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2. A prognostic model for use before elective surgery to estimate the risk of postoperative pulmonary complications (GSU-Pulmonary Score): a development and validation study in three international cohorts
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Bravo, Laura, Simões, Joana FF, Cardoso, Victor R, Adisa, Adewale, Aguilera, Maria L, Arnaud, Alexis, Biccard, Bruce, Calvache, Jose, Chernbumroong, Saisakul, Elhadi, Muhammed, Ghosh, Dhruv, Gujjuri, Rohan, Harrison, Ewen, Ho, Michael WS, Kasivisvanathan, Veerappan, Kouli, Omar, Lederhuber, Hans, Li, Elizabeth, Löffler, Markus W, Isik, Arda, Marcus, Hani, Martin, Janet, McLean, Kenneth A, Minaya-Bravo, Ana, Modolo, María Marta, Nepogodiev, Dmitri, Pellino, Gianluca, Picciochi, Maria, Pockney, Peter, van Ramshorst, Gabriëlle, Riad, Aya, Sayyed, Raza, Sund, Malin, Gkoutos, Georgios, Bhangu, Aneel A, Glasbey, James C, Bravo, L, Cardoso, V, Glasbey, J, Chernbumroong, S, Nepogodiev, D, Simoes, JFF, Mclean, KA, Bhangu, AA, Gkoutos, G, Kadir, B, Omar, O, Revell, E, Bahrami-Hessari, M, Ahmed, Waheed-Ul-Rahman, Argus, Leah, Ball, Alasdair, Bhangu, Aneel, Bywater, Edward P, Blanco-Colino, Ruth, Brar, Amanpreet, Chaudhry, Daoud, Dawson, Brett E, Duran, Irani, Gujjuri, Rohan R, Jones, Conor S, Harrison, Ewen M, Kamarajah, Sivesh K, Keatley, James M, Lawday, Samuel, Mann, Harvinder, Marson, Ella J, Mclean, Kenneth A, Norman, Lisa, Ots, Riinu, Outani, Oumaima, Santos, Irène, Shaw, Catherine, Taylor, Elliott H, Trout, Isobel M, Varghese, Chris, Venn, Mary L, Xu, William, Dajti, Irida, Gjata, Arben, Kacimi, Salah Eddine Oussama, Boccalatte, Luis, Cox, Daniel, Aigner, Felix, Kronberger, Irmgard Elisabeth, Samadov, Elgun, Alderazi, Amer, Padmore, Greg, van Ramshorst, Gabrielle, Lawani, Ismaïl, Cerovac, Anis, Delibegovic, Samir, Baiocchi, Glauco, Gomes, Gustavo Mendonça Ataíde, Lima Buarque, Igor, Gohar, Muhammad, Slavchev, Mihail, Nwegbu, Chukwuemeka, Agarwal, Arnav, Ng-Kamstra, Joshua, Olivos, Maricarmen, Lou, Wenhui, Ren, Dong-Lin, Calvache, Jose Andres, Perez Rivera, Carlos J, Danic Hadzibegovic, Ana, Kopjar, Tomislav, Mihanovic, Jakov, Avilés Jiménez, Pablo Mijahil, Gouvas, Nikolaos, Klat, Jaroslav, Novysedlák, René, Amisi, Nicolas, Christensen, Peter, El-Hussuna, Alaa, Batista, Sylvia, Lincango-Naranjo, Eddy, Emile, Sameh, Arévalo Sandoval, Danilo Alfonso, Dhufera, Hailu, Hailu, Samuel, Mengesha, Mengistu G, Kauppila, Joonas H, Arnaud, Alexis P, Demetrashvili, Zaza, Albertsmeier, Markus, Löffler, Markus W., Kwesi Acquah, Daniel, Ofori, Bernard, Tabiri, Stephen, Metallidis, Symeon, Tsoulfas, Georgios, Aguilera-Arevalo, Maria-Lorena, Recinos, Gustavo, Mersich, Tamás, Wettstein, Dániel, Ghosh, Dhruva, Kembuan, Gabriele, Brouki Milan, Peiman, Khosravi, Mohammad Hossein, Mozafari, Masoud, Hilmi, Ahmed, Mohan, Helen, Zmora, Oded, Gallo, Gaetano, Pata, Francesco, Fujimoto, Yuki, Kuroda, Naoto, Satoi, Sohei, Abou Chaar, Mohamad K., Ayasra, Faris, Fakhradiyev, Ildar, Hamdun, Intisar Hisham Said, Jin-Young, Jang, Jamal, Mohammad, Karout, Lina, Gulla, Aiste, Rasoaherinomenjanahary, Fanjandrainy, Samison, Luc Hervé, Roslani, April Camilla, Durán Sánchez, Iran Irani, Gonzalez, Diana Samantha, Martinez, Laura, Martínez, María José, Nayen, Alejandra, Ramos-De la 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R, Nissi, Kannaiyan, Lavanya, Matai, Dr. Anil, Misra, Sanjeev, Bhende, Vishal, Muthu, Sathish, Ghosh, Indranil, Sharma, Abhishek, Bajaj, Ankur, Rajan, Shiv, Agarwal, Gaurav, Pawar, Pranay, Alexander, Philip, Vijayakumar, M, Hameed, BM Zeeshan, L, Badareesh, Chaudhry, Navneet Kumar, Baliarsing, Lipika, Dharap, Satish, Kulkarni, Amruta, Thyavihally, Yuvaraja, Pramesh, C S, Soni, Rajesh, Dube, Surya Kumar, Sharma, Shilpa, Singh, Harvinder, Bains, Lovenish, Ghodke, Rahul, Kumar, Ashwani, Sodhai, Vivek, Maji, Suvendu, Basu, Somprakas, Mahakalkar, Chandrashekhar, Kannan, Ravi, Mehraj, Asif, N, Ranganath, Phadnis, Ashish, Yadev, I, Kavalakat, Alfie, Mittal, Rohin, Vallam, Karthik Chandra, Akhavizadegan, Hamed, Rezghi Maleki, Esmaeil, Yousefzadeh Kandevani, Naser, Ikele, Hilary, McNestry, Catherine, Fleming, Christina, O’Brien, Stephen, Abd Elwahab, Sami, Davis, Niall, Javadpour, Mohsen, McDonnell, Brendan, O Connor, Clare, Bolger, Jarlath, Clancy, Cillian, Croghan, Stefanie M, Donlon, Noel, 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Andrés-Asenjo, Beatriz, Tejero-Pintor, Francisco J, Vallve-Bernal, Marc, Vazquez Melero, Alba, Sánchez Blasco, Laura, Escartin, Jorge, Duque Mallén, Victoria, Srishankar, Selvaratnam, Sooriyabandara, Charitha, Basnayake, Oshan, Gunawansa, Nalaka, Weeraddana, Prabuth dulanjan, Vimalakanthan, Thanusan, Gishanthan, Shanthamoorthy, Chandrasinghe, Pramodh, Sauvain, Marc-Olivier, Ghazal, Ahmad, Al-Sabbagh, Yusra, Alhassoun, Turki, Maa Albared, Sara, Naem, Antoine, Alnahr, Hareth, Jisry, Ghassan, Hammed, Ali, Francis Xaviour, Okedi, Turinawe, Gaston, Mubezi, Isaac, Sikakulya, Franck K., Kakeeto, Andrew, Kabweru, Dr. Wilberforce M., Kiweewa, Ronald, Matovu, Paul, Isaac, Otolia, Mashhour, Mohamed, El Helw, Amin, Mohamed, Awadelkarim, Abbas, Ferial Mohamed Ali, Mohammed, Diary, Aldlyami, Ehab, Kundra, Rakesh, Michael, Antony Louis Rex, Khalil, Kareem S., Dbeis, Rachel, Shaikh, Shafaque, Ferry, Jenny, Jamal, Aiman, Siddique, Haleema, Das, Rishi, Ponugoti, Nikhil, Rattanasirivilai, Pornjittra, Sultana, Asma, Mosley, Frances, Chan, Matthew, Bateman, Antony, Davies-Jones, Gareth, Georgiades, Fanourios, Ahmadi, Navid, Coonar, Aman, Baig, Mariam, Khatri, Chetan, Surendran, Arthika, Sonksen, Julian, Sinnerton, Robert, Brennan, Caitlin, Gemma, Greenhalgh, Michael, Emerson, Hannah, Singisetti, Kiran, Totty, Joshua, Wilson, Michael, Lo, Terence, Corbett, Harriet, Okonkwo, Ijeoma, Arbane, Gill, El-Boghdadly, Kariem, Kerawala, Cyrus, Parmar, Chetan, Bath, Michael, Odejinmi, Funlayo, Sagar, Jayesh, Talwar, Rishi, Newman, Samuel, Hammond, John, Moir, John, Duric, Natalie, Szakmany, Tamas, Iftikhar Talib, Ahmar, Youssef, Mina, Lewis-Lloyd, Christopher, Lami, Mariam, Ayub, Khurram, Dean, Benjamin, Balasubramanya, Supriya, Lakpriya, Sathya, Rogers, Luke, Turner, Paul, Maher, Mark, Sigamoney, Kohila, El Kafsi, Jihène, Hardie, John, Johnson, David, Henein, Christin, Hollyman, Marianne, Agarwal, Ketan, Powell, Simon, Chauhan, Govind Singh, Patel, Rakesh, Gagnier, Joel, Carmichael, Heather, Olson, Kristofor A., Etchill, Eric, Hirji, Sameer, Naunheim, Matthew, Drake, Frederick, Reinke, Caroline, Alecci, Anna, Vaysburg, Dennis, Rodriquez, Jennifer, Shih, Emily, Ban, Vin Shen, Coleman, Julia, Kaups, Krista, Giorgakis, Emmanouil, DiNome, Maggie, Bhutiani, Neal, Aiken, Taylor, Diehl, Thomas, Gosain, Ankush, Rattan, Rishi, Ghani, Muhammad Owais Abdul, Ruzgar, Nensi Melissa, Liveris, Anna, Glass, Nina, Paranjape, Charu, Chin, Theresa, Meola, Antonio, Nicholson, Kristina, Squiers, John, Lueckel, Stephanie, Reisenauer, Janani, Callcut, Rachael, Mansour, Ahmed, Berndtson, Allison, Kornblith, Lucy, Seegert, Sara, Martins, Paulo, Al-Shehari, Mohammed, Al-Raimi, Ibrahim, Ngulube, Allan, Siamuchembu, Maphios, Mushiwokufa, Willard, Mlambo, Busisiwe, Chinyowa, Simbarashe, Bollano, Enton, Zijaj, Lorena, Fatima zohra, Belabbes, Bali, Oussama, Benallel, Nassim, Abdoun, Meriem, Bouchenak, Kamel, Soualili, Zineddine, Bastet, Joaquin, Capitaine, Daniel, Centeno Lozada, Jorge, Esquivel, Carlos, Figueroa, Rodrigo, García, Pablo Martín, Mondino, Jose, Traverso, Rogelio, Berber, Norberto, Di Vincenzo, Marcela, Grassano, Bianca, Maricic, Maximiliano, Zezular, Damaris Idara Anabel, Abuawad, Carla, Albani Forneris, Agustin, Brandariz, Rodrigo, Burchakchi, Arturo, Buttaro, Martin, Cano Busnelli, Virginia, Carminatti, Tomas, Cereghini, Julian, Crespi Amor, María Sol, Figari, Marcelo, Gonzalez Salazar, Esteban, Higuera, Felipe, Llano, Lionel, Lobos, Pablo, Mc Loughlin, Santiago, Mendoza, Efrain, Mercado, Pedro, Moyano, Pablo, Noll, Florencia, Odetto, Diego, Ramos, María Lourdes, Saadi, Jose, Scherñuk, Jordán, Slullitel, Pablo, Taboada, María Victoria, Vagni, Roberto, Montal, Silvina, Alvarez, Fernando Andres, Bruera, Nicolás, Parodi, Matias, Picon molina, Héctor, Belisle, Diego, Esteban, Agustin, García, José Sebastian, Granero, Lucas, Yaryura Montero, José Gabriel, D’Addino, José Luis, John, Brooke-Smith, Mark, Hii, Amanda, Kundu, Nikhil, Marshall-Webb, Matthew, McVeay, Christina, Ong, Bee Shan, Parker, Dominic, Barnett, Dylan Richard, Buddingh, Karel, Dobbins, Christopher, Dudi-Venkata, Nagendra, Haque, Izhar-Ul, Herath, Matheesha, Lim, Alicia, Moller, Cea-Cea, Neo, Eu Nice, Neo, Eu Ling, Russell, Christine, Tourani, Saam, Tran, Steven, Yin, Yijie, Watson, Matthew, Rugendyke, Anya, Sercombe, Ashleigh, Tee, Chin Li, Batstone, Martin, Field, Michael, Scott, Benjamin, Charlton, Gabriella, Chen, Si, Gauri, Neha, Hayhurst, Ross, Jang, Seyoung, Zhang, Helen, Nagra, Sonal, Rudock, Gareth, Tan, Hannah, Underwood, Kirk, Youssef, Daniel, Bengeri, Savitha, Cheah, Hock Ping, Chen, Paul, Clark, Sara, Devan, Eliya, Fitt, Sophia, Gaul, John, Jolly, Danielle, Kane, James Dimitri, Kwok, Kelvin, Laura, Sharon, Lee, Vivian, Liang, Ina, Luo, Haili, Miles, Stephanie, Noor, Anthony, Simpson, Renwick, Cox, Aram, Thani, Nova, Egoroff, Natasha, Farik, Shebani, Moss, Jana-Lee, Salama, Paul, Phan, Du, Townend, David, Barnett, Stephen, Hasan Kheslat, Hajar, Jamieson-Grigg, Chloe, Larner, Brett, Proud, David, Riddiough, Georgina, Seevanayagam, Sivendran, Zhao, Chris, An, Vinna, Drake, Anna, Hunt, Benjamin, Jain, Anshini, Lim, Christopher Seng Hong, Luck, Tara, Maroske, Georgia, Roubos, Nicholas, Sengupta, Shomik, Tsigaras, Zac, Ward, Salena, Zhou, Jessie, Coates, Amy, Nataraja, Ramesh, Gillard, Andrew, Petrakis, Nikki, Qureshi, Maryum, Seow, Sean Ezekiel, Vivian-Taylor, Josephine, Wheatley, Jennifer, O’Connor, Brendan, Teague, Warwick, Hii, Michael Wei, Isaacs, Anna, Johnson, Mary Ann, Price, Veronique, Rowcroft, Alistair, Shaw, Kalai, Stark, Claire, Lauritz, Brianne, Ong, Chui Foong, Tang, Howard, Davis, Amelia, Peerally, Bibi Nabeeha, Aubin, Anne-Marie, Lim, Zhi Ying, Alsoudani, Ali, Amico, Francesco, Anning, Rebecca, Dale, Tyson, Duggal, Anchal, Ennis, Brendan, Fu, Yi Xin Joanna, Gani, Jonathan, Gelzinnis, Scott, Hadlow, Claudia, Hardstaff, Ruth, Lau, Wayne Yan, Liang, Hsin-Ping, Lim, Daniel, Mcilwain, Elysse, Ming, Yan Joyce, Ocsan, Ryan James, Organ, Nicole, Paterson, Amanda, Perez Cerdeira, Marisol, Ting, Caleb, Willoughby, Ellisha, Bailey, Ashley, Narayanan, Viswanathan, Wilkes, Anna, Zhao, Jie, D’Souza, Carl, Forbes, Clara, Haddleton, Monique, Mohammed Abdul, Naseer, Archer, Leigh, Dilevska, Tina, Hans, Tasvinder, Kumar, Mohit, Pham, Terence, Picazo Pineda, Fernando, Suk-Udom, Supisara, Jeyamanoharan, Nivedan, Sala, Michael, Dravid, Madhulika, McCartney, Conor, Sam, Apostolou, Christos, Berney, Christophe, Lam, Andrew, Ma, Vanessa, Cope, Jennifer, Llorando, Alyssa, Mahin, Humaira Haider, Syed, Faisal, Ireland, Patrick, Zhu, Bonnie, Zitt, Matthias, Königsrainer, Ingmar, Allmer, Caterina, Mitteregger, Martin, Seitinger, Gerald, Duller, David, Mathew, Erwin, Skias, Christoph, Brinskelle, Petra, Holzmeister, Clemens, Kahn, Judith, Kresic, Josip, Lindenmann, Joerg, Lumenta, David, Puchwein, Paul, Singer, Georg, Wolf, Axel, Arco, Daniel, Kilic, Nura, Widschwendter, Peter, Freyschlag, Christian, Graber, Michael, Hirsch, Jakob, Kuen, Marlene, Liebensteiner, Michael, Messner, Franka, Messner, Alex, Naegele, Felix, Osl, Antonia, Ower, Cornelia, Rauchegger, Teresa, Reimer, Daniel, Sokolovski, Filipp, Süss, Markus, Thaler, Martin, Zehetner, Claus, Allerstorfer, Jakob, Haslhofer, David, Pisecky, Lorenz, Poier, Nikolaus, Schmolmüller, Christoph, Zwittag, Paul, Windischbauer, Amadeus, Fischer, Ines, Kirchweger, Patrick, Saini, Thomas, Berger, Julian, Fraz, Wolfgang, Nia, Arastoo, Kenez, Mihaly, Nichita, Margit, Noe, Chiara, Borhanian, Kurosch, Emmanuel, Klaus, Gantschnigg, Antonia, Grechenig, Michael, Gruber, Ricarda, Manzenreiter, Lisa, Schredl, Philipp, Weitzendorfer, Michael, Gruber, Michaela, Grünbart, Martin, Kemmetinger, Vanessa, Mosshammer, Victoria, Schaetz, Tobias, Lanner, Maximilian, Binder, Alf-Dorian, Bernardi, Martin