Activation and functional studies of the Type VI secretion systems in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a versatile and prevalent opportunistic pathogen. It encodes a large arsenal of pathogenicity factors, and secrets a plethora of proteins using specialised protein secretion systems. The type VI secretion system (T6SS) delivers proteins directly into neighbouring bacteria or eukaryotic cells using a mechanism homologous to the T4 bacteriophage tail spike. Three T6SS are encoded on the P. aeruginosa genome. The study of the H1-T6SS has been facilitated by the fact it can be activated by the manipulation of the RetS/Gac/Rsm regulatory cascade by deletion of retS. However, the precise signals required for activation of this cascade, resulting in H1-T6SS activation, are unknown. This work investigates the role of subinhibitory concentrations of antibiotics in activating the system, and shows that kanamycin is able to induce production of core H1-T6SS components. This activation requires a functional Gac/Rsm cascade, but it is not known if this is due to direct signalling via the cascade, or due to a dominant effect of RsmA repression. The H2-T6SS is characterized in this work. We highlight key differences between the H2-T6SS cluster in PAO1 and PA14, including the presence of additional core T6SS components and putative secreted effectors. A strain is generated in which expression of the PA14 H2-T6SS cluster can be activated and tightly controlled by arabinose inducible promoters. The activity of the promoters is confirmed by the H2-T6SS dependent secretion of Hcp2 specifically upon arabinose induction. We further consider two putative H2-T6SS secreted substrates, VgrG14 and Rhs14. Production of these proteins is observed following arabinose induction, but their secretion is not detected. The Rhs14 protein is characterised, and its possible role as a H2-T6SS dependent effector is discussed. Finally, the H2-T6SS system in PA14 is shown to inhibit the internalisation of P. aeruginosa PA14, in contrast to the previously published observations of the H2-T6SS promoting internalisation of PAO1.Open Acces

Développement de l'immunothérapie anti-tumorale médiée par vecteur bactérien vivant basé sur le système de sécrétion de type III de Pseudomonas aeruginosa

En raison de l'efficacité pour délivrer des antigènes directement dans le cytoplasme des CAPs in vivo, les vecteurs bactériens atténués et basés sur les propriétés du système de sécrétion de type 3 (SST3) attirent de plus en plus l'attention grâce à leur potentiel dans le développement des vaccins contre le cancer. Pseudomonas aeruginosa est un pathogène opportuniste responsable d'infections graves chez les personnes immunodéprimées, les grands brûlés et les patients atteints de la mucoviscidose. Cette pathogénicité repose sur de nombreux facteurs de virulence dont le SST3. Dans nos travaux précédents, le potentiel de souches atténuées de P. aeruginosa dans le domaine de la vaccination anti-tumorale a été démontré. Dans ce travail, nous avons optimisé des vecteurs vaccinaux basés sur le SST3 de P. aeruginosa pour des applications cliniques. Dans un