keratoepithelin (KE) protein encoded by the transforming growth factor beta induced/beta induced gene human clone 3 (TGFBI/BIGH3) gene, corneal dystrophies are restricted to the cornea, and no other tissues are affected. We investigated the role of TGFBI/BIGH3 in Groenouw corneal dystrophies by generating transgenic mice overexpressing TGFBI/BIGH3 containing the R555W mutation. METHODS: Transgenic animals expressing the Groenouw mutation of human TGFBI/BIGH3 were generated using lentiviral vectors. The line expressed TGFBI/BIGH3 containing the R555W mutation under the control of the phosphoglycerate kinase (PGK) promoter. Expression of the transgene was monitored by Southern and western blotting and by RT-PCR. Electroretinogram analysis was performed and four mice were subjected to complete necroscopy. RESULTS: Transgene expression was observed in different organs although without specific expression in the cornea. The overall morphology of the transgenic animals was not severely affected by KE overexpression. However, we observed an age-dependent retinal degeneration both functionally and histologically. Female-specific follicular hyperplasia in the spleen and increased levels of lipofuscin in the adrenal gland were also seen in transgenic animals. CONCLUSIONS: Cellular degeneration in the retina of transgenic animals suggest that perturbation of the transforming growth factor beta (TGFbeta) family regulation may affect photoreceptor survival and may induce possible accelerated aging in several tissues. No corneal phenotype could be observed, probably due to the lack of transgene expression in this tissue.

Geología del Montgrí

This paper, fundamentally stratigraphic, is based on the vertical distribution of fauna in sediments which constitute the Montgri Massif. The stratigraphic series is composed of two units: one allochtonous (Mesozoic) and a second one autochtonous (Paleogene). The latter crops out in front of the thrust belt and presents the typical facies described by PALLI (1972). The allochtonous part is formed by the following units (from bottom to top): variegated gypsiferous shales of Keuper age; marls, limestones and dolomites from the Lias (Domerian); gray breccia ted dolomites: supraliassic; and Cretaceous limestones with rare interbeds of calcareous marls and nodular shaly marls. In this unit the Neocomian, Barremian (?), Aptian, Albian, Cenomanian, Turonian (?) and Santonian haven been recognized. Structurally, the Montgri Massif is part of a thrust belt which has been divided into three parts of different development ang age. Both parts are affected by ample synclinal folds of NE-SW directions and fractures of NW-SE orientation. The fractures condition the torrential streams as well as the karstic phenomena present in the Massif. These and the eolian actions are the main causes of the present geomorphology of the Montgri

Quelle attitude face à une masse médiastinale antérieure chez l'adulte [Management of anterior mediastinal masses in adults].

The discovery of an anterior mediastinal mass requires careful management with specific consideration of the pathology. More than 50% of all mediastinal masses seen in adults are in the anterior mediastinum. The most frequent diagnoses are thymoma, lymphoma, teratoma and benign thyroid tumours. 60% of cases are malignant. Often the clinical and radiological findings do not allow a definitive diagnosis and a histological diagnosis is often required to select the optimal treatment modality. The choice of biopsy technique depends on the localization of the lesion, clinical factors, and the availability of special techniques and equipment. Biopsy may be obtained by trans-thoracic puncture under computed tomography or ultrasound guidance, or by a surgical approach (mediastinotomy or thoracoscopy).

A new infectious mammary tumor virus in the milk of mice implanted with C4 hyperplastic alveolar nodules.

We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates V beta 2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with V beta 3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.

Atuação de Acidithiobacillus na solubilização de fosfato natural em solo de tabuleiro cultivado com jacatupé (Pachyrhizus erosus)

