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1.  Specific roles for the GATA transcription factors end-1 and end-3 during C. elegans E-lineage development 
Developmental biology  2011;358(2):345-355.
end-1 and end-3 are GATA transcription factors important for specifying endoderm cell fate in Caenorhabditis elegans. Deletion of both factors together results in larval arrest, 0% survival and a fate change in the endoderm-specifying E lineage. Individual deletions of either factor, however, result in the development of viable, fertile adults, with 100% of worms developing to adults for end-1(−) and 95% for end-3(−). We sought to quantify the variable phenotypes seen in both deletions using automated cell lineaging. We quantified defects in cell lifetime, cell movement and division axis in end-3(−) embryos, while quantifying perturbations in downstream reporter gene expression in strains with homozygous deletions for either gene, showing that each deletion leads to a unique profile of downstream perturbations in gene expression and cellular phenotypes with a high correlation between early and late defects. Combining observations in both cellular and gene expression defects we found that misaligned divisions at the E2 stage resulted in ectopic expression of the Notch target ref-1 in end-3(−) embryos. Using a maximum likelihood phylogenetic approach we found end-1 and end-3 split to form two distinct clades within the Caenorhabditis lineage with distinct DNA-binding structures. These results indicate that end-1 and end-3 have each evolved into genes with unique functions during endoderm development, that end-3(−) embryos have a delay in the onset of E lineage cell fate and that end-1 has only a partially penetrant ability to activate E lineage fate.
doi:10.1016/j.ydbio.2011.08.002
PMCID: PMC4454387  PMID: 21854766
C. elegans; Endoderm; GATA factors; Gene expression; Cell fate; Cell migration; Gastrulation
2.  Remarkably Divergent Regions Punctuate the Genome Assembly of the Caenorhabditis elegans Hawaiian Strain CB4856 
Genetics  2015;200(3):975-989.
The Hawaiian strain (CB4856) of Caenorhabditis elegans is one of the most divergent from the canonical laboratory strain N2 and has been widely used in developmental, population, and evolutionary studies. To enhance the utility of the strain, we have generated a draft sequence of the CB4856 genome, exploiting a variety of resources and strategies. When compared against the N2 reference, the CB4856 genome has 327,050 single nucleotide variants (SNVs) and 79,529 insertion–deletion events that result in a total of 3.3 Mb of N2 sequence missing from CB4856 and 1.4 Mb of sequence present in CB4856 but not present in N2. As previously reported, the density of SNVs varies along the chromosomes, with the arms of chromosomes showing greater average variation than the centers. In addition, we find 61 regions totaling 2.8 Mb, distributed across all six chromosomes, which have a greatly elevated SNV density, ranging from 2 to 16% SNVs. A survey of other wild isolates show that the two alternative haplotypes for each region are widely distributed, suggesting they have been maintained by balancing selection over long evolutionary times. These divergent regions contain an abundance of genes from large rapidly evolving families encoding F-box, MATH, BATH, seven-transmembrane G-coupled receptors, and nuclear hormone receptors, suggesting that they provide selective advantages in natural environments. The draft sequence makes available a comprehensive catalog of sequence differences between the CB4856 and N2 strains that will facilitate the molecular dissection of their phenotypic differences. Our work also emphasizes the importance of going beyond simple alignment of reads to a reference genome when assessing differences between genomes.
doi:10.1534/genetics.115.175950
PMCID: PMC4512556  PMID: 25995208
C. elegans; genome assembly; evolution; variation
3.  Blinded by the light: why the treatment of metastatic melanoma has created a new paradigm for the management of cancer 
Until recently, treatment for metastatic melanoma was characterised by a limited availability of treatment options that offer objective survival benefit. Cytotoxic agents fundamentally lack the ability to achieve disease control and cytokine therapy with interleukin-2 has an unacceptably high – for the use across all patient cohorts – rate of toxicities. The validation of braf as an oncogene driving melanoma tumorigenesis, as well as the discovery of the role of CTLA-4 receptor in the evasion of anticancer immune response by melanoma, has revolutionised our treatment options against a disease with dismal prognosis. Quick implementation of translational discoveries brought about BRAF/MEK inhibition in clinic, while at the same time, wider experience with CTLA-4 blockade enabled clinicians to manage previously fatal immune-related toxicities with greater confidence. The suitability for clinical use of other oncogenic drivers such as NRAS and c-kit is currently being tested whilst the PD-1/PD-L1/PD-L2 axis has emerged as a new immunotherapy target with exciting early phase results. The recent exponential progress in treatment of melanoma has set an example of translational medicine and the current review aims to explain why, as well as suggesting new goals for the future.
