Identification of lignin genes and regulatory sequences involved in secondary cell wall formation in Acacia auriculiformis and Acacia mangium via de novo transcriptome sequencing

<p>Abstract</p> <p>Background</p> <p><it>Acacia auriculiformis </it>× <it>Acacia mangium </it>hybrids are commercially important trees for the timber and pulp industry in Southeast Asia. Increasing pulp yield while reducing pulping costs are major objectives of tree breeding programs. The general monolignol biosynthesis and secondary cell wall formation pathways are well-characterized but genes in these pathways are poorly characterized in <it>Acacia </it>hybrids. RNA-seq on short-read platforms is a rapid approach for obtaining comprehensive transcriptomic data and to discover informative sequence variants.</p> <p>Results</p> <p>We sequenced transcriptomes of <it>A. auriculiformis </it>and <it>A. mangium </it>from non-normalized cDNA libraries synthesized from pooled young stem and inner bark tissues using paired-end libraries and a single lane of an Illumina GAII machine. <it>De novo </it>assembly produced a total of 42,217 and 35,759 contigs with an average length of 496 bp and 498 bp for <it>A. auriculiformis </it>and <it>A. mangium </it>respectively. The assemblies of <it>A. auriculiformis </it>and <it>A. mangium </it>had a total length of 21,022,649 bp and 17,838,260 bp, respectively, with the largest contig 15,262 bp long. We detected all ten monolignol biosynthetic genes using Blastx and further analysis revealed 18 lignin isoforms for each species. We also identified five contigs homologous to R2R3-MYB proteins in other plant species that are involved in transcriptional regulation of secondary cell wall formation and lignin deposition. We searched the contigs against public microRNA database and predicted the stem-loop structures of six highly conserved microRNA families (miR319, miR396, miR160, miR172, miR162 and miR168) and one legume-specific family (miR2086). Three microRNA target genes were predicted to be involved in wood formation and flavonoid biosynthesis. By using the assemblies as a reference, we discovered 16,648 and 9,335 high quality putative Single Nucleotide Polymorphisms (SNPs) in the transcriptomes of <it>A. auriculiformis </it>and <it>A. mangium</it>, respectively, thus yielding useful markers for population genetics studies and marker-assisted selection.</p> <p>Conclusion</p> <p>We have produced the first comprehensive transcriptome-wide analysis in <it>A. auriculiformis </it>and <it>A. mangium </it>using <it>de novo </it>assembly techniques. Our high quality and comprehensive assemblies allowed the identification of many genes in the lignin biosynthesis and secondary cell wall formation in <it>Acacia </it>hybrids. Our results demonstrated that Next Generation Sequencing is a cost-effective method for gene discovery, identification of regulatory sequences, and informative markers in a non-model plant.</p

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines.

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo

Misfolded SOD1 Associated with Motor Neuron Mitochondria Alters Mitochondrial Shape and Distribution Prior to Clinical Onset

Mutations in superoxide dismutase (SOD1) are causative for inherited amyotrophic lateral sclerosis. A proportion of SOD1 mutant protein is misfolded onto the cytoplasmic face of mitochondria in one or more spinal cord cell types. By construction of mice in which mitochondrially targeted enhanced green fluorescent protein is selectively expressed in motor neurons, we demonstrate that axonal mitochondria of motor neurons are primary in vivo targets for misfolded SOD1. Mutant SOD1 alters axonal mitochondrial morphology and distribution, with dismutase active SOD1 causing mitochondrial clustering at the proximal side of Schmidt-Lanterman incisures within motor axons and dismutase inactive SOD1 producing aberrantly elongated axonal mitochondria beginning pre-symptomatically and increasing in severity as disease progresses. Somal mitochondria are altered by mutant SOD1, with loss of the characteristic cylindrical, networked morphology and its replacement by a less elongated, more spherical shape. These data indicate that mutant SOD1 binding to mitochondria disrupts normal mitochondrial distribution and size homeostasis as early pathogenic features of SOD1 mutant-mediated ALS

Building prognostic models for breast cancer patients using clinical variables and hundreds of gene expression signatures

