2024-03-29T01:43:17Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/2813532019-08-30T11:30:32Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2013-04-15T14:10:55Z
urn:hdl:10033/281353
Cellular immune reactions in the lung.
Hasenberg, Mike
Stegemann-Koniszewski, Sabine
Gunzer, Matthias
Institute of Experimental Immunology and Imaging, University of Duisburg/Essen, University Hospital, Essen, Germany.
The lung constantly interacts with the environment through thousands of liters of air that are inhaled daily. This continually transports toxic chemicals and particles or pathogenic microorganisms deep into the respiratory system, posing a challenge to physicochemical barriers and the local immune system. Thus, complex structures and mechanisms have evolved to recognize and fend off environmental dangers while at the same time allowing efficient gas exchange. Here we review our current knowledge regarding cellular mechanisms of the immune system in context with the highly specialized anatomical features of the airways and especially the alveolar compartment. The focus is on fungal and viral infections, merging anatomical aspects well known to pulmonologists with fundamental immunological concepts. We discuss the specialized morphological constraints of immune cells compressed under a continuous layer of the surfactant lining within alveoli as well as the importance of functional polarization of respiratory tract epithelia. Furthermore, we summarize the different types of innate and adaptive immune cells and their relative contribution to lung homeostasis with respect to localization. Finally, we provide a list of currently unresolved questions with high relevance for the field that might serve as food for thought regarding future research directions.
2013-04-15T14:10:55Z
2013-04-15T14:10:55Z
2013-01
Article
Cellular immune reactions in the lung. 2013, 251 (1):189-214 Immunol. Rev.
1600-065X
23278750
10.1111/imr.12020
http://hdl.handle.net/10033/281353
Immunological reviews
en
Archived with thanks to Immunological reviews
oai:repository.helmholtz-hzi.de:10033/3019042019-08-30T11:36:33Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2013-09-19T12:22:33Z
urn:hdl:10033/301904
The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems.
Chylinski, Krzysztof
Le Rhun, Anaïs
Charpentier, Emmanuelle
The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå, Sweden.
CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool.
2013-09-19T12:22:33Z
2013-09-19T12:22:33Z
2013-05
Article
The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. 2013, 10 (5):726-37 RNA Biol
1555-8584
23563642
10.4161/rna.24321
http://hdl.handle.net/10033/301904
RNA biology
en
Archived with thanks to RNA biology
oai:repository.helmholtz-hzi.de:10033/3064232019-08-30T11:24:31Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2013-12-06T10:19:05Z
urn:hdl:10033/306423
Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.
Fonfara, Ines
Le Rhun, Anaïs
Chylinski, Krzysztof
Makarova, Kira S
Lécrivain, Anne-Laure
Bzdrenga, Janek
Koonin, Eugene V
Charpentier, Emmanuelle
The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå S-90187, Sweden, Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig D-38124, Germany, Deptartment of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Vienna A-1030, Austria, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA and Hannover Medical School, Hannover D-30625, Germany.
The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.
2013-12-06T10:19:05Z
2013-12-06T10:19:05Z
2013-11-22
Article
Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. 2013: Nucleic Acids Res.
1362-4962
24270795
10.1093/nar/gkt1074
http://hdl.handle.net/10033/306423
Nucleic acids research
Archived with thanks to Nucleic acids research
oai:repository.helmholtz-hzi.de:10033/3443832019-08-30T11:37:00Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2015-02-12T09:06:19Z
urn:hdl:10033/344383
Low-density lipoprotein receptor-related protein-1 mediates endocytic clearance of tissue inhibitor of metalloproteinases-1 and promotes its cytokine-like activities.
