| 1. |
- Bratovic, Majda, et al.
(author)
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Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches
- 2020
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In: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 16:5, s. 587-595
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Journal article (peer-reviewed)abstract
- The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering. Tuning CRISPR-Cas9 nuclease specificity enables precision genome engineering. Identifying arginine residues along the bridge helix of SpCas9 that mediate Cas9 mismatch sensitivity enabled engineering of Cas9 with increased specificity in human cells.
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| 2. |
- Draaken, Markus, et al.
(author)
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Classic Bladder Exstrophy: Frequent 22q11.21 Duplications and Definition of a 414 kb Phenocritical Region
- 2014
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In: Birth Defects Research. Part A: Clinical and Molecular Teratology. - : Wiley. - 1542-0760 .- 1542-0752. ; 100:6, s. 512-517
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Journal article (peer-reviewed)abstract
- Background: Classic bladder exstrophy (CBE) is the most common form of the bladder exstrophy and epispadias complex. Previously, we and others have identified four patients with a duplication of 22q11.21 among a total of 96 unrelated CBE patients. Methods: Here, we investigated whether this chromosomal aberration was commonly associated with CBE/bladder exstrophy and epispadias complex in an extended case-control sample. Multiplex ligation-dependent probe amplification and microarray-based analysis were used to identify 22q11.21 duplications in 244 unrelated bladder exstrophy and epispadias complex patients (including 217 CBE patients) and 665 healthy controls. Results: New duplications of variable size were identified in four CBE patients and one control. Pooling of our previous and present data (eight duplications in 313 CBE patients) yielded a combined odds ratio of 31.86 (95% confidence interval, 4.24-1407.97). Array-based sequence capture and high-throughput targeted re-sequencing established that all breakpoints resided within the low-copy repeats 22A to 22D. Comparison of the eight duplications revealed a 414 kb phenocritical region harboring 12 validated RefSeq genes. Characterization of these 12 candidate genes through whole-mount in situ hybridization of mouse embryos at embryonic day 9.5 suggested that CRKL, THAP7, and LZTR1 are CBE candidate genes. Conclusion: Our data suggest that duplication of 22q11.21 increases CBE risk and implicate a phenocritical region in disease formation. (C) 2014 Wiley Periodicals, Inc.
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| 3. |
- Gossmann, Toni I., et al.
(author)
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Ice-Age Climate Adaptations Trap the Alpine Marmot in a State of Low Genetic Diversity
- 2019
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In: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 29:10, s. 1712-1720.e7
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Journal article (peer-reviewed)abstract
- © 2019 The Author(s) Some species responded successfully to prehistoric changes in climate [1, 2], while others failed to adapt and became extinct [3]. The factors that determine successful climate adaptation remain poorly understood. We constructed a reference genome and studied physiological adaptations in the Alpine marmot (Marmota marmota), a large ground-dwelling squirrel exquisitely adapted to the “ice-age” climate of the Pleistocene steppe [4, 5]. Since the disappearance of this habitat, the rodent persists in large numbers in the high-altitude Alpine meadow [6, 7]. Genome and metabolome showed evidence of adaptation consistent with cold climate, affecting white adipose tissue. Conversely, however, we found that the Alpine marmot has levels of genetic variation that are among the lowest for mammals, such that deleterious mutations are less effectively purged. Our data rule out typical explanations for low diversity, such as high levels of consanguineous mating, or a very recent bottleneck. Instead, ancient demographic reconstruction revealed that genetic diversity was lost during the climate shifts of the Pleistocene and has not recovered, despite the current high population size. We attribute this slow recovery to the marmot's adaptive life history. The case of the Alpine marmot reveals a complicated relationship between climatic changes, genetic diversity, and conservation status. It shows that species of extremely low genetic diversity can be very successful and persist over thousands of years, but also that climate-adapted life history can trap a species in a persistent state of low genetic diversity. Despite being highly abundant and well adapted, Gossmann et al. report that the Alpine marmot is among the least genetically diverse animal species. The low diversity is found to be the consequence of consecutive, climate-related events, including long-term extreme niche adaptation, that also greatly retarded the recovery of its genetic diversity.
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| 4. |
- Jax, Elinor, et al.
(author)
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Health monitoring in birds using bio-loggers and whole blood transcriptomics
- 2021
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Journal article (peer-reviewed)abstract
- Monitoring and early detection of emerging infectious diseases in wild animals is of crucial global importance, yet reliable ways to measure immune status and responses are lacking for animals in the wild. Here we assess the usefulness of bio-loggers for detecting disease outbreaks in free-living birds and confirm detailed responses using leukocyte composition and large-scale transcriptomics. We simulated natural infections by viral and bacterial pathogens in captive mallards (Anas platyrhynchos), an important natural vector for avian influenza virus. We show that body temperature, heart rate and leukocyte composition change reliably during an acute phase immune response. Using genome-wide gene expression profiling of whole blood across time points we confirm that immunostimulants activate pathogen-specific gene regulatory networks. By reporting immune response related changes in physiological and behavioural traits that can be studied in free-ranging populations, we provide baseline information with importance to the global monitoring of zoonotic diseases.
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| 5. |
- Koscielniak, Ewa, et al.
