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Overexpression of heat shock protein 90 as a potential therapeutic target for angiosarcoma
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Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
the potential use of L. pentosus GMNL-77 in the future treatment of psoriasis.yf5126. http://dx.doi.org/10.1016/j.jdermsci.2017.02.050 P01-48[O1-30] The therapeutic potential and molecular mechanism of flavonoid HCF-A on inflammatory skin diseases Hsin-Ju Li 1,∗ , Chi-Feng Hung 2 1 Department of Chemstry, Fu Jen Catholic University, New Taipei City, Taiwan 2 School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan In recent years, there is increasing incidence and prevalence of diverse chronic inflammatory skin diseases, such as psoriasis and atopic dermatitis. However, the current management is usually not satisfactory due to the possibility of poor efficacy, severe side effects, high expenses or frequent recurrence. Therefore, it is urgent and important to develop novel drugs in the treatment of these troublesome inflammatory skin diseases. With the recent advancement in the molecular and cellular biology, the molecular pathogenesis of these diseases has been gradually teased out. Previous studies have shown that they are mostly related to the dysregulation of immune responses and aberrant protein expression. Many studies also found that the production of reactive oxygen species, and release of inflammatory cytokines, such as IFNs, IL-17 and TNFalpha, play critical roles in the inflammatory skin diseases. The potential applications of Chinese herbal medicine in the treatment of these diseases have also been reported. Our results revealed that flavonoid HCF-A could reduce IL-17A or TNF-alphainduced MAPK phosphorylation, NF-kappa B activation, CCL20 release, and inflammatory makers mRNA expression from epidermal keratinocytes. Most importantly, flavonoid HCF-A could obviously attenuate imiquimoid-induced psoriasis-like skin lesions in mice, and improve the imiquimod-induced disrupted skin barrier function. We believe that exploration of the therapeutic potential of these herbal medicines will not only provide novel pharmacologic agents in the clinical treatment of inflammatory skin diseases but also contribute to the academic advancement in the field of Chinese herbal medicine.
http://dx.doi.org/10.1016/j.jdermsci.2017.02.051 Category 2 (P02): Carcinogenesis/Growth Factors/Signal Transduction/Cancer Genetics P02-01[I-1] Mechanistic insight into the ATP-induced fibrosis in systemic sclerosis Buddhini Perera ∗ , Akihiko Uchiyama, Akihito Uehara, Kazuya Yamada, Sachiko Ogino, Yoko Yokoyama, Osamu Ishikawa, Sei-ichiro Motegi Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan ATP is released from cells by tissue injury/hypoxia, and initiates an autocrine/paracrine signaling for the inflammation. It has been considered that vasculopathy induces hypoxia and oxidative stress in the dermis in SSc, suggesting that hypoxia-induced oxidative stress enhances ATP release from cells, such as fibroblasts
and endothelial cells. However, the role of ATP in the pathogenesis of skin fibrosis in SSc is unknown. Objective was to elucidate the role of ATP in skin fibrosis in SSc. IL-6 is a potent stimulator of collagen production in fibroblasts. ATP enhanced IL-6 production more significantly in SSc fibroblasts than in normal fibroblasts. ATP binds both P2X and P2Y receptors to activate the downstream signaling. There were no significant differences in the expression levels of P2X7, P2Y2 and P2Y4 receptors between normal and SSc fibroblasts treated with or without ATP. Furthermore, the ectonucleotidases CD39 and CD73 were not expressed in normal and SSc fibroblasts. Non-selective P2 receptor antagonist, suramin, inhibited ATP-induced IL-6 production and phosphorylation of p38 in SSc fibroblasts. ATP-induced IL-6 production was significantly inhibited by p 38 inhibitor, SB203580, suggesting that ATP-induced phosphorylation of p38 via P2 receptor enhances IL-6 production in SSc fibroblasts. We previously found that