| 1. |
- Altenhoff, Adrian M., et al.
(författare)
-
Standardized benchmarking in the quest for orthologs
- 2016
-
Ingår i: Nature Methods. - 1548-7091 .- 1548-7105. ; 13:5, s. 425-
-
Tidskriftsartikel (refereegranskat)abstract
- Achieving high accuracy in orthology inference is essential for many comparative, evolutionary and functional genomic analyses, yet the true evolutionary history of genes is generally unknown and orthologs are used for very different applications across phyla, requiring different precision-recall trade-offs. As a result, it is difficult to assess the performance of orthology inference methods. Here, we present a community effort to establish standards and an automated web-based service to facilitate orthology benchmarking. Using this service, we characterize 15 well-established inference methods and resources on a battery of 20 different benchmarks. Standardized benchmarking provides a way for users to identify the most effective methods for the problem at hand, sets a minimum requirement for new tools and resources, and guides the development of more accurate orthology inference methods.
|
|
| 2. |
- Anikeeva, Polina, et al.
(författare)
-
Voices in methods development
- 2019
-
Ingår i: Nature Methods. - : NATURE PUBLISHING GROUP. - 1548-7091 .- 1548-7105. ; 16:10, s. 945-951
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
|
|
| 3. |
- Canals, Isaac, et al.
(författare)
-
Rapid and efficient induction of functional astrocytes from human pluripotent stem cells
- 2018
-
Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 15:9, s. 693-696
-
Tidskriftsartikel (refereegranskat)abstract
- The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease.
|
|
| 4. |
- Chen, Yen Hsi, et al.
(författare)
-
The GAGOme : a cell-based library of displayed glycosaminoglycans
- 2018
-
Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 15:11, s. 881-888
-
Tidskriftsartikel (refereegranskat)abstract
- Glycosaminoglycans (GAGs) are essential polysaccharides in normal physiology and disease. However, understanding of the contribution of specific GAG structures to specific biological functions is limited, largely because of the great structural heterogeneity among GAGs themselves, as well as technical limitations in the structural characterization and chemical synthesis of GAGs. Here we describe a cell-based method to produce and display distinct GAGs with a broad repertoire of modifications, a library we refer to as the GAGOme. By using precise gene editing, we engineered a large panel of Chinese hamster ovary cells with knockout or knock-in of the genes encoding most of the enzymes involved in GAG biosynthesis, to generate a library of isogenic cell lines that differentially display distinct GAG features. We show that this library can be used for cell-based binding assays, recombinant expression of proteoglycans with distinct GAG structures, and production of distinct GAG chains on metabolic primers that may be used for the assembly of GAG glycan microarrays.
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Domingo-Almenara, Xavier, et al.
(författare)
-
CMS-MRM and METLIN-MRM : a cloud library and public resource for targeted analysis of small molecules
- 2018
-
Ingår i: Nature Methods. - : Nature Publishing Group. - 1548-7091 .- 1548-7105. ; 15:9, s. 681-
-
Tidskriftsartikel (refereegranskat)abstract
- We report XCMS-MRM and METLIN-MRM (http://xcmsonline-mrm.scripps.edu/ and http://metlin.scripps.edu/), a cloud-based data-analysis platform and a public multiple-reaction monitoring (MRM) transition repository for small-molecule quantitative tandem mass spectrometry. This platform provides MRM transitions for more than 15,500 molecules and facilitates data sharing across different instruments and laboratories.
|
|
| 9. |
|
|
| 10. |
|
|
| 11. |
- Frauenfeld, Jens, et al.
(författare)
-
A saposin-lipoprotein nanoparticle system for membrane proteins
- 2016
-
Ingår i: Nature Methods. - : Nature Publishing Group. - 1548-7091 .- 1548-7105. ; 13:4, s. 345-351
-
Tidskriftsartikel (refereegranskat)abstract
- A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptT(S02) by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state.
|
|
| 12. |
- Fuller, Franklin D, et al.