premier temps, la performance de ces vecteurs bactériens a été améliorée en utilisant différents modèles de tumeurs murines. Ceci par : 1) l'ajout d'un épitope spécifique des lymphocytes CD4+ Th aux vecteurs; 2) l'application d'un modèle d'expression bi-antigénique aux vecteurs; 3) la construction de vecteurs induisant une réponse humorale. Dans un deuxième temps, la performance thérapeutique du vecteur bactérien a été optimisée par la modulation de la fréquence des injections et l'intervalle qui les sépare. Cette performance a été confirmée dans des modèles différents de tumeurs murines. Dans un troisième temps, un candidat qui pourrait être appliqué en clinique a été généré par l'adaptation d'un mutant (CHA-OAL) de P. aeruginosa totalement avirulent dans un milieu chimiquement défini. La très faible infectiosité de cette souche a été surmontée par en vaccinant à des emplacements multiples. Par la suite, le potentiel du vecteur bactérien dans l'immunothérapie humaine a été également évalué- dans un premiers temps-dans un modèle de souris humanisées (HHD). Enfin, nous avons observé qu'une immunité anti-vecteur pré-existante n'a pas d'effet sur l'efficacité de la vaccination par le vecteur bactérien. L'ensemble de nos résultats a mis en évidence le potentiel de nos vecteurs vivants et atténués de P. aeruginosa pour des applications dans des essais cliniques pertinents.Due to the endowed effective ability to deliver antigen to cytoplasm of APCs in vivo, T3SS based attenuated bacterial vectors attracted more and more attention for their potential interest in cancer vaccine development. Pseudomonas aeruginosa est un pathogène opportuniste responsable d'infections graves chez les personnes immunodéprimées, les grands brûlés et les patients atteints de la mucoviscidose. Cette pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SSTT). In our previous work, the potential of attenuated P. aeruginosa strains as the carriers for anti-tumor vaccination purpose has been reported. In this work, we would like to strengthen P. aeruginosa T3SS based vaccine vectors and direct the development of these bacterial vectors toward clinical applications. First, the performance of these bacterial vectors has been improved in different murine cancer models by: 1) adding one CD4+ Th epitope to vectors; 2) applying bi-antigen expression pattern to vectors; 3) constructing potential humoral response inducing vectors. Second, the therapeutic performance of bacterial vector has been optimized by modulating injection frequency and interval and then be confirmed in murine tumor models. Third, one clinically applicable candidate has been generated by adapting one totally avirulent P. aeruginosa mutant (CHA-OAL) in a chemically defined medium and the poor infectivity of this new strain has been overcome by vaccinations at multiple loci. Fourth, the potential of bacterial vector for human immunotherapy has been further evaluated in one first level humanized mice (HHD) model. Finally, we observed that the pre-existing anti-vector immunity didn't impair the vaccination efficiency of bacteria vector. Taken together, our results highlight the potentials of our live attenuated P. aeruginosa vectors for applications in relevant clinical trials.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Investigation into the changing colonisation of skin bacteria during isotretinoin treatment for acne vulgaris