doi:10.1177/1758834014566619
PMCID: PMC4346213  PMID: 25755683
BRAF/MEK inhibition; ipilimumab; metastatic melanoma; molecularly targeted treatment; PD-1/PD-L1/PD-L2 axis
4.  Comparative Analysis of the Transcriptome across Distant Species 
Gerstein, Mark B. | Rozowsky, Joel | Yan, Koon-Kiu | Wang, Daifeng | Cheng, Chao | Brown, James B. | Davis, Carrie A | Hillier, LaDeana | Sisu, Cristina | Li, Jingyi Jessica | Pei, Baikang | Harmanci, Arif O. | Duff, Michael O. | Djebali, Sarah | Alexander, Roger P. | Alver, Burak H. | Auerbach, Raymond | Bell, Kimberly | Bickel, Peter J. | Boeck, Max E. | Boley, Nathan P. | Booth, Benjamin W. | Cherbas, Lucy | Cherbas, Peter | Di, Chao | Dobin, Alex | Drenkow, Jorg | Ewing, Brent | Fang, Gang | Fastuca, Megan | Feingold, Elise A. | Frankish, Adam | Gao, Guanjun | Good, Peter J. | Guigó, Roderic | Hammonds, Ann | Harrow, Jen | Hoskins, Roger A. | Howald, Cédric | Hu, Long | Huang, Haiyan | Hubbard, Tim J. P. | Huynh, Chau | Jha, Sonali | Kasper, Dionna | Kato, Masaomi | Kaufman, Thomas C. | Kitchen, Robert R. | Ladewig, Erik | Lagarde, Julien | Lai, Eric | Leng, Jing | Lu, Zhi | MacCoss, Michael | May, Gemma | McWhirter, Rebecca | Merrihew, Gennifer | Miller, David M. | Mortazavi, Ali | Murad, Rabi | Oliver, Brian | Olson, Sara | Park, Peter J. | Pazin, Michael J. | Perrimon, Norbert | Pervouchine, Dmitri | Reinke, Valerie | Reymond, Alexandre | Robinson, Garrett | Samsonova, Anastasia | Saunders, Gary I. | Schlesinger, Felix | Sethi, Anurag | Slack, Frank J. | Spencer, William C. | Stoiber, Marcus H. | Strasbourger, Pnina | Tanzer, Andrea | Thompson, Owen A. | Wan, Kenneth H. | Wang, Guilin | Wang, Huaien | Watkins, Kathie L. | Wen, Jiayu | Wen, Kejia | Xue, Chenghai | Yang, Li | Yip, Kevin | Zaleski, Chris | Zhang, Yan | Zheng, Henry | Brenner, Steven E. | Graveley, Brenton R. | Celniker, Susan E. | Gingeras, Thomas R | Waterston, Robert
Nature  2014;512(7515):445-448.
doi:10.1038/nature13424
PMCID: PMC4155737  PMID: 25164755
5.  Comparative analysis of regulatory information and circuits across distant species 
Nature  2014;512(7515):453-456.
Summary
Despite the large evolutionary distances, metazoan species show remarkable commonalities, which has helped establish fly and worm as model organisms for human biology1,2. Although studies of individual elements and factors have explored similarities in gene regulation, a large-scale comparative analysis of basic principles of transcriptional regulatory features is lacking. We mapped the genome-wide binding locations of 165 human, 93 worm, and 52 fly transcription-regulatory factors (RFs) generating a total of 1,019 data sets from diverse cell-types, developmental stages, or conditions in the three species, of which 498 (48.9%) are presented here for the first time. We find that structural properties of regulatory networks are remarkably conserved and that orthologous RF families recognize similar binding motifs in vivo and show some similar co-associations. Our results suggest that gene-regulatory properties previously observed for individual factors are general principles of metazoan regulation that are remarkably well-preserved despite extensive functional divergence of individual network connections. The comparative maps of regulatory circuitry provided here will drive an improved understanding in the regulatory underpinnings of model organism biology and how these relate to human biology, development, and disease.
doi:10.1038/nature13668
PMCID: PMC4336544  PMID: 25164757
Transcription Factor; Regulatory Information; Gene Regulation; Single Nucleotide Polymorphisms; ChIP-seq
6.  Isolation of Specific Neurons from C. elegans Larvae for Gene Expression Profiling 
PLoS ONE  2014;9(11):e112102.
