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Article ; Online: Small RNA-Sequencing for Analysis of Circulating miRNAs: Benchmark Study.

Androvic, Peter / Benesova, Sarka / Rohlova, Eva / Kubista, Mikael / Valihrach, Lukas

The Journal of molecular diagnostics : JMD

2022 Volume 24, Issue 4, Page(s) 386–394

Abstract: Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated ... ...

Abstract	Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated development of several novel methods. Here, we present comparison of all small RNA-Seq library preparation approaches that are commercially available for quantification of miRNAs in biofluids. Using synthetic and human plasma samples, we compared performance of traditional two-adaptor ligation protocols (Lexogen, Norgen), as well as methods using randomized adaptors (NEXTflex), polyadenylation (SMARTer), circularization (RealSeq), capture probes (EdgeSeq), or unique molecular identifiers (QIAseq). There was no single protocol outperforming others across all metrics. Limited overlap of measured miRNA profiles was documented between methods largely owing to protocol-specific biases. Methods designed to minimize bias largely differ in their performance, and contributing factors were identified. Usage of unique molecular identifiers has rather negligible effect and, if designed incorrectly, can even introduce spurious results. Together, these results identify strengths and weaknesses of all current methods and provide guidelines for applications of small RNA-Seq in biomarker research.
MeSH term(s)	Benchmarking ; Circulating MicroRNA/genetics ; High-Throughput Nucleotide Sequencing/methods ; Humans ; MicroRNAs/genetics ; Sequence Analysis, RNA/methods
Chemical Substances	Circulating MicroRNA ; MicroRNAs
Language	English
Publishing date	2022-01-23
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2000060-1
ISSN	1943-7811 ; 1525-1578
ISSN (online)	1943-7811
ISSN	1525-1578
DOI	10.1016/j.jmoldx.2021.12.006
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Article ; Online: High-Resolution RNA Sequencing from PFA-Fixed Microscopy Sections.

Ji, Hao / Besson-Girard, Simon / Androvic, Peter / Bulut, Buket / Liu, Lu / Wang, Yijing / Gokce, Ozgun

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

2023 Volume 2616, Page(s) 205–212

Abstract: Obtaining high-quality RNA sequencing results from archived biological tissues, such as paraformaldehyde (PFA)-fixed sections for microscopy, is challenging due to the incompatibility of current high-throughput RNA sequencing methods. Here, we present a ... ...

Abstract	Obtaining high-quality RNA sequencing results from archived biological tissues, such as paraformaldehyde (PFA)-fixed sections for microscopy, is challenging due to the incompatibility of current high-throughput RNA sequencing methods. Here, we present a low-input method for RNA sequencing from archived PFA-fixed sections. Using this method, we routinely obtain high-quality sequencing results from archived mouse brain sections that are prepared for imaging without any special care for avoiding RNA degradation. The PFA cross-linking locks and protects RNA from degradation but cross-linking is also hard to reverse. For this goal, we developed an effective decrosslinking protocol based on Proteinase K activity to retrieve PFA-cross-linked mRNAs which was followed up by a Smart-seq2 library preparation protocol. Our protocol enables spatially defined transcriptomic analysis of archived sections and allows the genomic analysis of PFA-fixed samples. Furthermore, our protocol inactivates pathogenic samples and allows working under regular biosafety levels.
MeSH term(s)	Animals ; Mice ; Microscopy ; RNA/genetics ; RNA, Messenger ; Sequence Analysis, RNA ; Gene Expression Profiling ; High-Throughput Nucleotide Sequencing
Chemical Substances	RNA (63231-63-0) ; RNA, Messenger
Language	English
Publishing date	2023-01-30
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2926-0_16
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Article: A view of the genetic and proteomic profile of extracellular matrix molecules in aging and stroke.

Chmelova, Martina / Androvic, Peter / Kirdajova, Denisa / Tureckova, Jana / Kriska, Jan / Valihrach, Lukas / Anderova, Miroslava / Vargova, Lydia

Frontiers in cellular neuroscience

2023 Volume 17, Page(s) 1296455

Abstract: Introduction: Modification of the extracellular matrix (ECM) is one of the major processes in the pathology of brain damage following an ischemic stroke. However, our understanding of how age-related ECM alterations may affect stroke pathophysiology and ...

