The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (≥75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice.

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Garlic, an economically important vegetable, spice, and medicinal crop, produces highly enlarged bulbs and unique organosulfur compounds. Here, we report a chromosome-level genome assembly for garlic, with a total size of approximately 16.24 Gb, as well as the annotation of 57 561 predicted protein-coding genes, making garlic the first Allium species with a sequenced genome. Analysis of this garlic genome assembly reveals a recent burst of transposable elements, explaining the substantial expansion of the garlic genome. We examined the evolution of certain genes associated with the biosynthesis of allicin and inulin neoseries-type fructans, and provided new insights into the biosynthesis of these two compounds. Furthermore, a large-scale transcriptome was produced to characterize the expression patterns of garlic genes in different tissues and at various growth stages of enlarged bulbs. The reference genome and large-scale transcriptome data generated in this study provide valuable new resources for research on garlic biology and breeding.

BACKGROUND & AIMS Hyperactivation of the RAS-RAF signaling pathway in colorectal tumors is associated with metastasis and poor outcomes of patients. Little is known about how RAS–RAF signaling is turned off once activated. We investigated how the pH domain and leucine-rich repeat protein phosphatases (PHLPPs) control RAS–RAF signaling and colorectal cancer (CRC) development. METHODS We used co-immunoprecipitation assays to identify substrates of PHLPP1 and PHLPP2.We studied phosphorylation of RAF1 in CRC cells that express transgenic PHLPP1 or PHLPP2, or lentiviral-based small hairpin (sh)RNAs against their transcripts; we measured effects on cell motility, migration, and invasion in vitro. Tumor progression and survival were analyzed in Phlpp1−/− mice, ApcMin mice, and ApcMin/Phlpp1−/− mice. Microarray data sets of colorectal tumor and non-tumor tissues were analyzed for PHLPP gene expression. RESULTS PHLPP1 and 2 were found to dephosphorylate RAF1 at S338, inhibiting its kinase activity in vitro and in CRC cells. In cells, shRNA knockdown of PHLPP1 or PHLPP2 increased the amplitude and duration of RAF-MEK-ERK signaling downstream of EGFR and KRAS, whereas overexpression had the opposite effect. Knockdown of PHLPP1 or PHLPP2 caused CRC cells to express markers of the epithelial-mesenchymal transition (EMT), and increased migration and invasion in vitro. ApcMin/Phlpp1−/− mice had decreased survival and developed larger intestinal and colon tumors than ApcMin mice, which developed mostly low-grade adenomas; in contrast, 20% of the tumors that developed in ApcMin/Phlpp1−/− mice were invasive adenocarcinomas. Normal villi and adenomas of ApcMin/Phlpp1−/− mice had significantly fewer apoptotic cells than ApcMin mice. Human CRC patient microarray data revealed that the expression of PHLPP1 or PHLPP2 is positively correlated with CDH1. CONCLUSIONS PHLPP1 and 2 dephosphorylate RAF1 to reduce its signaling, increase the invasive and migratory activities of CRC cells, and activate the EMT. In ApcMin mice, loss of PHLPP1 promotes tumor progression.

Surface biofunctional modification of coronary artery stents to prevent thrombosis and restenosis formation, as well as accelerate endothelialization, has become a new hot spot. However, bioactive coatings on implants are not yet sufficiently developed for long-term activity, as they quickly lose efficiency in vivo and finally fail. On the basis of a novel time-ordered concept of biofunctionality for vascular stents, heparin/poly l-lysine nanoparticle (NP) was developed and immobilized on a polydopamine-coated titanium surface, with the aim of regulating and maintaining the intravascular biological response within the normal range after biomaterial implantation. An in vitro dynamic release model was established to mimic the blood flow condition in vivo with three phases: (1) An early phase (1-7 days) with release of predominantly anticoagulant and anti-inflammatory substances and to a minor degree antiproliferative effects against smooth muscle cells (SMCs); (2) this is followed by a phase (7-14 days) of supported endothelial cell (ECs) proliferation and suppressed SMC proliferation with persisting high antithrombogenicity and anti-inflammatory properties of the surface. (3) Finally, a stable stage (14-28 days) with adequate biomolecules on the surface that maintain hemocompatibility and anti inflammation as well as inhibit SMCs proliferation and promote ECs growth. In vivo animal tests further confirmed that the NP-modified surface provides a favorable release behavior to apply a stage-adjusted remedy. We suggested that these observations provide important guidance and potential means for reasonable and suitable platform construction on a stent surface.

SummaryThe horse, like the majority of animal species, has a limited amount of species-specific expressed sequence data available in public databases. As a result, structural models for the majority of genes defined in the equine genome are predictions based on ab initio sequence analysis or the projection of gene structures from other mammalian species. The current study used Illumina-based sequencing of messenger RNA (RNA-seq) to help refine structural annotation of equine protein-coding genes and for a preliminary assessment of gene expression patterns. Sequencing of mRNA from eight equine tissues generated 293 758 105 sequence tags of 35 bases each, equalling 10.28 gbp of total sequence data. The tag alignments represent approximately 207· coverage of the equine mRNA transcriptome and confirmed transcriptional activity for roughly 90% of the protein-coding gene structures predicted by Ensembl and NCBI. Tag coverage was sufficient to refine the structural annotation for 11 356 of these predicted genes, while also identifying an additional 456 transcripts with exon/intron features that are not listed by either Ensembl or NCBI. Genomic locus data and intervals for the protein-coding genes predicted by the Ensembl and NCBI annotation pipelines were combined with 75 116 RNA-seq-derived transcriptional units to generate a consensus equine protein-coding gene set of 20 302 defined loci. Gene ontology annotation was used to compare the functional and structural categories of genes expressed in either a tissue-restricted pattern or broadly across all tissue samples.

Ovarian cancer is the third most common cancer in the female reproductive organs and epithelial ovarian cancer has the highest lethality of all gynecological cancers. Pomegranate fruit juice (PFJ) has been shown to inhibit the growth of several types of cancer other than ovarian cancer. In this study, we exposed the ovarian cancer cell line A2780 to PFJ and two of its components (ellagic acid and luteolin). MTT and wound healing assays demonstrated that all three treatments suppressed the proliferation and migration of the ovarian cancer cells. In addition, western blotting and ELISA assays showed that the expression levels of MMP2 and MMP9 gradually decreased after treatment with increasing concentrations of ellagic acid and luteolin. To confirm our findings in the in vitro experiments, we used another ovarian cancer cell line, ES-2, in nude mice experiments. All three treatments inhibited tumor growth without obvious side-effects. Furthermore, compared with the control group, the expression levels of MMP2 and MMP9 were depressed. Ellagic acid induced a greater effect than luteolin, suggesting that ellagic acid might be a promising candidate for further preclinical testing for treatment of human ovarian cancer.

Baseline data for Chinese cervical spine ICRs were established for the third through eighth decade of life, including age-related changes and the kinematic effects of degenerative change on the ICR in the functional spine unit. These findings should be considered in clinical practice and when designing disc prostheses.