Poster presentatio

Activation and functional studies of the Type VI secretion systems in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a versatile and prevalent opportunistic pathogen. It encodes a large arsenal of pathogenicity factors, and secrets a plethora of proteins using specialised protein secretion systems. The type VI secretion system (T6SS) delivers proteins directly into neighbouring bacteria or eukaryotic cells using a mechanism homologous to the T4 bacteriophage tail spike. Three T6SS are encoded on the P. aeruginosa genome. The study of the H1-T6SS has been facilitated by the fact it can be activated by the manipulation of the RetS/Gac/Rsm regulatory cascade by deletion of retS. However, the precise signals required for activation of this cascade, resulting in H1-T6SS activation, are unknown. This work investigates the role of subinhibitory concentrations of antibiotics in activating the system, and shows that kanamycin is able to induce production of core H1-T6SS components. This activation requires a functional Gac/Rsm cascade, but it is not known if this is due to direct signalling via the cascade, or due to a dominant effect of RsmA repression. The H2-T6SS is characterized in this work. We highlight key differences between the H2-T6SS cluster in PAO1 and PA14, including the presence of additional core T6SS components and putative secreted effectors. A strain is generated in which expression of the PA14 H2-T6SS cluster can be activated and tightly controlled by arabinose inducible promoters. The activity of the promoters is confirmed by the H2-T6SS dependent secretion of Hcp2 specifically upon arabinose induction. We further consider two putative H2-T6SS secreted substrates, VgrG14 and Rhs14. Production of these proteins is observed following arabinose induction, but their secretion is not detected. The Rhs14 protein is characterised, and its possible role as a H2-T6SS dependent effector is discussed. Finally, the H2-T6SS system in PA14 is shown to inhibit the internalisation of P. aeruginosa PA14, in contrast to the previously published observations of the H2-T6SS promoting internalisation of PAO1

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

This work was supported by the National Institute of General Medical Sciences [GM131919].In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.PostprintPeer reviewe

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

none2929siIn 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.openKlionsky, Daniel J; Abdel-Aziz, Amal Kamal; Abdelfatah, Sara; Abdellatif, Mahmoud; Abdoli, Asghar; Abel, Steffen; Abeliovich, Hagai; Abildgaard, Marie H; Abudu, Yakubu Princely; Acevedo-Arozena, Abraham; Adamopoulos, Iannis E; Adeli, Khosrow; Adolph, Timon E; Adornetto, Annagrazia; Aflaki, Elma; Agam, Galila; Agarwal, Anupam; Aggarwal, Bharat B; Agnello, Maria; Agostinis, Patrizia; Agrewala, Javed