Abstract	Introduction: Modification of the extracellular matrix (ECM) is one of the major processes in the pathology of brain damage following an ischemic stroke. However, our understanding of how age-related ECM alterations may affect stroke pathophysiology and its outcome is still very limited. Methods: We conducted an ECM-targeted re-analysis of our previously obtained RNA-Seq dataset of aging, ischemic stroke and their interactions in young adult (3-month-old) and aged (18-month-old) mice. The permanent middle cerebral artery occlusion (pMCAo) in rodents was used as a model of ischemic stroke. Altogether 56 genes of interest were chosen for this study. Results: We identified an increased activation of the genes encoding proteins related to ECM degradation, such as matrix metalloproteinases (MMPs), proteases of a disintegrin and metalloproteinase with the thrombospondin motifs (ADAMTS) family and molecules that regulate their activity, tissue inhibitors of metalloproteinases (TIMPs). Moreover, significant upregulation was also detected in the mRNA of other ECM molecules, such as proteoglycans, syndecans and link proteins. Notably, we identified 8 genes where this upregulation was enhanced in aged mice in comparison with the young ones. Ischemia evoked a significant downregulation in only 6 of our genes of interest, including those encoding proteins associated with the protective function of ECM molecules (e.g., brevican, Hapln4, Sparcl1); downregulation in brevican was more prominent in aged mice. The study was expanded by proteome analysis, where we observed an ischemia-induced overexpression in three proteins, which are associated with neuroinflammation (fibronectin and vitronectin) and neurodegeneration (link protein Hapln2). In fibronectin and Hapln2, this overexpression was more pronounced in aged post-ischemic animals. Conclusion: Based on these results, we can conclude that the ratio between the protecting and degrading mechanisms in the aged brain is shifted toward degradation and contributes to the aged tissues' increased sensitivity to ischemic insults. Altogether, our data provide fresh perspectives on the processes underlying ischemic injury in the aging brain and serve as a freely accessible resource for upcoming research.
Language	English
Publishing date	2023-11-30
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2452963-1
ISSN	1662-5102
ISSN	1662-5102
DOI	10.3389/fncel.2023.1296455
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Article ; Online: Circulating miRNA analysis for cancer diagnostics and therapy.

Valihrach, Lukas / Androvic, Peter / Kubista, Mikael

Molecular aspects of medicine

2019 Volume 72, Page(s) 100825

Abstract: Successful treatment of cancer depends on early diagnosis and effective monitoring of patients' response to therapy. Traditional tools based on tumor biopsies lack the sensitivity and specificity to capture cancer development in its early phases and are ... ...

Abstract	Successful treatment of cancer depends on early diagnosis and effective monitoring of patients' response to therapy. Traditional tools based on tumor biopsies lack the sensitivity and specificity to capture cancer development in its early phases and are not applicable for continuous monitoring. To overcome these barriers, liquid biopsies have been introduced as a minimally invasive and cost-efficient means of diagnosis with high level of specificity and sensitivity. Traditionally, liquid biopsy markers include circulating tumor cells and circulating tumor DNA. During the last decade, a new promising group of biomarkers has appeared and its utilization for cancer diagnosis and monitoring is intensively studied - the microRNAs (miRNAs). In this review, we provide a comprehensive overview of circulating miRNA analysis. We highlight the importance of sampling and quality control, discuss the technical aspects of miRNA extraction and quantification, summarize recommendations for downstream analysis and conclude with future perspectives. Taken together, we present the current state of knowledge in the field of miRNA analysis in liquid biopsies and the expected development and standardization.
MeSH term(s)	Biomarkers, Tumor/genetics ; Circulating MicroRNA/analysis ; Circulating MicroRNA/isolation & purification ; Humans ; Liquid Biopsy ; Neoplasms/diagnosis ; Neoplasms/genetics ; Neoplasms/therapy ; Quality Control ; Specimen Handling/methods
Chemical Substances	Biomarkers, Tumor ; Circulating MicroRNA
Language	English
Publishing date	2019-10-18
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	197640-0
ISSN	1872-9452 ; 0098-2997
ISSN (online)	1872-9452
ISSN	0098-2997
DOI	10.1016/j.mam.2019.10.002
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Article ; Online: Performance Comparison of Reverse Transcriptases for Single-Cell Studies.

