Ceracis furcifer species-group (Coleoptera: Ciidae) originally comprised nine species names: Ceracis cornifer ; C. cylindricus ; C. furcifer Mellié, 1849; C. hastifer ; C. monocerus Lawrence, 1967; C. ruficornis Pic, 1916; C. simplicicornis ; C. semipallidus Pic, 1922 and C. unicornis Gorham, 1898. Ceracis semipallidus was synonymised with C. furcifer and then no further changes were made to the composition of the group. Here, we provide a taxonomic revision of the Ceracis furcifer species-group and new data on the geographic distribution and host fungi of the included species. Lectotypes are designated for C. cornifer, C. furcifer, C. hastifer, C. ruficornis, C. semipallidus and C. unicornis. As results we: (i) synonymise C. cylindricus, C. monocerus, C. simplicicornis, C. unicornis with C. cornifer; (ii) confirm the synonymy of C. semipallidus with C. furcifer; (iii) redescribe C. cornifer, C. hastifer, C. furcifer and C. ruficornis; and (iv) provide an identification key for species in the furcifer group. The frontoclypeal horn and body coloration showed great intraspecific variation. We show that species in the furcifer group have distributions wider than previously known and use mainly Pycnoporus sanguineus as host fungus. Species of the furcifer group are the only animals specialized in feeding on basidiomes of P. sanguineus.The  Ceracis was described by Mellié in 1849 \25? [handwritten]\Brasilia [handwritten]\LECTOTYPE Ennearthron corniferum ”. Historical Material:Ceracis unicornis, lectotype male, here designated. (BMNH) “Type \type \St, Vincent, W.I., H.H., Smith [printed], 247. [handwritten]\W. Indies. 98.237. [printed]\Ceracis unicornis Gorh. ♂. [handwritten]”; 1 paralectotype female, here designated. (BMNH) “W. Indies. 98.237. [printed]\St, Vincent, W.I., H.H., Smith [printed], 247. [handwritten]\Paratype? \Ceracis unicornis Gorh. ♀. [handwritten]”; 1 specimen male (BMNH) “St, Vincent, H.H., Smith, 99-37. [printed]; 1 specimen female (BMNH) “St, Vincent, H.H., Smith, 99-37. [printed]\2 [pencilled note-details of collecting data]”. BRAZIL. 3 specimens   “BRAZIL J. Rick\J.F. Lawrence Lot. 1986\Polyporus haedinus\ex. USDA. Herbaria”. State of Minas Gerais: 13 specimens (CELC) “Brazil: MG, PN Serra do Cipó, próx. à Cachoeira da Farofa, -19.385724, -43. 585808, 11.v.2016, 758 m, Em grande tronco podre caído, leg. J. Chaul & E. Epifânio\ex Pycnoporus sanguineus; 51 specimens  “Brazil: MG, Juiz de Fora, Embrapa, 20.ix.2013, Pecci-Maddalena, Í.S.C. leg.\ex. Pycnoporus sanguineus”; 16 specimens  “Brazil: MG, Jequeri, Grota, 20.i.2011, Sandoval-Gómez, V.E. leg.\ex. Pycnoporus sanguineus”; 15 specimens  “Brazil: MG, Jequeri, Piscamba, vi.2010, Edigio, E.M. leg.\ex. Pycnoporus sanguineus”; 33 specimens  “Brazil: MG, Sacramento, Distrito de Manhuaçu, 15.vii.2010, Antunes-Carvalho, C. leg.\ex. Pycnoporus sanguineus”; 32 specimens  “Brazil: MG, Sacramento, Distrito de Manhuaçu, 18.vii.2010\Antunes-Carvalho, C. leg.\ex. Pycnoporus sanguineus”; 30 specimens  “Brazil: MG, Sacramento, Distrito de Manhuaçu, 31.vii.2010\Antunes-Carvalho, C. leg.\ex. Pycnoporus sanguineus”; 32 specimens  “Brazil: MG, Viçosa, UFV-Apiário, 09.vi.2010, Campos, L.A. leg.\ex. Pycnoporus sanguineus”; 13 specimens  “Brazil: MG, Viçosa, Campus UFV, 02.i.2008, Campos, L.A. leg.\ex. Pycnoporus sanguineus”; 1 specimen (CELC) “Brazil: MG, Viçosa, Violeira, 17.xii.2004, Zacaro, A.A. leg.”; 5 specimens  “Brazil: MG, Viçosa, Atrás do insetário, 14.xi.2003, Zacaro, A.A. & Lopes-Andrade, C. legs. [sic]”; 38 specimens  “Brazil: MG, Pains, 01.vi.2008, Soares, L.G.S. leg.\ex. Pycnoporus sanguineus”; 30 specimens  “Brazil: MG, Rio Paranaiba, Cerrado, ES Ponto 11, CE 11 Tronco caído 24 cm Pote2, 05.i.2011, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 30 specimens  “Brazil: MG, Rio Paranaiba, Cerrado, ES Ponto 11, CE 11 Tronco caído 18 cm Tempo sol, 05.i.2011\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 16 specimens  “Brazil: MG, Rio Paranaiba, Cerradão C2 (19 cm) Fotos 45-46 6.27 g, 26.xii.2011\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 1 specimen (CELC) “Brazil: MG, Rio Paranaiba, Cerradão C1 (27 cm) Fotos 47-48-49 107.79 g, 26.xii.2011\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 8 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerradão C6 (11 cm) Fotos 291-292 2.32 g, 28.xii.2011, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 7 specimens  “Brazil: MG, Rio Paranaiba, Cerradão C7 (18 cm) Fotos 52-53 13.54 g, 28.xii.2011\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 3 specimens  “Brazil: MG, Rio Paranaiba, Campo limpo P7 (20 cm) Fotos 25-26-27 56.22 g, 14.xii.2011\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 2 specimens  “Brazil: MG, Rio Paranaiba, Campo limpo P1 (30 cm) Fotos 5-6 4.82 g, 12.xii.2011, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 2 specimens (CELC) “Brazil: MG, Rio Paranaiba, Campo limpo P2 (14 cm), Fotos 8-9 12,18 cm, 12.xii.2011, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 2 specimens (CELC) “Brazil: MG, Rio Paranaiba, Campo limpo P1 (15 cm) Fotos 1-2-3-4 5.31 g, 12.xii.2011\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 10 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES Ponto 11 CE 11 tronco caído 24 cm tempo sol, 05.i.2011\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 10 specimens (CELC) “Brazil: MG, Rio Paranaiba, Ponto 11 CE 11 tronco caído 18 cm tempo sol, 05.i.2011\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 6 specimens  “Brazil: MG, Rio Paranaiba, Cerrado ES Ponto 10 CE 10 tronco caído 18 cm tempo sol, 05.i.2012, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 15 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES 10 CE 11 Fotos 57-58 18 cm 9.039 g, 05.i.2012, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 6 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES CE11 Fotos 1701-1702-1703 28 cm 1.08 g, 05.i.2012, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 14 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES CE11 Fotos 59-60 24 cm 45.57 g, 05.i.2012, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 11 specimens  “Brazil: MG, Rio Paranaiba, Cerrado ES CE8 (28 cm) Fotos 310-311 50.74 g, 03.i.2012, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 1 specimen (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES CE11 Fotos 217-218-219 16 cm 50.38 g, 05.i.2012, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 2 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES CE10 Fotos 315-316 18 cm 129.014 g, 05.i.2012\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 40 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES árvore vinho/tronco caído 20 cm Ponto 12 CE 12, 05.i.2012\Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 25 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES Ponto 10 CE 10 Tronco caído 18cm tempo sol, 05.i.2012, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 4 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES Ponto 10 Tronco caído 18cm, 05.i.2012, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 10 specimens (CELC) “Brazil: MG, Rio Paranaiba, Cerrado ES Ponto 10 (CE 10) tronco caído 18 cm, tempo sol, 05.i.2012, Resende, N.F. leg.\ex. Pycnoporus sanguineus”; 39 specimens  “Brazil: MG, Ipatinga, 2006, Nolasco, J.P. leg.\ex. Pycnoporus sanguineus”; 33 specimens  “Brazil: MG, Ipatinga, 2009, Nolasco, J.P. leg.\ex Pycnoporus sanguineus”; 2 specimens  “Brazil: MG, Ipatinga, RPPN ZACA, 07.xii.2011, Araújo, L.S. leg.\código: Trans: 2 Parcela: C Fungo 1”; 18 specimens  “Brazil: MG, Ipatinga, 2009, Nolasco, J.P. leg.\ex. Pycnoporus sanguineus”; 21 specimens  “Brazil: MG, Ubá, Faz. Córrego do Pari , viii.2000, Gumier-Costa, F. leg.\Ceracis cornifer  det. Cristiano Lopes-Andrade 2003”; 2 specimens (CELC) “Brazil: MG, Ubá, Faz. Córrego do Pari, x.2000, Gumier-Costa, F. leg.\Ceracis cornifer  det. Cristiano Lopes-Andrade 2003”; 2 specimens (CELC) “Brazil: MG, Ubá, Faz. Córrego do Pari, x.2000, Gumier-Costa, F. leg.\Ceracis cornifer  det. Cristiano Lopes-Andrade 2003\Fotografado em MEV-PV; Piracicaba SP, NAP/MEPA (ESALQ/USP), VII / 2002; C. Lopes-Andrade”; 22 specimens  “Brasil: MG, Piau, 22.vi.2014, Pecci-Maddalena, Í.S.C. leg.\ex. Trametes sp.”; 7 specimens (CELC) “Brasil: MG, Piau, 22.vi.2014, Pecci-Maddalena, Í.S.C. leg.\ex. Pycnoporus sanguineus”; 13 specimens  “Brasil: MG, Carrancas, Complexo da Zilda, cerrado e fragmento de mata 27-29.xii.2012, Oliveira, E.H. leg.”; 12 specimens  “Brasil: MG, Guaraciaba, Chalé do Turvo, 21.v.2012, Lopes-Andrade, C. leg.\ex. Pycnoporus sanguineus”; 20 specimens (CELC) “Brasil: MG, Texeiras, “saindo de Viçosa no restaurante da BR, 12.i.2014, Lopes-Andrade, C. leg.\ex. Pycnoporus sanguineus”; 10 specimens (CELC) “Brasil: MG, Ibitipoca, 03.iii.2014, Pecci-Maddalena, I.S.C. leg”. State of Rio de Janeiro: 73 specimens  “Brasil: RJ, Seropédica, 2004, Grossi, P.C. leg.\ex. Pycnoporus sanguineus”; 12 specimens (CELC) “Brasil: RJ, Porciúncula, 30.x.2016, Pecci-Maddalena, I.S.C. & Folly, C. leg.\ex Pycnoporus sanguineus”; 12 specimens  “Brasil: RJ, Rio das Ostras, Rebio União, 01.iii.2013, Aloquio, S. leg.\ex. Pycnoporus sanguineus”; 12 specimens  “Brasil: RJ, Grussaí, 01.xi.2003, Souto, L.S. leg.\ex. Pycnoporus sanguineus”; 4 specimens  “Brasil: RJ, Paraty, Ponta Negra, 5-6.iii.2011, Sandoval, V.E & Idrobo, C.J. leg.”. State of São Paulo: 3 specimens  “Ilha da Victoria, S. Paulo, Brazil, Dec. 1963, Exped. Dep. Zool.\Ceracis cornifer 9 (Mellié) 202”; 1 specimen dissected (CELC) “Peruibe, SP, 28.xi a 01.xii.84\Exp. MZUSP em fungo\ Ceracis ruficornis Pic. Gen 1 [handwritten]”; 4 specimens  “Raiz da Serra, SP, 28.ix.1907, Luderwaldt\Dep. Zool. São Paulo 1247\SP”; 3 specimens (CELC) “Brasil: SP, São José dos Campos, Parque da cidade, 09.x.2004, leg S.S.P. Almeida & G.S.P. Almeida\ex. Pycnoporus sanguineus”. State of Espírito Santo: 10 specimens  “Brasil: ES, Alto Bergamo, João Neiva, 11.v.2008, Furieri, leg.