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Article ; Online: Creating 2D Occupancy Plots Using plot2DO.

Beati, Paula / Chereji, Răzvan V

Methods in molecular biology (Clifton, N.J.)

2020 Volume 2117, Page(s) 93–108

Abstract: Chromatin organization and epigenetic marks play a critical role in stem cell pluripotency and differentiation. Chromatin digestion by micrococcal nuclease (MNase) followed by high-throughput sequencing (MNase-seq) is the most widely used genome-wide ... ...

Abstract	Chromatin organization and epigenetic marks play a critical role in stem cell pluripotency and differentiation. Chromatin digestion by micrococcal nuclease (MNase) followed by high-throughput sequencing (MNase-seq) is the most widely used genome-wide method for studying nucleosome organization, that is, the first level of DNA packaging into chromatin. Combined with chromatin immunoprecipitation (ChIP), MNase-ChIP-seq represents a high-resolution method for investigating both chromatin organization and the distribution of epigenetic marks and histone variants. The plot2DO package presented here is a flexible tool for evaluating the quality of MNase-seq and MNase-ChIP-seq data, and for visualizing the distribution of nucleosomes near the functional regions of the genome. The plot2DO package is open-source software, and it is freely available from https://github.com/rchereji/plot2DO under the MIT license.
MeSH term(s)	Animals ; Chromatin Immunoprecipitation ; Computational Biology/methods ; Computer Simulation ; Epigenesis, Genetic ; High-Throughput Nucleotide Sequencing ; Humans ; Nucleosomes/genetics ; Nucleosomes/metabolism ; Sequence Analysis, DNA ; Software
Chemical Substances	Nucleosomes
Language	English
Publishing date	2020-01-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Intramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-0301-7_5
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Article ; Online: Major Determinants of Nucleosome Positioning.

Chereji, Răzvan V / Clark, David J

Biophysical journal

2018 Volume 114, Issue 10, Page(s) 2279–2289

Abstract: The compact structure of the nucleosome limits DNA accessibility and inhibits the binding of most sequence-specific proteins. Nucleosomes are not randomly located on the DNA but positioned with respect to the DNA sequence, suggesting models in which ... ...

Abstract	The compact structure of the nucleosome limits DNA accessibility and inhibits the binding of most sequence-specific proteins. Nucleosomes are not randomly located on the DNA but positioned with respect to the DNA sequence, suggesting models in which critical binding sites are either exposed in the linker, resulting in activation, or buried inside a nucleosome, resulting in repression. The mechanisms determining nucleosome positioning are therefore of paramount importance for understanding gene regulation and other events that occur in chromatin, such as transcription, replication, and repair. Here, we review our current understanding of the major determinants of nucleosome positioning: DNA sequence, nonhistone DNA-binding proteins, chromatin-remodeling enzymes, and transcription. We outline the major challenges for the future: elucidating the precise mechanisms of chromatin opening and promoter activation, identifying the complexes that occupy promoters, and understanding the multiscale problem of chromatin fiber organization.
MeSH term(s)	Adenosine Triphosphate/metabolism ; Animals ; DNA/genetics ; DNA/metabolism ; Histones/genetics ; Histones/metabolism ; Humans ; Nucleosomes/metabolism ; Transcription, Genetic
Chemical Substances	Histones ; Nucleosomes ; Adenosine Triphosphate (8L70Q75FXE) ; DNA (9007-49-2)
Language	English
Publishing date	2018-04-06
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Intramural ; Review
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2018.03.015
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Article ; Online: Quantitative MNase-seq accurately maps nucleosome occupancy levels.

Chereji, Răzvan V / Bryson, Terri D / Henikoff, Steven

Genome biology

2019 Volume 20, Issue 1, Page(s) 198

Abstract: Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono- ... ...