norepinephrine (NE) also induced IL-6 production in SSc fibroblsts. Combined treatment with ATP and NE resulted in an additive increase in IL-6 production in SSc fibroblasts. These results suggest that vasculopathy-induced hypoxia and oxidative stress might enhance ATP release in the dermis in SSc, and ATP might contribute to the skin fibrosis via potentiation of IL-6 and collagen type I production from fibroblasts in SSc. http://dx.doi.org/10.1016/j.jdermsci.2017.02.052 P02-02[C12-1] Overexpression of heat shock protein 90 as a potential therapeutic target for angiosarcoma Saori Yamada-Kanazawa 1,∗ , Ikko Kajihara 1 , Satoshi Fukushima 1 , Masatoshi Jinnin 1 , Mamiko Masuzawa 2 , Mikio Masuzawa 3 , Yasuyuki Amoh 2 , Daichi Hoshina 4 , Riichiro Abe 5 , Hironobu Ihn 1 1 Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan 2 Department of Dermatology, Kitasato University School of Medicine, Japan 3 Department of Molecular Diagnostics, School of Allied Health Sciences, Kitasato University, Japan 4 Department of Dermatology, Hokkaido University Graduate School of Medicine, Japan 5 Department of Dermatology, Niigata University Graduate School of Medical and Dental Sciences, Japan The prognosis of angiosarcoma is very poor compared with that of other skin malignancies because advanced angiosarcoma is resistant to standard chemotherapy. Recently, heat shock protein 90 (HSP90) has been identified as a molecular chaperone to activate various cancer related proteins. In addition many clinical trials of HSP90 inhibitors are ongoing in several malignant tumors. Therefore, we investigated the possibility that inhibition of HSP90 had therapeutic potential to suppress angiosarcoma. HSP90 protein levels in cultured angiosarcoma cell lines were markedly increased compared to normal endothelial cells. Immunohistochemical analyses revealed the expression of HSP90 protein was strongly detected in angiosarcoma tissues compared with normal dermal vessels or senile angioma. Ganetespib, a HSP90 inhibitor, with or without taxanes inhibited the proliferation of angiosarcoma cells via apoptosis in dose-dependent manners. Inhibition of HSP90 suppressed migration and invasion of angiosarcoma cells.
Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
Knock down of HSP90 decreased several cancer-associated proteins including p-ERK and survivin. Taken together, HSP90 could be a novel therapeutic target for angiosarcoma. http://dx.doi.org/10.1016/j.jdermsci.2017.02.053 P02-03[C12-2] Class III phosphoinositide 3-kinase Vps34 is essential for epidermal homeostasis Takuya Nagai 1,∗ , Shin-Ichi Osada 1 , Yuta Mizusawa 1 , Tomoko Suzuki 1 , Satoshi Eguchi 2 , Junko Sasaki 2 , Takehiko Sasaki 2 , Motomu Manabe 1 1 Department of Dermatology and Plastic Surgery, Akita University Graduate School of Medicine, Akita, Japan 2 Department of Medical Biology, Akita University Graduate School of Medicine, Japan Vacuolar protein sorting 34 (Vps34) is the Class III phosphoinositide 3kinase that produces intracellular pools of phosphatidylinositol-3phosphate (PI3P), and regulates autophagosome and endosome formation by recruiting PI3P-binding proteins and additional factors. To examine the role of Vps34 in the epidermis, we generated Vps34 floxed (Vps34 flox/flox) mice, and deleted Vps34 specifically in the epidermis by crossing them with the keratin 5-Cre transgenic mice. Homozygous mutant mice died several hours after birth because of severe vacuolation in the epidermis, which hampered further investigation. To overcome the postneonatal death, we then generated CreERT2; Vps34 flox/flox mice, in which Vps34 could be deleted in a tamoxifen-dependent manner. Application of tamoxifen onto the mouse dorsal skin in 5 consecutive days led to a scaly thickened skin. Histopathological analysis revealed acanthosis, spongiosis and dyskeratotic cells in the epidermis and lymphocyte infiltration in the upper dermis, which resembled interface dermatitis. These results suggest that Vps34 is required for epidermal homeostasis and abnormal PI3P metabolism is involved in pathogenesis of inflammatory skin diseases.
