Browsing by Subject "CELL"

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  • Diosdi, Akos; Hirling, Dominik; Kovacs, Maria; Toth, Timea; Harmati, Maria; Koos, Krisztian; Buzas, Krisztina; Piccinini, Filippo; Horvath, Peter (2021)
    3D multicellular spheroids quickly emerged as in vitro models because they represent the in vivo tumor environment better than standard 2D cell cultures. However, with current microscopy technologies, it is difficult to visualize individual cells in the deeper layers of 3D samples mainly because of limited light penetration and scattering. To overcome this problem several optical clearing methods have been proposed but defining the most appropriate clearing approach is an open issue due to the lack of a gold standard metric. Here, we propose a guideline for 3D light microscopy imaging to achieve single-cell resolution. The guideline includes a validation experiment focusing on five optical clearing protocols. We review and compare seven quality metrics which quantitatively characterize the imaging quality of spheroids. As a test environment, we have created and shared a large 3D dataset including approximately hundred fluorescently stained and optically cleared spheroids. Based on the results we introduce the use of a novel quality metric as a promising method to serve as a gold standard, applicable to compare optical clearing protocols, and decide on the most suitable one for a particular experiment. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.
  • Verma, Arvind; Laakso, Into; Seppanen-Laakso, Tuulikki; Huhtikangas, Aarre; Riekkola, Marja-Liisa (2007)
  • Ouwerkerk, Janneke P.; van der Ark, Kees C. H.; Davids, Mark; Claassens, Nico J.; Finestra, Teresa Robert; de Vos, Willem M.; Belzer, Clara (2016)
    Akkermansia muciniphila colonizes the mucus layer of the gastrointestinal tract, where the organism can be exposed to the oxygen that diffuses from epithelial cells. To understand how A. muciniphila is able to survive and grow at this oxic-anoxic interface, its oxygen tolerance and response and reduction capacities were studied. A. muciniphila was found to be oxygen tolerant. On top of this, under aerated conditions, A. muciniphila showed significant oxygen reduction capacities and its growth rate and yield were increased compared to those seen under strict anaerobic conditions. Transcriptome analysis revealed an initial oxygen stress response upon exposure to oxygen. Thereafter, genes related to respiration were expressed, including those coding for the cytochrome bd complex, which can function as a terminal oxidase. The functionality of A. muciniphila cytochrome bd genes was proven by successfully complementing cytochrome-deficient Escherichia coli strain ECOM4. We conclude that A. muciniphila can use oxygen when it is present at nanomolar concentrations. IMPORTANCE This article explains how Akkermansia muciniphila, previously described as a strictly anaerobic bacterium, is able to tolerate and even benefit from low levels of oxygen. Interestingly, we measured growth enhancement of A. muciniphila and changes in metabolism as a result of the oxygen exposure. In this article, we discuss similarities and differences of this oxygen-responsive mechanism with respect to those of other intestinal anaerobic isolates. Taken together, we think that these are valuable data that indicate how anaerobic intestinal colonizing bacteria can exploit low levels of oxygen present in the mucus layer and that our results have direct relevance for applicability, as addition of low oxygen concentrations could benefit the in vitro growth of certain anaerobic organisms.
  • Costa, Clarinda; Liu, Zehua; Martins, João Pedro; Correia, Alexandra; Rahikkala, Antti Tuomas Antero; Li, Wei; Seitsonen, Jani; Ruokolainen, Janne; Hirvonen, Sami-Pekka; Aguiar- Ricardo, Ana; Corvo, M. Luísa; Santos, Hélder A. (2020)
    Here, a continuous two-step glass-capillary microfluidic technique to produce a multistage oral delivery system is reported. Insulin is successfully encapsulated into liposomes, which are coated with chitosan to improve their mucoadhesion. The encapsulation in an enteric polymer offers protection from the harsh gastric conditions. Insulin permeability is enhanced across an intestinal monolayer.
  • Hautala, Laura C.; Pang, Poh-Choo; Antonopoulos, Aristotelis; Pasanen, Annukka; Lee, Cheuk-Lun; Chiu, Philip C. N.; Yeung, William S. B.; Loukovaara, Mikko; Bützow, Ralf; Haslam, Stuart M.; Dell, Anne; Koistinen, Hannu (2020)
    Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Gal beta 1-4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins. Glycodelin is a major endometrial glycoprotein. The authors analyzed glycan structures of endometrial carcinoma associated glycodelin and established a novel glycodelin-glycoform specific histochemical staining method. With this, they showed that glycodelin is differentially glycosylated in endometrial carcinoma tissue, as compared to normal endometrium, representing a neoantigen with potential clinical applications.