Abstract	Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. Here, we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.
MeSH term(s)	Animals ; Base Sequence ; Cell Line ; Chromatin ; DNA/chemistry ; Drosophila melanogaster/genetics ; High-Throughput Nucleotide Sequencing ; Kinetics ; Micrococcal Nuclease ; Nucleosomes ; Sequence Analysis, DNA ; Transcription, Genetic
Chemical Substances	Chromatin ; Nucleosomes ; DNA (9007-49-2) ; Micrococcal Nuclease (EC 3.1.31.1)
Language	English
Publishing date	2019-09-13
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Intramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2040529-7
ISSN	1474-760X ; 1474-760X
ISSN (online)	1474-760X
ISSN	1474-760X
DOI	10.1186/s13059-019-1815-z
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Article: Quantitative MNase-seq accurately maps nucleosome occupancy levels

Chereji, Răzvan V / Bryson, Terri D / Henikoff, Steven

Genome biology. 2019 Dec., v. 20, no. 1

2019

Abstract	Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. Here, we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.
Keywords	DNA ; Drosophila ; enzymatic hydrolysis ; enzyme activity ; heterochromatin ; humans ; micrococcal nuclease ; nucleosomes ; prediction
Language	English
Dates of publication	2019-12
Size	p. 198.
Publishing place	BioMed Central
Document type	Article
ZDB-ID	2040529-7
ISSN	1474-760X ; 1465-6906
ISSN (online)	1474-760X
ISSN	1465-6906
DOI	10.1186/s13059-019-1815-z
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Article ; Online: Contrasting roles of the RSC and ISW1/CHD1 chromatin remodelers in RNA polymerase II elongation and termination.

Ocampo, Josefina / Chereji, Răzvan V / Eriksson, Peter R / Clark, David J

Genome research

2019 Volume 29, Issue 3, Page(s) 407–417

Abstract: Most yeast genes have a nucleosome-depleted region (NDR) at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. We have examined the interplay between RSC (a conserved essential SWI/SNF-type complex ... ...

Abstract	Most yeast genes have a nucleosome-depleted region (NDR) at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. We have examined the interplay between RSC (a conserved essential SWI/SNF-type complex that determines NDR size) and the ISW1, CHD1, and ISW2 nucleosome spacing enzymes in chromatin organization and transcription, using isogenic strains lacking all combinations of these enzymes. The contributions of these remodelers to chromatin organization are largely combinatorial, distinct, and nonredundant, supporting a model in which the +1 nucleosome is positioned by RSC and then used as a reference nucleosome by the spacing enzymes. Defective chromatin organization correlates with altered RNA polymerase II (Pol II) distribution. RSC-depleted cells exhibit low levels of elongating Pol II and high levels of terminating Pol II, consistent with defects in both termination and initiation, suggesting that RSC facilitates both. Cells lacking both ISW1 and CHD1 show the opposite Pol II distribution, suggesting elongation and termination defects. These cells have extremely disrupted chromatin, with high levels of closely packed dinucleosomes involving the second (+2) nucleosome. We propose that ISW1 and CHD1 facilitate Pol II elongation by separating closely packed nucleosomes.
MeSH term(s)	Adenosine Triphosphatases/genetics ; Adenosine Triphosphatases/metabolism ; Chromatin Assembly and Disassembly ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Gene Expression Regulation, Fungal ; Nucleosomes/genetics ; Nucleosomes/metabolism ; RNA Polymerase II/genetics ; RNA Polymerase II/metabolism ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Transcription Elongation, Genetic ; Transcription Factors/genetics ; Transcription Factors/metabolism ; Transcription Termination, Genetic
Chemical Substances	CHD1 protein, S cerevisiae ; DNA-Binding Proteins ; ISWI protein ; Nucleosomes ; RSC complex, S cerevisiae ; Saccharomyces cerevisiae Proteins ; Transcription Factors ; RNA Polymerase II (EC 2.7.7.-) ; Adenosine Triphosphatases (EC 3.6.1.-) ; ISW1 protein, S cerevisiae (EC 3.6.1.-)
Language	English
Publishing date	2019-01-25
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Intramural
ZDB-ID	1284872-4
ISSN	1549-5469 ; 1088-9051 ; 1054-9803
ISSN (online)	1549-5469
ISSN	1088-9051 ; 1054-9803
DOI	10.1101/gr.242032.118
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Article ; Online: Ubiquitous nucleosome crowding in the yeast genome.

Chereji, Răzvan V / Morozov, Alexandre V

Proceedings of the National Academy of Sciences of the United States of America

2014 Volume 111, Issue 14, Page(s) 5236–5241

Abstract: Nucleosomes may undergo a conformational change in which a stretch of DNA peels off the histone octamer surface as a result of thermal fluctuations or interactions with chromatin remodelers. Thus, neighboring nucleosomes may invade each other's ... ...