\ex. Pycnoporus sanguineus”; 23 specimens  “Brasil: ES, Santa Teresa, EBSL, 18-24.ix.2015, Pereira, M.R. leg., trilha atrás do alojamento\ex Pycnoporus sanguineus”; 3 specimens (CELC) “Brasil: ES, Santa Teresa, Reserva Biológica de Santa Lúcia, 03.iii.2003, Barreto, F.C.C & Furieri, K.S. leg.”; 10 specimens (CELC) “Brasil: ES, Santa Teresa, Rebio Augusto Ruschi, 17-19.vi.2013, Pereira, M.R. leg.\ex. Pycnoporus sanguineus”; 1 specimen (CELC) “Brasil: ES, Santa Teresa, EBSL, iii.2003, Furieri, K.S. leg.\ex. Pycnoporus sanguineus”; 1 specimen dissected (CELC) “Brasil: ES, Conceição da Barra, Rebio Córrego Grande, 14−19.xii.2005, Furieri, K.S., Loiola, G.R., Van de Koken, A.F. legs. [sic]”; 1 specimen dissected (CELC) “Brasil: ES, Conceição da Barra, Floresta Nacional do Rio Preto, 03.xii.2011, Araujo, L.S. leg.\Trans: 3, Parcela B, Fungo 1”; 2 specimens (CELC) “Brasil: ES, Conceição da Barra, Floresta Nacional do Rio Preto, 03.xii.2011, Araujo, L.S. leg.\Trans: 3, Parcela 0, Fungo 4”; 2 specimens (CELC) “Brasil: ES, Conceição da Barra, Floresta Nacional do Rio Preto, 03.xii.2011, Araujo, L.S. leg.\Trans: 1, Parcela 0, Fungo 1”; 1 specimen (CELC) “Brasil: ES, Conceição da Barra, Floresta Nacional do Rio Preto, 03.xii.2011, Araujo, L.S. leg.\Trans: 2, Parcela C, Fungo 1”; 1 specimen (CELC) “Brasil: ES, Conceição da Barra, Floresta Nacional do Rio Preto, 03.xii.2011, Araujo, L.S. leg.\Trans: 2, Parcela A, Fungo 5”; 1 specimen  “Brasil: ES, Aracruz, 04.i.2004, Furieri, K.S. leg.”. State of Bahia: 2 specimens  “Brasil: BA, Porto Seguro, RPPN Pau Brasil, 4−7 Janeiro 2010, Chamorro, J. leg.\ex. Pycnoporus sanguineus”. State of Mato Grosso do Sul: 9 specimens  “Brasil: MS, Paranhos, Chácara Santo Antônio, Pastagem, vi.2012, Puker, A. leg.\ex. Pycnoporus sanguineus”; 6 specimens  “Brasil: MS, Campo Grande, Cerradinho UFMS, 27.v.2015, Chamorro, J. leg.\ex. Pycnoporus sanguineus”. State of Paraná: 1 specimen (DZUP) “ANTONINA –PR, Reserva Sapitanduva, BRASIL 27.I.1987, Lev. Ent. PROFAUPAR, LÂMPADA [printed]\DZUP 273631 [printed]”; 1 specimen (DZUP) “ ANTONINA –PR, Reserva Sapitanduva, BRASIL 30.III.1987, Lev. Ent. PROFAUPAR, LÂMPADA [printed]\DZUP 273654 [printed]”; 1 specimen (DZUP) “GUARAPUAVA – PARANÁ, Est. Águas Sta. Clara, BRASIL 27.I.1987, Lev. Ent. PROFAUPAR, LÂMPADA [printed]\DZUP 273632”. State of Santa Catarina: 2 specimens (MNRJ) “Brasilien, Nova Teutonia, 27°11 B, 52°23’ L, Fritz Plaumann [printed]”; 3 specimens (MNRJ) “Brasilien, Nova Teutonia, 27°11 B, 52°23’ L, Fritz Plaumann [printed]”; 3 specimens (MNRJ) “9 [handwritten], 1936 [“6” handwritten and “193” printed], Brasilien, Nova Teutonia, 27°11 B, 52°23’ L, Fritz Plaumann [printed]”; 24 specimens  “Brasil, SC, Urubici, Estrada p/Serra do Corvo Branco em placa, 06.iii.2011, Grossi & Perigotto legs. [sic]\28°02’53’’S, 49°23’06’’W, 1022 m\ex. Pycnoporus sanguineus”; 30 specimens (CELC) “Brasil, SC, Urubici, Estrada p/Serra do Corvo Branco em placa, 06.iii.2011, Grossi & Perigotto legs.\28°02’53’’S, 49°23’06’’W, 1000 m\ex. Pycnoporus sanguineus”. State of Rio Grande do Sul: 3 specimens  “Brasil: RS, São Francisco de Paula, Flona SFP, 2006, Graf, L.V. leg.\ex. Pycnoporus sanguineus; 15 specimens (CELC) “Brasil: RS, São Francisco de Paula, Flona SFP, xii. 2006, Graf, L.V. leg.\Fungo 1970, sp 29”; 51 specimens  “Brasil: S. Fran. Paula, RS (FLONA) IX. 2006 L. V. Graf col.\Em Pycnoporus sanguineus”; 41 specimens (MCNZ) “Brasil: RS, Santa Tereza, 03.IX.2015, 26.X.2015, 10.II.2016, 18.III.2016, Mezzomo, A.G leg. ARGENTINA: 12 specimens  “Argentina, Misiones Prov. nr. Wanda, 25°58’S, 54°35’W, 22.x.2007 S. Neser\Ex. Orange bracket fungus on old pine logs\National coll. of insects\Pretoria, South Africa”; 3 specimens  “Arg: Salta Capital, July, 1971, L.A. Stange, Lot 14\ Pycnoporus cinnabarinus”; 2 specimens  “Arg. Tucuman, Rio Urueña, nr, Salta, Border, Dec 1970\L.A. Stange Lot 4(2)\Pycnoporus cinnabarinus”; 2 specimens  “Arg. Tucuman, Tafi del Valle, xii.17.1971\L.A. Stange Lot 15”; 2 specimens  “Tucuman, Arg.v.1926, R.C. Shannon\ex. Polyporus sanguineus\Ceracis (9) sp 448”; 2 specimens  Tucuman“Arg. Feb. ’29, R.C. Shannon\ex. Polyporus sanguineus”; 4 specimens  “Tucuman, Argentina, L. Castillon, Coll. J.F. Lawrence, Lot. 2007\ex. Polyporus sanguineus, ex U.S.D.A Herbaria”. URUGUAY: 7 specimens  “Montevideo, Uruguay, F. Felippone, J.F. Lawrence, Lot. 2025\ex. Trametes hispida, ex U.S.D.A Herbaria”. MEXICO: 6 specimens  “Cer furcifer gr. Det. J.F. Lawrence 19\Mazatlan Crotch”. UNITED STATES: 3 specimens  “4 mi. SE Lake Placid Highlands County Florida, June 30, 1965\J.F. Lawrence Lot 1532\Polyporus sanguineus”.Remarks. The names here synonymized with C. cornifer are of species described largely on differences in shape and length of the frontoclypeal horn of males and in dorsal coloration of individuals. However, a careful examination and comparison of specimens, especially of sclerites of male terminalia, showed that these differences represent intraspecific variation. In all examined populations of C. cornifer, the male frontoclypeal horn is extremely variable in length and even in shape. There are small male morphs with the apex of the frontoclypeal horn slightly bifurcated or truncated, intermediate morphs, and large morphs like the lectotype of C. cornifer , body shape and anatomy of male abdominal terminalia show that it is like the large morphs of C. cornifer from South American localities and is considered here to be a synonym of C. cornifer. Ceracis simplicicornis (Pic), also proposed as a new synonym of C. cornifer, was described as “nigro-piceus”  resemble C. ruficornis, with the horn apex slightly bifurcated, body uniformly shiny black and legs with a somewhat amber colour, but a more careful examination and dissection of males allowed reliable identification as C. cornifer. Examined specimens from Paranhos  are also black , the emargination on the outer portion of each lobe is more prominent than in specimens from Brazil. All observed variation in male terminalia was weak or inconsistent in a population or between populations, and was not consistent with the observed variation in body colour, and length and shape of the male frontoclypeal horn. Therefore no support could be found for maintaining C. cylindricus, C. monocerus, C. simplicicornis and C. unicornis as separate species.cornifer A−H. Smalcornifer C−F but bcornifer H. Some scornifer A−C. The -piceus” D but we Lawrence , speciesLawrence  and confso black , VII. AsDistribution.Ceracis cornifer is widely distributed in the southern, southeastern and southwestern Neotropical region, without records from the central and northern South America, but occurring in southwestern Mexico, in a few Caribbean islands and southern USA (C. furcifer in Paranhos (southwestern Brazil) and “Serra do Cipó” . In these two samples, C. cornifer and C. furcifer were found living in the same basidiomes of P. sanguineus. Ceracis cornifer and C. ruficornis are also sympatric in three localities in southeastern Brazil  (hern USA . This spictória) . Ceracis furcifer Mellié, 1849: 379, pl. 4, Fig24. Type locality: Cayenne, French Guyana \[red label] Lectotype Ceracis furcifer Mellie [handwritten]”. Historical Material: 1 specimen (MHNG) “Furcifer Kuntze Cayenne Mellié [handwritten]\[yellow label] PARALECTOTYPE [printed] Ceracis furcifer Mellié [handwritten]”; 1 specimen (MHNG) “Coll. Melly [printed]\[yellow label] PARALECTOTYPE [printed] Ceracis furcifer Mellié [handwritten]”; 2 specimens  “Furcifer Kuntze Perou Mellié [handwritten]\[yellow label] PARALECTOTYPE [printed] Ceracis furcifer Mellié [handwritten]”; 6 specimens (MHNG) “Coll. Melly [printed]\[yellow label] PARALECTOTYPE [printed] Ceracis furcifer Mellié [handwritten]; 2 specimens  “Furcifer Kuntze mexique Geory Mellié [handwritten]”; 1 specimen (MHNG) “furcifer Kuntze Suriname Geory Mellié [handwritten] ”; 1 dissected specimen (MFN) “[green label] Hist. Coll. (Coleoptera) Nr. 53172 (3.Ex.) Cis furcifer Kunze Zool. Mus. Berlin [printed]”; 1 dissected specimen (MNHN) “[green label] MUSEUM PARIS GUYANE FRANÇAISE St-Laurent du Maroni E. Le moult 1911?”; Ceracis semipallidus, lectotype male, here designated, dissected (MNHN) “[red label] LECTOTYPE [printed] Ceracis semipallidus Pic [handwritten]\Ceracis semipallidus mp? [handwritten]\Ciside n°241.3[handwritten]\Guadeloup [printed]\Ex. M. Pic Collection Générale (ex. Anceyiete) Exotique [handwritten]”. BRAZIL. State of Minas Gerais: 40 specimens  “Brazil: MG, PN Serra do Cipó, próx. à Cachoeira da Farofa, -19.385724, -43. 585808, 11.v.2016, 758 m, Em grande tronco podre caído, leg. J. Chaul & E. Epifânio\ex Pycnoporus sanguineus; State of Amazonas: 30 specimens  “Brasil: AM, Manaus, Campinara, 12.vii.2011, Pereira, M.R. leg.\ex. Pycnoporus sanguineus”; 28 specimens  “Brasil: AM, Manaus, Tarumã-Mirim Rio Negro, 10.vi.2011, Pereira, M.R. leg.\ex. Pycnoporus sanguineus”. State of Pará: 30 specimens  “Brasil: PA, Marabá, Reserva Biológica de Tapirapé, pastagem, 2003, Gumier-Costa, F. leg.”; 15 specimens (CELC) “Brasil: PA, Marabá, Reserva Biológica de Tapirapé, Amazônia Legal, 04.xii.2003, Gumier-Costa, F. leg.\Amostra extra n°09, Borda”; 12 specimens  “Brasil: PA, Marabá, Reserva Biológica de Tapirapé, Amazônia Legal, 03.xii.2003, Gumier-Costa, F. leg.\AMOSTRA n°59, Pasto”; 6 specimens (CELC) “Brasil: PA, Marabá, Reserva Biológica de Tapirapé, Amazônia Legal, 04.xii.2003, Gumier-Costa, F. leg.