http://dx.doi.org/10.1016/j.jdermsci.2017.02.054 P02-04[C12-3] Tumor-suppressive effects of interferon- through interleukin-24 in melanoma Yoshinori Watanabe ∗ , Yoshimasa Nobeyama, Munenari Itoh, Hidemi Nakagawa The Department of Dermatology, The Jikei University school of Medicine, Tokyo, Japan Background: Interferon beta (IFN-) is widespread used as a treatment against melanoma to improve its prognosis. Several literature has suggested that the sensitivity of melanoma cells to IFN- shows wide spectrum through in vivo and in vitro examinations including our previous study that RPMI-7951 and A375 is relatively sensitive to IFN-, while G361, SK-MEL-5 and SK-MEL-1 is relatively resistant. Interleukin24 (IL-24) has been reported to inhibit the tumor growth by inducing the tumor-selective apoptosis. This study was conducted to elucidate the relation with IL-24 in inhibition effects of IFN- on melanoma cells. Method: Quantitative real-time reverse transcription PCR (RTPCR) and enzyme-linked immunosorbent assay (ELISA) were carried out to detect amounts of IL-24 mRNA and proteins, respectively. Flow cytometry was performed to assess the tumor cell
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growth and apoptosis of melanoma cells treated with IL-24 using propidium iodide staining. Invasion assay was performed to evaluate an invasive capacity of the melanoma cells treated with IL-24. Result: RT-PCR revealed that IL-24 mRNA at day 5 after IFN- treatment was more expressed in IFN--sensitive cell lines (A375, RPMI-7951) than in IFN--resistant cell lines (G361, SK-MEL-5, SK-MEL1). ELISA revealed that IL-24 protein at day 5 after IFN- treatment was induced in 4.8-fold in RPMI-7951 and 1.9-fold in A375 cells, but the protein was not detected in either non-treated or treated G361 cells, SKMEL-5 cells or SK-MEL-1 cells. IL-24 treatment of terminal concentration of 100 g/ml decreased melanoma cells indicating G2/M period in the 5 melanoma cell lines. Conclusion: These results suggested that IFN- may inhibit melanoma cell proliferation through inducing IL-24 from melanoma cells. http://dx.doi.org/10.1016/j.jdermsci.2017.02.055 P02-05[C12-4] High expression of activation induced deaminase in recurrent or metastatic squamous cell carcinoma Takashi Yamaguchi ∗ , Daisuke Omoto, Yu Sawada, Sanehito Haruyama, Manabu Yoshioka, Etsuko Okada, Motonobu Nakamura Department of Dermatology, University of Occupational and Environmental Health, Japan Abnormally expressed activation-induced cytidine deaminase (AID), a member of a cytidine deaminase family, acts as a genome mutator that contributes to malignancy such as gastric cancer and hepatocellular carcinoma. We have recently shown that transgenic mice expressing AID in the epidermis under a keratin 14 promoter developed cutaneous squamous cell carcinoma (SCC) with Hras and Trp53 mutations spontaneously. However, the role of AID in human SCC had not been fully investigated. To explore the correlation between the AID expression and clinical manifestations of SCC, we performed an immunohistochemical study using anti-AID antibody for 97 Japanese SCC patients who visited our hospital during 2005–2015. In total, 49 cases (50.5%) were positive for anti-AID staining. In all 8 SCC cases with local recurrence, AID was expressed highly in the tumor cells. In all 14 SCC cases with lymph node metastasis and 2 patients with lung metastasis, AID staining was strongly positive. There was no difference in a frequency of AID expression between the female and male patients or between SCC taking place in sun-exposed and unexposed area. In 8 out of 9 SCC cases preceded by burn and a case of SCC caused by a contact dermatitis with oil, AID was highly expressed. In normal human keratinocytes, several cytokines, interleukin (IL)-17 and IL-22, induced the expression of AID mRNA besides LPS, IL-1 and TNF-␣. Taken together, high expression of AID may be one of the predicative markers for poor prognosis with metastasis and local recurrence of SCC by induction of mutations in a number of genes. Moreover, AID can become a potential therapeutic target for prevention of occurrence of SCC progressed from precancerous inflammatory skin conditions. http://dx.doi.org/10.1016/j.jdermsci.2017.02.056
Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
the potential use of L. pentosus GMNL-77 in the future treatment of psoriasis.yf5126. http://dx.doi.org/10.1016/j.jdermsci.2017.02.050 P01-48[O1-30] The therapeutic potential and molecular mechanism of flavonoid HCF-A on inflammatory skin diseases Hsin-Ju Li 1,∗ , Chi-Feng Hung 2 1 Department of Chemstry, Fu Jen Catholic University, New Taipei City, Taiwan 2 School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan In recent years, there is increasing incidence and prevalence of diverse chronic inflammatory skin diseases, such as psoriasis and atopic dermatitis. However, the current management is usually not satisfactory due to the possibility of poor efficacy, severe side effects, high expenses or frequent recurrence. Therefore, it is urgent and important to develop novel drugs in the treatment of these troublesome inflammatory skin diseases. With the recent advancement in the molecular and cellular biology, the molecular pathogenesis of these diseases has been gradually teased out. Previous studies have shown that they are mostly related to the dysregulation of immune responses and aberrant protein expression. Many studies also found that the production of reactive oxygen species, and release of inflammatory cytokines, such as IFNs, IL-17 and TNFalpha, play critical roles in the inflammatory skin diseases. The potential applications of Chinese herbal medicine in the treatment of these diseases have also been reported. Our results revealed that flavonoid HCF-A could reduce IL-17A or TNF-alphainduced MAPK phosphorylation, NF-kappa B activation, CCL20 release, and inflammatory makers mRNA expression from epidermal keratinocytes. Most importantly, flavonoid HCF-A could obviously attenuate imiquimoid-induced psoriasis-like skin lesions in mice, and improve the imiquimod-induced disrupted skin barrier function. We believe that exploration of the therapeutic potential of these herbal medicines will not only provide novel pharmacologic agents in the clinical treatment of inflammatory skin diseases but also contribute to the academic advancement in the field of Chinese herbal medicine.
http://dx.doi.org/10.1016/j.jdermsci.2017.02.051 Category 2 (P02): Carcinogenesis/Growth Factors/Signal Transduction/Cancer Genetics P02-01[I-1] Mechanistic insight into the ATP-induced fibrosis in systemic sclerosis Buddhini Perera ∗ , Akihiko Uchiyama, Akihito Uehara, Kazuya Yamada, Sachiko Ogino, Yoko Yokoyama, Osamu Ishikawa, Sei-ichiro Motegi Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan ATP is released from cells by tissue injury/hypoxia, and initiates an autocrine/paracrine signaling for the inflammation. It has been considered that vasculopathy induces hypoxia and oxidative stress in the dermis in SSc, suggesting that hypoxia-induced oxidative stress enhances ATP release from cells, such as fibroblasts
and endothelial cells. However, the role of ATP in the pathogenesis of skin fibrosis in SSc is unknown. Objective was to elucidate the role of ATP in skin fibrosis in SSc. IL-6 is a potent stimulator of collagen production in fibroblasts. ATP enhanced IL-6 production more significantly in SSc fibroblasts than in normal fibroblasts. ATP binds both P2X and P2Y receptors to activate the downstream signaling. There were no significant differences in the expression levels of P2X7, P2Y2 and P2Y4 receptors between normal and SSc fibroblasts treated with or without ATP. Furthermore, the ectonucleotidases CD39 and CD73 were not expressed in normal and SSc fibroblasts. Non-selective P2 receptor antagonist, suramin, inhibited ATP-induced IL-6 production and phosphorylation of p38 in SSc fibroblasts. ATP-induced IL-6 production was significantly inhibited by p 38 inhibitor, SB203580, suggesting that ATP-induced phosphorylation of p38 via P2 receptor enhances IL-6 production in SSc fibroblasts. We previously found that norepinephrine (NE) also induced IL-6 production