Abstract	Nucleosomes may undergo a conformational change in which a stretch of DNA peels off the histone octamer surface as a result of thermal fluctuations or interactions with chromatin remodelers. Thus, neighboring nucleosomes may invade each other's territories by DNA unwrapping and translocation, or through initial assembly in partially wrapped states. A recent high-resolution map of distances between dyads of neighboring nucleosomes in Saccharomyces cerevisiae reveals that nucleosomes frequently overlap DNA territories of their neighbors. This conclusion is supported by lower-resolution maps of S. cerevisiae nucleosome lengths based on micrococcal nuclease digestion and paired-end sequencing. The average length of wrapped DNA follows a stereotypical pattern in genes and promoters, correlated with the well-known distribution of nucleosome occupancy: nucleosomal DNA tends to be shorter in promoters and longer in coding regions. To explain these observations, we have developed a biophysical model that uses a 10-11-bp periodic histone-DNA binding energy profile. The profile is based on the pattern of histone-DNA contacts in nucleosome crystal structures, as well as the idea of linker length discretization caused by higher-order chromatin structure. Our model is in agreement with the observed genome-wide distributions of interdyad distances, wrapped DNA lengths, and nucleosome occupancies. Furthermore, our approach explains in vitro measurements of the accessibility of nucleosome-covered target sites and nucleosome-induced cooperativity between DNA-binding factors. We rule out several alternative scenarios of histone-DNA interactions as inconsistent with the genomic data.
MeSH term(s)	Chromatin/metabolism ; DNA, Fungal/genetics ; DNA, Fungal/metabolism ; Genome, Fungal ; Histones/metabolism ; Nucleosomes ; Saccharomyces cerevisiae/genetics
Chemical Substances	Chromatin ; DNA, Fungal ; Histones ; Nucleosomes
Language	English
Publishing date	2014-03-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1321001111
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Zs.A 1148: Show issues

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Article ; Online: Functional roles of nucleosome stability and dynamics.

Chereji, Răzvan V / Morozov, Alexandre V

Briefings in functional genomics

2014 Volume 14, Issue 1, Page(s) 50–60

Abstract: Nucleosome is a histone-DNA complex known as the fundamental repeating unit of chromatin. Up to 90% of eukaryotic DNA is wrapped around consecutive octamers made of the core histones H2A, H2B, H3 and H4. Nucleosome positioning affects numerous cellular ... ...

Abstract	Nucleosome is a histone-DNA complex known as the fundamental repeating unit of chromatin. Up to 90% of eukaryotic DNA is wrapped around consecutive octamers made of the core histones H2A, H2B, H3 and H4. Nucleosome positioning affects numerous cellular processes that require robust and timely access to genomic DNA, which is packaged into the tight confines of the cell nucleus. In living cells, nucleosome positions are determined by intrinsic histone-DNA sequence preferences, competition between histones and other DNA-binding proteins for genomic sequence, and ATP-dependent chromatin remodelers. We discuss the major energetic contributions to nucleosome formation and remodeling, focusing especially on partial DNA unwrapping off the histone octamer surface. DNA unwrapping enables efficient access to nucleosome-buried binding sites and mediates rapid nucleosome removal through concerted action of two or more DNA-binding factors. High-resolution, genome-scale maps of distances between neighboring nucleosomes have shown that DNA unwrapping and nucleosome crowding (mutual invasion of nucleosome territories) are much more common than previously thought. Ultimately, constraints imposed by nucleosome energetics on the rates of ATP-dependent and spontaneous chromatin remodeling determine nucleosome occupancy genome-wide, and shape pathways of cellular response to environmental stresses.
MeSH term(s)	Animals ; Chromatin Assembly and Disassembly ; Gene Expression Regulation ; Humans ; Models, Biological ; Nucleosomes/metabolism ; Protein Binding ; Thermodynamics
Chemical Substances	Nucleosomes
Language	English
Publishing date	2014-09-30
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2540916-5
ISSN	2041-2657 ; 2041-2649 ; 2041-2647
ISSN (online)	2041-2657
ISSN	2041-2649 ; 2041-2647
DOI	10.1093/bfgp/elu038
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Article ; Online: MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers.