Background
The simple and well-described structure of the C. elegans nervous system offers an unprecedented opportunity to identify the genetic programs that define the connectivity and function of individual neurons and their circuits. A correspondingly precise gene expression map of C. elegans neurons would facilitate the application of genetic methods toward this goal. Here we describe a powerful new approach, SeqCeL (RNA-Seq of C. elegans cells) for producing gene expression profiles of specific larval C. elegans neurons.
Methods and Results
We have exploited available GFP reporter lines for FACS isolation of specific larval C. elegans neurons for RNA-Seq analysis. Our analysis showed that diverse classes of neurons are accessible to this approach. To demonstrate the applicability of this strategy to rare neuron types, we generated RNA-Seq profiles of the NSM serotonergic neurons that occur as a single bilateral pair of cells in the C. elegans pharynx. These data detected >1,000 NSM enriched transcripts, including the majority of previously known NSM-expressed genes.
Significance
This work offers a simple and robust protocol for expression profiling studies of post-embryonic C. elegans neurons and thus provides an important new method for identifying candidate genes for key roles in neuron-specific development and function.
doi:10.1371/journal.pone.0112102
PMCID: PMC4221280  PMID: 25372608
7.  A genome scale resource for in vivo tag-based protein function exploration in C. elegans 
Cell  2012;150(4):855-866.
Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in Biology. To enable systematic protein function interrogation in a multicelluar context, we built a genome-scale transgenic platform for in vivo expression of fluorescent and affinity tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering and next generation sequencing to generate a resource of 14637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins
doi:10.1016/j.cell.2012.08.001
PMCID: PMC3979301  PMID: 22901814
8.  A High-Fidelity Cell Lineage Tracing Method for Obtaining Systematic Spatiotemporal Gene Expression Patterns in Caenorhabditis elegans 
G3: Genes|Genomes|Genetics  2013;3(5):851-863.
Advances in microscopy and fluorescent reporters have allowed us to detect the onset of gene expression on a cell-by-cell basis in a systematic fashion. This information, however, is often encoded in large repositories of images, and developing ways to extract this spatiotemporal expression data is a difficult problem that often uses complex domain-specific methods for each individual data set. We present a more unified approach that incorporates general previous information into a hierarchical probabilistic model to extract spatiotemporal gene expression from 4D confocal microscopy images of developing Caenorhabditis elegans embryos. This approach reduces the overall error rate of our automated lineage tracing pipeline by 3.8-fold, allowing us to routinely follow the C. elegans lineage to later stages of development, where individual neuronal subspecification becomes apparent. Unlike previous methods that often use custom approaches that are organism specific, our method uses generalized linear models and extensions of standard reversible jump Markov chain Monte Carlo methods that can be readily extended to other organisms for a variety of biological inference problems relating to cell fate specification. This modeling approach is flexible and provides tractable avenues for incorporating additional previous information into the model for similar difficult high-fidelity/low error tolerance image analysis problems for systematically applied genomic experiments.
doi:10.1534/g3.113.005918
PMCID: PMC3656732  PMID: 23550142
C. elegans; cell fate; gene expression; image analysis; lineage
9.  The effect of robenacoxib on the concentration of C-reactive protein in synovial fluid from dogs with osteoarthritis 
Background
Robenacoxib is a novel and highly selective inhibitor of COX-2 in dogs and cats and because of its acidic nature is regarded as being tissue-selective. Thirty four dogs with stifle osteoarthritis secondary to failure of the cranial cruciate ligament were recruited into this study. Lameness, radiographic features, synovial cytology and C-reactive protein concentrations in serum and synovial fluid were assessed before and 28 days after commencing a course of Robenacoxib at a dose of 1 mg/kg SID.