H., Dawoud, Christopher, Foessleitner, Philipp, Grimm, Christoph, Pesta, Maximilian, Pillerstorff, Robert, Riss, Stefan, Scheriau, Georg, Fisus, Andreea, Ruiss, Manuel, Meindl, Nikolaus, Primavesi, Florian, Steiner, Florian, Falkensammer, Eva, Knotzer, Hans, Trivik-Barrientos, Felipe, Alam, Mahmood, Mulla, Husain, Qader, Kawthar, Shirazi, Ahmed, Juma, Isam, Waheed Akbar Mohamad Akbar Nawab Deen, Fatema, AbdulQadir, Seemal, Jabr, Abdulla, Almoosa, Noora, Alam, Mohammed Shadrul, Basher, AKM Khairul, Islam, S.M. Nazmul, Khan, Sadia, Mannan, Iftekhar Ibne, Corbin, Sasha, O’Shea, Margaret, Phillips, Emil, Straltsov, Vladislav, Lyzikov, Alexei, Borges, Mafalda, Vleminckx, Nils, Bontinck, Julie, Van Haver, Anne-Sophie, Viste, Claire, Van Daele, Elke, Jansen, Yanina, Messaoudi, Nouredin, Ruyssers, Michael, Van Eetvelde, Ellen, Akpla, Marcellin, Agossou, Hermann, Aouagbe Behanzin, Hulrich, Yome, Hugues, Boukari, Alassan, Amossou, Labissi Francois, Dossou, Francis, Boras, Miran, Čuljak Blagojević, Ivana, Gunaric, Filip, Letica, Ludvig, Martinovic, Vlatka, Miskovic, Josip, Rastović, Pejana, Sesar, Irena, Favaretto Filho, Rodolfo Jose, Rodrigues, Rodrigo, Schalge Campioto, Débora, Alonso, Nivaldo, Fernandes Rezende, Ricardo, Marson, Fernando Augusto Lima, Accorsi Neto, Alfeu, Accorsi, Guilherme, Machareth, Camila, Viegas Moura, Barbara, Guarienti, Luciano, Becker, Karin, Dias, Andre, Girardi Fachin, Camila, Stychnicki, Adriano Seikiti, Avelino, Melissa, Elaine Francisca, Amorim, Robson, Bellotti Leão, Maria Eduarda, Melo da Camara, Isabelle, Silva Junior, Rubem Alves, Soares Da Silva Menezes, Agna, Cavazzola, Leandro Totti, Roloff Cardoso, Guilherme, Silva Neto, Brasil, Soares, Tilaê, Guerra, Enilde Eloena, Salem, Moacyr, de Campos Prado, Caio Antonio, Zani, Ana Carolina Tagliatti, Lourenço Gomes dos Santos, Carolina, Quintana, Silvana Maria, Veccechi Bijos Zaccaro, Marília, Lima, Leonardo, da Cunha Viana Júnior, Alonço, Trindade Martins, Isabela, R Oliveira, Priscila, Schmitz Nunes, Kamilla, Abdallah, Emne, Facure, Luciana, Kupper, Bruna, Leite, Fernanda, Marques, Narimã, Scintini Herbst, Ana Carolina, Soares Dos Santos, Silvana, Gonçalves, Rodrigo, Machado, Vinicius, Nadal, Luis Roberto, Pansani, Adrieli, Simonsen, Marcelo, Tsuchiya, Daniela, Vasconcelos, Vladimir, Kim, Nam Jin, Nunes, Rafael, Pinto, Fabio, Sigoli Pereira, Thiago Henrique, Volpe, Gustavo Jardim, Nardi, Caroline, Braz de Oliveira, Layze, Ribeiro, Ivonizete Pires, Sousa, Álvaro Francisco Lopes de, Freitas, Marcelo Augusto Faria, Rodrigues da Cunha, Juliano, Dimitrov, Dobromir, Gyokova, Elitsa, Neykov, Vasil, Atanasov, Boyko, Kamenova, Paolina, Yotsov, Tsanko, Spassov, Kolyo, Tsankov, Tsanko, Brown, James, Bali, Krittika, Bigam, David, Dajani, Khaled, Hong, Dennis, Luketic, Lea, Mayer, Robert, McGee, Jacob, Yang, Homer, Hanson, Melissa, Hopkins, Brent, Merchant, Richard, Belzile, Etienne, Boucher-Kovalik, Sofia, Corriveau-Durand, Simon, Cote, Mathieu, Côté, Mathilde, Demers, Suzanne, Desbiens, Christine, El Ouazzani, Mehdi, Gosselin, Karo, Maheux-Lacroix, Sarah, Nadeau, Sylvie, Nguyen, Sebastien, Pelet, Stéphane, Wood, Melissa, Atrey, Amit, Ellenbogen, Yosef, Kellett, Sorcha, Suresh, Hrishikesh, Gentili, Fred, St. Jacques, Leslie, Tabasinejad, Raha, Hsiao, Marvin, Broe, Claire, Grey, Rebecca, Lindberg, Andrew, Mata Gutierrez, Juan, Phillips, Drew, Fernández, Esteban, Ochoa Gaete, Katherine, Sandoval Tobar, Mauricio, Reyes, José Tomás, Rivera, Barbara, Cohn, David, Recabal, Pedro, Sandoval, Camilo, Pincheira, Camila, Torres, Janina, Valenzuela, Marco, Yang, Kun, Zhang, Yuexin, Cunchuan, Wang, Yang, Wah, Liang, Tingbo, Caicedo, Camilo, Perez Granados, Carolina, Serrano, Oscar, Vega Calvera, Andrea Juliana, Acosta Buitrago, Lina M., Cabrera Rivera, Paulo Andrés, Cormane Alfaro, Valeria, Guerrero-Becerra, Albert Franz, Kadamani Abiyomaa, Akram, Polanía Sandoval, Camilo Andrés, Roman Ortega, Carlos Fernando, Téllez, Manuela, Arias-Amézquita, Fernando, Escorcia, Elkin, Herrera-Almario, Gabriel, Monsalve, Guillermo, Caicedo Giraldo, María Alejandra, Castillo Florez, Maria Carolina, Cendales, María Angelica, Daniel, Mario, Espitia, Hernando, Navarro, Juan Carlos, Parra Abaunza, Katherine, Pulido, Eliana, Riveros Castillo, William Mauricio, Rubiano, Wilson, Salgado Tovar, Javier Mauricio, Trillos, Marcial, Villate leon, Juan pablo, Becerra Mendez, Luis, Beltran, Rafael Jose, Calderon Quiroz, Pedro Hernando, Cervera Bonilla, Sergio, Garcia Mora, Mauricio, Mariño, Ivan, Pinilla Morales, Raúl Eduardo, Puerto, Angela, Quintero-Ortíz, Ma. Andrea, Suarez, Raul, Velez Bernal, Jorge Luis, Camargo Gómez, Daniela, Christian kammerer, Acuna Saravia, Maria Fernanda, Arroyave, Roberto, Barreto Angulo, Jose Maria, Dueñas-Ramirez, Juan Carlos, Escobar Vidarte, Oscar Andres, Gomez, Laura, Gomez, Alejandro, Herrera Castañeda, Enrique, Hinaoui, Marisol, Idrobo Escobar, Anuar Armando, Llanos Lucero, Carlos Antonio, Montoya Casella, Antonio José, Perea Serna, Andrea Carolina, Ríos-Samper, Gabriel, Sandoval, John, Sarmiento Ramirez, Guillermo Alberto, Zuluaga Zuluaga, Mauricio, Figueroa, Juan Sebastian, Gonzalez Mosos, Maria Fernanda, Jaramillo Roncancio, Juan Jose, Mosos, Monica, Ramirez, Juan Sebastian, Idarraga Ramírez, Leidy Natalia, Marin Gonzalez, Ana Maria, Mejia, David Alejandro, Vanegas, Luis Emiro, Aruachan Vesga, Sandra, Martinez Estrada, Gustavo Antonio, Giraldo Velasquez, Angela Maria, Tovar, Jesus Hernan, Tovar, German Alirio, Buesaquillo, Christian Ali, Olave Montaño, Victor David, Genda, Eben-ezer, Eboma, Fabrice, Grulović, Karlo, Bosak Versic, Ana, Srsen Medancic, Suzana, Brkic, Lucija, Cokarić, Sara, Tomić, Josipa, Brzic, Domagoj, Dijan, Emilio, Grgec Dragicevic, Maja, Konjevoda, Suzana, Morović, Domagoj, Perišić, Gordan, Zuzul, Matea, Anzic, Srdan Ante, Carevic, Iva, Biočina, Bojan, Bobovec, Dino, Bumber, Boris, Duric Vukovic, Katarina, Jelčić, Ivan, Kastelan, Zeljko, Kolak, Juraj, Pasini, Miram, Penezić, Luka, Prstacic, Ratko, Romić, Ivan, Tomic, Mislav, Luksic, Ivica, Panayiotou, Yiannis, Stavrinidou, Olga, Yiallourou, Anneza, Michal, Burda, Lukáš, Gawel, Wladyslaw B., Lerch, Milan, Macečková, Nicole, Simetka, Ondrej, Vavra, Petr, Zelenik, Karol, Přibyl, Martin, Klíma, Karel, Pirmorad, Setareh, Hotová, Zuzana, Mladěnka, Aleš, Bozo, Nulvin, Hviid, Louise, Kristensen, Helle Ø, Paulsen, Laerke, Ebbehøj, Anders Lyng, Jönsson, Maria Lovisa, Krarup, Peter-Martin, Otte, Helena, Smith, Henry, Bælum, Jens Kristian, Ellebæk, Signe Bremholm, Collado Expósito, María Mabel, Crespo, Aldo, D’óleo García, Claudio Samuel, Figueroa, Jatnna, Garcia-Dubus Rodriguez, U, Guzman, Lillian, Lopez, Ariela, Maletta Francisco, Leeany, Munoz, Herisardy, Perez Fernandez, Maricely Ambar, Rodríguez, Ada, Soto, Joel, Ubinas, Raul, Villegas, Aaron, Acra-Tolari, Ricardo, Beltran, Larissa, Betances, Luis Fernand, Díaz Vásquez, Pedro Pablo, Fernandez, Damaris, Figueroa Germosen, Jiomar Manuel, Mejia De la Cruz, Dolores, Ortiz De La Cruz, Fabio, Pelletier, Gabriela, Próspero Enrique, Rodriguez Pumarol, Suero Almanzar, Irina, Negrete Ocampo, José Ricardo, Sallam, Ibrahim, Sherief, Mohamed, Abdou, Mostafa, Abo Shanab, Ahmed, Abodeeb, Aya, Aboelkhel, Roger, Abosamak, Nour Eldin, Abouelnagah, Yossof, Adel, Dina, Ahmed, Sara, Alberkamy, Abdelrahman, Ali, Ahmed, Ashraf, Olfat, Darwish, Sara, El kharashy, Rasha, Elbadawy, Seifeldin, Elsaka, Alromisaa, Ewedah, Moataz, Ezz, Yara, Gadelrab, Ziyad, Hafez, Nour, Hafez, Youssef, Hassanin, Mohamed, Hussein Aly Salama Aly, Hamza, Ibrahim, Abdelrahman, Mahmoud Abo shabana, Alaa, Mourad, Mohamed, Mubarek, Kamilia, Omar, Samaa, Ragal, Marawan, Romany, John, Salem, Alaa, Samir Abdelaal, Ahmed, Shehata, Abdelrahman, Shenit, Karim, Yehia, Nermin, Khaled Mohamed Eid, Omar, Ibrahim Elsayed, Omar, Shehata, Mostafa Ahmed, Abd El-Ghani, Wael, Abd Elazeem, Hossam Aldein S., Abdalla, Shimaa, Abdel-Aleem, Mahmoud, Abdelfattah, Mahmoud, Abdelhafez, Mohammed, Abdelkarem, Mohamed M., Abubeih, Hossam, Ahmed, Nagm Eldin Abu Elnga, Ahmed, Ahmed, Ahmed Saad, Sarah, Ali, Abdelrahman Ahmed Abdelrahman, Elmaghraby, Khaled, Elsdfy, Shady, Eltayeb, Almoutaz, Essam, Esraa, Gadelkareem, Rabea, Ghoneim, Ahmed, Hamada Takrouney, Mohammed, Hasan, Ahmad, Hassanin, Mohamed Abdelghafor, Mahran, Wesam, Mahrous, Doha, Ragab, Abdelrahman, Sabry, Aya, Safwat, Hadeer, Saleh, Ahmed, Samir, Ahmed, Sayed, Esraa, Taher, Mohamed Gamal, Yousof, Ebrahim Ahmed, Youssef, Ahmed, Zein Elabedeen, Omar, Ali, Samar, Elbehairy, Gehad, Eleisawy, Mahmoud, Elgendy, Abdelrahman, Elhawary, Rewan, Hamdy, Mahmoud, Hassan, Gehad, Nowar, Mostafa, Sabry, Hesham, Tarek Said-elnaby, Ahmed, Zahed, Mohamed, Zahran, Mohamed, Zoghary, Ahmed Zaki, Atef, Mohamed, Abdelhamid, Mahmoud Hossameldin Saad, El Fiky, Lobna, ElGarhy, Ibrahim, Wahba, Abdelrahman, Youssif Omar Fouad, Omar, Abdelsamed, Ahmed, Elsalhy, Mohamed, Eldesouky, Omnia, Wael Mostafa Khalil Bahi, Abdullah, Tawheed, Ahmed, Al-oribi, Saeid, Aly, Nuran Khaled, ElFiky, Mahmoud, Nabil, Ahmed, Samir Farahat, Ahmed, Soliman, Mostafa, Ghaly, Galal, Abbas, Alzhraa Salah, Ibraheem, Maher, Bakry, Abdelrahman, Jammal, Mohamed, M Makram, Abdelrahman, Adel abdelaty, Ahmed, Fayad, Elsayed A., Sallam, Moataz, Elfeki, Hossam, Elsaid, Mahmoud, Mostafa, Mohamed, Sakr, Ahmed, Shetiwy, Mohamed, Tawakl, Noura, Yunes, Asmaa, Atallah, Khalid, Shetiwy, Mosab, Abdelmawla, Ahmed, Abdrabou, Ahmed, Abubakr, Asmaa, Al Gohary, Rawda, Elsabagh, Alaa, Elshabrawy, Enas, Hafez, Ahmed, Mikhail, Pola, Mohamed, Mervat, Nada, Ahmed, Omran, Janna, Salem, Osama, Selim, Salma, Shalaby, Ghada, Wahbah, Mahmoud, Zakaria Abdelbary, Eman, Zayan, Ahmad Helmy, Afify, Abdelrahman, Mohamed Ahmed, Mostafa, Abdeldayem, Hesham, Abdelkader Salama, Ibrahim, Ammar, Khaled, Ayoub, Islam, Gad, Emad Hamdy, Hammad, Essam, Macshut, Mahmoud, Shoreem, Hany, Soliman, Hossam Eldeen, Eldaly, Abdullah, Mashaly, Sarah, Abd-Elsalam, Sherief, Abdel-Elsalam, Wafaa, Morsy, Mohamed Sherif, Nasreddin, Mohammed, Sarsik, Sameh, Kams, Liisa, Rätsep, Tõnu, Riips, Karolin, Abebe, Kirubel, Asfaw, Fitsum, Eshete, Mahder, Gedefaw, Yetsedaw, Menjeta, Abeje, Muleta, Mahteme Bekele, Sefera, Sena, Teklu, Daniel, Tirfe, Leake, Worku, Bereket, Eado, Yegeremu, Woldemariam, Mersha Abebe, Alemu, Megersa, Azmach, Dawit, Mamo, Dr.Mickyas, Menkir, Abel, Woldemariam, Dereje, Wubishet, Estifanos, Supha, Melka, Abebe, Nebyou, Chanie, Abera, Gebre, Hiwot, Laeke, Tsegazeab, Mammo, Tihitena Negussie, Sahlu, Abat, Tiruneh, Abraham Genetu, Zewdneh, Betelhem, Fentaw, Jibril, Tabore, Ketema, Addisalem, Abreha, Tassew, Abdissa, Desalegn, Tesfaw, Mequannet, Asmamaw, Dawit, Bedane, Dereje, Bekele, Ephrem, Getie, Aderaw, Girma, Adissu, Shimelash, Dagim, Ayalew, Tewabe, Kassahun, Berhanu, Mulugeta, Esubalew, Ibrahim, Adem, Mekete, Alem, Mohammed, Adnan Abdulkadir, Abdullahi, Yasir Younis, Adem, Ephrem, Siyoum, Eneyew Getachew, Zeleke, Ewunetu, Degefe, Derje Worku, Eliyas, Nebiyu, Deressa, Yadani, Melese, Lemi, Muleta, Lemesa, Teshome, Abraham, Tesso, Birhanu, Gebrekirstos, Dr.Gebreagziabher, Pitkänen, Joel, Qian, Cheng, Sinikumpu, Jaakko, Dakpé, Stéphanie, Devauchelle, Bernard, Lavagen, Nolwenn, Bastard, François, Bin, Kim, Moukoko, Didier, Alshawared, Fadi, Beyrne, Carlos Daniel, Lugans, Laurene, Doussot, Alexandre, Onorati, Ilaria, Radu, Dana, Marion, Herjean, Trilling, Bertrand, Boleslawski, Emmanuel, Noiret, Barbara, Aubry, Estelle, Christou, Niki, Usseglio, Julie, Denis, Waast, Regenet, Nicolas, Rigaud, Jerome, Anract, Philippe, Dohan, Anthony, Cazelles, Antoine, Chamouni, Alexandre, Karoui, Mehdi, Mejean, Arnaud, Thouement, Clermidi, Pauline, Langlais, Tristan, Leonelli, Lorenzo, Thomin, Anne, Athiel, Yoann, Cathala, Nathalie, Levy-Zauberman, Yael, Macek, Petr, Mombet, Annick, Ollat, Didier, Fabrice, Chartier-Kastler, Emmanuel, Chereau, Nathalie, Melot, Charlotte, Turco, Célia, Chouillard, Elie, Frasca, Denis, Livin, Marie, Mahmoud, Fedy, Sulpice, Laurent, Cazemajou, Clément