\AMOSTRA n°72, Pasto”. State of Góias: 10 specimens  “Brasil: GO, Mambaí, Fronteira GO/BA, maio 2012, Nilber leg.”; 7 specimens . State of Mato Grosso do Sul “Brasil: MS, Paranhos, Chácara Santo Antônio, Pastagem, vi.2012, Puker, A. leg.\ex. Pycnoporus sanguineus”; 1 specimen (CELC) “Brasil: MS, Paranhos, Chácara Santo Antônio, Pastagem, vi.2012, Puker, A. leg.”. State of Tocantins: 20 specimens (CELC) “Brasil: TO, Araguaína, Campos UFT/ EMUZ, vi.2015, Sandoval-Gómez, V.E. leg.\ex. Pycnoporus sanguineus.”; COLOMBIA: 20 specimens  “Colômbia, Casanare San Luis de Palenque, 260 m, 30.i.2010, Contreras, J. L leg.\ex. Pycnoporus sanguineus”; 4 specimens  “Colômbia: Valle Rio Jamundi entre Cali y Jamundi, 1000 m\slipt corn”. PANAMÁ: 4 specimens  “Madden Dam, Canal Zone, vii-18-1969, J.F. Lawrence\Lot 2901\ex. Daedalea elegans”; 5 specimens  “R. Panamá: Almirante, 1959, H.S. Dybas, FMNH (HD) # 59-154\Polyporus sanguineus”. JAMAICA: 1 specimen dissected (ANIC) “Kingston, Jamaica, W.I., A.H. Ritchie, J.F. Lawrence, Lot 1991\ex. Polyporus maximus ex USDA herbaria\Ceracis furcifer (8) mellié 203”. ANTIGUA: 2 specimens  “Gaynor’s Gut, Antigua, B.W.I, IX-11.65, A.P. Laska\J. F. Lawrence, Lot 1598\ex. Polyporus sanguineus”. COSTA RICA: 3 specimens  “Turrialba, Costa Rica, ix-5-66\JF Lawrence Lot 1851\Robin Andrews Collector\ex. Daedalea elegans”; 4 specimens  “Smi. S.W. Cañas Guanacaste, Costa Rica, Feb. 3-12.1967\J.F. Lawrence Lot.2153\ Robin Andrews Coll.\ex. Polyporus sanguineus”. MEXICO: 1 dissected specimen (ANIC) “Mex: Veracruz, Coatzacoalcos\mi.S.vii.10.63\ J. Doyen collector”.Remarks. We dissected the lectoypes of C. furcifer and C. semipallidus, compared their male genitalia and confirmed the synonymy proposed by Lawrence \[yellow label] LECTOTYPE [printed] Ennearthron hastiferum Mellié [handwritten]\[white label] GEN [printed]”. Historical Material: One dissected male (MHNG) “Hastifer Kuntze Colombie Mellié [handwritten]\[yellow label] PARALECTOTYPE [printed] Ennearthron hastiferum Mellié [handwritten]”; 1 specimen (MHNG) “Coll. Melly [printed]\[yellow label] PARALECTOTYPE [printed] Ennearthron hastiferum Mellié [handwritten]”; 1 specimen (MHNG) “Coll. Melly [printed]\[yellow label] PARALECTOTYPE [printed] Ennearthron hastiferum Mellié [handwritten]”; 2 specimens  “Hastifer Kuntze Perou ?Mellié [handwritten]”. COLOMBIA: 1 dissected specimen (CELC) “Colômbia, Casanare San Luis de Palenque\1247965-1092026, 290.9 m, 30.i.2010, Contreras, J. L. leg.\ex. Pycnoporus sanguineus”; 1 dissected specimen (MFN) “\[blue label] Columb Moritz [handwritten]\[red label] Hist.-Coll. (Coleoptera) Nr. 53171 (4. Ex.) Cis hastifer Kunze Peru – Columb. Zool. Mus. Berlin [printed]. PERU: 1 specimen (SNSD) “Peru, Coll. Maerkel. [printed]\Staatl. Museum für Tierkunde Dresden [printed]”; 1 specimen (SNSD) “Peru, Coll. Maerkel. [printed]\Staatl. Museum für Tierkunde Dresden [printed]\Cis hastifer K Z ♂♀ Peru [handwritten]”; 4 specimens  “Peru, Intercep Miami Florida 14576\ oct 19, 1960 ex Polyporid”. BRAZIL: 15 specimens (CELC) “Brasil: PA, Marabá, Reserva Biológica de Tapirapé, Amazônia Legal, 04.xii.2003, Gumier-Costa, F. leg.\Amostra extra n°09, Borda”; 1 dissected specimen (MPEG) “Brasil: AM, Tapurucuara, Rio-Negro, 1.2.1963, M.P.E. Goeldi, J & B. Bechyné”. Doubtfully included. 3 specimens  “São Roque (Mato Dentro) S. Paulo, Brazil, xi-1963”\N. Papavero, Coll.”.Remarks. We had few specimens in-hand, from a few localities. However, we observed important variation: the frontoclypeal horn of the male from Colombia  has an inflection about four-fifths its length and a square shaped apex. In large morphs, the frontoclypeal horn is most prominent and the greatest pronotal width is larger in comparison with individuals of C. cornifer , and Marabá , in the Amazon biome; and San Luis de Palenque , Savannah biome, where C. hastifer is syntopic with C. furcifer : TL 1.22, PL 0.49, PW 0.56, EL 0.73, EW 0.59, GD 0.51. Ratios: PL/PW 0.88, EL/EW 1.24, EL/PL 1.49, GD/EW 0.86, TL/EW 2.07. Body glabrous, elongate, subcylindrical, shiny, blackish on dorsum; venter blackish, except for yellowish-brown legs, mouthparts, funicle and basal antennomeres; procoxae and head  mostly reddish-brown; antennal club dark reddish-brown. Head barely visible from above; area immediately above horn base concave, glabrous, sparsely punctate; frontoclypeal ridge strongly produced forming a long horn directed upwardly  (Antennae (right antennae measured) with FL 0.07, CL 0.16, CL/FL 2.29, length of antennomeres I−VIII (in mm) as follows: 0.06, 0.04, 0.04, 0.02, 0.01, 0.04, 0.04, 0.08; sensillifers visible. Eyes coarsely faceted, with minute slender yellowish setae in intersections of ommatidia; GD 0.11 mm. Pronotum with anterior portion produced forward, concealing head when seen from above; anterior edge slightly emarginated, forming two very short rounded lobes; sides narrow, not visible from above; posterior edge sublinear; punctation fine, single, uniform and regularly distributed . Metaventrite moderately convex, subglabrous, microreticulate, bearing scattered slender setae; discrimen not visible. Abdominal ventrites microreticulate, punctation shallow and vestiture of scattered slender setae longer than those on dorsum; length of ventrites I−V : 0.17, 0.06, 0.07, 0.07, and 0.06. First abdominal ventrite bearing a circular, marginated pubescent sex patch at the centre  TL 1.11−1.29 (1.21 ± 0.05); PL 0.41−0.51 (0.46 ± 0.03); PW 0.5−0.58 (0.54 ± 0.02); EL 0.7−0.8 (0.76 ± 0.04); EW 0.54−0.61 (0.57 ± 0.02); GD 0.46−0.53 (0.49 ± 0.02); HL 0.09−0.3 (0.21 ± 0.08). Ratios: PL/PW 0.78−0.9 (0.84 ± 0.04); EL/EW 1.24−1.45 (1.31 ± 0.06); EL/PL 1.49−1.95 (1.66 ± 0.14); GD/EW 0.82−0.91 (0.86 ± 0.02); TL/EW 2−2.2 (2.1 ± 0.6). Females, measurements in mm (n = 7) TL 1.17−1.31 (1.23 ± 0.04); PL 0.42−0.5 (0.45 ± 0.02); PW 0.5−0.57 (0.53 ± 0.02); EL 0.72−0.85 (0.78 ± 0.05); EW 0.53−0.62 (0.57 ± 0.02); GD 0.47−0.51 (0.49 ± 0.01). Ratios: PL/PW 0.79−0.94 (0.85 ± 0.05); EL/EW 1.24−1.49 (1.37 ± 0.07); EL/PL 1.44−2.02 (1.76 ± 0.16); GD/EW 0.82−0.92 (0.86 ± 0.03); TL/EW 2.09−2.23 (2.16 ± 0.6). Specimens of the following localities were measured: Brazil: Rio de Janeiro ; São Paulo .Material examined. Lectotype male, here designated MNHN, F “[Blumenau. S.O. Brasilien. (Reitter)]\[Ceracis (antennes 8 art. Probabl.)]\[Type] yellow\[Type] red; [Ceracis ruficorne Pic]\Lectotype.\[Blumenau. S.O. Brasilien. (Reitter)]\[Ceracis (antennes 8 art. Probabl.)]\[Type] yellow\[Type] red; [Ceracis ruficorne Pic]\Paralectotype”. BRAZIL: 23 specimens  “Brasil: SP, Praia da Boraceia, 13.ix.2011, Sandoval-Gómez, V.E. leg.\ex. Pycnoporus sanguineus”; 11 specimens  “Ilha dos Buzios, São Paulo, Brazil, x.1963\Exped. Dep. Zool.\SP\ Ceracis ruficornis Pic Det. J.F. Lawrence”; 2 specimens  “Peruibe, SP, 28.xi/01.xii.1984, Exp. MZUSP col.\Ceracis ruficornis Pic Gen 1”; 4 specimens  “Ceracis (8) ruficornis Pic 447\Ilha da Victoria, S. Paulo, Brasil, 16-27.iii.1964\Exped. Dep. Zool.”; 4 specimens  “Brasil: RJ, Nova Friburgo, xi.2003, Grossi, E.J. leg.\Ceracis furcifer Mellié 1848; det. C. Lopes-Andrade”; 4 specimens  “Brasil: RJ, Paraty, Ponta Negra, 5-6.iii.2011, Sandoval, V.E & Idrobo, C.J. leg”.Remarks. We examined photographs of the lectotype , which is dark reddish-brown. As in other species of the furcifer group, the male frontoclypeal horn also varies in shape and length between morphs and, as in C. cornifer, the horn may be bifurcate at apex in small individuals with small horns , and in the examined material the closest locality was Peruíbe . However, specimens from Bertioga  were in better condition, so we used this for the redescription (one of them as a “plesiotype”). pe e.g., F. It difll horns N. Howevell horns M, which Distribution. Known only for a few localities close to the southeastern coast of Brazil, where it is sympatric with C. cornifer in Paraty (in the state of Rio de Janeiro), Peruíbe and Ilha da Victoria (in the state of São Paulo) (o Paulo) .Ceracis species, not included here.Other 1 Prosternal process laminate (furcifer group)….21’ Prosternal process thin but not laminate 4 Lateral contours of frontoclypeal horn usually with a pronounced inflection near base  species with eight antennomeres (C. furcifer and C. ruficornis), in which males usually have the frontoclypeal horn incised at apex forming two lobes and the tegmen is widened before apex, with an inflection on both outer apical edges, (ii) species with nine antennomeres (C. cornifer and C. hastifer), in which the male frontoclypeal horn is usually truncate or shallowly emarginate apically and the tegmen is almost parallel-sided, with both outer apical lobes rounded and each bearing a shallow emargination on the outer edge. Below we discuss the observed intraspecific variation in species of the furcifer group, their geographic distribution based on data provided here and their specialization on the host fungus Pycnoporus sanguineus.Species in the furcifer group than previously observed, mainly in the shape of the male frontoclypeal horn, between males of the same or of different populations . For(see Pic ), which Lawrence  is correfurcifer group species  from Buenos Ayres as “nigro-piceus”. This record represents precisely the most southern tip of the distribution and the area with the lowest mean temperature in our study  of Mid-West Brazil. Ceracis cornifer is also syntopic with C. furcifer in Serra do Cipó, a locality in the east limit of the Cerrado in Southeast Brazil, close to the Atlantic Forest biome. Ceracis hastifer and C. furcifer are syntopic in San Luis de Palenque (Colombian Savannah), and in Marabá (Amazon biome). Therefore, all syntopies observed by us are restricted to their distributional frontiers, which coincide with frontiers between biomes. Further