  • Rajendran, Jayasimman; Purhonen, Janne; Tegelberg, Saara; Smolander, Olli-Pekka; Mörgelin, Matthias; Rozman, Jan; Gailus-Durner, Valerie; Fuchs, Helmut; de Angelis, Martin Hrabe; Auvinen, Petri; Mervaala, Eero; Jacobs, Howard T.; Szibor, Marten; Fellman, Vineta; Kallijärvi, Jukka (2019)
    Alternative oxidase (AOX) is a non-mammalian enzyme that can bypass blockade of the complex III-IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)-deficient Bcs1l(p.S78G) knock-in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue-specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.
  • Blanco, Ignacio; Kuchenbaecker, Karoline; Cuadras, Daniel; Wang, Xianshu; Barrowdale, Daniel; Ruiz de Garibay, Gorka; Librado, Pablo; Sanchez-Gracia, Alejandro; Rozas, Julio; Bonifaci, Nuria; McGuffog, Lesley; Pankratz, Vernon S.; Islam, Abul; Mateo, Francesca; Berenguer, Antoni; Petit, Anna; Catala, Isabel; Brunet, Joan; Feliubadalo, Lidia; Tornero, Eva; Benitez, Javier; Osorio, Ana; Cajal, Teresa Ramon Y.; Nevanlinna, Heli; Aittomaki, Kristiina; Arun, Banu K.; Toland, Amanda E.; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; Greene, Mark H.; Mai, Phuong L.; Nussbaum, Robert L.; Andrulis, Irene L.; Domchek, Susan M.; Nathanson, Katherine L.; Rebbeck, Timothy R.; Barkardottir, Rosa B.; Jakubowska, Anna; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Claes, Kathleen; Van Maerken, Tom; Diez, Orland; Hansen, Thomas V.; Jonson, Lars; Gerdes, Anne-Marie; Ejlertsen, Bent; de la Hoya, Miguel; Caldes, Trinidad; Dunning, Alison M.; Oliver, Clare; Fineberg, Elena; Cook, Margaret; Peock, Susan; McCann, Emma; Murray, Alex; Jacobs, Chris; Pichert, Gabriella; Lalloo, Fiona; Chu, Carol; Dorkins, Huw; Paterson, Joan; Ong, Kai-Ren; Teixeira, Manuel R.; Teixeira,; Hogervorst, Frans B. L.; van der Hout, Annemarie H.; Seynaeve, Caroline; van der Luijt, Rob B.; Ligtenberg, Marjolijn J. L.; Devilee, Peter; Wijnen, Juul T.; Rookus, Matti A.; Meijers-Heijboer, Hanne E. J.; Blok, Marinus J.; van den Ouweland, Ans M. W.; Aalfs, Cora M.; Rodriguez, Gustavo C.; Phillips, Kelly-Anne A.; Piedmonte, Marion; Nerenstone, Stacy R.; Bae-Jump, Victoria L.; O'Malley, David M.; Ratner, Elena S.; Schmutzler, Rita K.; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hansjoerg J.; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Jensen, Uffe Birk; Thomassen, Mads; Kruse, Torben A.; Foretova, Lenka; Peterlongo, Paolo; Bernard, Loris; Peissel, Bernard; Scuvera, Giulietta; Manoukian, Siranoush; Radice, Paolo; Ottini, Laura; Montagna, Marco; Agata, Simona; Maugard, Christine; Simard, Jacques; Soucy, Penny; Berger, Andreas; Fink-Retter, Anneliese; Singer, Christian F.; Rappaport, Christine; Geschwantler-Kaulich, Daphne; Tea, Muy-Kheng; Pfeiler, Georg; John, Esther M.; Miron, Alex; Neuhausen, Susan L.; Terry, Mary Beth; Chung, Wendy K.; Daly, Mary B.; Goldgar, David E.; Janavicius, Ramunas; Dorfling, Cecilia M.; van Rensburg, Elisabeth J.; Fostira, Florentia; Konstantopoulou, Irene; Garber, Judy; Godwin, Andrew K.; Olah, Edith; Narod, Steven A.; Rennert, Gad; Paluch, Shani Shimon; Laitman, Yael; Friedman, Eitan; Liljegren, Annelie; Rantala, Johanna; Stenmark-Askmalm, Marie; Loman, Niklas; Imyanitov, Evgeny N.; Hamann, Ute; Spurdle, Amanda B.; Healey, Sue; Weitzel, Jeffrey N.; Herzog, Josef; Margileth, David; Gorrini, Chiara; Esteller, Manel; Gomez, Antonio; Sayols, Sergi; Vidal, Enrique; Heyn, Holger; Stoppa-Lyonnet, Dominique; Leone, Melanie; Barjhoux, Laure; Fassy-Colcombet, Marion; de Pauw, Antoine; Lasset, Christine; Ferrer, Sandra Fert; Castera, Laurent; Berthet, Pascaline; Cornelis, Francois; Bignon, Yves-Jean; Damiola, Francesca; Mazoyer, Sylvie; Sinilnikova, Olga M.; Maxwell, Christopher A.; Vijai, Joseph; Robson, Mark; Kauff, Noah; Corines, Marina J.; Villano, Danylko; Cunningham, Julie; Lee, Adam; Lindor, Noralane; Lazaro, Conxi; Easton, Douglas F.; Offit, Kenneth; Chenevix-Trench, Georgia; Couch, Fergus J.; Antoniou, Antonis C.; Angel Pujana, Miguel; BCFR; SWE-BRCA; KConFab Investigators; GEMO (2015)