(author)
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Which Patients With Rhabdomyosarcoma Need Radiotherapy? : Analysis of the Radiotherapy Strategies of the CWS-96 and CWS-2002P Studies and SoTiSaR Registry
- 2023
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In: Journal of Clinical Oncology. - : Lippincott Williams & Wilkins. - 0732-183X .- 1527-7755. ; 41:31, s. 4916-
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Journal article (peer-reviewed)abstract
- PURPOSETo analyze and compare the indications, doses, and application methods of radiotherapy (RT) and their influence on prognosis of patients with localized rhabdomyosarcoma (RMS).METHODSOne thousand four hundred seventy patients with localized RMS 21 years and younger entered on CWS-96, CWS-2002P, and SoTiSaR were eligible for the analysis. The median follow-up was 6.5 years (IQR, 3.3-9.5).RESULTSThe 5-year event-free survival (EFS) and local control survival (LCS) for 910 (62%) irradiated versus nonirradiated patients were 71% versus 69% and 78% versus 73% (P = .03), respectively. Ninety-five percent of patients in IRS I (90% embryonal RMS [eRMS]) were nonirradiated (EFS, 87%). Irradiated patients with IRS II had improved LCS (91% v 80%; P = .01) and EFS (not significant). In IRS III, EFS and LCS were significantly better for RT patients: 71% versus 56% (P = 3.1e-06) and 76% versus 61% (P = 4.1e-07). Patients with tumors in the head and neck region (orbita, parameningeal, and nonparameningeal) and in other sites had significantly better EFS and LCS and in parameningeal also overall survival (OS). The efficacy of low RT doses of 32 Gy (hyperfractionated, accelerated RT [HART]) and 36 and 41.4 Gy (conventional fractionated RT [CFRT]) in the favorable groups and higher doses of 44.8 Gy (HART) and 50.4 and 55.4 Gy (CFRT) in the unfavorable groups was comparable. Proton RT was used predominantly in head/neck-parameningeal (HN-PM) tumors, with similar EFS and LCS to photon RT.CONCLUSIONRT can be omitted in patients with IRS I eRMS. RT improves LCS and EFS in IRS II and III. RT improves OS in patients with HN-PM, with proton RT comparable with photon RT. Doses of 32 Gy (HART) or 36 and 41.4 Gy (CFRT) had comparable efficacy in patients with favorable risk profiles and 44.8 Gy (HART) or 50.4 and 55.8 Gy (CFRT) in the unfavorable groups.
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| 6. |
- Philippe, Herve, et al.
(author)
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Mitigating Anticipated Effects of Systematic Errors Supports Sister-Group Relationship between Xenacoelomorpha and Ambulacraria
- 2019
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In: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 29:11, s. 1818-1826.e6
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Journal article (peer-reviewed)abstract
- Xenoturbella and the acoelomorph worms (Xenacoe-lomorpha) are simple marine animals with controversial affinities. They have been placed as the sister group of all other bilaterian animals (Nephrozoa hypothesis), implying their simplicity is an ancient characteristic [1, 2]; alternatively, they have been linked to the complex Ambulacraria (echinoderms and hemichordates) in a Glade called the Xenambulacraria [3,5], suggesting their simplicity evolved by reduction from a complex ancestor. The difficulty resolving this problem implies the phylogenetic signal supporting the correct solution is weak and affected by inadequate modeling, creating a misleading non-phylogenetic signal. The idea that the Nephrozoa hypothesis might be an artifact is prompted by the faster molecular evolutionary rate observed within the Acoelomorpha. Unequal rates of evolution are known to result in the systematic artifact of long branch attraction, which would be predicted to result in an attraction between long-branch acoelomorphs and the outgroup, pulling them toward the root [6]. Other biases inadequately accommodated by the models used can also have strong effects, exacerbated in the context of short internal branches and long terminal branches [7]. We have assembled a large and informative dataset to address this problem. Analyses designed to reduce or to emphasize misleading signals show the Nephrozoa hypothesis is supported under conditions expected to exacerbate errors, and the Xenambulacraria hypothesis is preferred in conditions designed to reduce errors. Our reanalyses of two other recently published datasets [1, 2] produce the same result. We conclude that the Xenacoelomorpha are simplified relatives of the Ambulacraria.
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| 7. |
- Weise, Anja, et al.
(author)
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High-throughput sequencing of microdissected chromosomal regions.
- 2010
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In: European Journal of Human Genetics. - : Springer Science and Business Media LLC. - 1018-4813 .- 1476-5438. ; 18:4, s. 457-62
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Journal article (peer-reviewed)abstract
- The linkage of disease gene mapping with DNA sequencing is an essential strategy for defining the genetic basis of a disease. New massively parallel sequencing procedures will greatly facilitate this process, although enrichment for the target region before sequencing remains necessary. For this step, various DNA capture approaches have been described that rely on sequence-defined probe sets. To avoid making assumptions on the sequences present in the targeted region, we accessed specific cytogenetic regions in preparation for next-generation sequencing. We directly microdissected the target region in metaphase chromosomes, amplified it by degenerate oligonucleotide-primed PCR, and obtained sufficient material of high quality for high-throughput sequencing. Sequence reads could be obtained from as few as six chromosomal fragments. The power of cytogenetic enrichment followed by next-generation sequencing is that it does not depend on earlier knowledge of sequences in the region being studied. Accordingly, this method is uniquely suited for situations in which the sequence of a reference region of the genome is not available, including population-specific or tumor rearrangements, as well as previously unsequenced genomic regions such as centromeres.
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