Thierry, Nyangoh Timoh, Krystel, Renault, Annaëlle, Cherkaoui, Zineb, Vix, Michel, Gornes, Hugo, Berthoumieu, Pierre, Pierre, Trocard, Del, Mathilde, Vancon, Antoine, Machemedze, Solomon, Muhemi, Roger, Ndizeye, Olivier, O’Connor, Jennifer, Tchoba, Simplice, Schäfer, Benedikt, Wallqvist, Julia, Ziemann, Sebastian, Anthuber, Matthias, Broecheler, Tobias, Goßlau, Yvonne, Maksymiw, Florian, Sommer, Florian, Sommer, Björn, Loch, Florian N, Aghalarov, Ilgar, Braumann, Chris, Horn, Julian, Egger, Eva, Enderes, Jana, Strieth, Sebastian, Treede, Hendrik, Wittmann, Maria, Marche, Benedikt, Schulz, Sissy-Amelie, Fuchs, Hans, Meisel, Cornelia, Petzold, Andrea, Knoefel, Wolfram Trudo, Solodarenko, Oleksandra, Grützmann, Robert, Hackner, Danilo, Faqar-Uz-Zaman, Sara Fatima, Fung, Christian, Leiber, Christian, Neidert, Nicolas, Sandkamp, Richard, Strähle, Jakob, Becker, Johannes, Gonschor, Bernhard, Herbolzheimer, Marit, Hecker, Matthias, Reichert, Martin, Sander, Michael, Schmidt, Götz, Schneck, Emmanuel, Kleeff, Jorg, Lorenz, Kerstin, Thomssen, Christoph, Dumpies, Christian W., Fischer, Isabel, Gessner, Michael, Klauke, Friederike, Schmidt, Birte, Vinz, Franziska, Sander, Johannes, Betz, Christian Stephan, Bewarder, Julian, Burg, Simon, Klatte, Till Orla, König, Daniela, Köpke, Leon-Gordian, Stangenberg, Martin, Roux, Frederic, Hakami, Ibrahim Abdullah, Mall, Julian W, Graeb, Christian, Huber-Strößner, Kristin, Pempe, Christina, Kelly, Kathrin, Siebert, Julia, Agrawal, Rachit, Qureischie, Homeira, Meyer, Frank, Turial, Salmai, Al-Nawas, Bilal, Battista, Marco Johannes, Heider, Julia, Mueller, Lena Katharina, Jentschura, Sina- Louisa Patrizia, Kowalewski, Karl-Friedrich, Reissfelder, Christoph, Rotter, Nicole, Scherl, Claudia, Seyfried, Steffen, Fichter, Andreas, Schäffer, Christoph, Wegmann, Helmut, Aghamaliyev, Ughur, Böcker, Wolfgang, von Ehrlich-Treuenstätt, Viktor H., Ladurner, Roland, Schlager, Justin Gabriel, Smolka, Wenko, Glowalla, Claudio, Schneidmueller, Dorien, Henkel, Karl Wilhelm, Hölz, Katharina, Prantl, Lukas, Kröger, Marie, Niemeier, Andreas, Sras, Yara, Steidle, Christoph, Dayan, Davut, Greve, Jens, Löbig, Niklas, Schochter, Fabienne, Wezel, Felix, Schmidbauer, Stefan, Boenicke, Lars, Degener, Stephan, Seiberth, Rose, Smit, Marieke, von Rundstedt, Friedrich-Carl, Germer, Christoph-Thomas, Lock, Johan, Lotz, Christopher, Schlesinger, Tobias, Schmid, Benedikt, Treutlein, Agnes, Agyeman-Duah, Nana Kwaku, Appiah, Enoch, Gakpetor, Delali, Minlah Allah, Stephen, Adu-Aryee, Nii Armah, Agboadoh, Nelson, Bediako Bowan, Antoinette, Brown, George Darko, Clegg-Lamptey, Joe-Nat, Fenu, Benjamin Sena, Kumassah, Philemon, Hussey, Romeo, Agbeno, Evans Kofi, Amoako-Boateng, Mabel, Debrah, Samuel, Doku, Dr Kingsley, Mensah, Philip, Nortey, Michael, Opandoh, Isabella Naa Morkor, Yigah, Makafui, Jiagge, Nuna, Acquaye, Jane, Agyemang-Prempeh, Akosua, Amo-Antwi, Kwabena, Amoah, Michael, Amoah, George, Aning, Daniel Gyawu, Annor Mintah, Dominic, Arthur, Joshua, Darko- Asante, Regina, Gaveh, Valerie, Gyamfi, Frank Enoch, Jonathan, Boakye - Yiadom, Konney, Thomas Okpoti, Kyei, Ishmael, Nimako, Boateng, Ofosu-Barko, Ben Blay, Osabutey, Anita, Sagoe, Robert, Yifieyeh, Abiboye, Ansong, George, Abdul-Mumin, Alhassan, Amadu, Munira, Azahares Leal, German, Buunaaim, Alexis, Cheyuo, Ernest, Daboo Salifu, Latif, Issaka, Adamu, Yakubu, Musah, Grypiotis, Ioannis, Papadopoulou, Triada, Korkolis, Dimitrios, Thomakos, Nikolaos, Dellaportas, Dionysios, Dimitra, Papalouka, Hadjizacharias, Theodoros, Konstadoulakis, Manousos, Massaras, Dimitrios, Vezakis, Antonios, Avgerinos, 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Naskar, Upasana, Jain, Prateek, Jain, Deepak, Kewlani, Vishal, Priya, Noopur, Parijat, Anand, Anand, Akshay, Chaurasia, Akhilanand, Enny, Loreno E., Kumar, Ambrish, Mishra, Brijesh, Pandey, Amita, Roy, Ashutosh, Sachan, Rekha, Sankhwar, Pushp, Singh, Uma, Singh, Urmila, Srivastava, Chhitij, Verma, Manju Lata, Yadav, Awdhesh, Chanthar, K.m.m.vishvak, Hoysal, Dileep, Mishra, Anjali, Batra, Nitin, Bhatti, Arun, Dhar, Tapasya, Dutta, Rohini, Haque, Parvez David, Luther, Anil, Mahajan, Amit, Pargal, Dr Pinki, Samuel, Abhishek, Singh, Inderjot, Singh Grewal, Sarvpreet, Singhania, Anusha, Thomas, Josy, Krishna, Sunil, Singh, Narinder, Agarwal, Sunny, Balasubramaniam, Srikant, Mhamane, Rameshwar, Nirgude, Anand, Parab, Sandesh, Peswani, Amit, Sahu, Anjana, Samel, Sarika, Shah, Fagun, Abraham, Bejoy, Asari, Ashishkumar, Badhe, Vivek, Badhwar, Sanjiv, Bothara, Vipul, Dalal, Anuj, Date, Avinash, Deshpande, Mandar, Doshi, Anirudha, Gade, Maya, Gajjar, Shreyash, Ghugare, Dr bhavisha, Jain, Divakar, Jenasamant, Saumya Sekhar, Joshi, Vinay, Kalwadia, Neha, Kayal, Akshat, Kulkarni, Yogesh, Mistry, Rajesh, Mulchandani, Manoj, Parab, Dr. Mrunal, Pednekar, Abhinav, Rahmani, Faizan, Rajput, Sunil, Sandlas, Gursev, Sarkar, Hrishikesh, Shetty, Sanket sadanand, Shukla kulkarni, Anshumala, Thete, Raghavendraswami, Wasnik, Shruti, Dhumane, Parag, Sharma, Rahul Deo, Deshmukh, Anuja, Gupta, Stuti, Nayak, Prakash, Thiagarajan, Shivakumar, Tiwari, Virendra Kumar, Voppuru, Saiesh Reddy, Bajpai, Minu, Farooque, Kamran, Parshad, Rajinder, Sharma, Vijay, Bansal, Kuldeep, Bhardwaj, Rohit, Mittal, Abhishek, Ponnusamy, Sabarirajan, Himani, Gadekar, Anup, Panjwani, Taufiq, Kandwal, Pankaj, Kumar, Shashank, Poonia, Dharma Ram, Sadhasivam, Saravanan, Choudhury, Yousuf, Das, Devishmita, Goala, Subhadra, Singha, S Thoibisana, Singha, Damayanti, Parray, Fazl, Shah, Raahil, Wani, Rauf, Hegde, Siddhi, Ankadavar, Dr.sushil, Bafna, Darshan, Lad, Parag, Chisthi, Meer, Jothi, Harihara, C. Mathew, Godwin David, Abel, Livingston, Barla, Ravi Kishore, Benjamin, Santosh, Bliss, Jeremy, Ete, Geley, James, Deeptiman, Jesudason, Mark Ranjan, Karnan, Gomathi, Mark Samuel, Vasanth, Nathan, Senthil K, Selvaraj, Daniel, Surendran, Suraj, T D, Hariharan, Thangaraj, Santhosh Kumar, Thomas, Varghese, Dilmy, Mohammad Adya Firmansha, Pratama, Dedy, Sugiarto, Adhrie, Surya, Ilham Utama, Tango, Tamara, Hasyim, Andi, Susanti, Ade, Saputra, Teddy, Syarifuddin, Erwin, Islam, Andi Asadul, Anam, Khoirul, Sugianto, Julius Albert, Lestari, Mayang Indah, Amba, Erick Gamaliel, Apriawan, Tedy, Wathoni, Roidah Taqiyya Zahra, Widyastuti, Yunita, Yun Jufan, Akhmad, Etezadpour, Mohammad, Mehrad-Majd, Hassan, Rajaei, Parisa, Mehdizadeh, Mohammad, Soltani, Mina, Ali Moazami Pour, Gholamreza Azarnia, Alizadeh, Narges, Hossein Mokarami, Tehrani, Shahrzad, Alborzi, Majid, Bagheri, Hamed, Eshraghi, Mohsen, Fatemi manesh, Hassan, Hejazi, Seyed fakhreddin, Pezeshki Modarres, Mahdi, Shafiee, Ali, Vafaeimanesh, Jamshid, Hosseini, Elahe, Nikoupour, Hamed, Bastaninejad, Shahin, Firouzifar, Mohammadreza, Motasaddi zarandy, Masoud, Yazdani, Nasrin, Mortazavi, SM Javad, Ahmadi, Sayedali, Vosoughi, Fardis, Golbakhsh, Mohammadreza, Al-Isawi, Ali, Al-Masood, Mohammed, Gahtan, Yarub, Ahmed, Abdullah, Muhssein, Haidar, Ekwere, Bosom, Essajee, Murtaza, Chan Chin, Bruno, Edwards Murphy, Amy, Foley, Niamh, Gardiner, Padraig, Hechtl, Daniel, Kayyal, Mohd yasser, Lyons, Maria, Ng, Mei Yee, Oduola, Oladayo, Aljohmani, Lylas, Nugent, Timothy, Harris, Johnathon, Kearney, David, Keenan, Robert Anthony, Nolan, Deirdre, Salloum, Donohoe, Emma, Larkin, John, Lynch, Thomas Hugh, Manecksha, Rustom, Nagassima, Katharina, Barry, Mary, Dowdall, Joseph, Hurson, Conor, Kelly, Orlaith, Kennelly, Rory, Kiernan, Aoife, Murphy, Ben, Njeze, Nwabundo, Reynolds, Ian Sean, Winter, Des, Ravi, Akshaya, Ridgway, Paul, Fowler, Amy, McAnena, Peter, Bahadoor, Muhammad Usaama, Khalid, Mohamed Hamed, Mujtaba, Syed Nadeem, Sarwar, Muhammad assam, Sugrue, Michael, Varzgalis, Manvydas, Jones-Whiting, Larne, Lim, Seantee, Sara, Al-Mukhaizeem, Youssef, Crowley, Clare, Flanagan, Michael, Fogarty, Amy, Glynn, Orna, Hill, Rhodri, Khalid, Muhammad Abdullah, Majeed, Zubair, McCullough, Peter, Neary, Peter, Noone, Anthony, O’Kelly, John, Abu abed, Arsan, Haim, Nadav, Hershkovitz, Yehuda, Majadla, Omar, Rabin, Igor, Zmora, Osnat, Demma, Yonatan Avraham, Fishman, Yuri, Farkas, Amicur, Gozal, Yaacov, Greenman, Dmitry, Ostrovsky, Israel Alexander, Pardes, Rivka, Shahar, Tal, Teren, David, Yahud, Reuven, Yellinek, Shlomo, Cianci, Pasquale, Minafra, Marina, Restini, Enrico, Bussolin, Edoardo, De Prizio, Marco, Sulce, Rezart, Poiasina, Elia, Antonacci, Filippo, Arena, Alessandro, Belvedere, Angela, Bernagozzi, Fabio, Boussedra, Safia, Cescon, Matteo, Cipolli, Alessandro, Daddi, Niccolo’, De Crescenzo, Eugenia, De Iaco, Pierandrea, Del Gaudio, Massimo, Della Gatta, Anna Nunzia, Droghetti, Matteo, Giorgini, Federico A., Lanci Lanci, Antonio, Masetti, Michele, Minni, Francesco, Morezzi, Daniele, Perrone, Anna Myriam, Pignatti, Marco, Puglisi, Silvana Bernadetta, Rottoli, Matteo, Schiavina, Riccardo, Serenari, Matteo, Serra, Margherita, Tesei, Marco, Violante, Tommaso, Aspide, Raffaele, Carretta, Alessandro, Conti, Alfredo, De Vita, Carla, Friso, Filippo, La Corte, Emanuele, Rosetti, Vittoria, Sturiale, Carmelo, Donati, Davide Maria, Palmerini, Emanuela, Berselli, Bruno, Bolognesi, Silvia, Farnia, Francesco, Maremonti, Pietro, Razzaboni, Alessandra, La Mendola, Roberta, Arrigoni, Giulia, Baiocchi, Gian Luca, Molfino, Sarah, Panciani, Pier Paolo, Sartori, Enrico, Zanin, Luca, Perrone, Fabrizio, Cappellani, Alessandro, Giaquinta, Alessia, Gioco, Rossella, Veroux, Pierfrancesco, Cianci, Antonio, Lo Giudice, Arturo, Russo, Giorgio Ivan, Sarpietro, Giuseppe, Scandura, Carmen Emanuela, Barca, Ida, Carnevali, Adriano, De Paola, Gilda, Giannaccare, Giuseppe, Sammarco, Giuseppe, D’Andrea, Marcello, Tosatto, Luigino, Mucilli, Felice, Muraglia, Angelo, Giuliani, Domenico Benvenuto, Segalini, Edoardo, Daniele, Alberto, Gelarda, Enrico, Olearo, Elena, Puppo, Andrea, Testa, Valentina, Porta, Andrea, Birindelli, Arianna, Bernabei, Massimiliano, Feo, Carlo V., Virgilio, Edoardo, Bartolini, Ilenia, Bottari, Andrea, Checcucci, Carlotta, De Vincenti, Rosita, Fambrini, Massimiliano, Fortuna, Laura, Gallo, Oreste, Maltinti, Gherardo, Taddei, Antonio, Michelagnoli, Stefano, Maccagnano, Giuseppe, Pesce, Vito, Ercolani, Giorgio, Fappiano, Francesca, Barberis, Andrea, Filauro, Marco, Santoliquido, Matteo, Aprile, Alessandra, Barra, Fabio, De Rosa, Raffaele, Ferrero, Simone, Sparavigna, Marco, Vagge, Aldo, Gambardella, Denise, Tedesco, Manfredo, Spampinato, Marcello Giuseppe, Chiarelli, Marco, Grandi, Samuele, Lenzi, Riccardo, Muscatello, Luca, Germano’, Antonino Francesco, Giani, Alessandro, Bissacco, Daniele, Cassinotti, Elisa, Pignataro, Lorenzo, Torretta, Sara, Armellin, Daniele, Basato, Silvia, Carrano, Francesco Maria, Colombo, Giovanni, De Lucia, Francesca, De Virgilio, Armando, Ferreli, Fabio, La Raja, Carlotta, Milana, Flavio, Rossi, Vanessa, Russo, Elena, Tamburello, Sara, Zerbi, Alessandro, Bona, Davide, Adamoli, Laura, Ansarin, Mohssen, Chu, Francesco, De Berardinis, Rita, Pietrobon, Giacomo, Sedda, Giulia, Caputo, Maria, Cellerino, Paola, Crespi, Michele Achille, Danelli, Piergiorgio, Ferrara, Francesco, Baccellieri, Domenico, Candiani, Massimo, Casiraghi, Arianna, Cipriani, Federica, De Nardi, Paola, Mortini, Pietro, Nocera, Gianluca, Pozzoni, Mirko, Ruffolo, Alessandro Ferdinando, Spina, Alfio, Barberio, Cristina, Beretta, Luigi, Cavenago, Francesca, Fresilli, Stefano, Landoni, Giovanni, Lombardi, Gaetano, Bonomi, Stefano, Guaglio, Marcello, Sala, Laura, Segattini, Silvia, Piccoli, Micaela, Migliore, Marco, Sasia, Diego, Cereda, Marco, Dell’Oro, Cristina, Fogliati, Alessandro, Tamini, Nicolò, Castaldi, Antonio, Bracale, Umberto, Lionetti, Ruggero, Pagano, Gianluca, Peltrini, Roberto, Pirozzi, Nello, D’amico, Maria, Izzo, Francesco, Marra, Ester, Marte, Gianpaolo, Bianco, Francesco, Cappiello, Antonio, Iovino, Claudio, Menna, Maria Paola, Romano, Francesco