(författare)
-
Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers
- 2017
-
Ingår i: Nature Methods. - : Macmillan Publishers Ltd.. - 1548-7091 .- 1548-7105. ; 14, s. 443-449
-
Tidskriftsartikel (refereegranskat)abstract
- X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.
|
|
| 13. |
|
|
| 14. |
- Lentini, Antonio, et al.
(författare)
-
A reassessment of DNA-immunoprecipitation-based genomic profiling
- 2018
-
Ingår i: Nature Methods. - : NATURE PUBLISHING GROUP. - 1548-7091 .- 1548-7105. ; 15:7, s. 499-
-
Tidskriftsartikel (refereegranskat)abstract
- DNA immunoprecipitation followed by sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, the results of independent DIP-seq studies often show considerable variation between profiles of the same genome and between profiles obtained by alternative methods. Here we show that these differences are primarily due to the intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50-99% of regions identified as enriched for DNA modifications in DIP-seq data. Correction of this error profoundly altered DNA-modification profiles for numerous cell types, including mouse embryonic stem cells, and subsequently revealed novel associations among DNA modifications, chromatin modifications and biological processes. We conclude that both matched input and IgG controls are essential in order for the results of DIP-based assays to be interpreted correctly, and that complementary, non-antibody-based techniques should be used to validate DIP-based findings to avoid further misinterpretation of genome-wide profiling data.
|
|
| 15. |
|
|
| 16. |
- Ouyang, Wei, et al.
(författare)
-
Analysis of the Human Protein Atlas Image Classification competition
- 2019
-
Ingår i: Nature Methods. - : Springer Nature. - 1548-7091 .- 1548-7105. ; 16:12, s. 1254-
-
Tidskriftsartikel (refereegranskat)abstract
- Pinpointing subcellular protein localizations from microscopy images is easy to the trained eye, but challenging to automate. Based on the Human Protein Atlas image collection, we held a competition to identify deep learning solutions to solve this task. Challenges included training on highly imbalanced classes and predicting multiple labels per image. Over 3 months, 2,172 teams participated. Despite convergence on popular networks and training techniques, there was considerable variety among the solutions. Participants applied strategies for modifying neural networks and loss functions, augmenting data and using pretrained networks. The winning models far outperformed our previous effort at multi-label classification of protein localization patterns by similar to 20%. These models can be used as classifiers to annotate new images, feature extractors to measure pattern similarity or pretrained networks for a wide range of biological applications.
|
|
| 17. |
|
|
| 18. |
- Pennacchietti, Francesca, et al.
(författare)
-
Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy
- 2018
-
Ingår i: Nature Methods. - : Nature Publishing Group. - 1548-7091 .- 1548-7105. ; 15:8, s. 601-
-
Tidskriftsartikel (refereegranskat)abstract
- Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells.
|
|
| 19. |
- Potrzebowski, Wojciech, et al.
(författare)
-
Automated determination of fibrillar structures by simultaneous model building and fiber diffraction refinement.
- 2015
-
Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7105 .- 1548-7091. ; 12:7, s. 679-679
-
Tidskriftsartikel (refereegranskat)abstract
- For highly oriented fibrillar molecules, three-dimensional structures can often be determined from X-ray fiber diffraction data. However, because of limited information content, structure determination and validation can be challenging. We demonstrate that automated structure determination of protein fibers can be achieved by guiding the building of macromolecular models with fiber diffraction data. We illustrate the power of our approach by determining the structures of six bacteriophage viruses de novo using fiber diffraction data alone and together with solid-state NMR data. Furthermore, we demonstrate the feasibility of molecular replacement from monomeric and fibrillar templates by solving the structure of a plant virus using homology modeling and protein-protein docking. The generated models explain the experimental data to the same degree as deposited reference structures but with improved structural quality. We also developed a cross-validation method for model selection. The results highlight the power of fiber diffraction data as structural constraints.
|
|
| 20. |
- Shariatgorji, Mohammadreza, et al.