Thevenard, Jessica
Verzeaux, Laurie
Devy, Jerôme
Etique, Nicolas
Jeanne, Albin
Schneider, Christophe
Hachet, Cathy
Ferracci, Géraldine
David, Marion
Martiny, Laurent
Charpentier, Emmanuelle
Khrestchatisky, Michel
Rivera, Santiago
Dedieu, Stéphane
Emonard, Hervé
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the extracellular matrix turnover by inhibiting the proteolytic activity of matrix metalloproteinases (MMPs). TIMP-1 also displays MMP-independent activities that influence the behavior of various cell types including neuronal plasticity, but the underlying molecular mechanisms remain mostly unknown. The trans-membrane receptor low-density lipoprotein receptor-related protein-1 (LRP-1) consists of a large extracellular chain with distinct ligand-binding domains that interact with numerous ligands including TIMP-2 and TIMP-3 and a short transmembrane chain with intracellular motifs that allow endocytosis and confer signaling properties to LRP-1. We addressed TIMP-1 interaction with recombinant ligand-binding domains of LRP-1 expressed by CHO cells for endocytosis study, or linked onto sensor chips for surface plasmon resonance analysis. Primary cortical neurons bound and internalized endogenous TIMP-1 through a mechanism mediated by LRP-1. This resulted in inhibition of neurite outgrowth and increased growth cone volume. Using a mutated inactive TIMP-1 variant we showed that TIMP-1 effect on neurone morphology was independent of its MMP inhibitory activity. We conclude that TIMP-1 is a new ligand of LRP-1 and we highlight a new example of its MMP-independent, cytokine-like functions.
2015-02-12T09:06:19Z
2015-02-12T09:06:19Z
2014
Article
Low-density lipoprotein receptor-related protein-1 mediates endocytic clearance of tissue inhibitor of metalloproteinases-1 and promotes its cytokine-like activities. 2014, 9 (7):e103839 PLoS ONE
1932-6203
25075518
10.1371/journal.pone.0103839
http://hdl.handle.net/10033/344383
PloS one
en
oai:repository.helmholtz-hzi.de:10033/5584162019-08-30T11:26:42Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2015-06-22T13:36:21Z
urn:hdl:10033/558416
Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity.
Charpentier, Emmanuelle
Richter, Hagen
van der Oost, John
White, Malcolm F
Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany.
CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.
2015-06-22T13:36:21Z
2015-06-22T13:36:21Z
2015-05
Article
Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity. 2015, 39 (3):428-441 FEMS Microbiol. Rev.
1574-6976
25994611
10.1093/femsre/fuv023
http://hdl.handle.net/10033/558416
FEMS microbiology reviews
oai:repository.helmholtz-hzi.de:10033/6060262019-08-30T11:32:16Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2016-04-20T08:28:53Z
urn:hdl:10033/606026
RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.
Le Rhun, Anaïs
Beer, Yan Yan
Reimegård, Johan
Chylinski, Krzysztof
Charpentier, Emmanuelle
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.
2016-04-20T08:28:53Z
2016-04-20T08:28:53Z
2016-02
Article
RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes. 2016, 13 (2):177-95 RNA Biol
1555-8584
26580233
10.1080/15476286.2015.1110674
http://hdl.handle.net/10033/606026
RNA biology
en
oai:repository.helmholtz-hzi.de:10033/6157802019-08-30T11:33:26Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2016-07-08T08:38:31Z
urn:hdl:10033/615780
CRISPR-Cas9: how research on a bacterial RNA-guided mechanism opened new perspectives in biotechnology and biomedicine.
Charpentier, Emmanuelle
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
2016-07-08T08:38:31Z
2016-07-08T08:38:31Z
2015-04
Article
CRISPR-Cas9: how research on a bacterial RNA-guided mechanism opened new perspectives in biotechnology and biomedicine. 2015, 7 (4):363-5 EMBO Mol Med
1757-4684
25796552
10.15252/emmm.201504847
http://hdl.handle.net/10033/615780
EMBO molecular medicine
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6201342019-08-30T11:33:30Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2016-09-14T08:13:12Z
urn:hdl:10033/620134
Adaptation in CRISPR-Cas Systems.
Sternberg, Samuel H
Richter, Hagen
Charpentier, Emmanuelle
Qimron, Udi
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity.
2016-09-14T08:13:12Z
2016-09-14T08:13:12Z
2016-03-17
Article
Adaptation in CRISPR-Cas Systems. 2016, 61 (6):797-808 Mol. Cell
1097-4164
26949040
10.1016/j.molcel.2016.01.030
http://hdl.handle.net/10033/620134
Molecular cell
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6205572019-08-30T11:25:43Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2016-10-19T11:01:17Z
urn:hdl:10033/620557
Toward Whole-Transcriptome Editing with CRISPR-Cas9.
Heckl, Dirk
Charpentier, Emmanuelle
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Targeted regulation of gene expression holds huge promise for biomedical research. In a series of recent publications (Gilbert et al., 2014; Konermann et al., 2015; Zalatan et al., 2015), sophisticated, multiplex-compatible transcriptional activator systems based on the CRISPR-Cas9 technology and genome-scale libraries advance the field toward whole-transcriptome control.