maria, Rossi, Settimio, Sciaudone, Guido, Ascari, Francesca, Bellora, Paolo, Cerri, Cristina, D’Aloisio, Giordana, Ferrari, Maurizio, Chioffi, Franco, Ciccarino, Pietro, Nezi, Giulia, Bissolotti, Guido, Di Donna, Mariano Catello, Licari, Leo, Salamone, Giuseppe, Toia, Francesca, Annicchiarico, Alfredo, Berretta, Roberto, Capozzi, Vito Andrea, Freyrie, Antonio, Frusca, Tiziana, Arici, Vittorio, Bozzani, Antonio, Filardo, Matteo, de Manzoni Garberini, Andrea, Cauteruccio, Michele, Caristo, Giuseppe, Calabrò, Marcello, Andreani, Lorenzo, Antonio, D’arienzo, Berrettini, Stefano, Colangeli, Simone, Cremonini, Camilla, Dallan, Iacopo, Di Franco, Gregorio, Korasidis, Stylianos, Musetti, Serena, Neri, Carlo Maria, Palmeri, Matteo, Picariello, Miriana, Statuti, Erica, Tartaglia, Dario, Pinotti, Enrico, Basso, Stefano Maria Massimiliano, Ubiali, Paolo, Coiro, Saverio, Mele, Simone, Corbellini, Carlo, Masciandaro, Antonio, Carannante, Filippo, Denaro, Vincenzo, Mazzotta, Erica, Zampogna, Biagio, Cinquepalmi, Matteo, Del Basso, Celeste, Guglielmo, Nicola, Meniconi, Roberto Luca, Sinibaldi, Giovanni, Agnes, Annamaria, Belia, Francesco, Bianchi, Valentina, Cozza, Valerio, Fico, Valeria, Fransvea, Pietro, La Greca, Antonio, Litta, Francesco, Pascale, Marco Maria, Silvia, Tedesco, Benevolo, Maria, Campo, Flaminia, Dona’, Maria Gabriella, Marchesi, Paolo, Mastroianni, Riccardo, Mazzola, Francesco, Moretto, Silvia, Petruzzi, Gerardo, Pichi, Barbara, Tuderti, Gabriele, Bellato, Vittoria, Grande, Michele, Petagna, Lorenzo, Sensi, Bruno, Bruzzaniti, Placido, Ciccarone, Flavia, Cicerchia, Pierfranco Maria, Cirillo, Bruno, D’ambrosio, Giancarlo, De Toma, Giorgio, Familiari, Pietro, Fonsi, Giovanni Battista, Picchetto, Andrea, Salvati, Maurizio, Valentini, Valentino, Zambon, Martina, Zancana, Giuseppa, Zuppi, Emma, Ibrahim, Mohsen, Menna, Cecilia, Rendina, Erino Angelo, Accarino, Giulio, Giancarlo, Accarino, Froiio, Caterina, Copelli, Chiara, Di Maio, Pasquale, Giudice, Marco, Altana, Cristian, Ciccarello, Sandro, Cossu, Maria Laura, Dessole, Francesco, Dettori, Salvatora, Madonia, Massimo, Petrillo, Marco, Piredda, Franco, Rizzo, Davide, Scognamillo, Fabrizio, Soma, Damiano, Tanca, Anna Rita, Marano, Luigi, Pesce, Anna Lisa, Piccioni, Stefania Angela, Resca, Luca, Roviello, Franco, Abrate, Alberto, Clarizia, Guglielmo, Franzini, Marco, Fratto, Antonio, Grechi, Alessandro, Scarnecchia, Elisa, Maiuri, Vincenzo, Dalprà, Francesca, Battistella, Enrico, Romano, Maurizio, Aizza, Giada, de Manzini, Nicolò, Drigo, Davide, Mastronardi, Manuela, Palmisano, Silvia, Bardelli, Laura, Borroni, Giacomo, Livraghi, Lorenzo, Palumbo, Mara, Zullo, Alessandra, Vignotto, Chiara, Caravati, Andrea, De Cristofaro, Carlotta, Giuliani, Tommaso, Guglielmi, Alfredo, Pedrazzani, Corrado, Butturini, Giovanni, Moretto, Gianluigi, Mastriale, Francesco, Olmi, Stefano, Kushikata, Tetsuya, Oyama, Tasuku, Kamiyama, Mizue, Tarao, Kentaroh, Yamada, Takayuki, Shiga, Toshiya, Deguchi, Yoshihiko, Hoshino, Tatsuki, Inoue, Hiroyuki, Ito, Shingo, Takano, Emi, Yamamoto, Masae, Matsuki, Yuka, Matsumoto, Takashi, Sawada, Atsushi, Sato, Kozo, Makino, Jun, Mizota, Toshiyuki, Yonekura, Hiroshi, Ishikawa, Haruka, Kaiho, Yu, Ogawa, Munehiro, Endo, Shunji, Fujiwara, Yoshinori, Kubota, Hisako, Okada, Toshimasa, Hisamatsu, Yoji, Michiura, Taku, Satoshi, Okazaki, Fujita, Tomoyuki, Fukushima, Satsuki, Otsuka, Yuji, Sawada, Ikumi, Miyamoto, Morikazu, Isada, Tetsuro, Bani Amer, Qaed, Kuratani, Norifumi, Takada, Misa, Noro, Shusaku, Okamura, Yukiyasu, Uesaka, Katshuhiko, Kuroda, Kento, Abe, Nobutsugu, Ando, Tadao, Hirano, Kouichi, Kobayashi, Yoichi, Motoyasu, Akira, Noguchi, Hikari, Tajima, Atsushi, Toyama, Satoshi, Uchida, Tokujiro, Furukawa, Yuri, Hasumi, Yoko, Iida, Katsuyuki, Ikeda, Tatsuhiko, Kuzuhara, Shigeki, Yamamoto, Naoya, Yamamoto, Mari, Yokota, Toshiya, Yoshioka, Yuki, Kawai, Akira, Nakata, Yoshinori, Sawamura, Shigehito, Kobayashi, Takayuki, Alrabadi, Mera, Owais, Qais, Smadi, Aseel, Abunawas, Huthifa, Alhawatmeh, Mohammad, Alwardat, Abdel Rahman Mohannad Ahmad, Dhoon, Slsabela, Jaradat, Enas, Mubaydeen, Teeba, Alzraikat, Sayel H., Suradi, Haya H., Al Abdallah, Murad, Al Bdour, Zakaria, Al-thaher, Mohammad, Alnajjar, Tareq, AlZaatreh, Mohammad, Ghayada, Ibrahim, Hasanein, Khaled Moh’d Ahmed, Hassouneh, Anas, Hijazeen, Raid, Khasawneh, Barihan, Semrin, Qusai, Theab, Mohammad, Abazeed, Alaa, Alkurdieh, Moh’d Mujahed, Hammouri, Mohammad, Khader, Sereen, Abu Obead, Hamza, Abudari, Hadeel, Al-azzeh, Nimer, Al-Darabah, Ali, Al-Qannas, Mohammad, Ashour, Subhi, Chabaan, Reem, Fawzi, Ahmad, Haij, Manal, Hassouneh, Esraa, Ibrahim, Zainab, Mohamed, Ishak, Qandeel, Mahmoud, Shorman, Marwan, Nofal, Sandra, Salameh, Mohammed, Sanjuq, Ghaidaa, Sharabi, Alaa, Sweiti, Alaa, Baninasr, Eman, Al-latayfeh, Motasem, Al-Shudifat, Abdel-Ellah, Shaikh Ahmad, Abd Almonem, Abu Abed, Laith, Allouzi, Mohammad, Almi’ani, Sari, Alsaiad, Abdulhakim, Daoud, Maher, Dawod, Omar, Fadli, Saba, Qulaghassi, Zaki, Al-howthi, Mohammed, Ababneh, Laila, Ababneh, Roba, Al Khassawneh, Ahmad, Al Sharie, Ahmed, Al-Dabaa, Tawfik, Al-Jarrah, Salsabeel, Alshannaq, Qutaiba, Alsulaiman, Rima, Audat, Ziad, Bdour, Saja, Khatatbeh, Ro’a, Marji, Fares, Abu Ismail, Dima.Y, Alzoubi, Mai, Alzoubi, Malak, Hussein, Hasan, Rawashdeh, Shireen, Ababneh, Adnan, Abu-Eisheh, Basil, Al Shurman, Hana’, Al-atiyah, Hussam, Aljaiuossi, Anas, Hamdoni, Sara, Sakhov, Orazbek, Tanabayeva, Shynar, Amrayev, Sultan, Kukubassov, Yerlan, Parker, Robert, Mwaria, Claude, Kibunyi, Muthoni, Mailu, Claire, Osea, Lydia, Hirsi, Omar, Dashti, Mustafa, Aldalal, Shuaib, Alozairi, Ous, Al-Shaiji, Tariq, Alhunaidi, Omar, Aljewaied, Ali, Farag, Ahmed, Termos, Salah, Sivins, Armands, Apse, Ingus Arnolds, Kaminskis, Aleksejs, Pāvulāns, Jānis, Itani, Rania, Owiedat, Mustafa, Antonios, Ingrid, Milan Moussa, Rita, Abdel Sater, Ali H., Souleiman, Bassem, Ghiya, Peter, Fakih, Ghinwa, Abdulrahman, Taha, Omar Abunaaja, Hayat, Al Gasi, Abd El Jawad, Alsaeiti, Sara, Elkhafeefi, Fatimah, Alhouni, Tarek, Alkeelani, Hana, Alkuwafi, Rauoof, Elbargathe, Osama, Elfadli, Mustafa, Elmahgoub, Asma, Alhaje, Awatif, Haron, Aaya, Alsuwiyah, Suhir, Khel, Samer, Yousef, Rowaida, Abuhlaiga, Ma’aly, Abushahma, Ahmed omar, Aldelensi Alzubi, Alhosen Saleh M, Alnehum, Mohamed alnaser, Alzedam, Ahmad, Matoug, Faisel, Mohammed, Burooj, Sawalem, Mohamed, Kamoka, Elhusain, Salamah, Abdulrauf, Abdeewi, Saedah, Eshnaf, Mabroka, Yahmad, Mohammad, Aljuroushi, Omar, Attia, Hajer, Emran, Ayyah, Samer, Ashraf, Abdullah, Duha milad, Ben esmael, Esraa, Benmasoud, Fatma, Debri, Laila, Younis, Balkees, Elmabrouk, Amna, Hasan, Ameerah Mahdi Abraheem, Alshareea, Entisar, Aribi, Tomather, Lamari, Amani hamid, Mufth, Mohammed, Aburima, Sarah, Al shukri, Asmaa, Alarabi, Rehab, Alelwany, Aisha, Bashir, Rasha, Elhadi, Ahmed, Elkouba, Esraa, Elmsherghi, Nabiha, Haidar, Arwa, Helal, Hala, Kriem, Eslam, Turshani, Laila, Ben Hasan, Hayat, Shaban Ben Hasan, Najat, Shaban Ben Hasan, Rabab, Dambrauskas, Zilvinas, Dekeryte, Inga, Koženiauskaitė, Akvilė, Maleckas, Almantas, Venclauskas, Linas, Zilinskas, Justas, Syminas, Vilius, Aliosin, Oleg, Cizauskaite, Agne, Jurgaitis, Jonas, Mikutaitis, Vytenis, Samalavicius, Narimantas Evaldas, Slepavicius, Algirdas, Vasiliauskas, Bernardas, Dulskas, Audrius, Cekauskas, Albertas, Zelvys, Arunas, Noël, Audrey, Rajaonarivony, Maheriandrianina Fanambinana Voahary, Andriantsoa, Herimampionona E, Tsiambanizafy, Guillaume Odilon, Solo, Corinne Eulalie, Safiry Andofenohasina, Fanonjomahasoa, Sablon Herinirina, Angelin, Das, Andre, Osman, Mohd Firdauss, Soh, Jien Yen, Zakaria, Zaidi, Chan, Kheng Hooi, Johan, Syamim, Mah, Jin Jiun, Subramaniam, Sentilnathan, Ibrahim, Mohd Razali, Nazimi, Abd Jabar, Bal, Pavin, Fadzli, Ahmad nazran, Jamaris, Suniza, Lee, Yeong Sing, Lim, Jeffery Zk, Moh Pauzi, Siti Farhan, Nair, Ashvin Krishna, Ng, Doris Sin Wen, Ng We Yong, Yuki Julius, Saaid, Rahmah, Sethi, Neha, Syed Jafer Hussain Zaidi, Syeda Nureena, Teoh, Li Ying, Islah Munjih, Sainal, Mohd Yusof, Sarif, Mat Salleh, Chua, Richelle, Ahmad, Abdul Rauf bin, Lau, Yii Ling, Liew, Ka Yee, Palayan, Kandasami, Abdul Rahman, Noorneza, Firman, Fatin, Hazleigh, Nik Mohd, Mohd Miswan, Mohd Fairudz, Mohd Zahid, Akmal Zulayla, Yahaya, Mohd Yusoff bin Yahaya, Yusoff, Ahmad Ramzi, Darryl, Brincat, Svetlana, Caruana, Clifford, Chircop, Daniel Mario, Czok, Viktoria, Muscat, Esther, Roshan, Mohsin Hassan Khan, Schembri, Nicholas, Jonathan Rubén, Cedillo Rodrìguez, Rojas Aguilar, Antonio, Sosa Duran, Erik Efrain, Arroyo, Diego, Martinez-Said, Hector, Medina-Franco, Heriberto, Pérez de León Vázquez, Martha Patricia, Soto Hernández, Fidel, Stenner, Juan Ignacio, Zuloaga Fernandez del Valle, Carlos Jose, Colunga Tinajero, Carlos Andres, 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Felipe, González León, Juan Antonio, Guzman, Marlin, Herappe, Dorihela, Lerma, Ricardo, Mendoza Pedraza, Fabián Hilario, Noguez Castillo, Monica, Resendiz, Jose Anatolio, Fernandez Rios, Laura Elena, Frigerio, Pamela, Garcia, Iset, Mendoza Frías, Gloria Isela, Anzures Mendoza, Alejandra, Hdez Miguelena, Luis, Zarate Saenz, David, Zavala gonzalez, Miguel angel, Andrade González, Francisco, Cortes, Ludivina, Gurghiș, Radu, Sanjaa, Burmaa, Ait benhamou, Rita, Rghioui, Mounir, Saaf, Sarra, Letrache, Ahmed, Zekhnini, Meryem, Abichou, Wijden, Baroudi, Salma, Hassoun, Chiraz, Zaim, Ouijdane, Arkha, Yasser, Bechri, Hajar, Benchekroun Belabbes, Meryem, Benslimane, Zaineb, Boualila, Lina, El Ouahabi, Abdessamad, Essakalli Hossyni, Leila, Tagmouti, Adam, Belkhadir, Zakaria Houssaïn, Benkabbou, Amine, Boutayeb, Saber, El Ahmadi, Brahim, Ghannam, Abdelilah, Kabbaj, Taha, Majbar, Anass Mohammed, Ndatewapo, Fillipus, Njuki, Peter Ngungi, Abrahams, Jenevive, Bhusal, Subarna, Dwa, Yam, Bloemers, Frank, Besselink, Marc, van Amstel, Paul, Smeele, Ludi, Harlaar, Niels, van der Burg, Sjirk, Franken, Josephine, Oosterling, Steven, Nolte, Peter, Alwayn, Ian, Braat, Andries, Goudsmit, Ben, van de Leemkolk, Fenna E.M., Albers, Kim, Van Goor, Harry, van Ginhoven, Tessa M., Schweitzer, Donald, Leclercq, Wouter, Vancoillie, Peter-Jan, Konsten, Joop, den Boer, Frank, Djamali, Kaveh, Ghamri, Naji, Joseph, Reece, Lie, Samuel, Olliff, Sue, Wood, Daniel, Furkert, Chris, Lim, Eric, Mclauchlan, Jared, Ailabouni, Ramez, Alqassab, Mohammed, Hawkins, Robyn, Baker, Joseph, Ou, Cindy Xin Yi, Singh, Vidit, Frunt, Brytt, Lakshman, Prashant, Henderson, Nigel, Haimona, Maira, Jury, Steffanie, Weeratunga, George, Witcomb Cahill, Henry, Abdussalam, Hameedat Opeyemi, Ahmed, Aminat Olayinka, Adeyeye, Ademola, Kayode-Nissi, Victor, Kadiri, Innih, Oduah, Stella, Abdull, Mohammed, Abdullahi, Mohammed Kabir, Ja’afar, Abubakar Bappah, Ja’afaru, Umma Hani, Yakubu Mohammed, Husseini, Edetanlen, Ekaniyere, Iribhogbe, Oseihie, Irowa, Omorodion, Nwashilli, Nnamdi, Obahiagbon, Osarobo, Onyeagwara, Ngozi, Oseghale, Eustace, Osemwegie, Osarenkhoe, Osho, Philip, Akanni, Bolaji, Ezomike, Uchechukwu, Okoro, Kelechukwu, Okwara, Blasius, Abdulsalam, Khalifa, Agida, Eyaofun, Ajibola, Hafees, Benard, Adeka, Buba, Amina, Eniola, Sefiu, Isah, Aliyu, Olute, Anne, Osagie, Olabisi, Pius Ogolekwu, Idoko, Ademola, Samuel, Afuwape, Oludolapo, Daodu, Israel, Egbuchulem, Ifeanyichukwu Kelvin, Nwaorgu, Onyekwere, Obadan, Izegaegbe, Odeyemi, Olubunmi, Ogundoyin, Omowonuola, Okere, Oghenekevwe, Oladiran, Ajibola, Olagunju, Naomi, Olawoye, Olayinka, Olusanya, Adeola, Oyelakin, Oyeleye, Ugwu, Ebere, Atoyebi, Oluwole, Bode, Chris, Chibuike George, Ihediwa, Ezenwankwo, Francis, Fatuga, Adedeji, Ladipo-Ajayi, Oluwaseun, Ojewola, Rufus Wale, Okunowo, Adeyemi, Ugwu, Aloy Okechukwu, Adebara, Idowu, Adeyemo, Olabisi, Banjo, Oluseyi, Obateru, John, Ojo, Owolabi, Oluwafemi, Fatudimu, Adelaja, Aderinsola, Atobatele, Kazeem, Ayodele, Olabamidele, Bello, Funmi, Omisanjo, Olufunmilade, Aderounmu, Adewale, Ajekwu, Temitope, Ajekwu, Samuel, Akeem Aderogba, Adeleke, Balogun, Simon, Ojo, Olugbenga, Olasehinde, Olalekan, Olugbami, Adebayo, Mohammed, Tajudeen, Afolabi, Abdulrahman