N; Agrotis, Alexander; Aguilar, Patricia V; Ahmad, S Tariq; Ahmed, Zubair M; Ahumada-Castro, Ulises; Aits, Sonja; Aizawa, Shu; Akkoc, Yunus; Akoumianaki, Tonia; Akpinar, Hafize Aysin; Al-Abd, Ahmed M; Al-Akra, Lina; Al-Gharaibeh, Abeer; Alaoui-Jamali, Moulay A; Alberti, Simon; Alcocer-Gómez, Elísabet; Alessandri, Cristiano; Ali, Muhammad; Alim Al-Bari, M Abdul; Aliwaini, Saeb; Alizadeh, Javad; Almacellas, Eugènia; Almasan, Alexandru; Alonso, Alicia; Alonso, Guillermo D; Altan-Bonnet, Nihal; Altieri, Dario C; Álvarez, Élida M C; Alves, Sara; Alves da Costa, Cristine; Alzaharna, Mazen M; Amadio, Marialaura; Amantini, Consuelo; Amaral, Cristina; Ambrosio, Susanna; Amer, Amal O; Ammanathan, Veena; An, Zhenyi; Andersen, Stig U; Andrabi, Shaida A; Andrade-Silva, Magaiver; Andres, Allen M; Angelini, Sabrina; Ann, David; Anozie, Uche C; Ansari, Mohammad Y; Antas, Pedro; Antebi, Adam; Antón, Zuriñe; Anwar, Tahira; Apetoh, Lionel; Apostolova, Nadezda; Araki, Toshiyuki; Araki, Yasuhiro; Arasaki, Kohei; Araújo, Wagner L; Araya, Jun; Arden, Catherine; Arévalo, Maria-Angeles; Arguelles, Sandro; Arias, Esperanza; Arikkath, Jyothi; Arimoto, Hirokazu; Ariosa, Aileen R; Armstrong-James, Darius; Arnauné-Pelloquin, Laetitia; Aroca, Angeles; Arroyo, Daniela S; Arsov, Ivica; Artero, Rubén; Asaro, Dalia Maria Lucia; Aschner, Michael; Ashrafizadeh, Milad; Ashur-Fabian, Osnat; Atanasov, Atanas G; Au, Alicia K; Auberger, Patrick; Auner, Holger W; Aurelian, Laure; Autelli, Riccardo; Avagliano, Laura; Ávalos, Yenniffer; Aveic, Sanja; Aveleira, Célia Alexandra; Avin-Wittenberg, Tamar; Aydin, Yucel; Ayton, Scott; Ayyadevara, Srinivas; Azzopardi, Maria; Baba, Misuzu; Backer, Jonathan M; Backues, Steven K; Bae, Dong-Hun; Bae, Ok-Nam; Bae, Soo Han; Baehrecke, Eric H; Baek, Ahruem; Baek, Seung-Hoon; Baek, Sung Hee; Bagetta, Giacinto; Bagniewska-Zadworna, Agnieszka; Bai, Hua; Bai, Jie; Bai, Xiyuan; Bai, Yidong; Bairagi, Nandadulal; Baksi, Shounak; Balbi, Teresa; Baldari, Cosima T; Balduini, Walter; Ballabio, Andrea; Ballester, Maria; Balazadeh, Salma; Balzan, Rena; Bandopadhyay, Rina; Banerjee, Sreeparna; Banerjee, Sulagna; Bánréti, Ágnes; Bao, Yan; Baptista, Mauricio S; Baracca, Alessandra; Barbati, Cristiana; Bargiela, Ariadna; Barilà, Daniela; Barlow, Peter G; Barmada, Sami J; Barreiro, Esther; Barreto, George E; Bartek, Jiri; Bartel, Bonnie; Bartolome, Alberto; Barve, Gaurav R; Basagoudanavar, Suresh H; Bassham, Diane C; Bast, Robert C; Basu, Alakananda; Batoko, Henri; Batten, Isabella; Baulieu, Etienne E; Baumgarner, Bradley L; Bayry, Jagadeesh; Beale, Rupert; Beau, Isabelle; Beaumatin, Florian; Bechara, Luiz R G; Beck, George R; Beers, Michael F; Begun, Jakob; Behrends, Christian; Behrens, Georg M N; Bei, Roberto; Bejarano, Eloy; Bel, Shai; Behl, Christian; Belaid, Amine; Belgareh-Touzé, Naïma; Bellarosa, Cristina; Belleudi, Francesca; Belló Pérez, Melissa; Bello-Morales, Raquel; Beltran, Jackeline Soares de Oliveira; Beltran, Sebastián; Benbrook, Doris Mangiaracina; Bendorius, Mykolas; Benitez, Bruno A; Benito-Cuesta, Irene; Bensalem, Julien; Berchtold, Martin W; Berezowska, Sabina; Bergamaschi, Daniele; Bergami, Matteo; Bergmann, Andreas; Berliocchi, Laura; Berlioz-Torrent, Clarisse; Bernard, Amélie; Berthoux, Lionel; Besirli, Cagri