    While interplay between BRCA1 and AURKA-RHAMM-TPX2-TUBG1 regulates mammary epithelial polarization, common genetic variation in HMMR (gene product RHAMM) may be associated with risk of breast cancer in BRCA1 mutation carriers. Following on these observations, we further assessed the link between the AURKA-HMMR-TPX2-TUBG1 functional module and risk of breast cancer in BRCA1 or BRCA2 mutation carriers. Forty-one single nucleotide polymorphisms (SNPs) were genotyped in 15,252 BRCA1 and 8,211 BRCA2 mutation carriers and subsequently analyzed using a retrospective likelihood approach. The association of HMMR rs299290 with breast cancer risk in BRCA1 mutation carriers was confirmed: per-allele hazard ratio (HR) = 1.10, 95% confidence interval (CI) 1.04 - 1.15, p = 1.9 x 10(-4) (false discovery rate (FDR)-adjusted p = 0.043). Variation in CSTF1, located next to AURKA, was also found to be associated with breast cancer risk in BRCA2 mutation carriers: rs2426618 per-allele HR = 1.10, 95% CI 1.03 - 1.16, p = 0.005 (FDR-adjusted p = 0.045). Assessment of pairwise interactions provided suggestions (FDR-adjusted p(interaction) values > 0.05) for deviations from the multiplicative model for rs299290 and CSTF1 rs6064391, and rs299290 and TUBG1 rs11649877 in both BRCA1 and BRCA2 mutation carriers. Following these suggestions, the expression of HMMR and AURKA or TUBG1 in sporadic breast tumors was found to potentially interact, influencing patients' survival. Together, the results of this study support the hypothesis of a causative link between altered function of AURKA-HMMR-TPX2-TUBG1 and breast carcinogenesis in BRCA1/2 mutation carriers.
  • Gorvin, C.M.; Hannan, F.M.; Cranston, T.; Valta, Helena; Mäkitie, Outi; Schalin-Jäntti, Camilla; Thakker, R.V. (2018)
    G-protein subunit -11 (G(11)) couples the calcium-sensing receptor (CaSR) to phospholipase C (PLC)-mediated intracellular calcium (Ca-i(2+)) and mitogen-activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss-of-function G(11) mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous G(11) germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild-type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of G(11), which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC-mediated signaling. In vitro studies involving transient transfection of WT and mutant G(11) proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 G(11) protein to impair CaSR-mediated Ca-i(2+) and extracellular signal-regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the G(11) cleft region also impaired signaling by PLC. The loss-of-function associated with the Ser220 G(11) mutant was rectified by treatment of cells with cinacalcet, which is a CaSR-positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser G(11) mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel G(11) germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the G(11) hydrophobic cleft region for CaSR-mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss-of-function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. (c) 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
  • Koivuniemi, Raili; Hakkarainen, Tiina; Kiiskinen, Jasmi; Kosonen, Mika; Vuola, Jyrki; Valtonen, Jussi; Luukko, Kari; Kavola, Heli; Yliperttula, Marjo (2020)