(författare)
-
Comprehensive mapping of neurotransmitter networks by MALDI-MS imaging
- 2019
-
Ingår i: Nature Methods. - : NATURE PUBLISHING GROUP. - 1548-7091 .- 1548-7105. ; 16:10, s. 1021-1028
-
Tidskriftsartikel (refereegranskat)abstract
- We present a mass spectrometry imaging (MSI) approach for the comprehensive mapping of neurotransmitter networks in specific brain regions. Our fluoromethylpyridinium-based reactive matrices facilitate the covalent charge-tagging of molecules containing phenolic hydroxyl and/or primary or secondary amine groups, including dopaminergic and serotonergic neurotransmitters and their associated metabolites. These matrices improved the matrix-assisted laser desorption/ionization (MALDI)-MSI detection limit toward low-abundance neurotransmitters and facilitated the simultaneous imaging of neurotransmitters in fine structures of the brain at a lateral resolution of 10 mu m. We demonstrate strategies for the identification of unknown molecular species using the innate chemoselectivity of the reactive matrices and the unique isotopic pattern of a brominated reactive matrix. We illustrate the capabilities of the developed method on Parkinsonian brain samples from human post-mortem tissue and animal models. The direct imaging of neurotransmitter systems provides a method for exploring how various neurological diseases affect specific brain regions through neurotransmitter modulation.
|
|
| 21. |
- Uhlen, Mathias, et al.
(författare)
-
A proposal for validation of antibodies
- 2016
-
Ingår i: Nature Methods. - : Nature Publishing Group. - 1548-7091 .- 1548-7105. ; 13:10, s. 823-
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- We convened an ad hoc International Working Group for Antibody Validation in order to formulate the best approaches for validating antibodies used in common research applications and to provide guidelines that ensure antibody reproducibility. We recommend five conceptual 'pillars' for antibody validation to be used in an application-specific manner.
|
|
| 22. |
|
|
| 23. |
- Ulman, V, et al.
(författare)
-
An objective comparison of cell-tracking algorithms
- 2017
-
Ingår i: Nature Methods. - : NATURE PUBLISHING GROUP. - 1548-7091 .- 1548-7105. ; 14:12, s. 1141-
-
Tidskriftsartikel (refereegranskat)abstract
- We present a combined report on the results of three editions of the Cell Tracking Challenge, an ongoing initiative aimed at promoting the development and objective evaluation of cell segmentation and tracking algorithms. With 21 participating algorithms and a data repository consisting of 13 data sets from various microscopy modalities, the challenge displays today's state-of-the-art methodology in the field. We analyzed the challenge results using performance measures for segmentation and tracking that rank all participating methods. We also analyzed the performance of all of the algorithms in terms of biological measures and practical usability. Although some methods scored high in all technical aspects, none obtained fully correct solutions. We found that methods that either take prior information into account using learning strategies or analyze cells in a global spatiotemporal video context performed better than other methods under the segmentation and tracking scenarios included in the challenge.
|
|
| 24. |
- Vickovic, Sanja, et al.
(författare)
-
High-definition spatial transcriptomics for in situ tissue profiling
- 2019
-
Ingår i: Nature Methods. - : NATURE PUBLISHING GROUP. - 1548-7091 .- 1548-7105. ; 16:10, s. 987-
-
Tidskriftsartikel (refereegranskat)abstract
- Spatial and molecular characteristics determine tissue function, yet high-resolution methods to capture both concurrently are lacking. Here, we developed high-definition spatial transcriptomics, which captures RNA from histological tissue sections on a dense, spatially barcoded bead array. Each experiment recovers several hundred thousand transcriptcoupled spatial barcodes at 2-mu m resolution, as demonstrated in mouse brain and primary breast cancer. This opens the way to high-resolution spatial analysis of cells and tissues.
|
|
| 25. |
|
|