G; Besteiro, Sebastien; Betin, Virginie M; Beyaert, Rudi; Bezbradica, Jelena S; Bhaskar, Kiran; Bhatia-Kissova, Ingrid; Bhattacharya, Resham; Bhattacharya, Sujoy; Bhattacharyya, Shalmoli; Bhuiyan, Md Shenuarin; Bhutia, Sujit Kumar; Bi, Lanrong; Bi, Xiaolin; Biden, Trevor J; Bijian, Krikor; Billes, Viktor A; Binart, Nadine; Bincoletto, Claudia; Birgisdottir, Asa B; Bjorkoy, Geir; Blanco, Gonzalo; Blas-Garcia, Ana; Blasiak, Janusz; Blomgran, Robert; Blomgren, Klas; Blum, Janice S; Boada-Romero, Emilio; Boban, Mirta; Boesze-Battaglia, Kathleen; Boeuf, Philippe; Boland, Barry; Bomont, Pascale; Bonaldo, Paolo; Bonam, Srinivasa Reddy; Bonfili, Laura; Bonifacino, Juan S; Boone, Brian A; Bootman, Martin D; Bordi, Matteo; Borner, Christoph; Bornhauser, Beat C; Borthakur, Gautam; Bosch, Jürgen; Bose, Santanu; Botana, Luis M; Botas, Juan; Boulanger, Chantal M; Boulton, Michael E; Bourdenx, Mathieu; Bourgeois, Benjamin; Bourke, Nollaig M; Bousquet, Guilhem; Boya, Patricia; Bozhkov, Peter V; Bozi, Luiz H M; Bozkurt, Tolga O; Brackney, Doug E; Brandts, Christian H; Braun, Ralf J; Braus, Gerhard H; Bravo-Sagua, Roberto; Bravo-San Pedro, José M; Brest, Patrick; Bringer, Marie-Agnès; Briones-Herrera, Alfredo; Broaddus, V Courtney; Brodersen, Peter; Brodsky, Jeffrey L; Brody, Steven L; Bronson, Paola G; Bronstein, Jeff M; Brown, Carolyn N; Brown, Rhoderick E; Brum, Patricia C; Brumell, John H; Brunetti-Pierri, Nicola; Bruno, Daniele; Bryson-Richardson, Robert J; Bucci, Cecilia; Buchrieser, Carmen; Bueno, Marta; Buitrago-Molina, Laura Elisa; Buraschi, Simone; Buch, Shilpa; Buchan, J Ross; Buckingham, Erin M; Budak, Hikmet; Budini, Mauricio; Bultynck, Geert; Burada, Florin; Burgoyne, Joseph R; Burón, M Isabel; Bustos, Victor; Büttner, Sabrina; Butturini, Elena; Byrd, Aaron; Cabas, Isabel; Cabrera-Benitez, Sandra; Cadwell, Ken; Cai, Jingjing; Cai, Lu; Cai, Qian; Cairó, Montserrat; Calbet, Jose A; Caldwell, Guy A; Caldwell, Kim A; Call, Jarrod A; Calvani, Riccardo; Calvo, Ana C; Calvo-Rubio Barrera, Miguel; Camara, Niels Os; Camonis, Jacques H; Camougrand, Nadine; Campanella, Michelangelo; Campbell, Edward M; Campbell-Valois, François-Xavier; Campello, Silvia; Campesi, Ilaria; Campos, Juliane C; Camuzard, Olivier; Cancino, Jorge; Candido de Almeida, Danilo; Canesi, Laura; Caniggia, Isabella; Canonico, Barbara; Cantí, Carles; Cao, Bin; Caraglia, Michele; Caramés, Beatriz; Carchman, Evie H; Cardenal-Muñoz, Elena; Cardenas, Cesar; Cardenas, Luis; Cardoso, Sandra M; Carew, Jennifer S; Carle, Georges F; Carleton, Gillian; Carloni, Silvia; Carmona-Gutierrez, Didac; Carneiro, Leticia A; Carnevali, Oliana; Carosi, Julian M; Carra, Serena; Carrier, Alice; Carrier, Lucie; Carroll, Bernadette; Carter, A Brent; Carvalho, Andreia Neves; Casanova, Magali; Casas, Caty; Casas, Josefina; Cassioli, Chiara; Castillo, Eliseo F; Castillo, Karen; Castillo-Lluva, Sonia; Castoldi, Francesca; Castori, Marco; Castro, Ariel F; Castro-Caldas, Margarida; Castro-Hernandez, Javier; Castro-Obregon, Susana; Catz, Sergio D; Cavadas, Claudia; Cavaliere, Federica; Cavallini, Gabriella; Cavinato, Maria; Cayuela, Maria