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Chu V.T.,Max Delbruck Center for Molecular Medicine | Weber T.,Max Delbruck Center for Molecular Medicine | Wefers B.,Helmholtz Center for Environmental Research | Wefers B.,Deutsches Zentrum fur Neurodegenerative Erkrankungen e.V. | And 8 more authors.
Nature Biotechnology

The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells. © 2015 Nature America, Inc. All rights reserved. Source

El Amrani K.,Charite - Medical University of Berlin | Stachelscheid H.,Charite - Medical University of Berlin | Stachelscheid H.,Berlin Institute of Health | Lekschas F.,Charite - Medical University of Berlin | And 4 more authors.
BMC Genomics

Background: Identification of marker genes associated with a specific tissue/cell type is a fundamental challenge in genetic and cell research. Marker genes are of great importance for determining cell identity, and for understanding tissue specific gene function and the molecular mechanisms underlying complex diseases. Results: We have developed a new bioinformatics tool called MGFM (Marker Gene Finder in Microarray data) to predict marker genes from microarray gene expression data. Marker genes are identified through the grouping of samples of the same type with similar marker gene expression levels. We verified our approach using two microarray data sets from the NCBI's Gene Expression Omnibus public repository encompassing samples for similar sets of five human tissues (brain, heart, kidney, liver, and lung). Comparison with another tool for tissue-specific gene identification and validation with literature-derived established tissue markers established functionality, accuracy and simplicity of our tool. Furthermore, top ranked marker genes were experimentally validated by reverse transcriptase-polymerase chain reaction (RT-PCR). The sets of predicted marker genes associated with the five selected tissues comprised well-known genes of particular importance in these tissues. The tool is freely available from the Bioconductor web site, and it is also provided as an online application integrated into the CellFinder platform (http://cellfinder.org/analysis/marker ). Conclusions:MGFM is a useful tool to predict tissue/cell type marker genes using microarray gene expression data. The implementation of the tool as an R-package as well as an application within CellFinder facilitates its use. © 2015 El Amrani et al. Source

Hong J.B.,Charite - Medical University of Berlin | Leonards C.O.,Charite - Medical University of Berlin | Endres M.,Charite - Medical University of Berlin | Endres M.,Berlin Institute of Health | And 3 more authors.

Background and Purpose - The ankle-brachial index (ABI) is a fast, cheap, noninvasive indicator of atherosclerotic burden that may also be a predictor of stroke recurrence. In this systematic review and meta-analysis, we sought to explore ABI's merit as a marker for stroke recurrence and vascular risk by synthesizing the data currently available in stroke literature. Methods-We searched Embase, MEDLINE, and Pubmed databases for prospective cohort studies that included consecutive patients with stroke and transient ischemic attack, measured ABI at baseline, and performed a follow-up assessment at least 12 months after initial stroke or transient ischemic attack. The following end points were chosen for our analysis: recurrent stroke and combined vascular end point (recurrent vascular event or vascular death). Crude risk ratios and adjusted Cox proportional hazard ratios were combined separately using the random-effects model. Study-level characteristics (eg, percent of cohort with a history of hypertension, average cohort age, level of adjustment, and mean follow-up duration) were included as covariates in a metaregression analysis. Results-We identified 11 studies (5374 patients) that were not significantly heterogeneous. Pooling adjusted hazard ratios showed that low ABI was associated with both an increased hazard of recurrent stroke (hazard ratio, 1.70; 95% confidence interval, 1.10-2.64) and an increased risk of vascular events or vascular death (hazard ratio, 2.22; 95% confidence interval, 1.67-2.97). Conclusion-Our results confirm the positive association between ABI and stroke recurrence. Further studies are needed to see whether inclusion of ABI will help improve the accuracy of prediction models and management of stroke patients. © 2016 American Heart Association, Inc. Source

Mackowiak S.D.,Max Delbruck Center for Molecular Medicine | Zauber H.,Max Delbruck Center for Molecular Medicine | Bielow C.,Max Delbruck Center for Molecular Medicine | Bielow C.,Berlin Institute of Health | And 8 more authors.
Genome Biology

Background: There is increasing evidence that transcripts or transcript regions annotated as non-coding can harbor functional short open reading frames (sORFs). Loss-of-function experiments have identified essential developmental or physiological roles for a few of the encoded peptides (micropeptides), but genome-wide experimental or computational identification of functional sORFs remains challenging. Results: Here, we expand our previously developed method and present results of an integrated computational pipeline for the identification of conserved sORFs in human, mouse, zebrafish, fruit fly, and the nematode C. elegans. Isolating specific conservation signatures indicative of purifying selection on amino acid (rather than nucleotide) sequence, we identify about 2,000 novel small ORFs located in the untranslated regions of canonical mRNAs or on transcripts annotated as non-coding. Predicted sORFs show stronger conservation signatures than those identified in previous studies and are sometimes conserved over large evolutionary distances. The encoded peptides have little homology to known proteins and are enriched in disordered regions and short linear interaction motifs. Published ribosome profiling data indicate translation of more than 100 novel sORFs, and mass spectrometry data provide evidence for more than 70 novel candidates. Conclusions: Taken together, we identify hundreds of previously unknown conserved sORFs in major model organisms. Our computational analyses and integration with experimental data show that these sORFs are expressed, often translated, and sometimes widely conserved, in some cases even between vertebrates and invertebrates. We thus provide an integrated resource of putatively functional micropeptides for functional validation in vivo. © 2015 Mackowiak et al. Source

Dirnagl U.,Charite - Medical University of Berlin | Dirnagl U.,German Center for Neurodegenerative Diseases | Dirnagl U.,German Center for Cardiovascular Diseases | Dirnagl U.,Berlin Institute of Health | Przesdzing I.,Charite - Medical University of Berlin

Every professional doing active research in the life sciences is required to keep a laboratory notebook. However, while science has changed dramatically over the last centuries, laboratory notebooks have remained essentially unchanged since pre-modern science. We argue that the implementation of electronic laboratory notebooks (eLN) in academic research is overdue, and we provide researchers and their institutions with the background and practical knowledge to select and initiate the implementation of an eLN in their laboratories. In addition, we present data from surveying biomedical researchers and technicians regarding which hypothetical features and functionalities they hope to see implemented in an eLN, and which ones they regard as less important. We also present data on acceptance and satisfaction of those who have recently switched from paper laboratory notebook to an eLN. We thus provide answers to the following questions: What does an electronic laboratory notebook afford a biomedical researcher, what does it require, and how should one go about implementing it? © 2016 Dirnagl U and Przesdzing I. Source

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