Zucha, Daniel / Androvic, Peter / Kubista, Mikael / Valihrach, Lukas

Clinical chemistry

2019 Volume 66, Issue 1, Page(s) 217–228

Abstract: Background: Recent advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on reverse transcription (RT). However, RT is recognized as a known ... ...

Abstract	Background: Recent advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on reverse transcription (RT). However, RT is recognized as a known source of variability, particularly with low amounts of RNA. Recently, several new reverse transcriptases (RTases) with the potential to decrease the loss of information have been developed, but knowledge of their performance is limited. Methods: We compared the performance of 11 RTases in quantitative reverse transcription PCR (RT-qPCR) on single-cell and 100-cell bulk templates, using 2 priming strategies: a conventional mixture of random hexamers with oligo(dT)s and a reduced concentration of oligo(dT)s mimicking common single-cell RNA-sequencing protocols. Depending on their performance, 2 RTases were further tested in a high-throughput single-cell experiment. Results: All tested RTases demonstrated high precision (R2 > 0.9445). The most pronounced differences were found in their ability to capture rare transcripts (0%-90% reaction positivity rate) and in their absolute reaction yield (7.3%-137.9%). RTase performance and reproducibility were compared with Z scores. The 2 best-performing enzymes were Maxima H- and SuperScript IV. The validity of the obtained results was confirmed in a follow-up single-cell model experiment. The better-performing enzyme (Maxima H-) increased the sensitivity of the single-cell experiment and improved resolution in the clustering analysis over the commonly used RTase (SuperScript II). Conclusions: Our comprehensive comparison of 11 RTases in low RNA input conditions identified 2 best-performing enzymes. Our results provide a point of reference for the improvement of current single-cell quantification protocols.
MeSH term(s)	Animals ; DNA Primers/metabolism ; Humans ; RNA/metabolism ; RNA-Directed DNA Polymerase/metabolism ; Real-Time Polymerase Chain Reaction/methods ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Single-Cell Analysis ; Superoxide Dismutase-1/genetics
Chemical Substances	DNA Primers ; RNA (63231-63-0) ; Superoxide Dismutase-1 (EC 1.15.1.1) ; RNA-Directed DNA Polymerase (EC 2.7.7.49)
Language	English
Publishing date	2019-11-04
Publishing country	England
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	80102-1
ISSN	1530-8561 ; 0009-9147
ISSN (online)	1530-8561
ISSN	0009-9147
DOI	10.1373/clinchem.2019.307835
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Article ; Online: Platforms for Single-Cell Collection and Analysis.

Valihrach, Lukas / Androvic, Peter / Kubista, Mikael

International journal of molecular sciences

2018 Volume 19, Issue 3

Abstract: Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, ... ...

Abstract	Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.
MeSH term(s)	Animals ; Flow Cytometry/instrumentation ; Flow Cytometry/methods ; Humans ; Microfluidics/instrumentation ; Microfluidics/methods ; Single-Cell Analysis/instrumentation ; Single-Cell Analysis/methods
Language	English
Publishing date	2018-03-11
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms19030807
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Article ; Online: Spatial Transcriptomics-correlated Electron Microscopy maps transcriptional and ultrastructural responses to brain injury.

Androvic, Peter / Schifferer, Martina / Perez Anderson, Katrin / Cantuti-Castelvetri, Ludovico / Jiang, Hanyi / Ji, Hao / Liu, Lu / Gouna, Garyfallia / Berghoff, Stefan A / Besson-Girard, Simon / Knoferle, Johanna / Simons, Mikael / Gokce, Ozgun

Nature communications

2023 Volume 14, Issue 1, Page(s) 4115

Abstract: Understanding the complexity of cellular function within a tissue necessitates the combination of multiple phenotypic readouts. Here, we developed a method that links spatially-resolved gene expression of single cells with their ultrastructural ... ...

