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Iseli C, Searle SM, Mortazavi A, Sammeth M, Gerstein M, Leng J, Valsesia A, Schulz MH, Rozowsky J, Zerbino D, Bertone P, Kahles A, Du J, Solorzano ND, Stanke M, Lewis S, Ratsch G, Hubbard TJ, Wu TD, Cloonan N, Gingeras TR, Derrien T, Stockinger H, Guigo R, Stevenson BJ, Antonarakis SE, Engstrom PG, Abril JF, Zeller G, Djebali S, Sboner A, Richard H, Wold BJ, Habegger L, Niermann P, Akerman M, Wu J, Jean G, Zhang MQ, Steijger T, Harrow J, Rougemont J, Kokocinski F, Solovyev V, Reymond A, Weese D, Behr J, Bucher P, Gonzalez D, Grimmond SM, Ambrosini G, Ribeca P, Alioto T, Bohnert R, White S, Dudoit S, Lefebvre G, Lagarde J
[
Nat Methods,
2013]
We evaluated 25 protocol variants of 14 independent computational methods for exon identification, transcript reconstruction and expression-level quantification from RNA-seq data. Our results show that most algorithms are able to identify discrete transcript components with high success rates but that assembly of complete isoform structures poses a major challenge even when all constituent elements are identified. Expression-level estimates also varied widely across methods, even when based on similar transcript models. Consequently, the complexity of higher eukaryotic genomes imposes severe limitations on transcript recall and splice product discrimination that are likely to remain limiting factors for the analysis of current-generation RNA-seq data.
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[
J Comput Biol,
2011]
Biological networks reconstruction is a crucial step towards the functional characterization and elucidation of living cells. Computational methods for inferring the structure of these networks are of paramount importance since they provide valuable information regarding organization and behavior of the cell at a system level and also enable careful design of wet-lab experiments. Despite many recent advances, according to the scientific literature, there is room for improvements from both the efficiency and the accuracy point of view in link prediction algorithms. In this article, we propose a new method for the inference of biological networks that makes use of a notion of similarity between graph vertices within the framework of graph regularization for ranking the links to be predicted. The proposed approach results in more accurate classification rates in a wide range of experiments, while the computational complexity is reduced by two orders of magnitude with respect to many current state-of-the-art algorithms.
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[
Dev Biol,
2017]
The Caenorhabditis elegans germline is widely used as a model to study stem cell development, chromosome dynamics and apoptosis. Major readouts of germline phenotypes such as cell counting and protein expression profiling are routinely analysed manually and in a two-dimensional manner. The major disadvantages of the existing approaches are 1) they are time-consuming and laborious and 2) there is an inability to study the effects of genetic mutations in three dimensions. Here, we demonstrate a rapid, automated method for analysing the three-dimensional distribution of proteins, germline nuclei and cytoskeletal structures in the C. elegans germline. Using this method, we have revealed previously unappreciated germline organisation and cytoskeletal structures that will have a major impact on the characterisation of germline phenotypes. To conclude, our new method dramatically enhances the efficiency and resolution of C. elegans germline analysis and may be applied to other cellular structures.
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Schuster S, Zarse K, Brandes S, Zamboni N, Gebauer J, SchmeiBer K, Waschina S, Mansfeld J, Gentsch C, Ristow M, Klimmasch L, Kaleta C, Schauble S
[
Cell Syst,
2016]
We present a genome-scale model of Caenorhabditis elegans metabolism along with the public database ElegCyc
(http://elegcyc.bioinf.uni-jena.de:1100), which represents a reference for metabolic pathways in the worm and allows for the visualization as well as analysis of omics datasets. Ourmodel reflects the metabolic peculiarities of C.elegans that make it distinct from other higher eukaryotes and mammals, including mice and humans. We experimentally verify one of these peculiarities by showing that the lifespan-extending effect of L-tryptophan supplementation is dose dependent (hormetic). Finally, we show the utility of our model for analyzing omics datasets through predicting changes in amino acid concentrations after genetic perturbations and analyzing metabolic changes during normal aging as well as during twodistinct, reactive oxygen species (ROS)-related lifespan-extending treatments. Our analyses reveal a notable similarity in metabolic adaptation between distinct lifespan-extending interventions and point to key pathways affecting lifespan in nematodes.
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[
Parasit Vectors,
2016]
BACKGROUND: In the present study, we reconstructed the insulin/insulin-like growth factor 1 signalling (IIS) pathway for Haemonchus contortus, which is one of the most important eukaryotic pathogens of livestock worldwide and is related to the free-living nematode Caenorhabditis elegans. METHODS: We curated full-length open-reading frames from assembled transcripts, defined the complement of genes that encode proteins involved in this pathway and then investigated the transcription profiles of these genes for all key developmental stages of H. contortus. RESULTS: The core components of the IIS pathway are similar to their respective homologs in C. elegans. However, there is considerable variation in the numbers of isoforms between H. contortus and C. elegans and an absence of AKT-2 and DDL-2 homologs from H. contortus. Interestingly, DAF-16 has a single isoform in H. contortus compared with 12 in C. elegans, suggesting novel functional roles in the parasitic nematode. Some IIS proteins, such as DAF-18 and SGK-1, vary in their functional domains, indicating distinct roles from their homologs in C. elegans. CONCLUSIONS: This study paves the way for the further characterization of key signalling pathways in other socioeconomically important parasites and should help understand the complex mechanisms involved in developmental processes.