    Objective: Skin graft donor site management is a concern particularly for elderly patients and patients with poor wound healing competence, and also because donor sites are a source of pain and discomfort. Although different types of dressings exist, there is no consensus regarding optimal dressing type on donor site care to promote healing, reduce pain, and improve patients' comfort. Approach: This prospective, single-center clinical trial evaluated the performance of nanofibrillar cellulose (NFC) wound dressing (FibDex (R) by UPM-Kymmene Corporation) for treatment of donor sites compared with a polylactide-based copolymer dressing. The study enrolled 24 patients requiring skin grafting with mean age of 49 +/- 18. The primary outcome measure was wound healing time. Secondary outcomes, the epithelialization, subjective pain, the scar appearance assessed using the Patient and Observer Scar Assessment Scale (POSAS), and skin elasticity and transepidermal water loss (TEWL), were evaluated at 1 and 6 months postoperatively. Results: No statistically significant differences were observed between NFC and copolymer dressings regarding wound healing time, epithelialization, experience of pain, or TEWL. Significant differences were observed in the POSAS results for thickness and vascularity in the Observer score, in the favor of NFC over copolymer dressing. Moreover, skin elasticity was significantly improved with NFC dressing in terms of viscoelasticity and elastic modulus at 1 month postoperatively. Innovation: NFC dressing is a new, green sustainable product for wound treatment without animal or human-origin components. Conclusion: NFC dressing provides efficient wound healing at skin graft donor sites and is comparable or even preferable compared with the copolymer dressing.
  • Bauleth-Ramos, Tomás; Feijão, Tália; Gonçalves, André; Shahbazi, Mohammad-Ali; Liu, Zehua; Barrias, Cristina; Oliveira, Maria Jose; L. Granja, Pedro; Santos, Hélder A.; Sarmento, Bruno (2020)
    Colorectal cancer (CRC) is the third most common and the second deadliest type of cancer worldwide, urging the development of more comprehensive models and of more efficient treatments. Although the combination of nanotechnology with chemo- and immuno-therapy has represented a promising treatment approach, its translation to the clinic has been hampered by the absence of cellular models that can provide reliable and predictive knowledge about the in vivo efficiency of the formulation. Herein, a 3D model based on CRC multicellular tumor spheroids (MCTS) model was developed by combining epithelial colon cancer cells (HCT116), human intestinal fibroblasts and monocytes. The developed MCTS 3D model mimicked several tumor features with cells undergoing spatial organization and producing extracellular matrix, forming a mass of tissue with a necrotic core. Furthermore, monocytes were differentiated into macrophages with an anti-inflammatory, pro-tumor M2-like phenotype. For a combined chemoimmunotherapy effect, spermine-modified acetalated dextran nanoparticles (NPs) loaded with the chemotherapeutic Nutlin-3a (Nut3a) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were produced and tested in 2D cultures and in the MCTS 3D model. NPs were successfully taken-up by the cells in 2D, but in a significant less extent in the 3D model. However, these NPs were able to induce an anti-proliferative effect both in the 2D and in the 3D models. Moreover, Nut3a was able to partially shift the polarization of the macrophages present in the MCTS 3D model towards an anti-tumor M1-like phenotype. Overall, the developed MCTS 3D model showed to recapitulate key features of tumors, while representing a valuable model to assess the effect of combinatorial nano-therapeutic strategies in CRC. In addition, the developed NPs could represent a promising approach for CRC treatment.