Abstract	Understanding the complexity of cellular function within a tissue necessitates the combination of multiple phenotypic readouts. Here, we developed a method that links spatially-resolved gene expression of single cells with their ultrastructural morphology by integrating multiplexed error-robust fluorescence in situ hybridization (MERFISH) and large area volume electron microscopy (EM) on adjacent tissue sections. Using this method, we characterized in situ ultrastructural and transcriptional responses of glial cells and infiltrating T-cells after demyelinating brain injury in male mice. We identified a population of lipid-loaded "foamy" microglia located in the center of remyelinating lesion, as well as rare interferon-responsive microglia, oligodendrocytes, and astrocytes that co-localized with T-cells. We validated our findings using immunocytochemistry and lipid staining-coupled single-cell RNA sequencing. Finally, by integrating these datasets, we detected correlations between full-transcriptome gene expression and ultrastructural features of microglia. Our results offer an integrative view of the spatial, ultrastructural, and transcriptional reorganization of single cells after demyelinating brain injury.
MeSH term(s)	Male ; Animals ; Mice ; Transcriptome ; In Situ Hybridization, Fluorescence ; Microscopy, Electron ; Brain Injuries/genetics ; Lipids
Chemical Substances	Lipids
Language	English
Publishing date	2023-07-11
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-39447-9
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Article ; Online: Studies on regulation of the cell cycle in fission yeast.

Miroslava Požgajová / Peter Androvič / Anna Trakovická

Scientific Papers Animal Science and Biotechnologies, Vol 48, Iss 1, Pp 173-

2015 Volume 177

Abstract: All living organisms including plants and animals are composed of millions of cells. These cells perform different functions for the organism although they possess the same chromosomes and carry the same genetic information. Thus, to be able to ... ...

Abstract	All living organisms including plants and animals are composed of millions of cells. These cells perform different functions for the organism although they possess the same chromosomes and carry the same genetic information. Thus, to be able to understand multicellular organism we need to understand the life cycle of individual cells from which the organism comprises. The cell cycle is the life cycle of a single cell in the plant or animal body. It involves series of events in which components of the cell doubles and afterwards equally segregate into daughter cells. Such process ensures growth of the organism, and specialized reductional cell division which leads to production of gamets, assures sexual reproduction. Cell cycle is divided in the G1, S, G2 and M phase. Two gap-phases (G1 and G2) separate S phase (or synthesis) and M phase which stays either for mitosis or meiosis. Essential for normal life progression and reproduction is correct chromosome segregation during mitosis and meiosis. Defects in the division program lead to aneuploidy, which in turn leads to birth defects, miscarriages or cancer. Even thou, researchers invented much about the regulation of the cell cycle, there is still long way to understand the complexity of the regulatory machineries that ensure proper segregation of chromosomes. In this paper we would like to describe techniques and materials we use for our studies on chromosome segregation in the model organism Schizosaccharomyces pombe.
Keywords	meiosis ; mitosis ; live cell imaging ; schizosaccharomycesn pombe ; Agriculture ; S ; Technology ; T ; Science ; Q
Subject code	571
Language	English
Publishing date	2015-05-01T00:00:00Z
Publisher	Agroprint Timisoara
Document type	Article ; Online
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Article ; Online: Platforms for Single-Cell Collection and Analysis

Lukas Valihrach / Peter Androvic / Mikael Kubista

International Journal of Molecular Sciences, Vol 19, Iss 3, p

2018 Volume 807

Abstract	Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.
Keywords	single cell ; collection ; isolation ; analysis ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2018-03-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Two-tailed RT-qPCR panel for quality control of circulating microRNA studies.

Androvic, Peter / Romanyuk, Nataliya / Urdzikova-Machova, Lucia / Rohlova, Eva / Kubista, Mikael / Valihrach, Lukas

Scientific reports

2019 Volume 9, Issue 1, Page(s) 4255

Abstract: Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to ... ...

Abstract	Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).
MeSH term(s)	Animals ; Biomarkers/blood ; Circulating MicroRNA/blood ; Circulating MicroRNA/isolation & purification ; Cost-Benefit Analysis ; Feasibility Studies ; Healthy Volunteers ; Humans ; Quality Control ; Rats ; Reagent Kits, Diagnostic/standards ; Real-Time Polymerase Chain Reaction/economics ; Real-Time Polymerase Chain Reaction/instrumentation ; Real-Time Polymerase Chain Reaction/methods ; Real-Time Polymerase Chain Reaction/standards
Chemical Substances	Biomarkers ; Circulating MicroRNA ; Reagent Kits, Diagnostic
Language	English
Publishing date	2019-03-12
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-019-40513-w
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