O., Aremu, Isiaka, Ibraheem, Gbadebo, Odeyemi, Peter Olalekan, Olabinjo, Afusat, Raji, Hadijat Olaide, Salawu, Hafeez, Uche-Okonkwo, Kenechukwu, Anipole, Olalekan, Adesina, Oluwaseyi, Enwerem, Kenneth, Isichei, Mercy, Aghadi, Ifeanyi, Lawal, Jamila, Taingson, Matthew, Yusuf, Nuhu, Takai, Idris Usman, Aji, Sani Ali, Bello-Muhammad, Nafisatu, Garzali, Ibrahim Umar, Hasheem, Muhammad Ghazali, Hassan, Sadiq, Ibrahim Usman, Mustapha, Magashi, Mahmoud Kawu, Mukhtar, Ibrahim Aliyu, Nwachukwu, Callistus, Suleiman, Ibrahim, A, Maryam, Mahmood, Farrukh, Salihu, Mohammed, Alegbeleye, Justina Omoikhefe, Awopeju, Abimbola, Fyneface-Ogan, Sotonye, Kasso, Terhemen, Onwuagha, Ijeoma, Salami, Babatunde, Ajagha, Okeoghene, Awaisu, Mudi, Nwabuoku, Stanley Emeka, 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Khan, Muhammad Osama, Kumari, Sameeta, Malik, Muhammad Jehangir, Mansoor, Maheen, Mubarik, Areeba, Naz, Falak, Qamar, Javeria Bilal, Sadia, Haleema, Safri, Mohammad, Sattar, abida K., Siddiqui, Muhammad Ahsan Iqbal, Waqar, Usama, Ghufran, Muhammad Ali, Memon, Amjad Siraj, Samo, Khursheed Ahmed, Aswani, Manisha, Manzoor, Shahneela, Muhammad, Shoaib, Moin, Inshrah, Najjad, Muhammad Kazim Rahim, Zia, Kashif, Ali Shaikh, Shafqat, Hussaini, Dr, Kamal, Wajahat, Mughal, Zeeshan Uddin, Nadeem, Areeba, Nayyer, Fatima, Nisa, Kanwal, Rauf, Yaseen, Saleem, Shah Masabat, Shah, Aaqil, Siddiqui, Safia, Tariq, Nida, Zamir, Mehroz, Hafiz Muhammad Arif, Ahmed, Ehsan, Ahmed, Jawad, Akbar, Bilal, Ali, Abrar Ashraf, Iqbal, Zafar, Jameel, Muhammad Khurram, Janjua, Muhammad Haris, Janjua, Azwa, Naqi, Syed Asghar, Naumeri, Fatima, Nazir, Umer, Rafi, Yaseen, Saleem, Irfan, Shaukat, Ayesha, Sikandar, Hafiz Saqib, Tu zahara, Fatima, Ali, Ahsan, Jawad, Muhammad, Noman, Hamza, Taha, Dr Hassan, Dr. Gul e Raana, 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3. Evaluation of prognostic risk models for postoperative pulmonary complications in adult patients undergoing major abdominal surgery: a systematic review and international external validation cohort study
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Kouli, O, Murray, V, Bhatia, S, Cambridge, WA, Kawka, M, Shafi, S, Knight, SR, Kamarajah, SK, McLean, KA, Glasbey, JC, Khaw, RA, Ahmed, W, Akhbari, M, Baker, D, Borakati, A, Mills, E, Thavayogan, R, Yasin, I, Raubenheimer, K, Ridley, W, Sarrami, M, Zhang, G, Egoroff, N, Pockney, P, Richards, T, Bhangu, A, Creagh-Brown, B, Edwards, M, Harrison, EM, Lee, M, Nepogodiev, D, Pinkney, T, Pearse, R, Smart, N, Vohra, R, Sohrabi, C, Jamieson, A, Nguyen, M, Rahman, A, English, C, Tincknell, L, Kakodkar, P, Kwek, I, Punjabi, N, Burns, J, Varghese, S, Erotocritou, M, McGuckin, S, Vayalapra, S, Dominguez, E, Moneim, J, Salehi, M, Tan, HL, Yoong, A, Zhu, L, Seale, B, Nowinka, Z, Patel, N, Chrisp, B, Harris, J, Maleyko, I, Muneeb, F, Gough, M, James, CE, Skan, O, Chowdhury, A, Rebuffa, N, Khan, H, Down, B, Fatimah Hussain, Q, Adams, M, Bailey, A, Cullen, G, Fu, YXJ, McClement, B, Taylor, A, Aitken, S, Bachelet, B, Brousse de Gersigny, J, Chang, C, Khehra, B, Lahoud, N, Lee Solano, M, Louca, M, 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Wilde, S, Dickinson, M, Rockall, T, Senn, D, Wcislo, K, Zalmay, P, Adelekan, K, Allen, K, Bajaj, M, Gatumbu, P, Hang, S, Hashmi, Y, Kaur, T, Kawesha, A, Kisiel, A, Woodmass, M, Adelowo, T, Ahari, D, Alhwaishel, K, Atherton, R, Clayton, B, Cockroft, A, Curtis Lopez, C, Hilton, M, Ismail, N, Kouadria, M, Lee, L, MacConnachie, A, Monks, F, Mungroo, S, Nikoletopoulou, C, Pearce, L, Sara, X, Shahid, A, Suresh, G, Wilcha, R, Atiyah, A, Davies, E, Dermanis, A, Gibbons, H, Hyde, A, Lawson, A, Lee, C, Leung-Tack, M, Li Saw Hee, J, Mostafa, O, Nair, D, Pattani, N, Plumbley-Jones, J, Pufal, K, Ramesh, P, Sanghera, J, Saram, S, Scadding, S, See, S, Stringer, H, Torrance, A, Vardon, H, Wyn-Griffiths, F, Brew, A, Kaur, G, Soni, D, Tickle, A, Akbar, Z, Appleyard, T, Figg, K, Jayawardena, P, Johnson, A, Kamran Siddiqui, Z, Lacy-Colson, J, Oatham, R, Rowlands, B, Sludden, E, Turnbull, C, Allin, D, Ansar, Z, Azeez, Z, Dale, VH, Garg, J, Horner, A, Jones, S, Knight, S, McGregor, C, McKenna, J, McLelland, T, Packham-Smith, A, Rowsell, K, Spector-Hill, I, Adeniken, E, Baker, J, Bartlett, M, Chikomba, L, Connell, B, Deekonda, P, Dhar, M, Elmansouri, A, Gamage, K, Goodhew, R, Hanna, P, Knight, J, Luca, A, Maasoumi, N, Mahamoud, F, Manji, S, Marwaha, PK, Mason, F, Oluboyede, A, Pigott, L, Razaq, AM, Richardson, M, Saddaoui, I, Wijeyendram, P, Yau, S, Atkins, W, Liang, K, Miles, N, Praveen, B, Ashai, S, Braganza, J, Common, J, Cundy, A, Davies, R, Guthrie, J, Handa, I, Iqbal, M, Ismail, R, Jones, C, Jones, I, Lee, KS, Levene, A, Okocha, M, Olivier, J, Smith, A, Subramaniam, E, Tandle, S, Wang, A, Watson, A, Wilson, C, Chan, XHF, Khoo, E, Montgomery, C, Norris, M, Pugalenthi, PP, Common, T, Cook, E, Mistry, H, Shinmar, HS, Agarwal, G, Bandyopadhyay, S, Brazier, B, Carroll, L, Goede, A, Harbourne, A, Lakhani, A, Lami, M, Larwood, J, Martin, J, Merchant, J, Pattenden, S, Pradhan, A, Raafat, N, Rothwell, E, Shammoon, Y, Sudarshan, R, Vickers, E, Wingfield, L, Ashworth, I, Azizi, S, Bhate, R, Chowdhury, T, Christou, A, Davies, L, Dwaraknath, M, Farah, Y, Garner, J, Gureviciute, E, Hart, E, Jain, A, Javid, S, Kankam, HK, Kaur Toor, P, Kaz, R, Kermali, M, Khan, I, Mattson, A, McManus, A, Murphy, M, Nair, K, Ngemoh, D, Norton, E, Olabiran, A, Parry, L, Payne, T, Pillai, K, Price, S, Punjabi, K, Raghunathan, A, Ramwell, A, Raza, M, Ritehnia, J, Simpson, G, Smith, W, Sodeinde, S, Studd, L, Subramaniam, M, Thomas, J, Towey, S, Tsang, E, Tuteja, D, Vasani, J, Vio, M, Badran, A, Adams, J, Anthony Wilkinson, J, Asvandi, S, Austin, T, Bald, A, Bix, E, Carrick, M, Chander, B, Chowdhury, S, Cooper Drake, B, Crosbie, S, D Portela, S, Francis, D, Gallagher, C, Gillespie, R, Gravett, H, Gupta, P, Ilyas, C, James, G, Johny, J, Jones, A, Kinder, F, MacLeod, C, Macrow, C, Maqsood-Shah, A, Mather, J, McCann, L, McMahon, R, Mitham, E, Mohamed, M, Munton, E, Nightingale, K, O'Neill, K, Onyemuchara, I, Senior, R, Shanahan, A, Sherlock, J, Spyridoulias, A, Stavrou, C, Stokes, D, Tamang, R, Taylor, E, Trafford, C, Uden, C, Waddington, C, Yassin, D, Zaman, M, Bangi, S, Cheng, T, Chew, D, Hussain, N, Imani-Masouleh, S, Mahasivam, G, McKnight, G, Ng, HL, Ota, HC, Pasha, T, Ravindran, W, Shah, K, Vishnu K, S, Zaman, S, Carr, W, Cope, S, Eagles, EJ, Howarth-Maddison, M, Li, CY, Reed, J, Ridge, A, Stubbs, T, Teasdaled, D, Umar, R, Worthington, J, Dhebri, A, Kalenderov, R, Alattas, A, Arain, Z, Bhudia, R, Chia, D, Daniel, S, Dar, T, Garland, H, Girish, M, Hampson, A, Kyriacou, H, Lehovsky, K, Mullins, W, Omorphos, N, Vasdev, N, Venkatesh, A, Waldock, W, Bhandari, A, Brown, G, Choa, G, Eichenauer, CE, Ezennia, K, Kidwai, Z, Lloyd-Thomas, A, Macaskill Stewart, A, Massardi, C, Sinclair, E, Skajaa, N, Smith, M, Tan, I, Afsheen, N, Anuar, A, Azam, Z, Bhatia, P, Davies-kelly, N, Dickinson, S, Elkawafi, M, Ganapathy, M, Gupta, S, Khoury, EG, Licudi, D, Mehta, V, Neequaye, S, Nita, G, Tay, VL, Zhao, S, Botsa, E, Cuthbert, H, Elliott, J, Furlepa, M, Lehmann, J, Mangtani, A, Narayan, A, Nazarian, S, Parmar, C, Shah, D, Shaw, C, Zhao, Z, Beck, C, Caldwell, S, Clements, JM, French, B, Kenny, R, Kirk, S, Lindsay, J, McClung, A, McLaughlin, N, Watson, S, Whiteside, E, Alyacoubi, S, Arumugam, V, Beg, R, Dawas, K, Garg, S, Lloyd, ER, Mahfouz, Y, Manobharath, N, Moonesinghe, R, Morka, N, Patel, K, Prashar, J, Yip, S, Adeeko, ES, Ajekigbe, F, Bhat, A, Evans, C, Farrugia, A, Gurung, C, Long, T, Malik, B, Manirajan, S, Newport, D, Rayer, J, Ridha, A, Ross, E, Saran, T, Sinker, A, Waruingi, D, Allen, R, Al Sadek, Y, Alves do Canto Brum, H, Asharaf, H, Ashman, M, Balakumar, V, Barrington, J, Baskaran, R, Berry, A, Bhachoo, H, Bilal, A, Boaden, L, Chia, WL, Covell, G, Crook, D, Dadnam, F, Davis, L, De Berker, H, Doyle, C, Fox, C, Gruffydd-Davies, M, Hafouda, Y, Hill, A, Hubbard, E, Hunter, A, Inpadhas, V, Jamshaid, M, Jandu, G, Jeyanthi, M, Jones, T, Kantor, C, Kwak, SY, Malik, N, Matt, R, McNulty, P, Miles, C, Mohomed, A, Myat, P, Niharika, J, Nixon, A, O'Reilly, D, Parmar, K, Pengelly, S, Price, L, Ramsden, M, Turnor, R, Wales, E, Waring, H, Wu, M, Yang, T, Ye, TTS, Zander, A, Zeicu, C, Bellam, S, Francombe, J, Kawamoto, N, Rahman, MR, Sathyanarayana, A, Tang, HT, Cheung, J, Hollingshead, J, Page, V, Sugarman, J, Wong, E, Chiong, J, Fung, E, Kan, SY, Kiang, J, Kok, J, Krahelski, O, Liew, MY, Lyell, B, Sharif, Z, Speake, D, Alim, L, Amakye, NY, Chandrasekaran, J, Chandratreya, N, Drake, J, Owoso, T, Thu, YM, Abou El Ela Bourquin, B, Alberts, J, Chapman, D, Rehnnuma, N, Ainsworth, K, Carpenter, H, Emmanuel, T, Fisher, T, Gabrel, M, Guan, Z, Hollows, S, Hotouras, A, Ip Fung Chun, N, Jaffer, S, Kallikas, G, Kennedy, N, Lewinsohn, B, Liu, FY, Mohammed, S, Rutherfurd, A, Situ, T, Stammer, A, Taylor, F, Thin, N, Urgesi, E, Zhang, N, Ahmad, MA, Bishop, A, Bowes, A, Dixit, A, Glasson, R, Hatta, S, Hatt, K, Larcombe, S, Preece, J, Riordan, E, Fegredo, D, Haq, MZ, Li, C, McCann, G, Stewart, D, Baraza, W, Bhullar, D, Burt, G, Coyle, J, Deans, J, Devine, A, Hird, R, Ikotun, O, Manchip, G, Ross, C, Storey, L, Tan, WWL, Tse, C, Warner, C, Whitehead, M, Wu, F, Court, EL, Crisp, E, Huttman, M, Mayes, F, Robertson, H, Rosen, H, Sandberg, C, Smith, H, Al Bakry, M, Ashwell, W, Bajaj, S, Bandyopadhyay, D, Browlee, O, Burway, S, Chand, CP, Elsayeh, K, Elsharkawi, A, Evans, E, Ferrin, S, Fort-Schaale, A, Iacob, M, I, K, Impelliziere Licastro, G, Mankoo, AS, Olaniyan, T, Otun, J, Pereira, R, Reddy, R, Saeed, D, Simmonds, O, Singhal, G, Tron, K, Wickstone, C, Williams, R, Bradshaw, E, De Kock Jewell, V, Houlden, C, Knight, C, Metezai, H, Mirza-Davies, A, Seymour, Z, Spink, D, and Wischhusen, S
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- 2022
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4. Culture development for human embryonic stem cell propagation: molecular aspects and challenges
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Rao, BM and Zandstra, PW
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- 2005
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5. Quantification of Genotoxic Impurity 4-Chloro-1-Hydroxy Butane Sulfonic Acid Sodium Salt by LCMS/MS in Sumatriptan Succinate
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Chandrasekhar Kb, Rao Bm, and Narayana Mbv
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Detection limit ,chemistry.chemical_classification ,Chromatography ,Formic acid ,Sodium ,Pharmaceutical Science ,Salt (chemistry) ,chemistry.chemical_element ,Butane ,Sulfonic acid ,Ion source ,chemistry.chemical_compound ,chemistry ,Impurity - Abstract
The objective of present research work is to develop a suitable LCMS/MS method for the quantitative determination of genotoxic impurity 4-Chloro-1-Hydroxy Butane Sulfonic Acid Sodium Salt at ppm level present in Sumariptan dug substance. The LCMS/MS method was developed on Zorbax SB-C8 column using the mobile phase consists a mixture of 0.05% (v/v) Formic acid in water and Acetonirile using a isocratic composition of 90:10 (v/v) at a flow rate of 0.8 mL/min. Ion source is electospray ionization (ESI), source temperature is 325°C, gas flow is 8 L/min, Nebuliser pressure is 40 psi, capillary voltage is 4000 V. Under these conditions impurity was quantified by selecting most stable MRM pair (187/81). The limit of detection and the limit of quantitation for the impurity were established. Validation of the developed LCMS/MS method was carried out as per ICH requirements and the data shows that the proposed method is specific, linear, accurate, precise and robust. This method has been tested in a number of Sumatriptan samples and used successfully for quantification of the impurity at ppm level. The developed LCMS/MS method was found to be suitable to quantify the genotoxic impurity 4-Chloro-1-Hydroxy Butane Sulfonic Acid Sodium Salt at ppm level present in Sumatriptan Succinate.