  • Menden, Michael P.; Wang, Dennis; Mason, Mike J.; Szalai, Bence; Bulusu, Krishna C.; Guan, Yuanfang; Yu, Thomas; Kang, Jaewoo; Jeon, Minji; Wolfinger, Russ; Nguyen, Tin; Zaslavskiy, Mikhail; Abante, Jordi; Abecassis, Barbara Schmitz; Aben, Nanne; Aghamirzaie, Delasa; Aittokallio, Tero; Akhtari, Farida S.; Al-lazikani, Bissan; Alam, Tanvir; Allam, Amin; Allen, Chad; de Almeida, Mariana Pelicano; Altarawy, Doaa; Alves, Vinicius; Amadoz, Alicia; Anchang, Benedict; Antolin, Albert A.; Ash, Jeremy R.; Aznar, Victoria Romeo; Ba-alawi, Wail; Bagheri, Moeen; Bajic, Vladimir; Ball, Gordon; Ballester, Pedro J.; Baptista, Delora; Bare, Christopher; Bateson, Mathilde; Bender, Andreas; Bertrand, Denis; Wijayawardena, Bhagya; Boroevich, Keith A.; Bosdriesz, Evert; Bougouffa, Salim; Bounova, Gergana; Brouwer, Thomas; Bryant, Barbara; Calaza, Manuel; Calderone, Alberto; Calza, Stefano; Capuzzi, Stephen; Carbonell-Caballero, Jose; Carlin, Daniel; Carter, Hannah; Castagnoli, Luisa; Celebi, Remzi; Cesareni, Gianni; Chang, Hyeokyoon; Chen, Guocai; Chen, Haoran; Chen, Huiyuan; Cheng, Lijun; Chernomoretz, Ariel; Chicco, Davide; Cho, Kwang-Hyun; Cho, Sunghwan; Choi, Daeseon; Choi, Jaejoon; Choi, Kwanghun; Choi, Minsoo; Cock, Martine De; Coker, Elizabeth; Cortes-Ciriano, Isidro; Cserzö, Miklós; Cubuk, Cankut; Curtis, Christina; Daele, Dries Van; Dang, Cuong C.; Dijkstra, Tjeerd; Dopazo, Joaquin; Draghici, Sorin; Drosou, Anastasios; Dumontier, Michel; Ehrhart, Friederike; Eid, Fatma-Elzahraa; ElHefnawi, Mahmoud; Elmarakeby, Haitham; van Engelen, Bo; Engin, Hatice Billur; de Esch, Iwan; Evelo, Chris; Falcao, Andre O.; Farag, Sherif; Fernandez-Lozano, Carlos; Fisch, Kathleen; Flobak, Asmund; Fornari, Chiara; Foroushani, Amir B. K.; Fotso, Donatien Chedom; Fourches, Denis; Friend, Stephen; Frigessi, Arnoldo; Gao, Feng; Gao, Xiaoting; Gerold, Jeffrey M.; Gestraud, Pierre; Ghosh, Samik; Gillberg, Jussi; Godoy-Lorite, Antonia; Godynyuk, Lizzy; Godzik, Adam; Goldenberg, Anna; Gomez-Cabrero, David; Gonen, Mehmet; de Graaf, Chris; Gray, Harry; Grechkin, Maxim; Guimera, Roger; Guney, Emre; Haibe-Kains, Benjamin; Han, Younghyun; Hase, Takeshi; He, Di; He, Liye; Heath, Lenwood S.; Hellton, Kristoffer H.; Helmer-Citterich, Manuela; Hidalgo, Marta R.; Hidru, Daniel; Hill, Steven M.; Hochreiter, Sepp; Hong, Seungpyo; Hovig, Eivind; Hsueh, Ya-Chih; Hu, Zhiyuan; Huang, Justin K.; Huang, R. Stephanie; Hunyady, László; Hwang, Jinseub; Hwang, Tae Hyun; Hwang, Woochang; Hwang, Yongdeuk; Isayev, Olexandr; Don’t Walk, Oliver Bear; Jack, John; Jahandideh, Samad; Ji, Jiadong; Jo, Yousang; Kamola, Piotr J.; Kanev, Georgi K.; Karacosta, Loukia; Karimi, Mostafa; Kaski, Samuel; Kazanov, Marat; Khamis, Abdullah M.; Khan, Suleiman Ali; Kiani, Narsis A.; Kim, Allen; Kim, Jinhan; Kim, Juntae; Kim, Kiseong; Kim, Kyung; Kim, Sunkyu; Kim, Yongsoo; Kim, Yunseong; Kirk, Paul D. W.; Kitano, Hiroaki; Klambauer, Gunter; Knowles, David; Ko, Melissa; Kohn-Luque, Alvaro; Kooistra, Albert J.; Kuenemann, Melaine A.; Kuiper, Martin; Kurz, Christoph; Kwon, Mijin; van Laarhoven, Twan; Laegreid, Astrid; Lederer, Simone; Lee, Heewon; Lee, Jeon; Lee, Yun