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[
Biotechniques,
1998]
We present a novel approach to the viewing and analysis of 4-dimensional (4-D) data sets recorded from live fluorescent samples. With stereo-4-D reconstructions, the observer manipulates a rotatable projection of the full 3-dimensional (3-D) specimen while simultaneously controlling animation of the recording forward or backward in time. The result is a unique lifelike perspective on the development of an entire living subject. Here, we apply this technique to the observation of the cell membranes of developing Caenorhabditis elegans. Embryos labeled with the vital plasma membrane probe FM 4-64 were imaged by multiphoton laser scanning fluorescence microscopy, yielding 4-D data sets of entire embryos over several hours of development. Stereo 4-D and standard focal-plane 4-D viewing of these novel time-lapse recordings provide the observer with detail at both the subcellular and whole-animal level from a single data set and produce a unique record of the lineage, cell shape changes, cell contacts and morphogenetic dynamics that make up embryogenesis. The procedures by which stereo-4-D reconstructions are created and viewed rely on public domain software running on a personal computer and should therefore be accessible by a general audience. Data output utilizes the versatile and well-supported QuickTime animation format. Additional features allow for stereo-4-D reconstruction of isolated 3-D volumes of interest from within the larger specimen.
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[
J Cell Sci,
2010]
Centrioles are highly conserved structures that fulfil important cellular functions, such as nucleation of cilia and flagella (basal-body function) and organisation of pericentriolar material to form the centrosome. The evolution of these functions can be inferred from the distribution of the molecular components of extant centrioles and centrosomes. Here, we undertake an evolutionary analysis of 53 proteins known either for centriolar association or for involvement in cilia-associated pathologies. By linking protein distribution in 45 diverse eukaryotes with organism biology, we provide molecular evidence to show that basal-body function is ancestral, whereas the presence of the centrosome is specific to the Holozoa. We define an ancestral centriolar inventory of 14 core proteins, Polo-like-kinase, and proteins associated with Bardet-Biedl syndrome (BBS) and Meckel-Gruber syndrome. We show that the BBSome is absent from organisms that produce cilia only for motility, predicting a dominant and ancient role for this complex in sensory function. We also show that the unusual centriole of Caenorhabditis elegans is highly divergent in both protein composition and sequence. Finally, we demonstrate a correlation between the presence of specific centriolar proteins and eye evolution. This correlation is used to predict proteins with functions in the development of ciliary, but not rhabdomeric, eyes.
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[
Evol Dev,
2022]
Pristionchus pacificus is a nematode model for the developmental genetics of morphological polyphenism, especially at the level of individual cells. Morphological polyphenism in this species includes an evolutionary novelty, moveable teeth, which have enabled predatory feeding in this species and others in its family (Diplogastridae). From transmission electron micrographs of serial thin sections through an adult hermaphrodite of P. pacificus, we three-dimensionally reconstructed all epithelial and myoepithelial cells and syncytia, corresponding to 74 nuclei, of its face, mouth, and pharynx. We found that the epithelia that produce the predatory morphology of P. pacificus are identical to Caenorhabditis elegans in the number of cell classes and nuclei. However, differences in cell form, spatial relationships, and nucleus position correlate with gross morphological differences from C. elegans and outgroups. Moreover, we identified fine-structural features, especially in the anteriormost pharyngeal muscles, that underlie the conspicuous, left-right asymmetry that characterizes the P. pacificus feeding apparatus. Our reconstruction provides an anatomical map for studying the genetics of polyphenism, feeding behavior, and the development of novel form in a satellite model to C. elegans.
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[
Nat Methods,
2012]
Anatomy of large biological specimens is often reconstructed from serially sectioned volumes imaged by high-resolution microscopy. We developed a method to reassemble a continuous volume from such large section series that explicitly minimizes artificial deformation by applying a global elastic constraint. We demonstrate our method on a series of transmission electron microscopy sections covering the entire 558-cell Caenorhabditis elegans embryo and a segment of the Drosophila melanogaster larval ventral nerve cord.
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[
Methods Cell Biol,
2010]
The roundworm Caenorhabditis elegans is one of the major model organisms in modern cell and developmental biology. Here, we present methods for the three-dimensional (3D) reconstruction of the worm ultrastructure. We describe the use of (1) serial-section analysis, (2) electron tomography, and (3) serial block face imaging by scanning electron microscopy (SEM). Sample preparation for high-pressure freezing/freeze substitution (HPF/FS) has been extensively covered in a previous volume of this "Methods in Cell Biology" series and will only be described briefly. We will discuss these 3D methods in light of recent research activities related to worm and early embryo biology.