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- 2012
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6. Evaluation of Bearing Capacity Improvement Using Composite Grids
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Link, RE, primary, Link, RE, additional, Sivakumar Babu, GL, additional, Srinivasa Rao, BM, additional, and Sridharan, A, additional
- Published
- 2002
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7. Surgical repair of hip abductors. A new technique using Graft Jacket allograft acellular human dermal matrix.
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Rao BM, Kamal TT, Vafaye J, Taylor L, Rao, Biyyam M, Kamal, Tamer T, Vafaye, John, and Taylor, Lee
- Abstract
Purpose: Avulsion of the abductors from the hip can be an infrequent but debilitating complication after total hip arthroplasty performed through a trans-gluteal approach. This can result in intractable pain, limp, Trendelenberg lurch and instability of the hip. There have been various methods described for repairing or reconstruction of this abductor muscle complex including direct trans-osseous repair, muscle transfers, muscle and tendon sling, bone tendon allograft reconstruction and endoscopic repair techniques.Methods: In a prospective study at our institution we evaluated the results of a surgical technique in 12 patients using a trans-osseous repair of gluteus medius and minimus insertions augmented by a Graft Jacket allograft acellular human dermal matrix (Graft Jacket; Wright Medical Technology, Arlington, TN) over the anterior and anterolateral aspects of the greater trochanter. Diagnosis of hip abductor avulsions was made by evaluation of the history of presenting complaint, clinical examination and confirmed by ultrasound or MRI scans.Results: Evaluation of results included pain scoring, gait evaluation, Trendelenberg test, and the Harris hip score. There was a significant improvement in pain (VAS mean values 8.25 to 2.33; p value < 0.0001), limp and gait along with abductor strength. The Trendelenberg test became negative in all but one. At the mean follow up of 22 months Harris hip scores improved from 34.05 to 81.26 (p value <0.0001).Conclusion: Overall this procedure appears to be safe and associated with high patient satisfaction, without the morbidity of any tendon or muscle transfers. [ABSTRACT FROM AUTHOR]- Published
- 2012
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8. Evaluation of Bearing Capacity Improvement Using Composite Grids
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Sivakumar Babu, GL, Srinivasa Rao, BM, and Sridharan, A
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Recently, composite reinforcements in which combinations of materials and material forms such as strips, grids, and strips and anchors, depending on requirements, have proven to be effective in various ground improvement applications. Composite geogrids studied in this paper belong to the category of composite reinforcements and are useful for bearing capacity improvement. The paper presents evaluation of results of bearing capacity tests conducted on a composite geogrid, made of composite reinforcement consisting of steel and cement mortar. The study shows that the behavior of composite reinforcements follows the general trends observed in the case of conventional geogrids, with reference to the depth of first layer below the footing, number of layers of reinforcement, and vertical spacing of the reinforcement. Results show that the performance is comparable to that of a conventional polymer geogrid.
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- 2002
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9. Reduction In Pathogenic And Saprophytic Fungi In Maize, Chilli And Paddy Seeds By Hydrochloric-Acid Vapor And Soaking In Hcl Solution Treatment
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Shethna, YI and Rao, BM
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Microbiology & Cell Biology - Published
- 1981
10. Derivation of human trophoblast stem cells from placentas at birth.
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Karakis V, Britt JW, Jabeen M, Miguel AS, and Rao BM
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Human trophoblast stem cells (hTSCs) have emerged as a powerful tool for modeling the placental cytotrophoblast (CTB) in vitro. hTSCs were originally derived from CTBs of the first trimester placenta or blastocyst-stage embryos in trophoblast stem cell medium (TSCM) that contains epidermal growth factor (EGF), the glycogen synthase kinase-beta (GSK3β) inhibitor CHIR99021, the transforming growth factor-beta (TGFβ) inhibitors A83-01 and SB431542, valproic acid (VPA), and the Rho-associated protein kinase (ROCK) inhibitor Y-27632. Here we show that hTSCs can be derived from CTBs isolated from the term placenta, using TSCM supplemented with a low concentration of mitochondrial pyruvate uptake inhibitor UK5099 and lipid-rich albumin (TUA medium). Notably, hTSCs could not be derived from term CTBs using TSCM alone, or in the absence of either UK5099 or lipid-rich albumin. Strikingly, hTSCs cultured in TUA medium for a few passages could be transitioned into TSCM and cultured thereafter in TSCM. hTSCs from term CTBs cultured in TUA medium as well as those transitioned into and cultured in TSCM thereafter could be differentiated to the extravillous trophoblast and syncytiotrophoblast lineages and exhibited high transcriptome similarity with hTSCs derived from first trimester CTBs. We anticipate that these results will enable facile derivation of hTSCs from normal and pathological placentas at birth with diverse genetic backgrounds and facilitate in vitro mechanistic studies in trophoblast biology.
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- 2024
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11. Widespread occurrence of Tilapia parvovirus in farmed Nile tilapia Oreochromis niloticus from India.
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Rajendran KV, Sood N, Rao BM, Valsalam A, Bedekar MK, Jeena K, Pradhan PK, Paria A, Swaminathan TR, Verma DK, and Sood NK
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Tilapia parvovirus (TiPV) has been associated with heavy mortalities in tilapia as a single infection or in co-infection with Tilapia lake virus (TiLV). In this study, TiPV was detected in farmed Nile tilapia, Oreochromis niloticus, from two geographical regions of India, Maharashtra and Uttar Pradesh. TiPV-specific polymerase chain reaction (PCR) reported earlier was used in the screening. Tilapia collected from Maharashtra showed characteristic clinical signs, and TiPV was detected along with TiLV and/or Aeromonas spp. However, fish from Uttar Pradesh were apparently healthy and only TiPV could be detected in these samples. A high prevalence of TiPV was recorded from both the geographical locations, Maharashtra and Uttar Pradesh (59.6% and 95.0% respectively). The virus could be detected in tissues such as the spleen, liver, kidney, brain and mucus. The spleen appeared to be the best tissue for detecting TiPV in apparently healthy tilapia. The presence of TiPV was further confirmed through sequencing the PCR products, isolation of the virus in the cell line and electron microscopy. Sequences of the NS1 gene of the two TiPV isolates showed similarity to the earlier reported TiPV isolates. The virus could be successfully propagated in O. niloticus Liver (OnL) cell line, and cytopathic effect was observed as early as 3 days post-infection. Furthermore, the presence of non-enveloped icosahedral to round virus particles measuring about 26-35 nm could be demonstrated in the cytoplasm and nucleus of infected OnL cells in transmission electron microscopy. With this confirmation of the presence of the virus, India is the third country to report TiPV after China and Thailand. The detection of TiPV in co-infection cases with TiLV and in apparently healthy Nile tilapia suggests its wide distribution and potential synergistic effect in co-infection cases. Therefore, this emerging virus needs holistic attention to understand its virulence, host-specificity and epidemiological risk factors., (© 2023 John Wiley & Sons Ltd.)
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- 2023
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12. Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions.
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Karakis V, Jabeen M, Britt JW, Cordiner A, Mischler A, Li F, San Miguel A, and Rao BM
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- Humans, Colforsin pharmacology, Colforsin metabolism, HLA-G Antigens genetics, HLA-G Antigens metabolism, Transforming Growth Factor beta metabolism, Culture Media chemistry, Hypoxia-Inducible Factor 1, alpha Subunit genetics, Gene Expression Regulation, Developmental drug effects, Cell Differentiation drug effects, Laminin pharmacology, Stem Cells cytology, Stem Cells drug effects, Trophoblasts cytology, Trophoblasts metabolism, Cytological Techniques methods
- Abstract
Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFβ) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue-laminin-111-switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1
+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFβ signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFβ inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFβ inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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13. Surgical outcomes of non-periprosthetic distal femur fragility fractures treated with a locking plate.
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Liu J, Hein D, Huffman C, Rao BM, Cooper J, and Ebraheim NA
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Background: Management of distal femur fractures can be challenging, particularly in the aged female population. This retrospective study aims to analyze the surgical outcomes of aged female patients with non-periprosthetic distal femur fractures treated with a locking plate., Methods: This is an IRB approved retrospective review conducted at a level 1 trauma center. Fifty-five female patients (mean age of 71 years) with non-periprosthetic distal femur fractures underwent open reduction internal fixation using a locking plate from 2005 to 2019. The average follow-up time was 67 weeks from the date of injury. Criteria used for diagnosis of nonunion included one or more of the following: (I) three consecutive months without progression of healing on postoperative radiographs, (II) a total of nine months postoperative without complete healing, or (III) the physician diagnosed nonunion using clinical judgement. Outcome data was analyzed and compared amongst patients with and without obesity or diabetes. Statistical analysis was performed utilizing Microsoft Excel 2022 Data Analysis ToolPak with a standard statistically significant P value of <0.05., Results: Thirty-two patients (58%) with distal femur fractures achieved union after initial treatment while 23 patients (42%) were diagnosed with nonunion. Fourteen patients (61%) underwent revision with 9 of these patients (64%) achieving union while 5 patients (36%) had persistent nonunion. Average healing time from initial treatment to union was 29 weeks, while average time from definitive treatment to union was 22 weeks. Obese patients [body mass index (BMI) >30 kg/m
2 ] had a nonunion rate of 65%, while non-obese patients had a nonunion rate of 28%. Patients with diabetes had a nonunion rate of 65%, while patients without diabetes had a nonunion rate of 28%., Conclusions: Union can be successfully achieved in aged female patients with distal femur fractures treated with locking plates; however, the risks for nonunion and revision remain high. Patients with obesity and diabetes appear to be at an increased risk of nonunion (P=0.008 and 0.008, respectively). However, further research should be conducted with a prospective study or multivariate analysis and increased number of patients to reaffirm this data., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://aoj.amegroups.com/article/view/10.21037/aoj-22-15/coif). JL serves as an unpaid editorial board member of Annals of Joint from April 2022 to March 2024. The other authors have no conflicts of interest to declare., (2022 Annals of Joint. All rights reserved.)- Published
- 2022
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14. A systematic review of the surgical outcomes of interprosthetic femur fractures.
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Rao BM, Stokey P, Tanios M, Liu J, and Ebraheim NA
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Background: Interprosthetic femur fractures (IFFs) are rare, but the treatment is challenging. Currently, there are many treatment methods used in practice, but an updated systematic review of comparison of common different surgical outcomes has not been thoroughly inspected., Methods: A systematic review of retrospective studies was conducted. The resource databases of PubMed, Cochrane, and Embase were searched using a combination of the keywords involving IFFs and surgical outcomes from inception through June 2021. Data collected included patient demographics, intraoperative data, and postoperative outcomes. Outcomes were measured based on healing time, revision rate, complication rate, and functional scores., Results: Forty studies were included for review with a total of 508 patients. Average reported age of patients was 78.7 years old and 403 (79.3%) were females. Overall union rate was 74.0% with 376 of 508 patients achieving fracture union after primary treatment of IFF. Only 271 patients had reported healing times of fractures with a mean of 5.15 months. The plate, prosthetic revision, nail/rod, and external fixator groups had mean healing times of 4.69, 8.73, 6.5, and 5.1 months, respectively. Revision rates were highest in the femur replacement treatment group with 9 (32.1%) patients needing at least one reoperation surgery for any reason. Overall, hardware failure and non-unions were the most reported complications in treatment of IFFs. Postoperative functional outcome scores were available for 242 patients. Harris Hip Scores for the plate, revision, replacement, nail/rod, and plate + revision groups were 76.84, 77.14, 69.9, 77, and 78.4, respectively., Conclusion: Each treatment method should be carefully considered by the surgeon depending on the patient. Locking plate was the most common method for the treatment of the patients with IFFs. Half of them combined with cerclage wires/cables. Around two thirds' patients could achieve union with the fastest mean healing time around 4.69 months. Other less common methods included prosthetic revision, femur replacement, nail/rod, external fixator, etc. A small number of patients treated with Ilizarov external fixator, and it has proven to be a viable option with few complications and high union rates., Competing Interests: No conflicts of interest related to the study., (© 2022 Professor P K Surendran Memorial Education Foundation. Published by Elsevier B.V. All rights reserved.)
- Published
- 2022
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15. Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase.
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McArthur N, Cruz-Teran C, Thatavarty A, Reeves GT, and Rao BM
- Abstract
The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular "mix-and-read" enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting nonoverlapping epitopes on the target analyte were each fused to nonactive fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provides the foundation for the development of split luciferase assays for detection and quantification of various targets., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)
- Published
- 2022
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16. Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity.
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Meanor JN, Keung AJ, and Rao BM
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- HEK293 Cells, Humans, Jurkat Cells, K562 Cells, Protein Array Analysis, Protein Binding, Protein Engineering, Protein Processing, Post-Translational, Saccharomyces cerevisiae, Histone Code, Histones metabolism
- Abstract
Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.
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- 2022
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17. Discovery of Cyclic Peptide Binders from Chemically Constrained Yeast Display Libraries.
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Bacon K, Menegatti S, and Rao BM
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- Cyclization, Peptide Library, Peptides chemistry, Peptides, Cyclic chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
Cyclic peptides with engineered protein-binding activity have great potential as therapeutic and diagnostic reagents owing to their favorable properties, including high affinity and selectivity. Cyclic peptide binders have generally been isolated from phage display combinatorial libraries utilizing panning based selections. As an alternative, we have developed a yeast surface display platform to identify and characterize cyclic peptide binders from genetically encoded combinatorial libraries. Through a combination of magnetic selection and fluorescence-activated cell sorting (FACS), high-affinity cyclic peptide binders can be efficiently isolated from yeast display libraries. In this platform, linear peptide precursors are expressed as yeast surface fusions. To achieve cyclization of the linear precursors, the cells are incubated with disuccinimidyl glutarate, which crosslinks amine groups within the displayed linear peptide sequence. Here, we detail protocols for cyclizing linear peptides expressed as yeast surface fusions. We also discuss how to synthesize a yeast display library of linear peptide precursors. Subsequently, we provide suggestions on how to utilize magnetic selections and FACS to isolate cyclic peptide binders for target proteins of interest from a peptide combinatorial library. Lastly, we detail how yeast surface displayed cyclic peptides can be used to obtain efficient estimates of binding affinity, eliminating the need for chemically synthesized peptides when performing mutant characterization., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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18. Isolation of Single-Domain Antibodies to Transmembrane Proteins Using Magnetized Yeast Cell Targets.
- Author
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Bacon K, Menegatti S, and Rao BM
- Subjects
- Flow Cytometry, Ligands, Membrane Proteins genetics, Membrane Proteins metabolism, Peptide Library, Saccharomyces cerevisiae metabolism, Single-Domain Antibodies genetics, Single-Domain Antibodies metabolism
- Abstract
The isolation of binding ligands from yeast-displayed combinatorial libraries has typically relied on the use of a soluble, recombinantly expressed form of the target protein when performing magnetic selections or fluorescence-activated cell sorting. When identifying binding ligands, appropriate target protein expression and subsequent purification represents a significant bottleneck. As an alternative, we describe the use of target proteins expressed on the surface of magnetized yeast cells in the selection of yeast-displayed nanobody libraries. In this approach, yeast cells displaying the target protein also co-express an iron oxide-binding protein; incubation with iron oxide nanopowder results in magnetization of target-displaying cells. Alternatively, target-displaying cells are magnetized by nonspecific adsorption of iron oxide nanopowder. Subsequently, any library cells that interact with the magnetized target cells can be isolated using a magnet. Here, we detail protocols for the isolation of binders to membrane protein targets from a yeast display nanobody library using magnetized yeast cell targets. We provide guidance on how to generate magnetic yeast cell targets as well as library selection conditions to bias the isolation of high affinity binders. We also discuss how to assess the affinity and specificity of the isolated nanobodies using flow cytometry., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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19. Yeast Display Guided Selection of pH-Dependent Binders.
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Meanor JN, Keung AJ, Rao BM, and Gera N
- Subjects
- Cell Separation, Gene Library, Hydrogen-Ion Concentration, Antibodies, Saccharomyces cerevisiae genetics
- Abstract
pH-dependent antigen binding has proven useful in engineering next-generation therapeutics specifically via antibody recycling technology. This technology allows for half-life extension, thereby lowering the amount and frequency of dosing of therapeutics. Cell sorting, coupled with display techniques, has been used extensively for the selection of high-affinity binders. Herein, we describe a cell sorting methodology utilizing yeast surface display for selection of binding proteins with strong binding at physiological pH and weak to no binding at acidic pH. This methodology can be readily applied to engineer proteins and/or antibodies that do not have pH-dependent binding or for selection of de novo pH-dependent binders using library-based methods., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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20. Mapping the residue specificities of epigenome enzymes by yeast surface display.
- Author
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Waldman AC, Rao BM, and Keung AJ
- Subjects
- E1A-Associated p300 Protein genetics, Humans, E1A-Associated p300 Protein metabolism, Epigenome genetics, Saccharomyces cerevisiae metabolism
- Abstract
Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here, we describe a versatile and scalable approach which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first use this system to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, a large panel of commercially available anti-acetylation antibodies are screened for their specificities, identifying many suitable and unsuitable reagents. Furthermore, this approach enables efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4 and uncovers residue interdependencies affecting p300 activity. These results show that using yeast surface display to study histone modifications is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2021
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21. Worse Preoperative Pain and Higher Activity Levels Predict Patient Choice of Formal Physical Therapy After Primary Anterior Total Hip Arthroplasty.