L; Cebollada Rica, Paula; Cecarini, Valentina; Cecconi, Francesco; Cechowska-Pasko, Marzanna; Cenci, Simone; Ceperuelo-Mallafré, Victòria; Cerqueira, João J; Cerutti, Janete M; Cervia, Davide; Cetintas, Vildan Bozok; Cetrullo, Silvia; Chae, Han-Jung; Chagin, Andrei S; Chai, Chee-Yin; Chakrabarti, Gopal; Chakrabarti, Oishee; Chakraborty, Tapas; Chakraborty, Trinad; Chami, Mounia; Chamilos, Georgios; Chan, David W; Chan, Edmond Y W; Chan, Edward D; Chan, H Y Edwin; Chan, Helen H; Chan, Hung; Chan, Matthew T V; Chan, Yau Sang; Chandra, Partha K; Chang, Chih-Peng; Chang, Chunmei; Chang, Hao-Chun; Chang, Kai; Chao, Jie; Chapman, Tracey; Charlet-Berguerand, Nicolas; Chatterjee, Samrat; Chaube, Shail K; Chaudhary, Anu; Chauhan, Santosh; Chaum, Edward; Checler, Frédéric; Cheetham, Michael E; Chen, Chang-Shi; Chen, Guang-Chao; Chen, Jian-Fu; Chen, Liam L; Chen, Leilei; Chen, Lin; Chen, Mingliang; Chen, Mu-Kuan; Chen, Ning; Chen, Quan; Chen, Ruey-Hwa; Chen, Shi; Chen, Wei; Chen, Weiqiang; Chen, Xin-Ming; Chen, Xiong-Wen; Chen, Xu; Chen, Yan; Chen, Ye-Guang; Chen, Yingyu; Chen, Yongqiang; Chen, Yu-Jen; Chen, Yue-Qin; Chen, Zhefan Stephen; Chen, Zhi; Chen, Zhi-Hua; Chen, Zhijian J; Chen, Zhixiang; Cheng, Hanhua; Cheng, Jun; Cheng, Shi-Yuan; Cheng, Wei; Cheng, Xiaodong; Cheng, Xiu-Tang; Cheng, Yiyun; Cheng, Zhiyong; Chen, Zhong; Cheong, Heesun; Cheong, Jit Kong; Chernyak, Boris V; Cherry, Sara; Cheung, Chi Fai Randy; Cheung, Chun Hei Antonio; Cheung, King-Ho; Chevet, Eric; Chi, Richard J; Chiang, Alan Kwok Shing; Chiaradonna, Ferdinando; Chiarelli, Roberto; Chiariello, Mario; Chica, Nathalia; Chiocca, Susanna; Chiong, Mario; Chiou, Shih-Hwa; Chiramel, Abhilash I; Chiurchiù, Valerio; Cho, Dong-Hyung; Choe, Seong-Kyu; Choi, Augustine M K; Choi, Mary E; Choudhury, Kamalika Roy; Chow, Norman S; Chu, Charleen T; Chua, Jason P; Chua, John Jia En; Chung, Hyewon; Chung, Kin Pan; Chung, Seockhoon; Chung, So-Hyang; Chung, Yuen-Li; Cianfanelli, Valentina; Ciechomska, Iwona A; Cifuentes, Mariana; Cinque, Laura; Cirak, Sebahattin; Cirone, Mara; Clague, Michael J; Clarke, Robert; Clementi, Emilio; Coccia, Eliana M; Codogno, Patrice; Cohen, Ehud; Cohen, Mickael M; Colasanti, Tania; Colasuonno, Fiorella; Colbert, Robert A; Colell, Anna; Čolić, Miodrag; Coll, Nuria S; Collins, Mark O; Colombo, María I; Colón-Ramos, Daniel A; Combaret, Lydie; Comincini, Sergio; Cominetti, Márcia R; Consiglio, Antonella; Conte, Andrea; Conti, Fabrizio; Contu, Viorica Raluca; Cookson, Mark R; Coombs, Kevin M; Coppens, Isabelle; Corasaniti, Maria Tiziana; Corkery, Dale P; Cordes, Nils; Cortese, Katia; Costa, Maria do Carmo; Costantino, Sarah; Costelli, Paola; Coto-Montes, Ana; Crack, Peter J; Crespo, Jose L; Criollo, Alfredo; Crippa, Valeria; Cristofani, Riccardo; Csizmadia, Tamas; Cuadrado, Antonio; Cui, Bing; Cui, Jun; Cui, Yixian; Cui, Yong; Culetto, Emmanuel; Cumino, Andrea C; Cybulsky, Andrey V; Czaja, Mark J; Czuczwar, Stanislaw J; D'Adamo, Stefania; D'Amelio, Marcello; D'Arcangelo, Daniela; D'Lugos, Andrew C; D'Orazi, Gabriella; da Silva, James A; Dafsari, Hormos Salimi; Dagda, Ruben K; Dagdas, Yasin; Daglia, Maria; Dai, Xiaoxia; Dai, Yun; Dai, Yuyuan; Dal