<p>Abstract</p> <p>Background</p> <p>Multiple breast cancer gene expression profiles have been developed that appear to provide similar abilities to predict outcome and may outperform clinical-pathologic criteria; however, the extent to which seemingly disparate profiles provide additive prognostic information is not known, nor do we know whether prognostic profiles perform equally across clinically defined breast cancer subtypes. We evaluated whether combining the prognostic powers of standard breast cancer clinical variables with a large set of gene expression signatures could improve on our ability to predict patient outcomes.</p> <p>Methods</p> <p>Using clinical-pathological variables and a collection of 323 gene expression "modules", including 115 previously published signatures, we build multivariate Cox proportional hazards models using a dataset of 550 node-negative systemically untreated breast cancer patients. Models predictive of pathological complete response (pCR) to neoadjuvant chemotherapy were also built using this approach.</p> <p>Results</p> <p>We identified statistically significant prognostic models for relapse-free survival (RFS) at 7 years for the entire population, and for the subgroups of patients with ER-positive, or Luminal tumors. Furthermore, we found that combined models that included both clinical and genomic parameters improved prognostication compared with models with either clinical or genomic variables alone. Finally, we were able to build statistically significant combined models for pathological complete response (pCR) predictions for the entire population.</p> <p>Conclusions</p> <p>Integration of gene expression signatures and clinical-pathological factors is an improved method over either variable type alone. Highly prognostic models could be created when using all patients, and for the subset of patients with lymph node-negative and ER-positive breast cancers. Other variables beyond gene expression and clinical-pathological variables, like gene mutation status or DNA copy number changes, will be needed to build robust prognostic models for ER-negative breast cancer patients. This combined clinical and genomics model approach can also be used to build predictors of therapy responsiveness, and could ultimately be applied to other tumor types.</p

Energetic eruptions leading to a peculiar hydrogen-rich explosion of a massive star

Every supernova so far observed has been considered to be the terminal explosion of a star. Moreover, all supernovae with absorption lines in their spectra show those lines decreasing in velocity over time, as the ejecta expand and thin, revealing slower-moving material that was previously hidden. In addition, every supernova that exhibits the absorption lines of hydrogen has one main light-curve peak, or a plateau in luminosity, lasting approximately 100 days before declining1. Here we report observations of iPTF14hls, an event that has spectra identical to a hydrogen-rich core-collapse supernova, but characteristics that differ extensively from those of known supernovae. The light curve has at least five peaks and remains bright for more than 600 days; the absorption lines show little to no decrease in velocity; and the radius of the line-forming region is more than an order of magnitude bigger than the radius of the photosphere derived from the continuum emission. These characteristics are consistent with a shell of several tens of solar masses ejected by the progenitor star at supernova-level energies a few hundred days before a terminal explosion. Another possible eruption was recorded at the same position in 1954. Multiple energetic pre-supernova eruptions are expected to occur in stars of 95 to 130 solar masses, which experience the pulsational pair instability2,3,4,5. That model, however, does not account for the continued presence of hydrogen, or the energetics observed here. Another mechanism for the violent ejection of mass in massive stars may be required

Development of high-throughput SNP-based genotyping in <it>Acacia auriculiformis</it> x <it>A</it>. <it>mangium</it> hybrids using short-read transcriptome data

<p>Abstract</p> <p>Background</p> <p>Next Generation Sequencing has provided comprehensive, affordable and high-throughput DNA sequences for Single Nucleotide Polymorphism (SNP) discovery in <it>Acacia auriculiformis</it> and <it>Acacia mangium</it>. Like other non-model species, SNP detection and genotyping in <it>Acacia</it> are challenging due to lack of genome sequences. The main objective of this study is to develop the first high-throughput SNP genotyping assay for linkage map construction of <it>A</it>. <it>auriculiformis</it> x <it>A</it>. <it>mangium</it> hybrids.</p> <p>Results</p> <p>We identified a total of 37,786 putative SNPs by aligning short read transcriptome data from four parents of two <it>Acacia</it> hybrid mapping populations using Bowtie against 7,839 <it>de novo</it> transcriptome contigs. Given a set of 10 validated SNPs from two lignin genes, our <it>in silico</it> SNP detection approach is highly accurate (100%) compared to the traditional <it>in vitro</it> approach (44%). Further validation of 96 SNPs using Illumina GoldenGate Assay gave an overall assay success rate of 89.6% and conversion rate of 37.5%. We explored possible factors lowering assay success rate by predicting exon-intron boundaries and paralogous genes of <it>Acacia</it> contigs using <it>Medicago truncatula</it> genome as reference. This assessment revealed that presence of exon-intron boundary is the main cause (50%) of assay failure. Subsequent SNPs filtering and improved assay design resulted in assay success and conversion rate of 92.4% and 57.4%, respectively based on 768 SNPs genotyping. Analysis of clustering patterns revealed that 27.6% of the assays were not reproducible and flanking sequence might play a role in determining cluster compression. In addition, we identified a total of 258 and 319 polymorphic SNPs in <it>A</it>. <it>auriculiformis</it> and <it>A</it>. <it>mangium</it> natural germplasms, respectively.</p> <p>Conclusion</p> <p>We have successfully discovered a large number of SNP markers in <it>A</it>. <it>auriculiformis</it> x <it>A</it>. <it>mangium</it> hybrids using next generation transcriptome sequencing. By using a reference genome from the most closely related species, we converted most SNPs to successful assays. We also demonstrated that Illumina GoldenGate genotyping together with manual clustering can provide high quality genotypes for a non-model species like <it>Acacia</it>. These SNPs markers are not only important for linkage map construction, but will be very useful for hybrid discrimination and genetic diversity assessment of natural germplasms in the future.</p