- Author
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Rao BM, Cieslewicz TJ, Sochacki KR, Kohlrieser DA, Moylan DD, and Ellis TJ
- Subjects
- Humans, Pain, Patient Preference, Physical Therapy Modalities, Treatment Outcome, Arthroplasty, Replacement, Hip, Osteoarthritis, Hip surgery
- Abstract
Background: The purpose of this randomized controlled trial is (1) to compare the efficacy of supervised formal physical therapy (PT) and self-directed home exercises and (2) to identify independent predictors of transitioning from self-directed home exercises to supervised formal PT following total hip arthroplasty (THA) via an anterior approach., Methods: After Institutional Review Board approval, 147 patients undergoing primary unilateral THA through anterior approach were enrolled and randomized to receive either clinic or home-based PT. Surveys with Hip Disability Osteoarthritis Outcome Scores (HOOS) and Short Form-12 Health Survey scores for both groups were obtained before surgery and at 6, 14, and 24 weeks after surgery. Patients had the option to transition into the other study group at their 6-week follow-up visit., Results: Of the 147 patients enrolled, final analysis included scores for 136 patients. Forty-two (63.6%) of the 66 patients randomized to the supervised formal PT group crossed over to self-directed home exercise group and 12 (17.1%) of the 70 patients randomized to the self-directed home exercise group crossed over to supervised formal PT group. There were no significant (P > .05) differences between groups at all time points for the overall HOOS and Short Form-12 Health Survey. Subjects with worse preoperative pain scores (P = .005) and higher HOOS preoperative activity of daily living scores (P = .015) were more likely to transition from the home exercise group to formal PT group., Conclusion: There were no significant outcome score differences between subjects undergoing supervised formal PT and self-directed home exercises. However, patients reporting worse preoperative pain and higher preoperative activity levels may prefer supervised formal PT compared to self-directed home exercises after undergoing primary anterior THA., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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22. Geriatric assistive devices improve older patient engagement and clinical care in an emergency department.
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Fareed N, Southerland LT, Rao BM, and Sieck CJ
- Subjects
- Activities of Daily Living psychology, Aged, Emergency Service, Hospital organization & administration, Geriatrics methods, Geriatrics standards, Geriatrics statistics & numerical data, Humans, Patient Participation statistics & numerical data, Old Age Assistance, Patient Participation psychology
- Published
- 2021
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23. Quantitative Yeast-Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein-Protein Interactions.
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Bacon K, Blain A, Bowen J, Burroughs M, McArthur N, Menegatti S, and Rao BM
- Subjects
- Gene Library, Genes, Reporter, Protein Binding, Protein Domains, Saccharomyces cerevisiae genetics, Smad3 Protein chemistry, Transcription Factors chemistry, Two-Hybrid System Techniques, Protein Interaction Maps, Saccharomyces cerevisiae metabolism, Smad3 Protein metabolism, Transcription Factors metabolism
- Abstract
Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities ( K
D ). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.- Published
- 2021
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24. Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands.
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Bowen J, Schneible J, Bacon K, Labar C, Menegatti S, and Rao BM
- Subjects
- Binding Sites, Combinatorial Chemistry Techniques, Endoplasmic Reticulum metabolism, Flow Cytometry, Ligands, Peptides, Cyclic pharmacology, Protein Binding, Protein Engineering methods, Transglutaminases metabolism, YAP-Signaling Proteins, Yeasts genetics, Yeasts growth & development, Adaptor Proteins, Signal Transducing chemistry, Adaptor Proteins, Signal Transducing metabolism, Albumins metabolism, Muramidase metabolism, Peptides, Cyclic isolation & purification, Transcription Factors chemistry, Transcription Factors metabolism
- Abstract
We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[ E- LYLAYPAH -K ] featured a K
D of 1.75 μM for YAP and 0.68 μM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.- Published
- 2021
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25. Two distinct trophectoderm lineage stem cells from human pluripotent stem cells.
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Mischler A, Karakis V, Mahinthakumar J, Carberry CK, San Miguel A, Rager JE, Fry RC, and Rao BM
- Subjects
- Cell Culture Techniques, Cell Differentiation, Cell Lineage, Culture Media, Embryonic Stem Cells metabolism, Female, Humans, Placenta metabolism, Pluripotent Stem Cells metabolism, Pregnancy, Trophoblasts metabolism, CDX2 Transcription Factor metabolism, Embryonic Stem Cells cytology, Placenta cytology, Pluripotent Stem Cells cytology, Trophoblasts cytology
- Abstract
The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2
- stem cell comparable with placenta-derived TS cells-they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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26. Mandatory Nicotine Cessation for Elective Orthopedic Hip Procedures Results in Reduction in Postoperative Nicotine Use.
- Author
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Rao BM, Moylan DD, Sochacki KR, Kollmorgen RC, Atwal L, and Ellis TJ
- Abstract
Purpose To determine the efficacy of mandatory preoperative nicotine cessation on postoperative nicotine use, and to identify independent predictors of nicotine use relapse in subjects undergoing hip preservation surgery or total hip arthroplasty by a single fellowship-trained orthopedic surgeon. Methods Consecutive subjects that underwent hip surgery from November 2014 to December 2017 were reviewed. Subjects who self-reported nicotine use, quit prior to surgery, and completed a minimum one-year follow-up were included. Multiple linear regression models were constructed to determine the effect of independent variables on nicotine use relapse following surgery. Results Sixty subjects were included in the study (mean follow-up 35.1 months (17-57 months), mean age 44.9 years (20-82 years), and 23 (38.3%) males). Twenty-eight subjects (46.7%) remained nicotine-free at final follow-up. The mean number of cigarettes per day decreased from 13.4 preoperatively to 8.4 postoperatively in the subjects who relapsed (P=0.002). The mean time to return to nicotine postoperatively was 2.4 months. The number of preoperative cigarettes per day was the only independent predictor of tobacco use relapse (P=0.005). Conclusion Mandatory preoperative nicotine cessation prior to elective hip surgery demonstrates a 46.7% nicotine-free survivorship at final follow-up with the number of preoperative cigarettes per day found to be the only independent predictor of nicotine use relapse. Level of evidence The level of evidence of this research study is Level III since it is a non-experimental study with a cohort of patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020, Rao et al.)
- Published
- 2020
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27. Use of Target-Displaying Magnetized Yeast in Screening mRNA-Display Peptide Libraries to Identify Ligands.
- Author
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Bacon K, Bowen J, Reese H, Rao BM, and Menegatti S
- Subjects
- Ligands, Models, Molecular, Mitochondrial Membrane Transport Proteins chemistry, Peptide Library, RNA, Messenger chemistry, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae Proteins chemistry, Transcription Factors chemistry
- Abstract
This work presents the first use of yeast-displayed protein targets for screening mRNA-display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity ( K
D ) of select WW-YAP-binding peptides: KD values of 37 and 4 μM were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 and 3 μM for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22+ yeast compared to control cells. This work demonstrates that integrating mRNA- and yeast-display accelerates the discovery of peptide ligands.- Published
- 2020
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28. Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.
- Author
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Bowen J, Schloop AE, Reeves GT, Menegatti S, and Rao BM
- Subjects
- Animals, Cell Line, Cell-Penetrating Peptides chemistry, Cell-Penetrating Peptides genetics, Drosophila melanogaster embryology, Gene Library, Humans, Mice, Models, Molecular, NIH 3T3 Cells, Peptides, Cyclic chemistry, Peptides, Cyclic genetics, Permeability, Cell-Penetrating Peptides pharmacokinetics, Peptides, Cyclic pharmacokinetics, RNA, Messenger genetics
- Abstract
Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[ Glut -MRKRHASRRE- K *] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.
- Published
- 2020
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29. Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display.
- Author
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Bacon K, Blain A, Burroughs M, McArthur N, Rao BM, and Menegatti S
- Subjects
- Biophysical Phenomena, Cyclization, Flow Cytometry, Humans, Molecular Docking Simulation, Peptide Library, Protein Binding, Protein Conformation, Protein Engineering, Saccharomyces cerevisiae ultrastructure, Structure-Activity Relationship, Surface Properties, Cell Membrane chemistry, Interleukin-17 chemistry, Peptides, Cyclic isolation & purification, Recombinant Proteins chemistry, Saccharomyces cerevisiae chemistry
- Abstract
Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated cross-linking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides using magnetic selection, followed by fluorescence activated cell sorting (FACS) to isolate binders for a model target (lysozyme) with low micromolar binding affinity ( K
D ∼ 1.2-3.7 μM). The isolated peptides bind lysozyme selectively and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides can be used to efficiently obtain quantitative estimates of binding affinity, circumventing the need for chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that likely binds IL-17 in its receptor binding region with moderate apparent affinity ( KD ∼ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.- Published
- 2020
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30. Genetically encoded live-cell sensor for tyrosinated microtubules.
- Author
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Kesarwani S, Lama P, Chandra A, Reddy PP, Jijumon AS, Bodakuntla S, Rao BM, Janke C, Das R, and Sirajuddin M
- Subjects
- Colchicine pharmacology, Cytoskeleton drug effects, Cytoskeleton genetics, HEK293 Cells, Humans, Nocodazole pharmacology, Protein Processing, Post-Translational drug effects, Protein Processing, Post-Translational genetics, Tyrosine drug effects, Vincristine pharmacology, Microtubule-Associated Proteins genetics, Microtubules genetics, Tubulin genetics, Tyrosine genetics
- Abstract
Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells., (© 2020 Kesarwani et al.)
- Published
- 2020
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31. Harnessing the power of social media to support medical training during COVID-19.
- Author
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Hirniak J, Huddart D, Sethi R, Dibblin C, Hayer G, Rao BM, Jenkins C, Hueso B, Zaman ME, and Sethi S
- Abstract
Twitter offers a powerful means to share information, suggest ways to help and highlight useful initiatives during the global COVID-19 pandemic. We describe one successful Twitter campaign focusing on the role of medical students (#MedStudentCovid), led by the volunteer organisation Becoming A Doctor with support from leaders at the General Medical Council, Health Education England, NHS England and the World Health Organization., (© Royal College of Physicians 2020. All rights reserved.)
- Published
- 2020
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32. Validation of Self-Reported Smoking Status Among Orthopedic Hip Surgery Patients.
- Author
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Ellis ST, Rao BM, Kohlrieser D, Kollmorgen RC, and Sochacki KR
- Abstract
Purpose The purpose of this study was to determine the accuracy of self-reported non-smoking status in subjects undergoing elective orthopedic surgery as confirmed by serum cotinine levels. Methods Institutional Review Board approval was obtained for this retrospective review of consecutive subjects that underwent elective orthopedic surgery by a single fellowship-trained orthopedic surgeon. All patients provided smoking history (active, former, or non-smoker). Serum cotinine levels defined each subject as "non-smoker", "passive tobacco exposure", or "active smoker". Self-reported non-smokers were eligible for inclusion. Subjects were excluded if they failed to provide smoking history, reported themselves as "smokers", and/or had unavailable serum cotinine levels. Self-reported non-smoking status accuracy was determined by dividing the total number of included subjects by the number of subjects that were defined as "non-smoker" or "passive tobacco exposure" on their serum cotinine test. Results A total of 378 patients (mean age of 42.5 (13-78) years and 68% female) self-reported as non-smokers and were included. A total of 369 subjects had serum cotinine levels consistent with "non-smoking" resulting in a self-reported non-smoking status accuracy of 97.6%. None of the former smokers had cotinine levels consistent with active smoker status. Conclusion Subjects undergoing elective orthopedic surgery self-report as non-smokers with an accuracy of 97.6%. This suggests that routine serum cotinine testing of non-smokers in this patient population may not be necessary., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020, Ellis et al.)
- Published
- 2020
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33. Past, Present, and Future of Affinity-based Cell Separation Technologies.
- Author
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Bacon K, Lavoie A, Rao BM, Daniele M, and Menegatti S
- Subjects
- Cell Separation, Chromatography, Affinity, Flow Cytometry, Ligands, Regenerative Medicine
- Abstract
Progress in cell purification technology is critical to increase the availability of viable cells for therapeutic, diagnostic, and research applications. A variety of techniques are now available for cell separation, ranging from non-affinity methods such as density gradient centrifugation, dielectrophoresis, and filtration, to affinity methods such as chromatography, two-phase partitioning, and magnetic-/fluorescence-assisted cell sorting. For clinical and analytical procedures that require highly purified cells, the choice of cell purification method is crucial, since every method offers a different balance between yield, purity, and bioactivity of the cell product. For most applications, the requisite purity is only achievable through affinity methods, owing to the high target specificity that they grant. In this review, we discuss past and current methods for developing cell-targeting affinity ligands and their application in cell purification, along with the benefits and challenges associated with different purification formats. We further present new technologies, like stimuli-responsive ligands and parallelized microfluidic devices, towards improving the viability and throughput of cell products for tissue engineering and regenerative medicine. Our comparative analysis provides guidance in the multifarious landscape of cell separation techniques and highlights new technologies that are poised to play a key role in the future of cell purification in clinical settings and the biotech industry. STATEMENT OF SIGNIFICANCE: Technologies for cell purification have served science, medicine, and industrial biotechnology and biomanufacturing for decades. This review presents a comprehensive survey of this field by highlighting the scope and relevance of all known methods for cell isolation, old and new alike. The first section covers the main classes of target cells and compares traditional non-affinity and affinity-based purification techniques, focusing on established ligands and chromatographic formats. The second section presents an excursus of affinity-based pseudo-chromatographic and non-chromatographic technologies, especially focusing on magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Finally, the third section presents an overview of new technologies and emerging trends, highlighting how the progress in chemical, material, and microfluidic sciences has opened new exciting avenues towards high-throughput and high-purity cell isolation processes. This review is designed to guide scientists and engineers in their choice of suitable cell purification techniques for research or bioprocessing needs., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier Ltd.)
- Published
- 2020
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34. A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells.
- Author
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Mukherjee A, Singh R, Udayan S, Biswas S, Reddy PP, Manmadhan S, George G, Kumar S, Das R, Rao BM, and Gulyani A
- Subjects
- Animals, Cell Line, Cell Physiological Phenomena genetics, Fluorescence Resonance Energy Transfer, Fluorescent Dyes metabolism, HEK293 Cells, Humans, Magnetic Resonance Spectroscopy, Mice, Phosphorylation genetics, Yeasts genetics, Biosensing Techniques, Proto-Oncogene Proteins c-fyn chemistry, Proto-Oncogene Proteins c-fyn genetics, Proto-Oncogene Proteins c-fyn metabolism, Signal Transduction genetics
- Abstract
Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor ( FynSensor ) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate., Competing Interests: AM, RS, SU, SB, PR, SM, GG, SK, RD, BR, AG No competing interests declared
- Published
- 2020
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35. Simultaneous Soluble Secretion and Surface Display of Proteins in Saccharomyces cerevisiae Using Inefficient Ribosomal Skipping.
- Author
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Cruz-Teran CA, Bacon K, and Rao BM
- Subjects
- Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins genetics, Peptide Library, Peptides metabolism, Protein Biosynthesis, Ribosomes metabolism, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
Combinatorial library screening platforms, such as yeast surface display, typically identify several candidate proteins that need further characterization and validation using soluble recombinant protein. However, recombinant production of these candidate proteins involves tedious and time-consuming subcloning steps. This, in turn, limits the number of candidate proteins that can be characterized. To address this bottleneck, we have developed a platform that exploits inefficient ribosomal skipping by the F2A peptide for simultaneous soluble secretion and cell surface display of protein in the yeast Saccharomyces cerevisiae. Here we provide detailed protocols utilizing this F2A-based yeast display system. We discuss specific recommendations for the purification of the secreted protein. Additionally, we provide suggestions for testing the functionality and binding specificity of the soluble secreted proteins using flow cytometry analysis.
- Published
- 2020
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36. Screening Yeast Display Libraries against Magnetized Yeast Cell Targets Enables Efficient Isolation of Membrane Protein Binders.
- Author
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Bacon K, Burroughs M, Blain A, Menegatti S, and Rao BM
- Subjects
- Combinatorial Chemistry Techniques, Drug Evaluation, Preclinical, Ferric Compounds chemistry, Magnetic Phenomena, Mitochondria drug effects, Mitochondria metabolism, Mitochondrial Membrane Transport Proteins isolation & purification, Mitochondrial Membrane Transport Proteins metabolism, Protein Binding drug effects, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins isolation & purification, Saccharomyces cerevisiae Proteins metabolism, Small Molecule Libraries chemistry, Ferric Compounds pharmacology, Mitochondrial Membrane Transport Proteins antagonists & inhibitors, Nanoparticles chemistry, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae Proteins antagonists & inhibitors, Small Molecule Libraries pharmacology
- Abstract
When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is coexpressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder-target pair with modest binding affinity ( K
D ≈ 400 nM), we showed that a binder present at low frequency (1 in 105 ) could be enriched more than 100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 ( KD ≈ 272-1934 nM) and the extracellular domain of the c-Kit receptor ( KD ≈ 93 to KD > 2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion-in contrast to recombinant soluble expression-makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.- Published
- 2019
- Full Text
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37. Methicillin-Resistant Staphylococcus aureus in Seafood: Prevalence, Laboratory Detection, Clonal Nature, and Control in Seafood Chain.
- Author
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Vaiyapuri M, Joseph TC, Rao BM, Lalitha KV, and Prasad MM
- Subjects
- Animals, Anti-Bacterial Agents pharmacology, China epidemiology, Consumer Product Safety, Fish Diseases epidemiology, Fishes, Humans, Methicillin-Resistant Staphylococcus aureus classification, Methicillin-Resistant Staphylococcus aureus drug effects, Methicillin-Resistant Staphylococcus aureus genetics, Seafood analysis, Seafood microbiology, Staphylococcal Infections epidemiology, Fish Diseases microbiology, Food Contamination analysis, Methicillin-Resistant Staphylococcus aureus isolation & purification, Staphylococcal Infections veterinary
- Abstract
Methicillin-resistant Staphylococcus aureus (MRSA), a versatile pathogen bearing multiple virulence determinants, is increasingly being detected in various food-producing animals, including fish. In addition, it is a potential food poisoning agent. MRSA is not an inherent microbiota of fish; its presence is attributed to pre- or postharvest contamination through fish handlers, water, ice, and processing equipment. Several reviews have been written on MRSA in clinical as well as the food animal-producing sector, but information specific to MRSA in seafood is scant. This review puts forth insights on MRSA detection in seafood, antibiotic resistance, diversity of clones in seafood, and possible control measures in seafood production chain. Emphasis has been given on assessing the variations in the protocols employed for isolation and identification in different food matrices and lay the foundation for researchers to develop optimized procedure., (© 2019 Institute of Food Technologists®.)