Chereji, Răzvan V / Ocampo, Josefina / Clark, David J

Molecular cell

2017 Volume 65, Issue 3, Page(s) 565–577.e3

Abstract: Micrococcal nuclease (MNase) is commonly used to map nucleosomes genome-wide, but nucleosome maps are affected by the degree of digestion. It has been proposed that many yeast promoters are not nucleosome-free but instead occupied by easily digested, ... ...

Abstract	Micrococcal nuclease (MNase) is commonly used to map nucleosomes genome-wide, but nucleosome maps are affected by the degree of digestion. It has been proposed that many yeast promoters are not nucleosome-free but instead occupied by easily digested, unstable, "fragile" nucleosomes. We analyzed the histone content of all MNase-sensitive complexes by MNase-ChIP-seq and sonication-ChIP-seq. We find that yeast promoters are predominantly bound by non-histone protein complexes, with little evidence for fragile nucleosomes. We do detect MNase-sensitive nucleosomes elsewhere in the genome, including at transcription termination sites. However, they have high A/T content, suggesting that MNase sensitivity does not indicate instability, but rather the preference of MNase for A/T-rich DNA, such that A/T-rich nucleosomes are digested faster than G/C-rich nucleosomes. We confirm our observations by analyzing ChIP-exo, chemical mapping, and ATAC-seq data from other laboratories. Thus, histone ChIP-seq experiments are essential to distinguish nucleosomes from other DNA-binding proteins that protect against MNase.
MeSH term(s)	Base Composition ; Micrococcal Nuclease/metabolism ; Nucleosomes ; Promoter Regions, Genetic ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics
Chemical Substances	Nucleosomes ; Saccharomyces cerevisiae Proteins ; Micrococcal Nuclease (EC 3.1.31.1)
Language	English
Publishing date	2017-02-03
Publishing country	United States
Document type	Journal Article
ZDB-ID	1415236-8
ISSN	1097-4164 ; 1097-2765
ISSN (online)	1097-4164
ISSN	1097-2765
DOI	10.1016/j.molcel.2016.12.009
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Article: MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers

Chereji, Răzvan V / Ocampo, Josefina / Clark, David J

Molecular cell. 2017 Feb. 02, v. 65, no. 3

2017

Abstract	Micrococcal nuclease (MNase) is commonly used to map nucleosomes genome-wide, but nucleosome maps are affected by the degree of digestion. It has been proposed that many yeast promoters are not nucleosome-free but instead occupied by easily digested, unstable, “fragile” nucleosomes. We analyzed the histone content of all MNase-sensitive complexes by MNase-ChIP-seq and sonication-ChIP-seq. We find that yeast promoters are predominantly bound by non-histone protein complexes, with little evidence for fragile nucleosomes. We do detect MNase-sensitive nucleosomes elsewhere in the genome, including at transcription termination sites. However, they have high A/T content, suggesting that MNase sensitivity does not indicate instability, but rather the preference of MNase for A/T-rich DNA, such that A/T-rich nucleosomes are digested faster than G/C-rich nucleosomes. We confirm our observations by analyzing ChIP-exo, chemical mapping, and ATAC-seq data from other laboratories. Thus, histone ChIP-seq experiments are essential to distinguish nucleosomes from other DNA-binding proteins that protect against MNase.
Keywords	DNA ; DNA-binding proteins ; genome ; histones ; micrococcal nuclease ; nucleosomes ; transcription termination ; yeasts
Language	English
Dates of publication	2017-0202
Size	p. 565-577.e3.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1415236-8
ISSN	1097-4164 ; 1097-2765
ISSN (online)	1097-4164
ISSN	1097-2765
DOI	10.1016/j.molcel.2016.12.009
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Article ; Online: Quantitative MNase-seq accurately maps nucleosome occupancy levels

Răzvan V. Chereji / Terri D. Bryson / Steven Henikoff

Genome Biology, Vol 20, Iss 1, Pp 1-

2019 Volume 18

Abstract: Abstract Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and ... ...

Abstract	Abstract Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. Here, we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.
Keywords	Chromatin ; Linker DNA ; Nuclease digestion kinetics ; Biology (General) ; QH301-705.5 ; Genetics ; QH426-470
Language	English
Publishing date	2019-09-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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