in SSc fibroblsts. Combined treatment with ATP and NE resulted in an additive increase in IL-6 production in SSc fibroblasts. These results suggest that vasculopathy-induced hypoxia and oxidative stress might enhance ATP release in the dermis in SSc, and ATP might contribute to the skin fibrosis via potentiation of IL-6 and collagen type I production from fibroblasts in SSc. http://dx.doi.org/10.1016/j.jdermsci.2017.02.052 P02-02[C12-1] Overexpression of heat shock protein 90 as a potential therapeutic target for angiosarcoma Saori Yamada-Kanazawa 1,∗ , Ikko Kajihara 1 , Satoshi Fukushima 1 , Masatoshi Jinnin 1 , Mamiko Masuzawa 2 , Mikio Masuzawa 3 , Yasuyuki Amoh 2 , Daichi Hoshina 4 , Riichiro Abe 5 , Hironobu Ihn 1 1 Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan 2 Department of Dermatology, Kitasato University School of Medicine, Japan 3 Department of Molecular Diagnostics, School of Allied Health Sciences, Kitasato University, Japan 4 Department of Dermatology, Hokkaido University Graduate School of Medicine, Japan 5 Department of Dermatology, Niigata University Graduate School of Medical and Dental Sciences, Japan The prognosis of angiosarcoma is very poor compared with that of other skin malignancies because advanced angiosarcoma is resistant to standard chemotherapy. Recently, heat shock protein 90 (HSP90) has been identified as a molecular chaperone to activate various cancer related proteins. In addition many clinical trials of HSP90 inhibitors are ongoing in several malignant tumors. Therefore, we investigated the possibility that inhibition of HSP90 had therapeutic potential to suppress angiosarcoma. HSP90 protein levels in cultured angiosarcoma cell lines were markedly increased compared to normal endothelial cells. Immunohistochemical analyses revealed the expression of HSP90 protein was strongly detected in angiosarcoma tissues compared with normal dermal vessels or senile angioma. Ganetespib, a HSP90 inhibitor, with or without taxanes inhibited the proliferation of angiosarcoma cells via apoptosis in dose-dependent manners. Inhibition of HSP90 suppressed migration and invasion of angiosarcoma cells.
Abstracts from the 41st Annual Meeting / Journal of Dermatological Science 86 (2017) e1–e95
Knock down of HSP90 decreased several cancer-associated proteins including p-ERK and survivin. Taken together, HSP90 could be a novel therapeutic target for angiosarcoma. http://dx.doi.org/10.1016/j.jdermsci.2017.02.053 P02-03[C12-2] Class III phosphoinositide 3-kinase Vps34 is essential for epidermal homeostasis Takuya Nagai 1,∗ , Shin-Ichi Osada 1 , Yuta Mizusawa 1 , Tomoko Suzuki 1 , Satoshi Eguchi 2 , Junko Sasaki 2 , Takehiko Sasaki 2 , Motomu Manabe 1 1 Department of Dermatology and Plastic Surgery, Akita University Graduate School of Medicine, Akita, Japan 2 Department of Medical Biology, Akita University Graduate School of Medicine, Japan Vacuolar protein sorting 34 (Vps34) is the Class III phosphoinositide 3kinase that produces intracellular pools of phosphatidylinositol-3phosphate (PI3P), and regulates autophagosome and endosome formation by recruiting PI3P-binding proteins and additional factors. To examine the role of Vps34 in the epidermis, we generated Vps34 floxed (Vps34 flox/flox) mice, and deleted Vps34 specifically in the epidermis by crossing them with the keratin 5-Cre transgenic mice. Homozygous mutant mice died several hours after birth because of severe vacuolation in the epidermis, which hampered further investigation. To overcome the postneonatal death, we then generated CreERT2; Vps34 flox/flox mice, in which Vps34 could be deleted in a tamoxifen-dependent manner. Application of tamoxifen onto the mouse dorsal skin in 5 consecutive days led to a scaly thickened skin. Histopathological analysis revealed acanthosis, spongiosis and dyskeratotic cells in the epidermis and lymphocyte infiltration in the upper dermis, which resembled interface dermatitis. These results suggest that Vps34 is required for epidermal homeostasis and abnormal PI3P metabolism is involved in pathogenesis of inflammatory skin diseases.