studies shall evaluate whether the split of species of the furcifer group was related with the separation of the Atlantic and Amazon biomes. This event, which occurred in the Pliocene period, explains the currently distribution patterns of several organisms, e.g., mammals, birds, insects and plants [Species of the l ranges  but therl ranges . Ceracisd plants ,53,54,55furcifer group in other fungi  accepted basidiomes of P. sanguineus as food under laboratory conditions, although in the field it was observed harvesting basidiomes of Agrocybe [Although there are records of ungi see , they arnguineus . Aside f as food . For insAgrocybe . P. sanguineus by humans were isolated and regard principally to South American indigenous tribes [P. sanguineus as medicine, e.g., for treating against aphtha or insolation, in both cases diluting basidiomes in water and drinking [P. sanguineus in three Brazilian indigenous tribes from the Xingu National Park, state of Mato Grosso. In only one of these indigenous tribe (Txucarramãe) there were reports of people eating P. sanguineus. Humans use basidiomes of P. sanguineus in the early decay stages [P. sanguineus also have high concentration of cinnabarins, toxic substances with antibiotic action [P. sanguineus by ciid beetles suggests that they may be resistant to antibiotic and toxic compounds of this fungus [C. cornifer can colonize basidiomes of P. sanguineus before they begin to sporulate [P. sanguineus (mainly breeding records) demonstrates that the furcifer group constitute the only truly specialized animals feeding on basidiomes of P. sanguineus. Therefore, these beetles perform a great environmental service, removing basidiomes of P. sanguineus, which would persist in nature for years. The cases of consumption of s tribes ,58,59, wdrinking  studied y stages ,61, in wy stages . Basidioc action ,26. Suchs fungus . Populatfurcifer group in an evolutionary stasis: in all areas occupied by their populations, they use the same resource, which is widely available in most open areas in the Neotropical region. On the other hand, populations of polyphagous species can undergo subsequent specialization, becoming oligophagous or even monophagous, which may act as evolutionary constraint and lead to rapid radiation. For instance, that was possibly the case for polyphagous ciid species of the Ceracis cucullatus group [Cis taurus group [C. cucullatus and Cis taurus groups, there are a few widespread polyphagous species and several geographically restricted oligophagous ones [furcifer group is a hypothesis that may explain why disjunct populations of C. cornifer have not undergone speciation, or at least evolution has not lead to conspicuous morphological differences in individuals of disjunct populations. One of the ecological bases for evolution of specialization is environmental constancy . The useus group ,6 and alus group . In bothous ones ,6,10,19.furcifer group now encompasses four species: C. cornifer C. hastifer, C. furcifer and C. ruficornis. The male frontoclypeal horn and body coloration in these species have great intraspecific variation; therefore, these are not good diagnostic features. We concluded that examination of sclerites of male terminalia is the most accurate way to identify species of the furcifer group. We show that the furcifer group are widely distributed along the Neotropical region and constitute the only specialized animals that feed on basidiomes of P. sanguineus. The


The Wnt/β‐catenin signalling pathway is pivotal for stem cell function and the control of cellular differentiation, both during embryonic development and tissue homeostasis in adults. Its activity is carefully controlled through the concerted interactions of concentration‐limited pathway components and a wide range of post‐translational modifications, including phosphorylation, ubiquitylation, sumoylation, poly(ADP‐ribosyl)ation (PARylation) and acetylation. Regulation of Wnt/β‐catenin signalling by PARylation was discovered relatively recently. The PARP tankyrase PARylates AXIN1/2, an essential central scaffolding protein in the β‐catenin destruction complex, and targets it for degradation, thereby fine‐tuning the responsiveness of cells to the Wnt signal. The past few years have not only seen much progress in our understanding of the molecular mechanisms by which PARylation controls the pathway but also witnessed the successful development of tankyrase inhibitors as tool compounds and promising agents for the therapy of Wnt‐dependent dysfunctions, including colorectal cancer. Recent work has hinted at more complex roles of tankyrase in Wnt/β‐catenin signalling as well as challenges and opportunities in the development of tankyrase inhibitors. Here we review some of the latest advances in our understanding of tankyrase function in the pathway and efforts to modulate tankyrase activity to re‐tune Wnt/β‐catenin signalling in colorectal cancer cells.http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetocThis article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit  APCadenomatous polyposis coliARCankyrin repeat clusterARTDDiphtheria‐toxin‐like ADP‐ribosyltransferaseAXINaxis inhibition proteinB9LB‐cell CLL/lymphoma 9‐like proteinCRCcolorectal cancerDKK1Dickkopf‐related protein 1DVLDishevelledFRAPfluorescence recovery after photobleachingGSK3glycogen synthase kinase 3ISCintestinal stem cellLRP5/6LDL receptor‐related protein 5/6+NADnicotinamide adenine dinucleotidePARdUPAR‐dependent ubiquitylationRINGreally interesting new geneRNFRING fingerSAMsterile α motifTBMtankyrase‐binding motifTNKS/TNKS2tankyraseTNKSitankyrase inhibitorV5epitope tag derived from the P and V proteins of paramyxovirus and simian virus 5, respectivelyWg/WNTWingless and its vertebrate orthologueWWEdomain named after a motif containing two conserved Trp (W) residues and one conserved Glu (E)β‐TRCPβ‐transducin repeats‐containing proteinet al.,β‐catenin  and casein kinase 1 and β‐catenin  for substrate binding are followed by a polymerizing sterile α motif (SAM) domain and a C‐terminal PARP catalytic domain  results in a mild (two‐ to threefold) increase in endogenous Axin abundance in larvae, without measurable developmental effects  in the midgut /Arrow‐containing Wnt/Wg signalosomes  or its adenosine or nicotinamide portions. The first potent toolbox TNKSi, XAV939  , while IWR‐1 is more specific for the tankyrases (no measurable IC50 for full‐length PARPs 1 and 2)  suggests that, in principle, a certain degree of such selectivity can be achieved  are hemizygous for C‐terminal truncations of APC  resistance  in the gut epithelium gives rise to similar toxicity as highly dosed G007‐LK  provides another possible strategy for increasing the effectiveness of TNKSi while ensuring their safety  suggests that full inhibition of signalling might not be required for a therapeutic effect; so what degree of β‐catenin inhibition translates into a biological effect? Moreover, different dosage and timing regimes could be implemented to manage known toxicities (see Meric‐Bernstam and Mills, et al.,et al.,et al.,et al.,et al.,et al.,et al.,et al.,in vivo consequences of tankyrase inhibition, at least with regards to Wnt/β‐catenin signalling and telomere maintenance. In rats, the somewhat stronger deviation of the TBM will require a prior validation of a telomeric role for tankyrase (Muramatsu et al.,Drosophila occurs via a transposon‐mediated mechanism rather than telomerase (Villasante et al.,Drosophila, some mechanistic aspects may be different, for example given that Drosophila Axin only bears a single TBM (Figure A deeper knowledge of the responses and toxicities elicited by Wnt/β‐catenin pathway modulators in different species is much needed to exploit β‐catenin dependencies in cancer. With the vast range of known and putative tankyrase targets (Guettler M Figure C.The past few years have seen a rapid progress in our understanding of how Wnt/β‐catenin signalling is regulated by PARylation and tankyrase. Tankyrase is now an established core component of the Wnt/β‐catenin network. Nonetheless, we are still far from a full understanding of the complex roles that tankyrase plays in the pathway. How does tankyrase promote Wnt/β‐catenin signalling non‐catalytically, and do these mechanisms contribute to TNKSi resistance? Does scaffolding through tankyrase directly control β‐catenin degradasome assembly, and how is this process regulated? How does AXIN PARylation promote the function of the Wnt signalosome? It will be interesting to explore the consequences of AXIN PARylation on both its conformation and interactions with components of both the signalosome and degradasome complexes. Given early indications of a role for tankyrase in APC‐regulated destabilization of AXIN, additional work is needed to decipher how APC limits AXIN abundance. TNKSi have now reached a remarkable specificity. The continued exploration of their pharmacodynamics, the identification of potential biomarkers for their therapeutic implementation and the in‐depth analysis of emerging resistance and toxicity mechanisms are important avenues of further research. The latter will require a careful choice of model systems. The development of alternative tankyrase inhibition strategies through interfering with the non‐catalytic, scaffolding functions of tankyrase, in particular substrate binding, will undoubtedly offer new and exciting opportunities to understand tankyrase function and explore alternative therapeutic strategies.http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al.,et al.,Key protein targets and ligands in this article are hyperlinked to corresponding entries in The authors declare no conflicts of interest.