Realizou-se um experimento em casa de vegetação para avaliar a atuação do enxofre (S) inoculado com Acidithiobacillus na disponibilidade de fósforo (P) de fosfato natural (FN), em diferentes modos de aplicação do fertilizante, em um Espodossolo Ferrocárbico Órtico, do tabuleiro costeiro da Zona da Mata de Pernambuco, cultivado com jacatupé (Pachyrhizus erosus). As plantas foram inoculadas com rizóbio (NFB 747 e NFB 748) e adicionado tratamento-controle sem inoculação com rizóbio. Os tratamentos com P foram: (1) FN revestindo o enxofre inoculado com Acidithiobacillus (FN S*), (2) FN revestido com S e com Acidithiobacillus (S* FN), (3) mistura FN com S e com Acidithiobacillus (FN + S*), (4) mistura FN com S e sem Acidithiobacillus (FN + S), (5) superfosfato triplo (ST) e (6) sem aplicação de fósforo (P0). Os tratamentos foram aplicados: (a) na superfície, (b) em sulco 10 cm abaixo da semente, e (c) em sulco 10 cm abaixo e ao lado da semente. A biomassa nodular foi maior no tratamento (FN + S*) com melhor efeito quando aplicado ao lado e abaixo das sementes. O (FN + S*) também aumentou a altura das plantas, a biomassa da parte aérea e das túberas e o N total da parte aérea, especialmente quando aplicado 10 cm abaixo das sementes. O P total da parte aérea foi mais elevado com (S* FN) aplicado 10 cm abaixo das sementes. Os melhores teores de P no solo foram obtidos com (FN + S*) e (S* FN), com efeito mais evidente quando aplicados 10 cm abaixo das sementes.

Role of Notch signaling in epidermis and appendages

Résumé Dans la peau, il a été montré que Notch1 induit l'arrêt de la prolifération et la différentiation des keratinocytes. L'inactivation de Notch1 cause une hyperplasie de l'épiderme et la formation de carcinomes basaux cellulaires. Notre groupe a principalement identifié deux voies de signalisations, la voie Shh et la voie Wnt, qui sont dérégulées en conséquence de l'inactivation de Notch1 dans la peau. Nous avons démontré l'habilité de Notch1 à réprimer la voie Wnt induite par ß-catenin dans les keratinocytes primaires ainsi que dans d'autres types de cellules épithéliales humaines. De plus, nous avons pu déterminer que Notch1 régule cette voie, probablement en favorisant la phosphorylation de ß-catenin par le complexe axin/APC/GSK-3ß. La protéine faisant partie de la voie Wnt, ou la protéine affectant la voie Wnt, qui est régulée par Notch1 est sujette à de plus amples investigations. Un autre but de cette étude a été l'identification de potentiels gènes cibles de Notch1 autres que ceux faisant partie des voies de signalisation Shh et Wnt précédemment évoquées. Ce projet fut abordé par l'analyse de puces à ADN (ISREC et Affymetrix) qui ont été utilisées pour des expériences de gain et de perte de fonction de Notch1 dans des keratinocytes prúmaires. En plus de l'hyperplasie épidermale, les souris Notch1 déficiente ont une perte importante de poils. Nous avons montré que Notch1 est nécessaire pour le développement et l'homéostasie des follicules pileux. En effet, l'inactivation du gène Notch1 mediée par l'activation des kératines 5 ou 14 dans l'épiderme, cause des défauts du cycle ainsi que de la structure des poils. De plus, d'autres appendices de la peau, comme les glandes sudoripares et de Meibomius, ont une structure anormale et sont non fonctionnelles dans les souris Notch1 déficiente. Finalement, nous avons observé que la déficience de Notch1 dans l'épithélium cornéen mène à la formation d'une plaque épidermale opaque sur la cornée. Basé sur l'hypothèse que le défaut des glandes de Meibomius des souris Notch1 déficientes cause des lésions de la surface oculaire, nous avons montré que Notch1 est essentiel pour la cicatrisation de la cornée. Lorsque Notch1 est absent, les cellules souches de l'épithélium cornéen ne sont plus capables de se différentier en cellules cornéennes, mais réparent la blessure en se différentiant en épiderme. Ce résultat indique que Notch1 est essentiel pour la différentiation de cellules souches de la cornée qui sont spécifiquement impliquées dans la réparation de la cornée. De plus, nous avons montré que l'expression de CRBP1 dans l'épithélium cornéen est diminuée en l'absence de Notch1, ceci étant possiblement à l'origine de la formation de la plaque épidermale. Abstract: In the skin, Notch1 has been shown to trigger cell growth arrest and differentiation of keratinocytes. Notch1 inactivation results in epidermal hyperplasia and subsequent formation of basal cell carcinoma-like (BCC-like) tumors. So far our group has identified two main pathways, the Shh and the Wnt pathway, that are deregulated as a consequence of Notch1 inactivation in the skin. We showed the ability of Notch1 to represses ß-catenin-mediated Wnt signaling in primary keratinocytes as well as in other types of human epithelial cells. In addition we were able to determine that Notch1 regulates this pathway possibly by enhancing ß-catenin phosphorylation by the axin/APC/GSK-3ß complex. The exact target protein of the Wnt pathway or target protein that affects the Wnt pathway, and that is regulated by Notch1, is subject of current investigation. Another aim of this study was the identification of possible Notch1 target genes in addition to those of the Shh and Wnt signaling pathways. This was addressed by gene chip analysis using ISREC as well as Affymetrix microarrays for gain and loss of function of Notch1 in mouse primary keratinocytes. In addition to epidermal hyperplasia, Notch1 deficient mice show an important hair loss. We showed that Notch1 is required for postnatal development and homeostasis of hair follicles. Indeed, keratin5 or keratinl4-driven Cre recombinase-mediated inactivation of the Notch1 gene in the epidermis causes perturbations of the hair cycle and structural defects of the hair follicle. Moreover, other skin appendages, like the sweat and Meibomian glands show abnormal morphology and are not functional in the Notch 1 deficient mice. Finally, we observed that Notch1 deficiency in the corneal epithelium leads to the formation of an epidermal corneal plaque. Based on the hypothesis that the Meiboinian gland defect in the Notch1 deficient mice results in lesions of the eye surface, we showed that Notch1 is essential for wound-healing of the cornea. In absence of Notch1 the stem cells of the corneal epithelium are no longer able to differentiate in the corneal fate but instead repair the wound by differentiating into skin-like epidermis. This result indicated that Notch1 is essential for the differentiation of corneal stem cells specifically implicated in corneal wound-healing. Moreover, we showed that CRBP1 expression in the corneal epithelium was lost in the absence of Notch1, possibly being at the origin of plaque formation.