Results
There was a significant reduction in the lameness score (P < 0.01) and an increase in the radiographic score (P < 0.05) between pre- and post-treatment assessments. There was no difference between pre- (median 1.49 mg/l; Q1-Q3 0.56-4.24 mg/L) and post – (1.10 mg/L; 0.31-1.78 mg/L) treatment serum C-reactive protein levels although synovial fluid levels were significantly reduced (pre- : 0.44 mg/L; 0.23-1.62 mg/L; post- : 0.17 mg/L; 0.05-0.49 mg/L) (P < 0.05). There was no correlation between C-reactive protein concentrations in serum and matched synovial fluid samples.
Conclusions
Robenacoxib proved effective in reducing lameness in dogs with failure of the cranial cruciate ligament and osteoarthritis of the stifle joint. The drug also reduced levels of C-reactive protein in the synovial fluid taken from the affected stifle joint. Robenacoxib appears to reduce articular inflammation as assessed by C-reactive protein which supports the concept that Robenacoxib is a tissue-selective non-steroidal anti-inflammatory drug.
doi:10.1186/1746-6148-9-42
PMCID: PMC3610148  PMID: 23452411
Stifle; Osteoarthritis; Cruciate disease; C-reactive protein; Synovial fluid; Robenacoxib
10.  Evidence for compensatory upregulation of expressed X-linked genes in mammals, Caenorhabditis elegans and Drosophila melanogaster 
Nature genetics  2011;43(12):1179-1185.
Many animal species use a chromosome-based mechanism of sex determination, which has led to the coordinate evolution of dosage-compensation systems. Dosage compensation not only corrects the imbalance in the number of X chromosomes between the sexes but also is hypothesized to correct dosage imbalance within cells that is due to monoallelic X-linked expression and biallelic autosomal expression, by upregulating X-linked genes twofold (termed ‘Ohno’s hypothesis’). Although this hypothesis is well supported by expression analyses of individual X-linked genes and by microarray-based transcriptome analyses, it was challenged by a recent study using RNA sequencing and proteomics. We obtained new, independent RNA-seq data, measured RNA polymerase distribution and reanalyzed published expression data in mammals, C. elegans and Drosophila. Our analyses, which take into account the skewed gene content of the X chromosome, support the hypothesis of upregulation of expressed X-linked genes to balance expression of the genome.
doi:10.1038/ng.948
PMCID: PMC3576853  PMID: 22019781
11.  Genetic Control of Vulval Development in Caenorhabditis briggsae 
G3: Genes|Genomes|Genetics  2012;2(12):1625-1641.
The nematode Caenorhabditis briggsae is an excellent model organism for the comparative analysis of gene function and developmental mechanisms. To study the evolutionary conservation and divergence of genetic pathways mediating vulva formation, we screened for mutations in C. briggsae that cause the egg-laying defective (Egl) phenotype. Here, we report the characterization of 13 genes, including three that are orthologs of Caenorhabditis elegans unc-84 (SUN domain), lin-39 (Dfd/Scr-related homeobox), and lin-11 (LIM homeobox). Based on the morphology and cell fate changes, the mutants were placed into four different categories. Class 1 animals have normal-looking vulva and vulva-uterine connections, indicating defects in other components of the egg-laying system. Class 2 animals frequently lack some or all of the vulval precursor cells (VPCs) due to defects in the migration of P-cell nuclei into the ventral hypodermal region. Class 3 animals show inappropriate fusion of VPCs to the hypodermal syncytium, leading to a reduced number of vulval progeny. Finally, class 4 animals exhibit abnormal vulval invagination and morphology. Interestingly, we did not find mutations that affect VPC induction and fates. Our work is the first study involving the characterization of genes in C. briggsae vulva formation, and it offers a basis for future investigations of these genes in C. elegans.
doi:10.1534/g3.112.004598
PMCID: PMC3516484  PMID: 23275885
C. briggsae; C. elegans; vulva; development; cell proliferation; differentiation; morphogenesis; egg-laying defective
12.  Sensory neuropathy as part of the cerebellar ataxia neuropathy vestibular areflexia syndrome 
Neurology  2011;76(22):1903-1910.
Objective:
The syndrome of cerebellar ataxia with bilateral vestibulopathy was delineated in 2004. Sensory neuropathy was mentioned in 3 of the 4 patients described. We aimed to characterize and estimate the frequency of neuropathy in this condition, and determine its typical MRI features.
Methods:
Retrospective review of 18 subjects (including 4 from the original description) who met the criteria for bilateral vestibulopathy with cerebellar ataxia.