Col, Jessica; Dalhaimer, Paul; Dalla Valle, Luisa; Dallenga, Tobias; Dalmasso, Guillaume; Damme, Markus; Dando, Ilaria; Dantuma, Nico P; Darling, April L; Das, Hiranmoy; Dasarathy, Srinivasan; Dasari, Santosh K; Dash, Srikanta; Daumke, Oliver; Dauphinee, Adrian N; Davies, Jeffrey S; Dávila, Valeria A; Davis, Roger J; Davis, Tanja; Dayalan Naidu, Sharadha; De Amicis, Francesca; De Bosscher, Karolien; De Felice, Francesca; De Franceschi, Lucia; De Leonibus, Chiara; de Mattos Barbosa, Mayara G; De Meyer, Guido R Y; De Milito, Angelo; De Nunzio, Cosimo; De Palma, Clara; De Santi, Mauro; De Virgilio, Claudio; De Zio, Daniela; Debnath, Jayanta; DeBosch, Brian J; Decuypere, Jean-Paul; Deehan, Mark A; Deflorian, Gianluca; DeGregori, James; Dehay, Benjamin; Del Rio, Gabriel; Delaney, Joe R; Delbridge, Lea M D; Delorme-Axford, Elizabeth; Delpino, M Victoria; Demarchi, Francesca; Dembitz, Vilma; Demers, Nicholas D; Deng, Hongbin; Deng, Zhiqiang; Dengjel, Joern; Dent, Paul; Denton, Donna; DePamphilis, Melvin L; Der, Channing J; Deretic, Vojo; Descoteaux, Albert; Devis, Laura; Devkota, Sushil; Devuyst, Olivier; Dewson, Grant; Dharmasivam, Mahendiran; Dhiman, Rohan; di Bernardo, Diego; Di Cristina, Manlio; Di Domenico, Fabio; Di Fazio, Pietro; Di Fonzo, Alessio; Di Guardo, Giovanni; Di Guglielmo, Gianni M; Di Leo, Luca; Di Malta, Chiara; Di Nardo, Alessia; Di Rienzo, Martina; Di Sano, Federica; Diallinas, George; Diao, Jiajie; Diaz-Araya, Guillermo; Díaz-Laviada, Inés; Dickinson, Jared M; Diederich, Marc; Dieudé, Mélanie; Dikic, Ivan; Ding, Shiping; Ding, Wen-Xing; Dini, Luciana; Dinić, Jelena; Dinic, Miroslav; Dinkova-Kostova, Albena T; Dionne, Marc S; Distler, Jörg H W; Diwan, Abhinav; Dixon, Ian M C; Djavaheri-Mergny, Mojgan; Dobrinski, Ina; Dobrovinskaya, Oxana; Dobrowolski, Radek; Dobson, Renwick C J; Đokić, Jelena; Dokmeci Emre, Serap; Donadelli, Massimo; Dong, Bo; Dong, Xiaonan; Dong, Zhiwu; Dorn Ii, Gerald W; Dotsch, Volker; Dou, Huan; Dou, Juan; Dowaidar, Moataz; Dridi, Sami; Drucker, Liat; Du, Ailian; Du, Caigan; Du, Guangwei; Du, Hai-Ning; Du, Li-Lin; du Toit, André; Duan, Shao-Bin; Duan, Xiaoqiong; Duarte, Sónia P; Dubrovska, Anna; Dunlop, Elaine A; Dupont, Nicolas; Durán, Raúl V; Dwarakanath, Bilikere S; Dyshlovoy, Sergey A; Ebrahimi-Fakhari, Darius; Eckhart, Leopold; Edelstein, Charles L; Efferth, Thomas; Eftekharpour, Eftekhar; Eichinger, Ludwig; Eid, Nabil; Eisenberg, Tobias; Eissa, N Tony; Eissa, Sanaa; Ejarque, Miriam; El Andaloussi, Abdeljabar; El-Hage, Nazira; El-Naggar, Shahenda; Eleuteri, Anna Maria; El-Shafey, Eman S; Elgendy, Mohamed; Eliopoulos, Aristides G; Elizalde, María M; Elks, Philip M; Elsasser, Hans-Peter; Elsherbiny, Eslam S; Emerling, Brooke M; Emre, N C Tolga; Eng, Christina H; Engedal, Nikolai; Engelbrecht, Anna-Mart; Engelsen, Agnete S T; Enserink, Jorrit M; Escalante, Ricardo; Esclatine, Audrey; Escobar-Henriques, Mafalda; Eskelinen, Eeva-Liisa; Espert, Lucile; Eusebio, Makandjou-Ola; Fabrias, Gemma; Fabrizi, Cinzia; Facchiano, Antonio; Facchiano, Francesco; 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Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field