mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system.[Supplemental material is available online at http://www.genome.org. Sequence reads from this study have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE20895. Software is available for download from http://www.th-wildau.de/bioinformatics/wios/ and http://mayoresearch.mayo.edu/ mayo/research/beutler_lab/wios.cfm.]Microarray-based transcriptome studies have provided a productive approach to the discovery of therapeutic targets in neurological disorders. For example, gene expression profiling of the dorsal root ganglion (DRG) in chronic pain (Griffin et al. 2003) identified several altered genes (Costigan et al. 2002), some of which were subsequently shown to be key modulators of pain (Tegeder et al. 2006). Gene expression analysis using microarray technology suffers from well-known limitations including poor sensitivity and dynamic range, a requirement for substantial requisite amounts of RNA, and a limited capacity to identify new transcripts or RNA splice sites. Given these limitations, it is unlikely that microarray analysis can reveal the full extent of transcriptome reprogramming underlying neurological disorders, such as chronic pain.Ultra-high-throughput RNA sequencing has emerged as a revolutionary technology with superior...

There is evidence that blockade of the reticuloendothelial system (RES) by sequestration of autologous red blood cells (RBC) leads to an elevation of platelet counts in immune thrombocytopenia. To substantiate this hypothesis, 10 Rh0(D)-positive adult patients (9 female, 1 male) with chronic autoimmune thrombocytopenic purpura (ITP) (1 to 21 years duration) were treated with low doses of intravenous IgG-anti-Rh0(D) (200 to 1,000 micrograms per dose; 300 to 3,600 micrograms per course; administration within 1 to 5 days). All patients improved clinically as indicated by cessation of bleeding. In eight out of ten patients there was a rise in platelet count. Platelet increments were excellent (greater than 100 X 10(9)/l) in one, good (50-100 X 10(9)/l) in three, fair (20-50 X 10(9)/1) in two and low (10-20 X 10(9)/1) in two patients. Splenectomized patients (N = 4) had a poorer response than non-splenectomized patients (N = 6) with mean increments of 16 X 10(9)/l (range 5-43 X 10(9)/l) versus 60 X 10(9)/l (range 10-110 X 10(9)/l). The increase in platelet counts persisted for seven to over 150 days. Transient and slight signs of haemolysis developed in seven out of ten patients (haemoglobin remained stable; increase of lactate dehydrogenase (greater than 250 IU/l) in four, decrease of haptoglobin (less than 60 mg/dl) in five patients). The direct antiglobulin test became positive in all cases due to IgG1 without complement fixation. We conclude that the interaction of antibody-coated RBC with macrophages (and, probably, other means of RBC alteration) is a feasible therapeutic approach in selected cases of ITP and related conditions.

Cancer resistance is a major cause for longevity of the naked mole-rat. Recent liver transcriptome analysis in this animal compared to wild-derived mice revealed higher expression of alpha2-macroglobulin (A2M) and cell adhesion molecules, which contribute to the naked mole-rat’s cancer resistance. Notably, A2M is known to dramatically decrease with age in humans. We hypothesize that this might facilitate tumour development. Here we found that A2M modulates tumour cell adhesion, migration and growth by inhibition of tumour promoting signalling pathways, e.g. PI3K / AKT, SMAD and up-regulated PTEN via down-regulation of miR-21, in vitro and in tumour xenografts. A2M increases the expression of CD29 and CD44 but did not evoke EMT. Transcriptome analysis of A2M-treated tumour cells, xenografts and mouse liver demonstrated a multifaceted regulation of tumour promoting signalling pathways indicating a less tumorigenic environment mediated by A2M. By virtue of these multiple actions the naturally occurring A2M has strong potential as a novel therapeutic agent.

The use of a priori knowledge in the alignment of targeted sequencing data is investigated using computational experiments. Adapting a Needleman–Wunsch algorithm to incorporate the genomic position information from the targeted capture, we demonstrate that alignment can be done to just the target region of interest. When in addition use is made of direct string comparison, an improvement of up to a factor of 8 in alignment speed compared to the fastest conventional aligner (Bowtie) is obtained. This results in a total alignment time in targeted sequencing of around 7 min for aligning approximately 56 million captured reads. For conventional aligners such as Bowtie, BWA or MAQ, alignment to just the target region is not feasible as experiments show that this leads to an additional 88% SNP calls, the vast majority of which are false positives (∼92%).