Results:
The reported age at onset range was 39–71 years, and symptom duration was 3–38 years. The syndrome was identified in one sibling pair, suggesting that this may be a late-onset recessive disorder, although the other 16 cases were apparently sporadic. All 18 had sensory neuropathy with absent sensory nerve action potentials, although this was not apparent clinically in 2, and the presence of neuropathy was not a selection criterion. In 5, the loss of pinprick sensation was virtually global, mimicking a neuronopathy. However, findings in the other 11 with clinically manifest neuropathy suggested a length-dependent neuropathy. MRI scans showed cerebellar atrophy in 16, involving anterior and dorsal vermis, and hemispheric crus I, while 2 were normal. The inferior vermis and brainstem were spared.
Conclusions:
Sensory neuropathy is an integral component of this syndrome. It may result in severe sensory loss, which contributes significantly to the disability. The MRI changes are nonspecific, but, coupled with loss of sensory nerve action potentials, may aid diagnosis. We propose a new name for the condition: cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Neurology® 2011;76:1903–1910
doi:10.1212/WNL.0b013e31821d746e
PMCID: PMC3115806  PMID: 21624989
13.  Integrative Analysis of the Caenorhabditis elegans Genome by the modENCODE Project 
Gerstein, Mark B. | Lu, Zhi John | Van Nostrand, Eric L. | Cheng, Chao | Arshinoff, Bradley I. | Liu, Tao | Yip, Kevin Y. | Robilotto, Rebecca | Rechtsteiner, Andreas | Ikegami, Kohta | Alves, Pedro | Chateigner, Aurelien | Perry, Marc | Morris, Mitzi | Auerbach, Raymond K. | Feng, Xin | Leng, Jing | Vielle, Anne | Niu, Wei | Rhrissorrakrai, Kahn | Agarwal, Ashish | Alexander, Roger P. | Barber, Galt | Brdlik, Cathleen M. | Brennan, Jennifer | Brouillet, Jeremy Jean | Carr, Adrian | Cheung, Ming-Sin | Clawson, Hiram | Contrino, Sergio | Dannenberg, Luke O. | Dernburg, Abby F. | Desai, Arshad | Dick, Lindsay | Dosé, Andréa C. | Du, Jiang | Egelhofer, Thea | Ercan, Sevinc | Euskirchen, Ghia | Ewing, Brent | Feingold, Elise A. | Gassmann, Reto | Good, Peter J. | Green, Phil | Gullier, Francois | Gutwein, Michelle | Guyer, Mark S. | Habegger, Lukas | Han, Ting | Henikoff, Jorja G. | Henz, Stefan R. | Hinrichs, Angie | Holster, Heather | Hyman, Tony | Iniguez, A. Leo | Janette, Judith | Jensen, Morten | Kato, Masaomi | Kent, W. James | Kephart, Ellen | Khivansara, Vishal | Khurana, Ekta | Kim, John K. | Kolasinska-Zwierz, Paulina | Lai, Eric C. | Latorre, Isabel | Leahey, Amber | Lewis, Suzanna | Lloyd, Paul | Lochovsky, Lucas | Lowdon, Rebecca F. | Lubling, Yaniv | Lyne, Rachel | MacCoss, Michael | Mackowiak, Sebastian D. | Mangone, Marco | McKay, Sheldon | Mecenas, Desirea | Merrihew, Gennifer | Miller, David M. | Muroyama, Andrew | Murray, John I. | Ooi, Siew-Loon | Pham, Hoang | Phippen, Taryn | Preston, Elicia A. | Rajewsky, Nikolaus | Rätsch, Gunnar | Rosenbaum, Heidi | Rozowsky, Joel | Rutherford, Kim | Ruzanov, Peter | Sarov, Mihail | Sasidharan, Rajkumar | Sboner, Andrea | Scheid, Paul | Segal, Eran | Shin, Hyunjin | Shou, Chong | Slack, Frank J. | Slightam, Cindie | Smith, Richard | Spencer, William C. | Stinson, E. O. | Taing, Scott | Takasaki, Teruaki | Vafeados, Dionne | Voronina, Ksenia | Wang, Guilin | Washington, Nicole L. | Whittle, Christina M. | Wu, Beijing | Yan, Koon-Kiu | Zeller, Georg | Zha, Zheng | Zhong, Mei | Zhou, Xingliang | Ahringer, Julie | Strome, Susan | Gunsalus, Kristin C. | Micklem, Gos | Liu, X. Shirley | Reinke, Valerie | Kim, Stuart K. | Hillier, LaDeana W. | Henikoff, Steven | Piano, Fabio | Snyder, Michael | Stein, Lincoln | Lieb, Jason D. | Waterston, Robert H.