Flexible gastro-intestinal (GI) endoscopy is an integral diagnostic and therapeutic tool in clinical gastroenterology. High quality standards for safety, patients' comfort, and efficiency have already been achieved. Clinical challenges and technical approaches are discussed in this short review.Image enhanced endoscopy for further characterization of mucosal and vascular patterns includes dye-spray or virtual chromoendoscopy. For confocal laser endoscopy, endocytoscopy, and autofluorescence clinical value has not yet been finally evaluated. An extended viewing field provided by additional cameras in new endoscopes can augment detection of polyps behind folds. Attachable caps, flaps, or balloons can be used to flatten colonic folds for better visualization and stable position.Variable stiffness endoscopes, radiation-free visualization of endoscope position, and different overtube devices help reducing painful loop formation in clinical routine. Computer assisted and super flexible self-propelled colonoscopes for painless sedation-free endoscopy need further research. Single-use devices might minimize the risk of infection transmission in the future.Various exchangeable accessories are available for resection, dissection, tunneling, hemostasis, treatment of stenosis and closure of defects, including dedicated suturing devices. Multiple arm flexible devices controlled via robotic platforms for complex intraluminal and transmural endoscopic procedures require further improvement. Standard procedure for GI endoscopy has not changed much during the last decades. The tip of the flexible insertion tube can be bent vertically and horizontally by steering wheels via Bowden cables. Manual Insertion and retraction can be combined with rotation of the entire endoscope. Light is transmitted from the connected processor to the endoscope tip from where a chip sends back image signals from the lens to a monitor. Channels allow insufflation of the GI lumen, aspiration of fluid content, and rinsing of the lens. A larger instrumentation channel accommodates various diagnostic and therapeutic accessories. Design and cover of the endoscope allow efficient disinfection before re-use. Flexible endoscopy combines accurate mucosal visualization and therapy for the price of higher invasiveness than imaging techniques and wireless capsule endoscopy. Recommendations for technical and clinical quality standards in GI endoscopy have been established Advanced optical systems for precise visual diagnosis and multiple diagnostic and therapeutic accessories which can be applied and exchanged on demand are further important features. Robotic platforms for steering of multidimensional endoscopes have already been developed.Screening for colorectal cancer (CRC), one of the three most common types of cancer worldwide, is one of the major indications for flexible GI endoscopy Other indications for GI endoscopy are gastro-esophageal reflux disease including potentially premalignant Barrett's esophagus, gastro-duodenal ulcers, gastric cancer, treatment of small bowel bleeding, and diagnosis of inflammatory bowel disease.Clinical tasks in flexible endoscopy are optimal characterization of lesions for targeted management, improving adenoma detection, avoiding incomplete endoscopy, reducing pain and need for sedation, infection prevention, and improvements in therapeutic endoscopy. This short review discusses clinical needs and technical solutions already achieved or under development , address22.1high definition (HD) endoscopy  in narrow band imaging  . Other modalities like i-scan , Flexible Spectral Imaging Color Enhancement , and Storz Professional Image Enhancement System  involve real-time electronic post-processing algorithms for spectral color selection. By blue light imaging    c and in I; Fuji) e narrow I; Fuji) d combineConfocal laser endoscopy (CLE) uses blue laser light brought to direct contact with the mucosa after intravenous injection of fluorescein  allows visualization of details down the level of nuclei by contact light microscopy after staining the mucosa with methylene blue and crystal violet. The system was proposed as a miniprobe device and has also been integrated into flexible endoscopes (Olympus). A novel computer aided diagnosis system provided automatic classification of colonic polyps based on identification and characterization of nuclei during processing of EC images HD endoscopy is recommended for routine CRC screening, and real or virtual chromoendoscopy of the entire colon in high risk situations as surveillance of long standing ulcerative colitis or polyposis syndromes 3Up to 20–40% of adenomas are missed during standard colonoscopy Approaches to detect adenomas hidden behind colonic folds by expanding the standard forward-viewing angle of 140–170° or by mechanic manipulation of folds during endoscopy are described below and summarized in 3.1Full Spectrum Endoscopy(FUSE) colonoscopy platform  uses a standard colonoscope with two additional cameras and light sources build into the left and right side of the distal end . A single image is combined from a standard forward viewing lens and the additional convex shaped lens. A feasibility study suggested potential for a higher ADR In a similar matter, Olympus developed a prototype colonoscope with an Omniview modus of the single use, self-propelled pneumatic Aer-O-Scope  combines a 57° forward viewing lens and a 44° lateral view circular around the entire central axis. However, in a first clinical trial there was no diagnostic benefit over standard colonoscopy Third Eye Retroscope  is an auxiliary device with camera and light source. After insertion through the working channel of a standard colonoscope it is angulated 180° allowing an additional retrograde view of the colon with significant increase in adenoma detection of 11–23% Third Eye Panoramic (Avantis) with two side viewing cameras and light sources is attached to the tip of a colonoscope  is a single use silicone rubber device attached to the distal end of the colonoscope. Flexible flaps stretch and straighten out folds during withdrawal  is a similar single use device using rubber arms instead of flaps to straighten out the mucosa  has a balloon permanently integrated in the distal end of a standard colonoscope. If inflated during withdrawal it straightens out folds similar to EndoRings or Endocuff. A multicenter randomized study reported an 16% increased ADR compared to a standard colonoscope The cap attached to the endoscope tip had no benefit in ADR in a large randomized trial study Colonoscopy assisted by a transparent 3.3Auto fluorescence imaging  has been described as a tool to highlight GI neoplasia. A metaanalysis found no increase in ADR with AFI compared to WLE Endoscopy with 3.4To increase complete visualization of colonic mucosa, optic feedback on missed areas could be useful. For this purpose, an algorithm creating 3D images with color-coding of improperly visualized areas in a synthetic colon model based on brightness intensity analysis of endoscopic images has been proposed Technical aids discussed in 4Loop formation and luminal distension are main reasons for incomplete or painful colonoscopy. Technical approaches to tackle this issue are discussed below and summarized in 4.1Sedation, manual external compression, positioning of the patient and straightening and rotation of the endoscope may be useful in preventing loops and obtaining complete investigations.Variable stiffness colonoscopes  allow manual control of stiffness by applying tension to integrated cables. After passing a flexure with a more flexible endoscope, increasing rigidity can prevent loop formation during further advancement. The rate of complete colonoscopies was significantly higher compared to a standard colonoscope Additionally, 4.2ScopeGuide (Olympus) is a positioning system emitting electromagnetic signals from special colonoscopes or inserted miniprobe to a detector beside the patient. From the signals a 3D real-time localization image of the colonoscope is displayed on a monitor resembling a virtual X-ray visualization The commercially available NeoGuide Endoscopy System Inc.  developed a computer assisted prototype colonoscope consisting of multiple fully articulated segments in the insertion tube. A computer algorithm calculates their position in relation to each other to generate a real-life 3D image of the insertion tube to reduce looping 4.3Device assisted endoscopy (DAE) methods have been developed to reduce loops by pleating the long and hardly assessable small bowel over an overtube Single balloon endoscopy, Olympus) or on the tip of both overtube and endoscope  used to keep position by gripping the small bowel. A spiral endoscopy overtube  translates continuous rotation into forward movement while pleating the small bowel. A new prototype endoscope with a motor driven, force controlled rotating distal spiral segment (Olympus) demonstrated feasibility in patients, allowing complete enteroscopy with a single oral approach in some of them balloon assisted enteroscopy  with a standard colonoscope assisted by a through the scope (TTS) balloon has been reported Several DAE methods are the gold standard for therapeutic endoscopy of the small bowel. They may also be applied in case of incomplete colonoscopy to facilitate complete inspection of the entire colon. Variable stiffness colonoscopes are widely used in routine, Scope guide is available commercially.4.4Insufflation of CO2 instead of air through the scope diminishes patient's discomfort and pain during colonoscopy as it is absorbed up to 160 times faster Extensive insufflation of air causes pain and hampers endoscope progression due to a larger lumen. 4.5Another approach to reduce pain and discomfort during colonoscopy is the development of super flexible self-propelling devices. They potentially avoid necessity of sedation by smoothly following the loops without painful stretching of the bowel.Invendo SC20 , a single-use colonoscope including a working channel and controlled by motor rollers  is a super-flexible self-propelling and self-steering endoscope tip advancing through the colon loops by controlled air pressure between inflated balloon in the rectum and at the tip of the endoscopy. Mucosa is inspected during manual withdrawal of the scope. Biopsy and polypectomy are not possible. Complete colonoscopy was possible in a clinical study in 55/56 patients without sedation Endotics System  operates in a similar manner. A remotely controlled, single-use colonoscopy probe crawls through the colon by repeatedly adjusting its length mimicking an inch-worm  are sinColonoSight  a hybrid solution had been developed. The reusable colonoscope does not need disinfection as a single-use cover including the working channel prevents the endoscope from contact with potentially infectious agent. No bacterial contamination of endoscope was found With the However, none of these single use flexible endoscopes has entered clinical routine yet.6Flexible endoscopy is indispensable for treatment of GI lesions. The therapeutic spectrum has increasingly been extended, during routine procedures as well as by referrals to complex interventions potentially avoiding surgery. Through the scope (TTS), over the scope (OTS) and over the wire (OTW) accessories are available for various purposes.6.1Polypectomy of adenomatous polyps detected at colonoscopy is integral part of screening procedures. Standard TTS instruments are biopsy forceps for histology including complete removal of lesions up to 5 mm, and polypectomy snares with electrocautery for larger polyps. Flat and laterally spreading adenomas can be lifted by submucosal injection of saline before endoscopic mucosal resection (EMR) with a snare. Stain added to the saline may be useful for better delineation of the margins. Larger lesions have to be resected in parts . Although IEE can correctly classify most lesions accurate, histology is still important for correct classification . After injection of fluid into the submucosal space and circular incision of the mucosa around the lesion, the submucosa is dissected underneath the lesion using various types of TTS knifes for incision and dissection, needles for repeated injection, and graspers for coagulation