Woo; Lepp_aho, Eemeli; Lewis, Richard; Li, Jing; Li, Lang; Liley, James; Lim, Weng Khong; Lin, Chieh; Liu, Yiyi; Lopez, Yosvany; Low, Joshua; Lysenko, Artem; Machado, Daniel; Madhukar, Neel; Maeyer, Dries De; Malpartida, Ana Belen; Mamitsuka, Hiroshi; Marabita, Francesco; Marchal, Kathleen; Marttinen, Pekka; Mason, Daniel; Mazaheri, Alireza; Mehmood, Arfa; Mehreen, Ali; Michaut, Magali; Miller, Ryan A.; Mitsopoulos, Costas; Modos, Dezso; Moerbeke, Marijke Van; Moo, Keagan; Motsinger-Reif, Alison; Movva, Rajiv; Muraru, Sebastian; Muratov, Eugene; Mushthofa, Mushthofa; Nagarajan, Niranjan; Nakken, Sigve; Nath, Aritro; Neuvial, Pierre; Newton, Richard; Ning, Zheng; Niz, Carlos De; Oliva, Baldo; Olsen, Catharina; Palmeri, Antonio; Panesar, Bhawan; Papadopoulos, Stavros; Park, Jaesub; Park, Seonyeong; Park, Sungjoon; Pawitan, Yudi; Peluso, Daniele; Pendyala, Sriram; Peng, Jian; Perfetto, Livia; Pirro, Stefano; Plevritis, Sylvia; Politi, Regina; Poon, Hoifung; Porta, Eduard; Prellner, Isak; Preuer, Kristina; Pujana, Miguel Angel; Ramnarine, Ricardo; Reid, John E.; Reyal, Fabien; Richardson, Sylvia; Ricketts, Camir; Rieswijk, Linda; Rocha, Miguel; Rodriguez-Gonzalvez, Carmen; Roell, Kyle; Rotroff, Daniel; de Ruiter, Julian R.; Rukawa, Ploy; Sadacca, Benjamin; Safikhani, Zhaleh; Safitri, Fita; Sales-Pardo, Marta; Sauer, Sebastian; Schlichting, Moritz; Seoane, Jose A.; Serra, Jordi; Shang, Ming-Mei; Sharma, Alok; Sharma, Hari; Shen, Yang; Shiga, Motoki; Shin, Moonshik; Shkedy, Ziv; Shopsowitz, Kevin; Sinai, Sam; Skola, Dylan; Smirnov, Petr; Soerensen, Izel Fourie; Soerensen, Peter; Song, Je-Hoon; Song, Sang Ok; Soufan, Othman; Spitzmueller, Andreas; Steipe, Boris; Suphavilai, Chayaporn; Tamayo, Sergio Pulido; Tamborero, David; Tang, Jing; Tanoli, Zia-ur-Rehman; Tarres-Deulofeu, Marc; Tegner, Jesper; Thommesen, Liv; Tonekaboni, Seyed Ali Madani; Tran, Hong; Troyer, Ewoud De; Truong, Amy; Tsunoda, Tatsuhiko; Turu, Gábor; Tzeng, Guang-Yo; Verbeke, Lieven; Videla, Santiago; Consortium, AstraZeneca-Sanger Drug Combination DREAM (2019)
    The effectiveness of most cancer targeted therapies is short-lived. Tumors often develop resistance that might be overcome with drug combinations. However, the number of possible combinations is vast, necessitating data-driven approaches to find optimal patient-specific treatments. Here we report AstraZeneca’s large drug combination dataset, consisting of 11,576 experiments from 910 combinations across 85 molecularly characterized cancer cell lines, and results of a DREAM Challenge to evaluate computational strategies for predicting synergistic drug pairs and biomarkers. 160 teams participated to provide a comprehensive methodological development and benchmarking. Winning methods incorporate prior knowledge of drug-target interactions. Synergy is predicted with an accuracy matching biological replicates for >60% of combinations. However, 20% of drug combinations are poorly predicted by all methods. Genomic rationale for synergy predictions are identified, including ADAM17 inhibitor antagonism when combined with PIK3CB/D inhibition contrasting to synergy when combined with other PI3K-pathway inhibitors in PIK3CA mutant cells.