Science (New York, N.Y.)  2010;330(6012):1775-1787.
We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.
doi:10.1126/science.1196914
PMCID: PMC3142569  PMID: 21177976
14.  A Comprehensive Analysis of Gene Expression Changes Provoked by Bacterial and Fungal Infection in C. elegans 
PLoS ONE  2011;6(5):e19055.
While Caenorhabditis elegans specifically responds to infection by the up-regulation of certain genes, distinct pathogens trigger the expression of a common set of genes. We applied new methods to conduct a comprehensive and comparative study of the transcriptional response of C. elegans to bacterial and fungal infection. Using tiling arrays and/or RNA-sequencing, we have characterized the genome-wide transcriptional changes that underlie the host's response to infection by three bacterial (Serratia marcescens, Enterococcus faecalis and otorhabdus luminescens) and two fungal pathogens (Drechmeria coniospora and Harposporium sp.). We developed a flexible tool, the WormBase Converter (available at http://wormbasemanager.sourceforge.net/), to allow cross-study comparisons. The new data sets provided more extensive lists of differentially regulated genes than previous studies. Annotation analysis confirmed that genes commonly up-regulated by bacterial infections are related to stress responses. We found substantial overlaps between the genes regulated upon intestinal infection by the bacterial pathogens and Harposporium, and between those regulated by Harposporium and D. coniospora, which infects the epidermis. Among the fungus-regulated genes, there was a significant bias towards genes that are evolving rapidly and potentially encode small proteins. The results obtained using new methods reveal that the response to infection in C. elegans is determined by the nature of the pathogen, the site of infection and the physiological imbalance provoked by infection. They form the basis for future functional dissection of innate immune signaling. Finally, we also propose alternative methods to identify differentially regulated genes that take into account the greater variability in lowly expressed genes.
doi:10.1371/journal.pone.0019055
PMCID: PMC3094335  PMID: 21602919
15.  Oxaliplatin-induced coronary artery spasm: first report of an important side-effect 
BMJ Case Reports  2009;2009:bcr06.2008.0334.
A 67-year-old woman with metastatic colorectal cancer was given her first oxaliplatin infusion as part of the XELOX protocol. She developed chest pain with ECG changes leading subsequently to a diagnosis of coronary artery spasm. To our knowledge, this is the first report of oxaliplatin-induced coronary artery spasm.
doi:10.1136/bcr.06.2008.0334
PMCID: PMC3028146  PMID: 21686854
16.  A Negative Regulatory Loop between MicroRNA and Hox Gene Controls Posterior Identities in Caenorhabditis elegans 
PLoS Genetics  2010;6(9):e1001089.
MicroRNAs (miRNAs) have been found to regulate gene expression across eukaryotic species, but the function of most miRNA genes remains unknown. Here we describe how the analysis of the expression patterns of a well-conserved miRNA gene, mir-57, at cellular resolution for every minute during early development of Caenorhabditis elegans provided key insights in understanding its function. Remarkably, mir-57 expression shows strong positional bias but little tissue specificity, a pattern reminiscent of Hox gene function. Despite the minor defects produced by a loss of function mutation, overexpression of mir-57 causes dramatic posterior defects, which also mimic the phenotypes of mutant alleles of a posterior Hox gene, nob-1, an Abd homolog. More importantly, nob-1 expression is found in the same two posterior AB sublineages as those expressing mir-57 but with an earlier onset. Intriguingly, nob-1 functions as an activator for mir-57 expression; it is also a direct target of mir-57. In agreement with this, loss of mir-57 function partially rescues the nob-1 allele defects, indicating a negative feedback regulatory loop between the miRNA and Hox gene to provide positional cues. Given the conservation of the miRNA and Hox gene, the regulatory mechanism might be broadly used across species. The strategy used here to explore mir-57 function provides a path to dissect the regulatory relationship between genes.