of vessels Superficial malignant lesions of the mucosa can be resected en-bloc by 6.2Small perforations of the GI wall including those after endoscopic resection or after surgery can be treated endoscopically in selected patients. Multiple TTS metallic clips are useful to adapt wound margins. Larger defects can be closed with a TTS loop used to adapt several clips at both sides of the defect. Alternatively, a larger clip mounted over the scope clip (OTSC) may close margins sucked into the cap of the device c, d. A f6.3GI strictures can often be dilated with hydrostatic balloons advanced through the scope. Bougies are advanced over a wire placed through the endoscope after withdrawal of the scope, as well as larger pneumatic dilation balloons or self-expanding metal stents POEM) has been introduced as alternative to surgery of hypertrophic lower esophageal sphincter in achalasia causing dysphagia. The esophageal submucosa is dissected after incision of the superficial mucosa to create a tunnel allowing advancing the endoscope distally for incision of the deep muscular layer of the hypertrophic sphincter. Finally, the mucosal defect is closed by clips Recently, PerOral Endoscopic Myotomy  system Transoral Gastroplasty  OverstitchEndomina,  Endoscopy is used for a long time to guide percutaneous placement of feeding tubes in patients with jeopardized nutrition. However, nowadays, obesity and diabetes are increasing challenges. Hence, similar tubes have even been used to empty the stomach in obese patients. Endoscopically placed gastric balloons reduce the gastric lumen temporarily. Permanent gastroplasty is typically performed surgically but has also been performed endoscopically by stapling devices as experimental articulating circular endoscopic  improved obesity and diabetes, but proximal fixation hooks in the duodenum may cause adverse events The endoscopically implanted duodeno-jejunal bypass sleeve 6.6Complex endoscopic resection of large or unfavorably positioned lesions is augmented by a stable endoscope position. A robotic platform  additionally uses an intuitive interface to control motorized endoscope movements and deflection of the tip via joystick or touch pad A double channel endoscope  allows limited triangulation with an additional grasper. Procedure times for ESD could be shortened in a Japanese series Natural Orifice Transluminal Endoscopic Surgery (NOTES) aims to reduce invasiveness of traditional transabdominal surgery by accessing the peritoneal cavity through natural lumina as stomach, colon or vagina 2 requires higher flow rates than provided by standard endoscopes for distension of the GI lumen. Adopting high flow devices with pressure control from laparoscopy could be helpful Anubiscope™ (Storz) EndoSamurai (Olympus) robotic platform translates bimanual actions into movements of the two flexible small caliber instruments in a flexible endoscope. This system outperformed a conventional dual channel flexible endoscope The Cobra and TransPort flexible endoscopy systems  provide a platform for transluminal endoscopy. The TransPort device with 4 working channels allows simultaneous application of flexible endoscopes and surgical instruments Master And Slave Transluminal Endoscopic Robot (MASTER) platform translates bimanual steering to a flexible endoscope with two instrumentation arms with nine degrees of freedom Direct drive endoscopic system (DDES)  allows to bimanually directing two instruments in an endoscopic sleeve via a robotic platform by one operator Endoeye Flex 3D, Olympus) The principle of a semi flexible laparoscope creating 3D images from two separate cameras (Most cases of NOTES have been performed as hybrid transvaginal cholecystectomy still with limited transabdominal augmentation. Transgastric and transcolonic access have also been used but are not yet ready for use in clinical routine 7Wireless technique of capsule endoscopy, the first line diagnostic tool for the small bowel is already available for diagnostic endoscopy of the upper and lower GI tract. However, detailed characterization of lesions including histology and therapy will require flexible endoscopy as gold standard for the next years. An endoscope combining possible advantages would be a single-use, super flexible, self-propelled device for a pain free procedure without sedation. Optional increase in rigidity could provide a stable position for therapy. Ideally, a small caliber endoscope provides ample working channels with sufficient size to easily apply and exchange all appropriate accessories. Additionally, real-time image processing programs could assist in lesion detection and characterization. Dedicated robotic platforms might further augment intraluminal and transmural complex therapies augmented by 3D imaging.


V12 but not BRAFV600E in thyroid cells induced ß-catenin nuclear localization, increased ß-catenin-dependent transcriptional activity and inhibited GSK3ß. In a panel of human thyroid cancer cell lines representative of the main genetic events in thyroid cancer, ß-catenin activation was highly dependent on PI3K/AKT activity through its phosphorylation at S552, but not on MAPK. Silencing of ß-catenin expression in cell lines led to a dramatic reduction in proliferation due to an induction of senescence, which was concordant with a reduction in tumor size in nude mice. Moreover, ß-catenin silencing suppressed the expression of EMT-related genes and reduced the invasive capacity of the tumor cells. In conclusion, this work demonstrates that RAS-driven tumors induce PI3K/AKT-dependent ß-catenin activation.Mutations in ß-catenin are traditionally described as late events in thyroid cancer progression. However, the functional implications of ß-catenin dysregulation in the context of tumor initiating events remain unclear. The aim of this work was to investigate whether the two main oncogenic drivers in thyroid cancer, RAS and BRAF, could activate the Wnt/ß-catenin pathway. Expression of HRAS The most recent advances in thyroid cancer research emerge from an increased understanding of the mechanisms that control thyroid cell proliferation and differentiation, and the associated signal transduction pathways . The curAlthough no mutations in ß-catenin or Axin have been identified in PTC or FTC, ß-catenin dysregulation, particularly the reduction of ß-catenin plasma membrane levels as well as its aberrant nuclear localization, closely parallel loss of tumor differentiation and poor prognosis in human samples .These data suggest that additional mechanisms, including post-translational modifications, could be responsible for ß -catenin mislocalization in thyroid tumors in the absence of mutations in Wnt components. The PI3K/AKT pathway is traditionally linked to activation of the Wnt canonical pathway through the inhibition of GSK3ß , which iin vitro model of thyroid cancer, oncogenic RET/PTC, present only in PTC, induces ß-catenin stabilization and nuclear accumulation by a Wnt-independent mechanism involving activation of PI3K/AKT and MAPK signaling pathways . FT. FTV600EPI3K/AKT , 46. OvePI3K/AKT , 48. Thend lungs . By contIt has previously been demonstrated that oncogenic RET/PTC rearrangements stabilize ß-catenin, promoting its accumulation in the nucleus and enhancing its activity . SimilarAlthough human thyroid cancer cells with RAS mutations or PTEN loss show strong ß -catenin nuclear localization, there is only a modest activation of the ß-cat/TCF reporter relative to cells without ß-catenin in the nucleus Figure . ß-catenAnother interesting observation is that Cal62 cells, which harbor a K-RAS mutation, present ß-catenin in the membrane, while cells harboring H or N-RAS mutations show nuclear ß-catenin localization. RAS isoforms are functionally non-redundant, mainly due to their different subcellular localization, and it has been shown that K-RAS signals preferentially via BRAF/MAPK while the other isoforms have PI3K as their main effector .How ß-catenin is activated may have important functional implications for its potential use as a therapeutic target. In addition to GSK3ß inhibition, Castellone's work demonstrated RET-induced phosphorylation in ß-catenin at Tyr654 residue. This phosphorylation event is dependent on tyrosine kinase receptors, such as ERB and SRC , 54, butComponents of the MAPK and PI3K/AKT signaling pathways are important molecular targets for new cancer therapeutics. For example, selumetinib (a MEK inhibitor) and vemurafenib and dabrafenib (mutant BRAF inhibitors) are clinically beneficial in thyroid cancer patients with advanced metastatic disease. However, resistance to kinase inhibitors targeting components of these signaling pathways is a common phenomenon in patients with advanced radioactive iodine-refractory differentiated thyroid cancer . UnderstIn conclusion, we provide new insights into the mechanisms by which alterations in ß-catenin signaling impact thyroid cancer formation and progression. Depending on the genetic context (RAS-driven tumors and those where PI3K/AKT is predominantly activated such as PTEN-deficient tumors), ß-catenin is stabilized specifically through phosphorylation at Ser552 and this event protects cells from oncogene-induced senescence and contributes to EMT and cell invasion. ß-catenin targeting approaches could therefore have potential utility in thyroid cancer, particularly in combination with other therapies. Given the interconnectedness of ß-catenin signaling with other oncogenes and tumor suppressor pathways, further studies are clearly necessary to explore the potential value of combinatorial therapies. We believe that our study, together with those reported by Castellone et al.  and GujrV600E or HRASV12 [HeLa and HEK293T cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% and 10% fetal bovine serum (FBS), respectively. PC-BRAF and PC-HRAS were derived from PCCl3 rat thyroid follicular cells  to obtai HRASV12 , 59. Cel HRASV12 , PC-HRASThyroid cancer cell lines were from the following sources: C643, Hth7, Hth83 and SW1736 were originally from Dr. N.E. Heldin ; BCPAP was from Dr. M. Santoro ; FTC133 and Nthy-Ori-3.1 were obtained from the European Collection of Cell cultures; WRO-82-1 was from Dr. G. J. F. Juillard ; TPC1 was provided by Dr. A.P. Dackiw ; KTC-1 was provided by Junichi Kurebayashi ; CAL62, FRO and 8505c were obtained from the Leibniz-Institut DSMZ-German Collection of Microorganisms and Cell Cultures and T238 was from Dr. Lucia Roque . Cell lines were authenticated every 6 months by short tandem repeat (STR) profiles using the Applied Biosystems Identifier kit in the Genomic Facility at the Instituto de Investigaciones Biomédicas . Cell lines were grown at 37°C in RPMI 1640 with 10% FBS, except FTC133, which was cultured in DMEM. The following inhibitors were added to cells at the indicated concentrations: 10 μM UO126 , 10 μM AKT-i-VIII, 10 μM XAV939 and 10 μM bortezomib (Sigma).V12 and BRAFV600E expression vectors were used in transfection experiments as described [To measure ß-catenin transcriptional activity, cells were transfected with Super8xTop- and Fop-Luc luciferase vectors, which contain 8 optimized and 8 mutated TCF-binding sites, respectively (both kind gifts of R.T. Moon) . To measescribed .Thyroid cancer cells were grown in DMEM or RPMI and transfected with JetPEI transfection reagent (Polyplus). Twenty-four hours after transfection, cells were treated with the indicated compounds for 24 h and harvested for luciferase measurement . PC-BRAF and PC-HRAS cells were transfected in Coon's complete medium using JetPEI or by calcium phosphate coprecipitation , grown fTotal proteins were lysed in a buffer containing 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, supplemented with a protease inhibitor cocktail (Roche). Nuclear and cytoplasmic proteins were extracted as described . Protein−Δ Δ Ct method. The levels of PCR product were expressed as a function of actin. The primers used are listed in RNA was extracted using Trizol (Sigma) and equal amounts of RNA were added to a reverse-transcriptase reaction mix . Real time PCR was performed on an Mx3000 platform (Agilent) using the FastStart Universal Probe Master kit  and run for 40 cycles. Specificity of the reactions was determined by subsequent melting curve