- Published
- 2019
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38. Prevalence, molecular characterization, genetic heterogeneity and antimicrobial resistance of Listeria monocytogenes associated with fish and fishery environment in Kerala, India.
- Author
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Basha KA, Kumar NR, Das V, Reshmi K, Rao BM, Lalitha KV, and Joseph TC
- Subjects
- Ampicillin pharmacology, Animals, Clindamycin pharmacology, Erythromycin pharmacology, Fisheries, Fishes microbiology, Food Microbiology, Food Safety, Genetic Heterogeneity, Ice analysis, India, Listeria monocytogenes genetics, Listeria monocytogenes isolation & purification, Penicillins pharmacology, Prevalence, Serogroup, Tetracycline pharmacology, Anti-Bacterial Agents pharmacology, Drug Resistance, Multiple, Bacterial genetics, Listeria monocytogenes drug effects, Seafood microbiology, Shellfish microbiology
- Abstract
The prevalence of Listeria monocytogenes in the retail fish markets of the Kerala, India was investigated by screening 227 samples comprising of marine finfish (n = 97) shellfish (n = 19), ready-to-cook fish products (n = 47), ready-to-eat fish products (n = 10), dried fish (n = 11) and retail ice (n = 43). The prevalence of L. monocytogenes and L. innocua was 2·7% and 17·2% respectively. Sample category wise, prevalence of L. monocytogenes was higher in marine finfish (1·8%) and retail ice (0·9%). All the L. monocytogenes isolates carried virulent genes namely inlA, inlC, inlJ, hlyA, iap, plcA, prfA genes and majority (82%) belonged to 1/2a, 3a serogroups. L. monocytogenes isolates were multidrug-resistant and showed resistance to ampicillin, penicillin, erythromycin, tetracycline and clindamycin. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) delineated 58% genetic heterogeneity among the L. monocytogenes strains. The study reports that genetic similarities of the isolates were interlinked to their serogroup and sample origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence of Listeria monocytogenes, in the retail fish markets of Kerala, India was low but their relatively higher presence in marine finfish and retail ice and virulent nature of the isolates signifies food safety concerns. Moreover, multidrug-resistant nature of these isolates may potentially lead to spread of antimicrobial resistance. This study identified retail ice as a vehicle for entry of L. monocytogenes in retail fish and hence, there is a need to ensure quality of retail ice used for maintaining the cold-chain., (© 2019 The Society for Applied Microbiology.)
- Published
- 2019
- Full Text
- View/download PDF
39. Digital future in perioperative medicine: Are we there yet?
- Author
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Panchagnula U, Shanmugam M, and Rao BM
- Published
- 2019
- Full Text
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40. Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation.
- Author
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Tiruthani K, Mischler A, Ahmed S, Mahinthakumar J, Haugh JM, and Rao BM
- Subjects
- Animals, Cell Line, Endocytosis, ErbB Receptors genetics, Mice, Microscopy, Fluorescence, Phospholipase C gamma genetics, Phospholipase C gamma metabolism, Phosphorylation genetics, src Homology Domains, Biosensing Techniques, ErbB Receptors metabolism
- Abstract
Live-cell fluorescence microscopy is broadly applied to study the dynamics of receptor-mediated cell signaling, but the availability of intracellular biosensors is limited. A biosensor based on the tandem SH2 domains from phospholipase C-γ1 (PLCγ1), tSH2-WT, has been used to measure phosphorylation of the epidermal growth factor receptor (EGFR). Here, we found that tSH2-WT lacked specificity for phosphorylated EGFR, consistent with the known promiscuity of SH2 domains. Further, EGF-stimulated membrane recruitment of tSH2-WT differed qualitatively from the expected kinetics of EGFR phosphorylation. Analysis of a mathematical model suggested, and experiments confirmed, that the high avidity of tSH2-WT resulted in saturation of its target and interference with EGFR endocytosis. To overcome the apparent target specificity and saturation issues, we implemented two protein engineering strategies. In the first approach, we screened a combinatorial library generated by random mutagenesis of the C-terminal SH2 domain (cSH2) of PLCγ1 and isolated a mutant form (mSH2) with enhanced specificity for phosphorylated Tyr
992 (pTyr992 ) of EGFR. A biosensor based on mSH2 closely reported the kinetics of EGFR phosphorylation but retained cross-reactivity similar to tSH2-WT. In the second approach, we isolated a pTyr992 -binding protein (SPY992) from a combinatorial library generated by mutagenesis of the Sso7d protein scaffold. Compared to tSH2-WT and mSH2, SPY992 exhibited superior performance as a specific, moderate-affinity biosensor. We extended this approach to isolate a biosensor for EGFR pTyr1148 (SPY1148). This approach of integrating theoretical considerations with protein engineering strategies can be generalized to design and evaluate suitable biosensors for various phospho-specific targets., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)- Published
- 2019
- Full Text
- View/download PDF
41. An Engineered Sso7d Variant Enables Efficient Magnetization of Yeast Cells.
- Author
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Cruz-Teran CA, Bacon K, McArthur N, and Rao BM
- Subjects
- Amino Acid Sequence, Archaeal Proteins genetics, Cell Membrane metabolism, DNA-Binding Proteins genetics, Magnetic Phenomena, Mutation, Peptide Library, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Archaeal Proteins metabolism, DNA-Binding Proteins metabolism, Magnetite Nanoparticles chemistry, Saccharomyces cerevisiae metabolism
- Abstract
Magnetization using cheap and minimally toxic materials, such as iron oxide nanoparticles can enable easy separation of cells from culture medium and is relevant to several industrial applications. Here, we show that cell surface expression of a mutant protein that binds iron oxide can enable efficient magnetization of yeast cells. We screened a combinatorial library of mutants derived from the Sso7d protein scaffold to isolate proteins that exhibit preferential binding to iron oxide. One of the isolated mutants, SsoFe2, was chosen for further characterization. Yeast cells expressing SsoFe2 as fusions to a cell wall protein-but not other Sso7d mutants with similar overall protein charge or amino acid composition-preferentially bind iron oxide when present in a solution with high protein concentration and in the presence of 1000-fold excess of competitor yeast cells. Moreover, coexpression of cell surface SsoFe2 enables efficient magnetic capture and separation of yeast cells expressing an enzyme (glucose oxidase) on the cell surface from yeast culture medium, and solutions with high protein concentration or containing other metal oxides. Therefore, SsoFe2-enabled magnetization can enable a range of industrial and biotechnology applications, where easy separation of cells or organelles from complex media is desirable.
- Published
- 2018
- Full Text
- View/download PDF
42. Exfoliation of a non-van der Waals material from iron ore hematite.
- Author
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Puthirath Balan A, Radhakrishnan S, Woellner CF, Sinha SK, Deng L, Reyes CL, Rao BM, Paulose M, Neupane R, Apte A, Kochat V, Vajtai R, Harutyunyan AR, Chu CW, Costin G, Galvao DS, Martí AA, van Aken PA, Varghese OK, Tiwary CS, Malie Madom Ramaswamy Iyer A, and Ajayan PM
- Abstract
With the advent of graphene, the most studied of all two-dimensional materials, many inorganic analogues have been synthesized and are being exploited for novel applications. Several approaches have been used to obtain large-grain, high-quality materials. Naturally occurring ores, for example, are the best precursors for obtaining highly ordered and large-grain atomic layers by exfoliation. Here, we demonstrate a new two-dimensional material 'hematene' obtained from natural iron ore hematite (α-Fe
2 O3 ), which is isolated by means of liquid exfoliation. The two-dimensional morphology of hematene is confirmed by transmission electron microscopy. Magnetic measurements together with density functional theory calculations confirm the ferromagnetic order in hematene while its parent form exhibits antiferromagnetic order. When loaded on titania nanotube arrays, hematene exhibits enhanced visible light photocatalytic activity. Our study indicates that photogenerated electrons can be transferred from hematene to titania despite a band alignment unfavourable for charge transfer.- Published
- 2018
- Full Text
- View/download PDF
43. Design and Fabrication of Wettability Gradients with Tunable Profiles through Degrafting Organosilane Layers from Silica Surfaces by Tetrabutylammonium Fluoride.
- Author
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Miles J, Schlenker S, Ko Y, Patil R, Rao BM, and Genzer J
- Abstract
Surface-bound wettability gradients allow for a high-throughput approach to evaluate surface interactions for many biological and chemical processes. Here we describe the fabrication of surface wettability gradients on flat surfaces by a simple, two-step procedure that permits precise tuning of the gradient profile. This process involves the deposition of homogeneous silane SAMs followed by the formation of a surface coverage gradient through the selective removal of silanes from the substrate. Removal of silanes from the surface is achieved by using tetrabutylammonium fluoride which selectively cleaves the Si-O bonds at the headgroup of the silane. The kinetics of degrafting has been modeled by using a series of first order rate equations, based on the number of attachment points broken to remove a silane from the surface. Degrafting of monofunctional silanes exhibits a single exponential decay in surface coverage; however, there is a delay in degrafting of trifunctional silanes due to the presence of multiple attachment points. The effects of degrafting temperature and time are examined in detail and demonstrate the ability to reliably and precisely control the gradient profile on the surface. We observe a relatively homogeneous coverage of silane (i.e., without the presence of islands or holes) throughout the degrafting process, providing a much more uniform surface when compared to additive approaches of gradient formation. Linear gradients were formed on the substrates to demonstrate the reproducibility and tuneability of this subtractive approach.
- Published
- 2017
- Full Text
- View/download PDF
44. IFPA meeting 2016 workshop report I: Genomic communication, bioinformatics, trophoblast biology and transport systems.
- Author
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Albrecht C, Baker JC, Blundell C, Chavez SL, Carbone L, Chamley L, Hannibal RL, Illsley N, Kurre P, Laurent LC, McKenzie C, Morales-Prieto D, Pantham P, Paquette A, Powell K, Price N, Rao BM, Sadovsky Y, Salomon C, Tuteja G, Wilson S, and O'Tierney-Ginn PF
- Subjects
- Animals, Biological Transport, Biomedical Research trends, Computational Biology trends, DNA Methylation, Exome, Female, Genomics trends, Humans, International Agencies, Placenta cytology, Placenta pathology, Placenta physiopathology, Pregnancy, Pregnancy Complications pathology, Pregnancy Complications physiopathology, Societies, Scientific, Trophoblasts cytology, Trophoblasts pathology, Trophoblasts physiology, Biomedical Research methods, Computational Biology methods, Congresses as Topic, Genomics methods, Maternal-Fetal Exchange, Placenta physiology
- Abstract
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops covered innovative technologies applied to new and traditional areas of placental research: 1) genomic communication; 2) bioinformatics; 3) trophoblast biology and pathology; 4) placental transport systems., (Copyright © 2017 IFPA, Elsevier Ltd. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2017
- Full Text
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45. Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae.
- Author
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Cruz-Teran CA, Tiruthani K, Mischler A, and Rao BM
- Subjects
- Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Cell Adhesion Molecules genetics, Cell Adhesion Molecules metabolism, Gene Expression, Peptide Library, Peptides genetics, Peptides metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by "self-cleaving" 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence-with ∼50% efficiency of ribosomal skipping-between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein-Aga2 fusion that is tethered to the yeast cell surface. We show that binding proteins derived from the Sso7d scaffold and the homodimeric enzyme glucose oxidase can be simultaneously secreted solubly and expressed as yeast cell surface fusions using the F2A-based system. Furthermore, a combinatorial library of Sso7d mutants can be screened to isolate binders with higher affinity for a model target (lysozyme), and the pool of higher affinity binders can be characterized in soluble form. Significantly, we show that both N- and C-terminal fusions to Aga2 can be simultaneously secreted solubly and displayed on the cell surface; this is particularly advantageous because protein functionality can be affected by the specific position of Aga2 in the protein fusion. We expect that the F2A-based yeast surface display and secretion system will be a useful tool for protein engineering and enable efficient characterization of individual clones isolated from combinatorial libraries.
- Published
- 2017
- Full Text
- View/download PDF
46. Identification and characterization of a novel Sso7d scaffold-based binder against Notch1.
- Author
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Gocha T, Rao BM, and DasGupta R
- Subjects
- Animals, Archaeal Proteins genetics, Binding, Competitive drug effects, Carrier Proteins genetics, Carrier Proteins pharmacology, Cell Line, Tumor, Cell Membrane drug effects, Cell Membrane metabolism, Colorectal Neoplasms metabolism, Colorectal Neoplasms pathology, DNA-Binding Proteins genetics, HCT116 Cells, HEK293 Cells, Humans, MCF-7 Cells, Protein Binding, RNA Interference, Receptor, Notch1 antagonists & inhibitors, Receptor, Notch1 genetics, Spheroids, Cellular drug effects, Spheroids, Cellular metabolism, Archaeal Proteins metabolism, Carrier Proteins metabolism, DNA-Binding Proteins metabolism, Receptor, Notch1 metabolism
- Abstract
Notch signaling has important functions in regulating cell growth and development, misregulation of which has been implicated in various cancers. Monoclonal antibodies (mAbs) targeting Notch protein activity have already moved into clinical trials. However due to the limitations associated with cost and productivity of mAbs, there has been a surge in the development of complementary approaches that are based on non-antibody scaffolds. Non-antibody scaffolds are small proteins that are stable and can be engineered to develop high-affinity binders against specific targets of interest. Here we describe the isolation and characterization of a novel Notch1-binding protein, N9, obtained by screening of a combinatorial library based on the ultra-stable Sso7d scaffold. N9 targets the extracellular EGF-like repeats (ELR) 11-13 in Notch1, and therefore serves as a competitive inhibitor for Notch ligands to decrease expression of Notch target genes. We demonstrate that N9 recognizes surface expression of Notch1 on the plasma membrane and binds preferentially to cell lines misexpressing Notch1. Although N9 was selected against Notch1, we also observe cross-reactivity against other Notch receptors, including Notch2/3. Finally, we demonstrate that N9 inhibits proliferation and generation of tumorspheres in Notch expressing cancer cell lines, suggesting its potential as a therapeutic agent in Notch-associated malignancies.
- Published
- 2017
- Full Text
- View/download PDF
47. A case of ingested laryngoscope bulb during emergency endotracheal intubation.
- Author
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Muhamed S, Mathew S, Rao BM, and Arunkumar HD
- Abstract
Competing Interests: There are no conflicts of interest.
- Published
- 2017
- Full Text
- View/download PDF
48. Combinatorial Pairwise Assembly Efficiently Generates High Affinity Binders and Enables a "Mix-and-Read" Detection Scheme.
- Author
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Carlin KB, Cruz-Teran CA, Kumar JP, Gomes C, and Rao BM
- Subjects
- Archaeal Proteins genetics, Archaeal Proteins metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Escherichia coli genetics, Escherichia coli metabolism, Green Fluorescent Proteins metabolism, Mutagenesis, Plasmids genetics, Protein Conformation, Protein Engineering, Recombinant Proteins metabolism, Yeasts genetics, Yeasts metabolism, Green Fluorescent Proteins genetics, Peptide Library, Recombinant Proteins genetics
- Abstract
We show that a combinatorial library constructed by random pairwise assembly of low affinity binders can efficiently generate binders with increased affinity. Such a library based on the Sso7d scaffold, from a pool of low affinity binders subjected to random mutagenesis, contained putative high affinity clones for a model target (lysozyme) at higher frequency than a library of monovalent mutants generated by random mutagenesis alone. Increased binding affinity was due to intramolecular avidity generated by linking binders targeting nonoverlapping epitopes; individual binders of K
D ∼ 1.3 μM and 250 nM produced a bivalent binder with apparent KD ∼ 2 nM. Furthermore, the bivalent protein retained thermal stability (TM = 84.5 °C) and high recombinant expression yields in E. coli. Finally, when binders comprising the bivalent protein are fused to two of the three fragments of tripartite split-green fluorescent protein (GFP), target-dependent reconstitution of fluorescence occurs, thereby enabling a "mix-and-read" assay for target quantification.- Published
- 2016
- Full Text
- View/download PDF
49. Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins.
- Author
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Cruz-Teran CA, Carlin KB, Efimenko K, Genzer J, and Rao BM
- Subjects
- Adsorption, Animals, Archaeal Proteins chemistry, Archaeal Proteins genetics, Archaeal Proteins metabolism, Binding Sites, Chickens, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Immobilized Proteins metabolism, Immunoglobulins metabolism, Kinetics, Models, Molecular, Mutagenesis, Site-Directed, Mutant Proteins chemistry, Mutant Proteins genetics, Mutant Proteins metabolism, Peptide Library, Protein Binding, Silicon Dioxide, Surface Properties, Immobilized Proteins chemistry, Immobilized Proteins genetics
- Abstract
While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts.
- Published
- 2016
- Full Text
- View/download PDF
50. Identification of Epigenetic Factor Proteins Expressed in Human Embryonic Stem Cell-Derived Trophoblasts and in Human Placental Trophoblasts.
- Author
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Sarkar P, Mischler A, Randall SM, Collier TS, Dorman KF, Boggess KA, Muddiman DC, and Rao BM
- Subjects
- DNA (Cytosine-5-)-Methyltransferases genetics, Female, Gene Expression genetics, Humans, Placenta chemistry, Pregnancy, Transcription Factors genetics, Trophoblasts chemistry, Cell Differentiation, Epigenomics, Human Embryonic Stem Cells cytology, Placenta cytology, Trophoblasts cytology
- Abstract
Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development.
- Published
- 2016
- Full Text
- View/download PDF
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