  • Lukovac, Sabina; Belzer, Clara; Pellis, Linette; Keijser, Bart J.; de Vos, Willem M.; Montijn, Roy C.; Roeselers, Guus (2014)
  • Malyutina, Alina; Majumder, Muntasir Mamun; Wang, Wenyu; Pessia, Alberto; Heckman, Caroline A.; Tang, Jing (2019)
    High-throughput drug screening has facilitated the discovery of drug combinations in cancer. Many existing studies adopted a full matrix design, aiming for the characterization of drug pair effects for cancer cells. However, the full matrix design may be suboptimal as it requires a drug pair to be combined at multiple concentrations in a full factorial manner. Furthermore, many of the computational tools assess only the synergy but not the sensitivity of drug combinations, which might lead to false positive discoveries. We proposed a novel cross design to enable a more cost-effective and simultaneous testing of drug combination sensitivity and synergy. We developed a drug combination sensitivity score (CSS) to determine the sensitivity of a drug pair, and showed that the CSS is highly reproducible between the replicates and thus supported its usage as a robust metric. We further showed that CSS can be predicted using machine learning approaches which determined the top pharmaco-features to cluster cancer cell lines based on their drug combination sensitivity profiles. To assess the degree of drug interactions using the cross design, we developed an S synergy score based on the difference between the drug combination and the single drug dose-response curves. We showed that the S score is able to detect true synergistic and antagonistic drug combinations at an accuracy level comparable to that using the full matrix design. Taken together, we showed that the cross design coupled with the CSS sensitivity and S synergy scoring methods may provide a robust and accurate characterization of both drug combination sensitivity and synergy levels, with minimal experimental materials required. Our experimental-computational approach could be utilized as an efficient pipeline for improving the discovery rate in high-throughput drug combination screening, particularly for primary patient samples which are difficult to obtain.
  • Frismantas, Viktoras; Dobay, Maria Pamela; Rinaldi, Anna; Tchinda, Joelle; Dunn, Samuel H.; Kunz, Joachim; Richter-Pechanska, Paulina; Marovca, Blerim; Pail, Orrin; Jenni, Silvia; Diaz-Flores, Ernesto; Chang, Bill H.; Brown, Timothy J.; Collins, Robert H.; Uhrig, Sebastian; Balasubramanian, Gnana P.; Bandapalli, Obul R.; Higi, Salome; Eugster, Sabrina; Voegeli, Pamela; Delorenzi, Mauro; Cario, Gunnar; Loh, Mignon L.; Schrappe, Martin; Stanulla, Martin; Kulozik, Andreas E.; Muckenthaler, Martina U.; Saha, Vaskar; Irving, Julie A.; Meisel, Roland; Radimerski, Thomas; Von Stackelberg, Arend; Eckert, Cornelia; Tyner, Jeffrey W.; Horvath, Peter; Bornhauser, Beat C.; Bourquin, Jean-Pierre (2017)
    Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.
  • Pemmari, Toini; Ivanova, Larisa; May, Ulrike; Lingasamy, Prakash; Tobi, Allan; Pasternack, Anja; Prince, Stuart; Ritvos, Olli; Makkapati, Shreya; Teesalu, Tambet; Cairo, Mitchell S.; Järvinen, Tero A. H.; Liao, Yanling (2020)
    Systemic skin-selective therapeutics would be a major advancement in the treatment of diseases affecting the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene and manifests in transforming growth factor-beta (TGF-beta)-driven fibrosis and malignant transformation. Homing peptides containing a C-terminal R/KXXR/K motif (C-end rule [CendR] sequence) activate an extravasation and tissue penetration pathway for tumor-specific drug delivery. We have previously described a homing peptide CRKDKC (CRK) that contains a cryptic CendR motif and homes to angiogenic blood vessels in wounds and tumors, but it cannot penetrate cells or tissues. In this study, we demonstrate that removal of the cysteine from CRK to expose the CendR sequence confers the peptide novel ability to home to normal skin. Fusion of the truncated CRK (tCRK) peptide to the C terminus of an extracellular matrix protein de-corin (DCN), a natural TGF-beta inhibitor, resulted in a skin-homing therapeutic molecule (DCN-tCRK). Systemic DCN-tCRK administration in RDEB mice led to inhibition of TGF-beta signaling in the skin and significant improvement in the survival of RDEB mice. These results suggest that DCN-tCRK has the potential to be utilized as a novel therapeutic compound for the treatment of dermatological diseases such as RDEB.
  • Weissbrod, Omer; Hormozdiari, Farhad; Benner, Christian; Cui, Ran; Ulirsch, Jacob; Gazal, Steven; Schoech, Armin P.; van de Geijn, Bryce; Reshef, Yakir; Marquez-Luna, Carla; O'Connor, Luke; Pirinen, Matti; Finucane, Hilary K.; Price, Alkes L. (2020)
    Fine-mapping aims to identify causal variants impacting complex traits. We propose PolyFun, a computationally scalable framework to improve fine-mapping accuracy by leveraging functional annotations across the entire genome-not just genome-wide-significant loci-to specify prior probabilities for fine-mapping methods such as SuSiE or FINEMAP. In simulations, PolyFun + SuSiE and PolyFun + FINEMAP were well calibrated and identified >20% more variants with a posterior causal probability >0.95 than identified in their nonfunctionally informed counterparts. In analyses of 49 UK Biobank traits (average n = 318,000), PolyFun + SuSiE identified 3,025 fine-mapped variant-trait pairs with posterior causal probability >0.95, a >32% improvement versus SuSiE. We used posterior mean per-SNP heritabilities from PolyFun + SuSiE to perform polygenic localization, constructing minimal sets of common SNPs causally explaining 50% of common SNP heritability; these sets ranged in size from 28 (hair color) to 3,400 (height) to 2 million (number of children). In conclusion, PolyFun prioritizes variants for functional follow-up and provides insights into complex trait architectures. PolyFun is a computationally scalable framework for functionally informed fine-mapping that makes full use of genome-wide data. It prioritizes more variants than previous methods when applied to 49 complex traits from UK Biobank.