analysis. Stratagene analysis software was used to remove background fluorescence . The num® Gold antifade reagent with DAPI (Invitrogen) and observed under a confocal microscope with a 63× magnification objective (Leica).Cells were seeded onto coverslips and treated as indicated. Unfixed tumors were embedded in OCT (Tissue tek). Frozen tissue was sectioned  and mounted on SuperFrost Plus slides (Fisher). Cells and frozen sections were fixed with 4% paraformaldehyde in PBS pH 7.4 for 10 min, rinsed with PBS, permeabilized with 0.5% Triton X-100, and blocked with 1% BSA. After over-night incubation with primary ß-catenin (C-18) or phospho-Ser552 ß-catenin antibodies   the vector encoded an shRNA against human βcatenin . Control cells (shScramble) were infected with virus containing a non-coding control DNA . Cells expressing a dominant negative form of TCF (dnTCF4) were generated using the vector EdTP (vector was from Addgene and is described in ). VSV-G 5 cell per well in 24-well plates. Cells were fixed at the indicated times with 1% glutaraldehyde and stained with crystal violet. After extensive washing, crystal violet was resolubilized in 1% acetic acid and quantified at 595 nm as an indirect measure of cell number. DNA synthesis was determined using the Cell Proliferation ELISA BrdU Assay . Briefly, cells were seeded in 96-well plates (5 × 103 cells/well). Twenty-four hours later, cells were pulse labeled for 2 h with 10 μM BrdU, and measurements were carried on an ELISA reader at 450 nm. Experiments were performed three times in triplicate.To determine cell proliferation cells were plated at 2 × 105 cells/well). Twenty-four hours later, cells were fixed and assayed for SA-ß-gal activity using the Senescence Detection kit (BioVision). To obtain the percentage of SA-ß-gal positive cells, four images of each condition were captured using 10× magnification and quantified. Experiments were repeated at least three times.Thyroid cells were infected and selected and plated on 12-well plates (7 × 104) were placed on the upper chamber in DMEM plus 0.2% FBS. The lower chamber contained DMEM supplemented with 15% FBS. Cells were allowed to invade for 24 h at 37°C and 5% CO2. Nonmigrated cells on the upper chamber were removed with a cotton swab; filters were fixed in 4% paraformaldehyde and stained using the Diff-Quik staining kit (BD Biosciences). The total number of cells migrated to the lower surface was counted. The experiments were repeated three times.Invasion was measured in Transwell cell culture chambers using BD BioCoat Matrigel Invasion Chambers (BD Bioscience). Infected FTC133 cells  with 12-h light/dark cycles (lights on at 07:00 h) and had free access to food and water. For xenograft studies 9 × 10t-test was used to assess differences between measurements. Differences were considered statistically significant if p < 0.05.Results are presented as mean fold induction ± SEM from at least three independent biological experiments. Student's two-tailed


The healthy adult heart primarily relies on fatty acid oxidation (FAO) for energy production but instantaneously adapts its substrate preference in response to physiological or pathological challenges. Accurate FAO measurements are crucial to investigate early metabolic adaptations. While measurements in intact cardiomyocytes offer greater physiological relevance, current FAO protocols mainly employ cell-free systems and/or require expensive equipment. Here, we present an easy-to-use, inexpensive, and sensitive method to measure, compare and modulate FAO in various cardiomyocyte models. Basal FAO was 2-fold higher in fresh versus cultured adult rat cardiomyocytes (aRCM), while OXPHOS protein levels were maintained. Basal FAO was higher in cultured (3-fold) and fresh (8-fold) aRCM, versus widely used neonatal rat cardiomyocytes (nRCM) and mouse HL1 cardiomyocytes. Moreover, we utilized chemical and pharmacological treatments in order to modulate the FAO flux at different cellular signalling levels. Our data indicate that caution should be taken when studying metabolism in nRCM and HL1 cell models, as these display significantly lower FAO than aRCM. Accurate FAO measurement in cultured aRCM opens new avenues for studying the complex cardiomyocyte metabolic responses to mechanical, nutritional, pharmacological, and genetic manipulations. For instance, the development of heart failure usually is accompanied by an early shift towards a greater reliance on glycolytic substrates and a concomitant suppression of fatty acid oxidation (FAO). Conversely, among the early changes in the hearts of diabetic/obese patients is the almost exclusive reliance on FAO and the loss of ability to switch between substrates. Since changes in substrate preference often reflect cardiac pathologies, metabolic adaptation of cardiomyocytes has generated considerable research interest.Long-chain fatty acids (FA) constitute the main energy source of the heart under aerobic conditions and are the preferred substrate for mitochondrial oxidation at rest. Upon physiological and pathological challenges the heart is flexible to shift its substrate preferenceIn vitro and ex vivo models are essential means to investigate mechanisms of disease development and progression and provide efficient platforms for drug screening. Current protocols for measuring FAO often use isolated mitochondria or permeabilized cells and provide measures of mitochondrial FAO capacity under near optimal conditions. In intact cardiomyocytes, however, FAO flux depends on expression and functioning of the proteins involved in cellular FA uptake, mitochondrial import and β-oxidation of FA, and on tricarboxylic acid (TCA) cycle and respiratory chain activities under the prevailing (intra)cellular conditions. Therefore, the measurement of FAO flux in intact living cells is a preferred tool to assess cardiac metabolic state. Current measurements of FAO in intact cells are often estimations based on oxygen consumption rate. Besides that these methods require expensive equipment, it is hard to control which specific substrates are oxidized by the mitochondria. Moreover, while the medium can be supplemented with various substrates, cells can store and oxidize endogenous pools of substrates, making it difficult to determine which specific metabolic pathways are fuelling respiration. A direct and relatively inexpensive method to measure FAO is through the use of radiolabeled substrates. This allows for complete oxidation and precise determination of the metabolic fate of the specific substrate.14C]-labeled palmitate for use in cultured primary aRCM to allow for accurate measurement of metabolic adaptations to mechanical, nutritional, pharmacological, and genetic manipulations, thereby opening a new avenue for the study of the complex cardiomyocyte metabolism. We used freshly isolated aRCM, in which FAO kinetics were previously studied in detail by Luiken et al.2, as reference point. For comparison we measured FAO flux in intact fresh aRCM, cultured aRCM, and two commonly used cardiomyocyte models, i.e., neonatal rat cardiomyocytes (nRCM) and HL1 cells from the AT-1 mouse atrial myocyte tumor lineage. Additionally, we characterized the effect on FAO of several compounds known to modulate FAO. Furthermore, we studied the sensitivity of our protocol for the measurement of FAO flux in the cardiomyocytes of an in vivo model of pre-diabetes.An established protocol for measurement of FAO flux in cultured adult rat cardiomyocytes (aRCM) is currently missing. Here, we set-up a protocol with  palmitate label: as described previously5; Wash Solution: Medium M199, containing 0.9% of BSA; Day Label Oxidation Solution (DLOx): Wash Solution and 14C-labeled palmitate-albumin complex in a ratio of 0.23: 0.77, v/v, and pre-heated at 37 °C; Trapping Solution: mixing ethanolamine and ethylene glycol in a ratio of 1:2, v/v; Stop Solution: 3 M of perchloric acid. After scraping, cardiomyocytes were transferred into a 20-mL glass vial containing an Eppendorf tube with trapping solution  was gassed with carbogen (95% O2: 5% CO2) and the vials were immediately sealed with a rubber cap and lid. Subsequently, 0.5 mL of the pre-heated DLOx was added to the cells with a 1 mL syringe via the sealed rubber lid  (supplementary note 3). Upon injection of the DLOx, samples were placed for 30 min at 37 °C in a water bath with gentle shaking. The oxidation reaction was terminated by adding 0.4 mL Stop Solution to the cell suspension via the sealed rubber lid. The glass vials with cells were placed at 4 °C for at least 4 hours to allow trapping of 14CO2. Afterwards, the trapping solution (containing radioactive 14CO2) was transferred into a glass beta counter vial containing 10 mL Optifluor. Cells were lysed and total protein concentration was assessed. Radioactivity was measured by liquid scintillation counting using a 5 min counting protocol (Quench Indicator: tSIE/AEC). FAO fluxes were expressed as nmol/mg cell protein per min.The following solutions were prepared prior to performing the assay: ion Fig. . The welStimulation protocol: Oligomycin  or rotenone  were added to the cell suspensions before placing the samples in the water bath, allowing a total of 30 min incubation. Peroxisome proliferator activator receptor (PPAR)α ligand WY-14,643  was added to the cardiomyocytes already in the culture plates for 48 hours. In each experiment we included a so-called zero time control (ZTC) condition as a negative control (supplementary note 4).t-test for Gaussian data. For comparisons of >2 groups, one-way or two-way ANOVA was used followed by post hoc testing with Bonferroni correction. Values of p < 0.05 were considered statistically significant.Data are presented as average ± SEM. Comparisons between 2 groups were performed with the 2-tailed Student’s All data generated and analysed during this study are included in this article.14CO2 production from labeled palmitate was not detectable within the first 10 minute time-period of the assay2 (supplementary note 5). Nevertheless, these values are comparable to the palmitate oxidation rates measured in in vivo animals and in isolated perfused hearts using 14C/3H/13C techniques (0.2–1.3 μmol/g dry weight per min) summarized in ref6 (supplementary note 6).To analyze FAO fluxes in commonly used cardiomyocyte models, we compared fluxes in nRCM, aRCM and HL1 cells. We additionally investigated the effect of aRCM culturing on FAO fluxes. The FAO rate in freshly isolated aRCM amounted to 28 pmol/mg protein per min. This is an underestimation, since we previously showed that et al.7 who showed ~2.5-fold higher FAO rates in cardiomyocytes of 3, 10 and 52–78 week old rats as compared to nRCM, as estimated by oxygen consumption rate.After culturing cells remained viable and morphology was maintained Fig. . CultureTo investigate whether the different levels of FAO flux between different cardiomyocyte models could be explained by changes in expression of proteins constituting the electron transport chain, we measured protein expression levels of mitochondrial oxidative phosphorylation complexes (OXPHOS). After correcting for protein amount, fresh and cultured aRCM showed higher expression of OXPHOS as compared to nRCM and HL1 cells Fig. . This is9, we observed that short-term stimulation of cultured aRCM with oligomycin results in a 2-fold increase in FAO rate (p < 0.05).To further validate our FAO protocol, we used established short- and long-term stimulatory and inhibitory conditions Fig. . For thi10 and aRCM12. In nRCM it was shown to enhance FAO rate by 1.5-fold, both when measured as 14CO2 production10 or O2 consumption rate13. We here confirm the up-regulatory effect of PPARα-ligand WY-14,643 on the FA metabolism genes ucp2 (p < 0.05) and acsl1 (p < 0.05) and a trend to increased acox1 (p = 0.07) and cd36 (p = 0.07) in cultured aRCM  as compared to non-treated aRCM.14. Here we show that FAO rate is 3.6-fold higher in freshly isolated aRCM from ZF rats as compared to lean control Zucker Lean (ZL) rats  rat is a well-established genetic model of pre-diabetesats Fig. . These f ZL Fig. . The car ZL Fig. . These dTaken together, our data show that the presented protocol allows the monitoring of changes in FAO flux in cultured aRCM in response to both short- and