2016-10-19T11:01:17Z
2016-10-19T11:01:17Z
2015-05-21
Article
Toward Whole-Transcriptome Editing with CRISPR-Cas9. 2015, 58 (4):560-2 Mol. Cell
1097-4164
26000839
10.1016/j.molcel.2015.05.016
http://hdl.handle.net/10033/620557
Molecular cell
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6210602019-08-30T11:36:05Zcom_10033_264694com_10033_6815com_10033_6814com_10033_620644com_10033_621723col_10033_621724col_10033_264695col_10033_620650
2017-08-17T10:02:01Z
urn:hdl:10033/621060
A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum.
Fabiani, Florian D
Renault, Thibaud T
Peters, Britta
Dietsche, Tobias
Gálvez, Eric J C
Guse, Alina
Freier, Karen
Charpentier, Emmanuelle
Strowig, Till
Franz-Wachtel, Mirita
Macek, Boris
Wagner, Samuel
Hensel, Michael
Erhardt, Marc
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP-FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.
2017-08-17T10:02:01Z
2017-08-17T10:02:01Z
2017-08
Article
A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum. 2017, 15 (8):e2002267 PLoS Biol.
1545-7885
28771474
10.1371/journal.pbio.2002267
http://hdl.handle.net/10033/621060
PLoS biology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6210912019-08-30T11:32:41Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2017-09-05T11:36:06Z
urn:hdl:10033/621091
Identification of endoribonuclease specific cleavage positions reveals novel targets of RNase III in Streptococcus pyogenes.
Le Rhun, Anaïs
Lécrivain, Anne-Laure
Reimegård, Johan
Proux-Wéra, Estelle
Broglia, Laura
Della Beffa, Cristina
Charpentier, Emmanuelle
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
A better understanding of transcriptional and post-transcriptional regulation of gene expression in bacteria relies on studying their transcriptome. RNA sequencing methods are used not only to assess RNA abundance but also the exact boundaries of primary and processed transcripts. Here, we developed a method, called identification of specific cleavage position (ISCP), which enables the identification of direct endoribonuclease targets in vivo by comparing the 5΄ and 3΄ ends of processed transcripts between wild type and RNase deficient strains. To demonstrate the ISCP method, we used as a model the double-stranded specific RNase III in the human pathogen Streptococcus pyogenes. We mapped 92 specific cleavage positions (SCPs) among which, 48 were previously described and 44 are new, with the characteristic 2 nucleotides 3΄ overhang of RNase III. Most SCPs were located in untranslated regions of RNAs. We screened for RNase III targets using transcriptomic differential expression analysis (DEA) and compared those with the RNase III targets identified using the ISCP method. Our study shows that in S. pyogenes, under standard growth conditions, RNase III has a limited impact both on antisense transcripts and on global gene expression with the expression of most of the affected genes being downregulated in an RNase III deletion mutant.
2017-09-05T11:36:06Z
2017-09-05T11:36:06Z
2017-03-17
Article
Identification of endoribonuclease specific cleavage positions reveals novel targets of RNase III in Streptococcus pyogenes. 2017, 45 (5):2329-2340 Nucleic Acids Res.
1362-4962
28082390
10.1093/nar/gkw1316
http://hdl.handle.net/10033/621091
Nucleic acids research
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6213172019-08-30T11:28:51Zcom_10033_264694com_10033_6815com_10033_6814col_10033_264695
2018-03-09T10:47:03Z
urn:hdl:10033/621317
A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus.
Resch, Ulrike
Tsatsaronis, James Anthony
Le Rhun, Anaïs
Stübiger, Gerald
Rohde, M
Kasvandik, Sergo
Holzmeister, Susanne
Tinnefeld, Philip
Wai, Sun Nyunt
Charpentier, Emmanuelle
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to "anchorless surface proteins." Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response.
2018-03-09T10:47:03Z
2018-03-09T10:47:03Z
2016
Article
A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus. 2016, 7 (6) MBio
2150-7511
27803183
10.1128/mBio.00207-16
http://hdl.handle.net/10033/621317
mBio
en
http://creativecommons.org/licenses/by-nc-sa/4.0/