Author Summary
miRNAs are small RNAs found in many multi-cellular species that inhibit gene expression. Many of them play important roles in cancer and cell fate determination, but the function of most miRNAs is uncertain. Using live cell imaging and automated expression analysis, we found a miRNA gene, mir-57, is expressed in a position rather than tissue dependent way. Hox genes also regulate cell fate patterning along anterior-posterior (a-p) axis across different tissues. By investigating interactions between genes of these classes expressed in mir-57 expressing cells, we demonstrated by both genetic analysis and gene expression assays that a negative feedback loop between a posterior Hox gene, nob-1, and mir-57 regulates posterior cell fate determination in C. elegans. On the one hand, the Hox gene is required for normal activation of mir-57 expression, and on the other, the Hox gene functions as a direct target of and is repressed by the miRNA. Given the conservation of the two genes, a negative feedback loop between Hox and miRNA genes might be broadly used across species to regulate cell fate along the a-p axis. Detailed expression analysis may provide a general way to dissect the regulatory role of miRNAs.
doi:10.1371/journal.pgen.1001089
PMCID: PMC2932687  PMID: 20824072
17.  Comparison and calibration of transcriptome data from RNA-Seq and tiling arrays 
BMC Genomics  2010;11:383.
Background
Tiling arrays have been the tool of choice for probing an organism's transcriptome without prior assumptions about the transcribed regions, but RNA-Seq is becoming a viable alternative as the costs of sequencing continue to decrease. Understanding the relative merits of these technologies will help researchers select the appropriate technology for their needs.
Results
Here, we compare these two platforms using a matched sample of poly(A)-enriched RNA isolated from the second larval stage of C. elegans. We find that the raw signals from these two technologies are reasonably well correlated but that RNA-Seq outperforms tiling arrays in several respects, notably in exon boundary detection and dynamic range of expression. By exploring the accuracy of sequencing as a function of depth of coverage, we found that about 4 million reads are required to match the sensitivity of two tiling array replicates. The effects of cross-hybridization were analyzed using a "nearest neighbor" classifier applied to array probes; we describe a method for determining potential "black list" regions whose signals are unreliable. Finally, we propose a strategy for using RNA-Seq data as a gold standard set to calibrate tiling array data. All tiling array and RNA-Seq data sets have been submitted to the modENCODE Data Coordinating Center.
Conclusions
Tiling arrays effectively detect transcript expression levels at a low cost for many species while RNA-Seq provides greater accuracy in several regards. Researchers will need to carefully select the technology appropriate to the biological investigations they are undertaking. It will also be important to reconsider a comparison such as ours as sequencing technologies continue to evolve.
doi:10.1186/1471-2164-11-383
PMCID: PMC3091629  PMID: 20565764
18.  Improved Discrimination of Visual Stimuli Following Repetitive Transcranial Magnetic Stimulation 
PLoS ONE  2010;5(4):e10354.
Background
Repetitive transcranial magnetic stimulation (rTMS) at certain frequencies increases thresholds for motor-evoked potentials and phosphenes following stimulation of cortex. Consequently rTMS is often assumed to introduce a “virtual lesion” in stimulated brain regions, with correspondingly diminished behavioral performance.
Methodology/Principal Findings
Here we investigated the effects of rTMS to visual cortex on subjects' ability to perform visual psychophysical tasks. Contrary to expectations of a visual deficit, we find that rTMS often improves the discrimination of visual features. For coarse orientation tasks, discrimination of a static stimulus improved consistently following theta-burst stimulation of the occipital lobe. Using a reaction-time task, we found that these improvements occurred throughout the visual field and lasted beyond one hour post-rTMS. Low-frequency (1 Hz) stimulation yielded similar improvements. In contrast, we did not find consistent effects of rTMS on performance in a fine orientation discrimination task.
Conclusions/Significance
Overall our results suggest that rTMS generally improves or has no effect on visual acuity, with the nature of the effect depending on the type of stimulation and the task. We interpret our results in the context of an ideal-observer model of visual perception.
doi:10.1371/journal.pone.0010354
PMCID: PMC2860988  PMID: 20442776
19.  Unlocking the secrets of the genome 
Nature  2009;459(7249):927-930.
Despite the successes of genomics, little is known about how genetic information produces complex organisms. A look at the crucial functional elements of fly and worm genomes could change that.
doi:10.1038/459927a
PMCID: PMC2843545  PMID: 19536255
20.  Genome-Wide Identification of Binding Sites Defines Distinct Functions for Caenorhabditis elegans PHA-4/FOXA in Development and Environmental Response 
PLoS Genetics  2010;6(2):e1000848.
Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.
Author Summary
The C. elegans transcription factor PHA-4 is a member of the highly conserved FOXA family of transcription factors. These factors act as master regulators of organ development by controlling how genes are turned off and on as tissues are formed. Additionally they regulate genes in response to nutrient levels and control both longevity and survival of the organism. However, the extent to which these factors control similar or distinct gene targets for each of these functions is unknown. For this reason, we have used the technique of chromatin immunoprecipitation followed by deep sequencing (ChIP–Seq), to define the target binding sites of PHA-4 on a genome-wide scale, when it is either functioning as an organ identity regulator or in response to environmental stress. Our data clearly demonstrate distinct sets of biologically relevant target genes for the transcription factor PHA-4 under these two different conditions. Not only have we defined PHA-4 targets, but we established an experimental ChIP–Seq pipeline to facilitate the identification of binding sites for many transcription factors in the future.
doi:10.1371/journal.pgen.1000848
PMCID: PMC2824807  PMID: 20174564
21.  Using machine learning to speed up manual image annotation: application to a 3D imaging protocol for measuring single cell gene expression in the developing C. elegans embryo 
BMC Bioinformatics  2010;11:84.
Background
Image analysis is an essential component in many biological experiments that study gene expression, cell cycle progression, and protein localization. A protocol for tracking the expression of individual C. elegans genes was developed that collects image samples of a developing embryo by 3-D time lapse microscopy. In this protocol, a program called StarryNite performs the automatic recognition of fluorescently labeled cells and traces their lineage. However, due to the amount of noise present in the data and due to the challenges introduced by increasing number of cells in later stages of development, this program is not error free. In the current version, the error correction (i.e., editing) is performed manually using a graphical interface tool named AceTree, which is specifically developed for this task. For a single experiment, this manual annotation task takes several hours.
Results
In this paper, we reduce the time required to correct errors made by StarryNite. We target one of the most frequent error types (movements annotated as divisions) and train a support vector machine (SVM) classifier to decide whether a division call made by StarryNite is correct or not. We show, via cross-validation experiments on several benchmark data sets, that the SVM successfully identifies this type of error significantly. A new version of StarryNite that includes the trained SVM classifier is available at http://starrynite.sourceforge.net.
Conclusions
We demonstrate the utility of a machine learning approach to error annotation for StarryNite. In the process, we also provide some general methodologies for developing and validating a classifier with respect to a given pattern recognition task.
doi:10.1186/1471-2105-11-84
PMCID: PMC2838868  PMID: 20146825
22.  Why children's rights are central to international child health 
Archives of Disease in Childhood  2007;92(2):176-180.
The UN Convention on the Rights of the Child provides a framework for improving children's lives around the world. It covers both individual child health practice and public health and provides a unique and child‐centred approach to paediatric problems. The Convention applies to most child health problems and the articles are grouped into protection, provision and participation. Examples of the first are the right to protection from abuse, from economic exploitation and from illicit drugs. We examine one particular problem in each of these categories, specifically child labour, services for children with a disability and violence against children. The role of the paedialrician in applying a children's rights approach is discussed. Children's rights are increasingly being accepted around the world but still there is much more rhetoric paid to their value than genuine enforcement. Paediatricians can make a difference to the status of children worldwide by adopting a rights‐based approach.
doi:10.1136/adc.2006.098228
PMCID: PMC2083315  PMID: 17264288
23.  Comparative Analysis of Embryonic Cell Lineage between Caenorhabditis briggsae and C. elegans 
Developmental biology  2007;314(1):93-99.
Comparative genomic analysis of important signaling pathways in C. briggase and C. elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally-equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p < 0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.
doi:10.1016/j.ydbio.2007.11.015
PMCID: PMC2696483  PMID: 18164284
C. briggsae; C. elegans; embryo; cell lineage; signaling pathway
24.  Control of Cell Cycle Timing during C. elegans Embryogenesis 
Developmental biology  2008;318(1):65-72.
As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first eight out of ten rounds of embryonic cell division in C. elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P3 (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.
doi:10.1016/j.ydbio.2008.02.054
PMCID: PMC2442716  PMID: 18430415
statistics; single cell; fate differentiation; cdc25; Skp1-related

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