Eficiência e competitividade de variantes espontâneos isolados de estirpes de Bradyrhizobium spp recomendadas para a cultura da soja (Glycine max)

O cultivo sucessivo de soja inoculada numa mesma área proporcionou a adaptação de uma população de rizóbios, que podem não ser tão eficientes quanto à capacidade de fixação de N2, mas apresentam alta competitividade, dificultando a introdução de novas estirpes mais eficientes. Com a finalidade de avaliar o desempenho simbiótico (eficiência e competitividade) de variantes espontâneos isolados de estirpes de B. japonicum (SEMIA 5079 e SEMIA 5080) e B. elkanii (SEMIA 587 e SEMIA 5019), realizou-se um experimento em casa de vegetação onde os variantes foram inoculados isoladamente e em diferentes combinações entre os variantes e uma estirpe comprovadamente mais competitiva (SEMIA 587 ou SEMIA 5019) a partir da adição de inóculos mistos (1/1; v/v) no cultivar de soja BR-16. Por meio da avaliação das variáveis analisadas (nodulação, produção de matéria de seca da parte aérea, N total acumulado na parte aérea e ocupação nodular), foi possível constatar que o determinante da maior eficiência em tratamentos co-inoculados não foi a ocupação nodular de determinada estirpe ou variante presente no inóculo, mas, sim, o tipo de interação (sinérgica ou antagônica) predominante no tratamento co-inoculado e que é possível selecionar variantes eficientes e competitivos para a cultura da soja a partir de estirpes parentais que já apresentam características desejáveis para utilização em inoculantes comerciais.

Inflammation palpébrale pseudotumorale. A propos d'une observation anatomo-clinique [Pseudotumoral eyelid inflammation. Apropos of an anatomo-clinical case].

A clinicopathological case of a 76-year-old male patient with a chronic inflammatory change of the inferior left eyelid is reported. The inflammation appeared as a reddish area of the inner part of the eyelid, without sharp limits, but with loss of lashes. Numerous local treatments did not to cure this condition. As some true eyelid tumors may mimic an inflammation during growth and, for example, sebaceous carcinoma may clinically present as chronic unilateral blepharitis, a surgical excisional biopsy was performed on this left eyelid. Its histopathological study showed a granulomatous inflammation, which was typical of a simple chalazion. This case clearly illustrates that the chalazion may not always appear as a limited nodular inflammation of the eyelid, but may have a more diffuse clinical presentation.