long-term stimulatory as well as inhibitory conditions.2 production. A great advantage of this method is that it analyzes the metabolic fate of palmitate as specific carbon source. Moreover, this protocol allows the investigation of the effects of long-term pharmacological, pathophysiological and/or genetic manipulations on FAO. We utilized chemical and pharmacological treatments in order to modulate the FAO flux at different cellular signalling levels. Short-term incubation of cardiomyocytes with the mitochondrial F1F0-ATP synthase inhibitor oligomycin and the mitochondrial complex I inhibitor rotenone resulted in significant up- and down-regulation of FAO flux, respectively. Furthermore, increasing FAO capacity at the gene level via prior incubation with the PPARα ligand WY-14,643, indeed boosted FAO flux in cultured aRCM. Finally, we demonstrate that our protocol allows measuring differences in FAO flux in freshly isolated cardiomyocytes from disease models.This protocol describes a broadly applicable, easy-to-perform method for direct measurement of FAO fluxes in living cardiomyocytes, which does not require high instrument costs. In addition, by utilizing intact cells we avoid potential artefacts caused by mitochondrial isolation or cell permeabilization. Importantly, the latter assays do not take into account the regulation at the level of uptake and initial metabolic conversion of fatty acids, whereas our assay provides an integral measurement involving all steps: from uptake at sarcolemma to mitochondrial COin vitro cardiac disease models including pharmacological, pathophysiological and/or genetic manipulations of choice.Our data indicate that the commonly used nRCM and HL1 cardiomyocytes display FAO rates that are an order of magnitude lower than that of cultured primary adult rat cardiomyocytes. Caution should therefore be taken when using either neonatal cells or immortalized cell lines to study the impact of FA supply on the pathological and physiological mechanisms underlying the development of cardiac diseases such as heart failure. Furthermore, we conclude that OXPHOS protein content is not a good measure for FAO flux. Taken together, we have developed a straightforward method to measure FAO flux in living primary adult cardiomyocytes that is particularly suitable when using Supplementary information


The CX3C chemokine receptor CX3CR1 is expressed on monocytes as well as tissue resident cells, such as smooth muscle cells (SMCs). Its role in atherosclerotic tissue remodeling of the aorta after transplantation has not been investigated.Cx3cr1-/-Apoe-/- and Cx3cr1+/+Apoe-/- aortic segments into Apoe-/-mice, as well as Apoe-/- aortic segments into Cx3cr1-/-Apoe-/- mice. The intimal plaque size and cellular plaque composition of the transplanted aortic segment were analyzed after four weeks of atherogenic diet.We here have orthotopically transplanted infrarenal Cx3cr-/-Apoe-/- aortic segments into Apoe-/- mice resulted in reduced atherosclerotic plaque formation compared to plaque size in Apoe-/- or Cx3cr1-/-Apoe-/- mice after transplantation of Apoe-/- aortas. This reduction in lesion formation was associated with reduced numbers of lesional SMCs but not macrophages within the transplanted Cx3cr-/-Apoe-/- aortic segment. No differences in frequencies of proliferating and apoptotic cells could be observed.Transplantation of These results indicate that CX3CR1 on resident vessel wall cells plays a key role in atherosclerotic plaque formation in transplanted aortic grafts. Targeting of vascular CX3CL1/CX3CR1 may therefore be explored as a therapeutic option in vascular transplantation procedures. Atherosclerosis is an inflammatory disease of the arteries with a high rate of morbidity and mortality in the Western World. The immune reactions involved in atherosclerotic plaque formation are controlled by lipid mediators, costimulatory molecules and cytokines. Chemokines are a family of chemotactic cytokines that mediate immune cell recruitment and are of high relevance in atherogenesis , and tarFractalkine or CX3CL1 is a CX3C chemokine that is expressed in the vessel wall and mediates firm adhesion and chemotaxis of leukocytes expressing its G-protein-coupled receptor, CX3CR1. CX3CL1 is the only member of the CX3C chemokine family and is usually attached to the membrane through a mucin-like domain ,6.Cx3cr-/- mice) have previously been employed to investigate the function of monocyte survival and recruitment during atherogenesis [Fractalkine/CX3CL1 expression has been detected on activated endothelial cells, smooth muscle cells (SMCs), macrophages and platelets, and is involved in the development of numerous inflammatory pathologies including atherosclerosis, rheumatoid arthritis, or graft rejection –13. Whilogenesis ,15. The ogenesis , indicatogenesis . Moreoveogenesis ,11.in vivo, however, do not allow a discriminatation of local from systemic effects. In this regard, murine aortic transplant models have attracted great attention as these allow studying specific vascular responses caused by the graft itself separate from systemic factors related to the recipient (atherogenic) environment [Effects of a deficiency of CX3CL1/CX3CR1 or its blockade ironment –19.In this study, we focused on the function of CX3CR1 in the atherosclerotic response within an aortic segment graft orthotopically transplanted in mice. This allowed us to investigate the role of vascular CX3CR1 versus systemic CX3CR1 to gain further insight into the details of how this chemokine receptor controls atherosclerotic plaque formation.Apoe-/- mice (C57BL/6 background) were crossed with Cx3cr1-/- [Cx3cr1+/+Apoe-/- and Cx3cr1-/-Apoe-/- mice. 8–12 week old male mice were housed under standardized conditions in the Animal Facility of the University Hospital Aachen (Germany). The operating procedure was in accordance with European legislation and approved by local German authorities. Infrarenal abdominal Apoe-/- aorta segments were transplanted into Apoe-/- mice (n = 4), Cx3cr1-/-Apoe-/- aorta segments into Apoe-/- mice (n = 5), and Apoe-/- aorta segements were transplanted into Cx3cr1-/-Apoe-/- mice (n = 5). After transplantation, mice were placed on atherogenic diet  for four weeks.x3cr1-/-  mice to The abdominal aortic transplantation was performed as described previously using the sleeve technique ,21. Brie2  and vessels were flushed with phosphate-buffered saline (PBS) followed by 4% formaldehyde/PBS (pH 7.4) by cardiac puncture. Grafts were gently removed. After overnight fixation in 4% formaldehyde/PBS, specimens were further processed and embedded in paraffin. Serial sections (5 μm) throughout the transplanted aorta distal and proximal of the anastomosis sites were stained by hematoxylin and eosin staining (HE). Immunocytochemistry was performed using antibodies raised against macrophages , vascular smooth muscle cells  followed by anti-rat FITC  and anti-mouse-Cy3, respectively, antigen KI-67 for cellular proliferation  and TUNEL for detecting DNA fragmentation . Positively stained cells were counted in higher magnification (40x) and expressed as percent from total DAPI stained nuclei or as absolute numbers per mm2 .Data are presented as mean ± SEM. The significance of changes between experimental groups and different time points was determined by one-way ANOVA, with Tukey multiple comparison test. A p-value of <0.05 was considered significant. Statistical analyses were performed with GraphPad Prism 6.03 .Apoe-/- mice into Apoe-/- mice , of Cx3cr1-/-Apoe-/- aortic segments into Apoe-/- mice (n = 5), as well as Apoe-/- aortic segments into Cx3cr1-/-Apoe-/- mice (n = 5) were performed using the sleeve technique [Murine orthotopical transplantations of infrarenal aortic segments of echnique ,21, and Cx3cr1-/-Apoe-/- aortic segments in Apoe-/- recipients, whereas a trend towards reduced plaque burden was observed in Apoe-/- aortic segments transplanted into cx3cr1-/-Apoe-/- mice did not reach statistical significance, compared to control Apoe-/- aortic segments in Apoe-/- mice + SMCs in plaques of the aortic segment , this model allows to focus on the particular contribution of local (transferred) cells within the graft. Using this aorta transplantation model to discriminate between effects of circulating versus local vessel wall resident cells, we here are able to show that interference with the CX3CR1-driven signaling pathway in vessel wall cells inhibits atherosclerotic lesion formation.et al. and Lesnik et al. demonstrated a significant decrease in atherosclerotic lesions in mice lacking CX3CR1 [et al. showed that the deletion of CX3CL1 in CCR2-deficient mice reduced the accumulation of macrophages and the development of atherosclerosis [et al. demonstrated that the combined inhibition of CCL2, CX3CR1, and CCR5 in Apoe-/- mice was associated with a marked and additive reduction in atherosclerosis [Apoe-/- mice, and the CX3CL1/CX3CR1 signaling pathway was uncovered to exert pro-survival signals in macrophages, whose absence leads to the increased death of monocytes/foam cells during plaque growth [Cx3cr1-/-Apoe-/- mice transplanted with Apoe-/- mice that entail a deficiency in CX3CR1 in circulating leukocytes. This may be due to specificities of the transplantation model that produces plaques that predominantly contain SMCs rather than macrophages in the infrarenal abdominal aorta.Previous studies by Combadière g CX3CR1 ,24, assog CX3CR1 ,24. Saedclerosis . In accoclerosis . The site growth . In contvs normal diet fed Apoe-/- mice [The interaction of SMCs with monocytes induces a significant up-regulation of CX3CR1 gene and protein expression in both cell types . Moreove-/- mice . Notably-/- mice . Deficie-/- mice  may haveCx3cr1-/-Apoe-/- aortic segments was associated lower numbers of SMCs may indicate that inhibition of CX3CR1 may also have unfavorable effects on plaque stability. On the other hand, confining SMC proliferation is major challenge in vascular remodeling after percutanous transluminal coronary angioplasty and stent-implantation [Clinically, stability of an atherosclerotic plaque is an important characteristic . Unstablantation . In thisantation ,23,24 anet al. observed a prolonged in vivo survival of heterotopic murine cardiac allograft transplants in a major histocompatibility complex-mismatch model, when treated with CX3CR1-antibodies [An increased expression of CX3CL1 and CX3CR1 has been found in coronary arteries from patients after heart transplantation, and several studies have suggested a role of CX3CR1 in allograft rejection and dysfunction ,32–34. Ftibodies . In our Cx3cr1-/- donors and Cx3cr1-/- recipients is missing to evaluate the complete absence of Cx3cr1 and possible additive effects in our model. Taken together we here unveil an important function of CX3CR1 in resident vessel wall cells to promote atherosclerotic lesion growth in the transplanted abdominal aorta in Apoe-/- mice. Targeting CX3CL1/CX3CR1 may thus be viable therapeutic option in vascular transplantation procedures.A clear limitation of our study are the low n-numbers that may have precluded reaching more definite statistical conclusions. In particular, the trend of reduced plaque burden in Apoe-/- aortic segments transplanted into Cx3cr1-/- Apoe-/- mice compared to control Apoe-/- aortic segments in Apoe-/- mice may have substantiated when group sized were lager. Similarly, the cellular plaque characterization yielded values with a wide distribution, so that significant differences between groups may have been missed. Moreover, an additional group of mice with S1 Fig2. (E) No change is detected after staining with Ki-67 for proliferation and (F) TUNEL for apoptosis.(A) The positive stained sections for smooth muscle actin (SMA in red) was overlay with DAPI and the nuclei inside the stained area were counted (red arrow heads). Scale bar 50 μm. (B) Similar, nuclei inside the Mac2-stained area (green) are counted (green arrow heads). Immunohistochemistry and quantification of (C) SMCs and (D) macrophages in plaques of the transplanted aortic segment and expressed as cells/mm(TIF)Click here for additional data file.S1 TableThe original raw data table (in a Excel spreadsheet) that were used to generate our bar graphs.(XLSX)Click here for additional data file.