  • Kestilä, I.; Thevenot, J.; Finnilä, M. A.; Karhula, S. S.; Hadjab, I.; Kauppinen, S.; Garon, M.; Quenneville, E.; Haapea, M.; Rieppo, L.; Pritzker, K. P.; Buschmann, M. D.; Nieminen, H. J.; Saarakkala, S. (2018)
    Objective: The aims of this study were: to 1) develop a novel sample processing protocol to visualize human articular cartilage (AC) chondrons using micro-computed tomography (mu CT), 2) develop and validate an algorithm to quantify the chondron morphology in 3D, and 3) compare the differences in chondron morphology between intact and osteoarthritic AC. Method: The developed protocol is based on the dehydration of samples with hexamethyldisilazane (HMDS), followed by imaging with a desktop mCT. Chondron density and depth, as well as volume and sphericity, were calculated in 3D with a custom-made and validated algorithm employing semiautomatic chondron selection and segmentation. The quantitative parameters were analyzed at three AC depth zones (zone 1: 0-10%; zone 2: 10-40%; zone 3: 40-100%) and grouped by the OARSI histological grades (OARSI grades 0-1.0, n = 6; OARSI grades 3.0-3.5, n = 6). Results: After semi-automatic chondron selection and segmentation, 1510 chondrons were approved for 3D morphometric analyses. The chondrons especially in the deeper tissue (zones 2 and 3) were significantly larger (P Conclusion: We have developed a novel sample processing protocol for chondron imaging in 3D, as well as a high-throughput algorithm to semi-automatically quantify chondron/ chondrocyte 3D morphology in AC. Our results also suggest that 3D chondron morphology is affected by the progression of osteoarthritis (OA). (c) 2018 The Authors. Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
  • Sokka, Iris Katariina; Ekholm, Filip S.; Johansson, Mikael P. (2019)
    Monomethyl auristatin E and monomethyl auristatin F are widely used cytotoxic agents in antibody-drug conjugates (ADCs), a group of promising cancer drugs. The ADCs specifically target cancer cells, releasing the auristatins inside, which results in the prevention of mitosis. The auristatins suffer from a potentially serious flaw, however. In solution, the molecules exist in an equal mixture of two conformers, cis and trans. Only the trans-isomer is biologically active and the isomerization process, i.e., the conversion of cis to trans is slow. This significantly diminishes the efficiency of the drugs and their corresponding ADCs, and perhaps more importantly, raises concerns over drug safety. The potency of the auristatins would be enhanced by decreasing the amount of the biologically inactive isomer, either by stabilizing the transisomer or destabilizing the cis-isomer. Here, we follow the computer-aided design strategy of shifting the conformational equilibrium and employ high-level quantum chemical modeling to identify promising candidates for improved auristatins. Coupled cluster calculations predict that a simple halogenation in the norephedrine/phenylalanine residues shifts the isomer equilibrium almost completely toward the active trans-conformation, due to enhanced intramolecular interactions specific to the active isomer.
  • Hurler, Julia; Sorensen, Karen K.; Fallarero, Adyary; Vuorela, Pia; Skalko-Basnet, Natasa (2013)
  • Heybrock, Saskia; Kanerva, Kristiina; Meng, Ying; Ing, Chris; Liang, Anna; Xiong, Zi-Jian; Weng, Xialian; Kim, Young Ah; Collins, Richard; Trimble, William; Pomes, Regis; Prive, Gilbert G.; Annaert, Wim; Schwake, Michael; Heeren, Joerg; Lullmann-Rauch, Renate; Grinstein, Sergio; Ikonen, Elina; Saftig, Paul; Neculai, Dante (2019)
    The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol - like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.