Avaliação da biodiversidade de rizóbios simbiontes do feijoeiro (Phaseolus vulgaris L.) em Santa Catarina

Os solos brasileiros, em geral, apresentam uma população abundante de rizóbios capazes de nodular e fixar N2 em simbiose com o feijoeiro (Phaseolus vulgaris L.); contudo, a diversidade dessas bactérias ainda é pouco conhecida. Este estudo teve por objetivo conhecer a biodiversidade de microssimbiontes do feijoeiro em Santa Catarina e, para isso, foram obtidos 117 isolados de nódulos de plantas coletadas em campo, em 23 áreas do extremo oeste, do meio oeste e do planalto sul catarinense. Com base nos atributos morfofisiológicos, os isolados foram classificados em nove grupos. Pela análise dos perfis de DNA após a amplificação (PCR) com o "primer" BOX, que codifica regiões conservadas e repetidas do genoma, 107 perfis distintos foram agrupados em um nível final de similaridade de apenas 26,9 %. Os perfis obtidos pela amplificação do gene 16S ribossômico - referência na taxonomia atual de procariotos - seguida pela digestão com três enzimas de restrição (técnica de RFLP-PCR), resultaram em seis agrupamentos principais e cinco bactérias isoladas. As populações consistiram de 17,1 % de Rhizobium tropici, 35,9 % de R. etli, 32,5 % de R. leguminosarum, 1,7 % de R. giardinii e 12,8 % com perfis distintos das espécies descritas de rizóbios de feijoeiro. R. tropici predominou em solos ácidos do meio oeste e do planalto sul, R. leguminosarum não foi detectado no extremo oeste e R. etli ocorreu nas três regiões, essas duas últimas espécies em solos menos ácidos. Os resultados enfatizam a diversidade genética elevada de rizóbios, inter e intra-específica, nos solos catarinenses, inclusive com a indicação de novas espécies.

Specific (granulomatous) oral lesions of sarcoidosis: Report of two cases

Sarcoidosis is a multisystemic granulomatous disease of unknown etiology that usually presents with bilateral hiliar adenopathies, pulmonary infiltrations, and ocular and cutaneous lesions. The specific cutaneous lesions, or granulomatous, are produced in from 9% to 37% of patients with systemic sarcoidosis. Nevertheless, few patients have been described having specific lesions from sarcoidosis in the oral mucous membrane. We present 2 patients with systemic sarcoidosis who developed specific sarcoidosis lesions in the oral mucous membrane. The first patient presented a plaque on the tongue of a chronic nature associated with facial lupus pernio. The second patient presented a rapidly growing nodular lesion on the lower lip that was in fact the presentation sign of the disease. Although infrequent, oral lesions may constitute the first sign of systemic sarcoidosis. For this reason the oral mucous membrane needs to be explored when there is suspicion of sarcoidosis, and systemic sarcoidosis must be included in the differential diagnosis of oral granulomatous lesions.

Immunolocalization of neuropeptide Y in human pancreatic endocrine tumors.

Neuropeptide Y (NPY) is a 36 amino acid peptide known to inhibit glucose-stimulated insulin secretion. NPY has recently been shown to be synthetized within rat islets of Langerhans and to be secreted in a differentiated rat insulin-secreting cell line, and as to this date the localization of NPY in human endocrine pancreas has not been reported. As NPY shares high amino acid sequence homology with peptide YY (PYY) and pancreatic polypeptide (PP), the polyclonal antibodies raised against these peptides often cross-react with each other. To demonstrate the presence of NPY in the human endocrine pancreas, we used a highly specific monoclonal antibody raised against NPY and another against its C-flanking peptide (CPON). We studied three cases of hyperplasia of Langerhans islets and 11 cases of endocrine tumors of the pancreas. NPY and CPON were detected in all three cases of hyperplasia. For the 11 pancreatic tumors, five and nine of the tumors were positive for the antibodies NPY and CPON, respectively. The two negative tumors for CPON immunoreactivity were differentiated insulinomas, which showed no evidence of other hormonal secretion. In normal Langerhans islet, NPY and CPON immunoreactivities were colocalized in glucagon-producing cells (alpha-cells) and in a few insulin-secreting cell (beta-cells).(ABSTRACT TRUNCATED AT 250 WORDS)