http://dx.doi.org/10.1016/j.jdermsci.2017.02.054 P02-04[C12-3] Tumor-suppressive effects of interferon- through interleukin-24 in melanoma Yoshinori Watanabe ∗ , Yoshimasa Nobeyama, Munenari Itoh, Hidemi Nakagawa The Department of Dermatology, The Jikei University school of Medicine, Tokyo, Japan Background: Interferon beta (IFN-) is widespread used as a treatment against melanoma to improve its prognosis. Several literature has suggested that the sensitivity of melanoma cells to IFN- shows wide spectrum through in vivo and in vitro examinations including our previous study that RPMI-7951 and A375 is relatively sensitive to IFN-, while G361, SK-MEL-5 and SK-MEL-1 is relatively resistant. Interleukin24 (IL-24) has been reported to inhibit the tumor growth by inducing the tumor-selective apoptosis. This study was conducted to elucidate the relation with IL-24 in inhibition effects of IFN- on melanoma cells. Method: Quantitative real-time reverse transcription PCR (RTPCR) and enzyme-linked immunosorbent assay (ELISA) were carried out to detect amounts of IL-24 mRNA and proteins, respectively. Flow cytometry was performed to assess the tumor cell
e19
growth and apoptosis of melanoma cells treated with IL-24 using propidium iodide staining. Invasion assay was performed to evaluate an invasive capacity of the melanoma cells treated with IL-24. Result: RT-PCR revealed that IL-24 mRNA at day 5 after IFN- treatment was more expressed in IFN--sensitive cell lines (A375, RPMI-7951) than in IFN--resistant cell lines (G361, SK-MEL-5, SK-MEL1). ELISA revealed that IL-24 protein at day 5 after IFN- treatment was induced in 4.8-fold in RPMI-7951 and 1.9-fold in A375 cells, but the protein was not detected in either non-treated or treated G361 cells, SKMEL-5 cells or SK-MEL-1 cells. IL-24 treatment of terminal concentration of 100 g/ml decreased melanoma cells indicating G2/M period in the 5 melanoma cell lines. Conclusion: These results suggested that IFN- may inhibit melanoma cell proliferation through inducing IL-24 from melanoma cells. http://dx.doi.org/10.1016/j.jdermsci.2017.02.055 P02-05[C12-4] High expression of activation induced deaminase in recurrent or metastatic squamous cell carcinoma Takashi Yamaguchi ∗ , Daisuke Omoto, Yu Sawada, Sanehito Haruyama, Manabu Yoshioka, Etsuko Okada, Motonobu Nakamura Department of Dermatology, University of Occupational and Environmental Health, Japan Abnormally expressed activation-induced cytidine deaminase (AID), a member of a cytidine deaminase family, acts as a genome mutator that contributes to malignancy such as gastric cancer and hepatocellular carcinoma. We have recently shown that transgenic mice expressing AID in the epidermis under a keratin 14 promoter developed cutaneous squamous cell carcinoma (SCC) with Hras and Trp53 mutations spontaneously. However, the role of AID in human SCC had not been fully investigated. To explore the correlation between the AID expression and clinical manifestations of SCC, we performed an immunohistochemical study using anti-AID antibody for 97 Japanese SCC patients who visited our hospital during 2005–2015. In total, 49 cases (50.5%) were positive for anti-AID staining. In all 8 SCC cases with local recurrence, AID was expressed highly in the tumor cells. In all 14 SCC cases with lymph node metastasis and 2 patients with lung metastasis, AID staining was strongly positive. There was no difference in a frequency of AID expression between the female and male patients or between SCC taking place in sun-exposed and unexposed area. In 8 out of 9 SCC cases preceded by burn and a case of SCC caused by a contact dermatitis with oil, AID was highly expressed. In normal human keratinocytes, several cytokines, interleukin (IL)-17 and IL-22, induced the expression of AID mRNA besides LPS, IL-1 and TNF-␣. Taken together, high expression of AID may be one of the predicative markers for poor prognosis with metastasis and local recurrence of SCC by induction of mutations in a number of genes. Moreover, AID can become a potential therapeutic target for prevention of occurrence of SCC progressed from precancerous inflammatory skin conditions. http://dx.doi.org/10.1016/j.jdermsci.2017.02.056