Ultra-deep RNA sequencing (RNA-Seq) has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We previously developed multivariate analysis of transcript splicing (MATS), a statistical method for detecting differential alternative splicing between two RNA-Seq samples. Here we describe a new statistical model and computer program, replicate MATS (rMATS), designed for detection of differential alternative splicing from replicate RNASeq data. rMATS uses a hierarchical model to simultaneously account for sampling uncertainty in individual replicates and variability among replicates. In addition to the analysis of unpaired replicates, rMATS also includes a model specifically designed for paired replicates between sample groups. The hypothesis-testing framework of rMATS is flexible and can assess the statistical significance over any user-defined magnitude of splicing change. The performance of rMATS is evaluated by the analysis of simulated and real RNA-Seq data. rMATS outperformed two existing methods for replicate RNA-Seq data in all simulation settings, and RT-PCR yielded a high validation rate (94%) in an RNA-Seq dataset of prostate cancer cell lines. Our data also provide guiding principles for designing RNA-Seq studies of alternative splicing. We demonstrate that it is essential to incorporate biological replicates in the study design. Of note, pooling RNAs or merging RNA-Seq data from multiple replicates is not an effective approach to account for variability, and the result is particularly sensitive to outliers. The rMATS source code is freely available at rnaseq-mats.sourceforge. net/. As the popularity of RNA-Seq continues to grow, we expect rMATS will be useful for studies of alternative splicing in diverse RNA-Seq projects.RNA sequencing | alternative splicing | exon | isoform | transcriptome A lternative splicing generates tremendous transcriptomic and proteomic complexity in higher eukaryotes (1-4). Changes in alternative splicing underlie gene regulation in diverse biological and disease processes (5-7). However, it has been challenging to globally determine and compare gene splicing profiles among biological states. The RNA sequencing (RNA-Seq) technology has become a powerful tool for quantitative profiling of alternative splicing (3,4,8). Due to the high cost, earlier RNA-Seq studies of alternative splicing typically did not incorporate replicates in the study design (9-12). Nonetheless, it is important to note that biological variability remains a critical issue in high-throughput sequencing studies (13). Furthermore, as the cost of sequencing continues to decline, it has become feasible and increasingly common to carry out RNA-Seq on a large number of samples, with sufficient coverage to quantify alternative splicing in each individual sample. This creates an urgent need for new and robust analytic tools to detect alternative splicing changes from replicate RNA-Seq data.Although a variety of computational methods have been developed for RNA-Seq analysis of alternati...

Tumor-associated immune suppression can lead to defective T cell-mediated antitumor immunity. Here, we identified a unique phenotype of exhausted T cells in mice with advanced acute myelogenous leukemia (AML). This phenotype is characterized by the coexpression of Tim-3 and PD-1 on CD8 ؉ T cells in the liver, the major first site of AML metastases. PD-1 and Tim-3 coexpression increased during AML progression. PD-1 ؉ Tim-3 ؉ CD8 ؉ T cells were deficient in their ability to produce IFN-␥, TNF-␣, and IL-2 in response to PD-1 ligand (PDL1) and Tim-3 ligand (galectin-9) expressing AML cells. PD-1 knockout (KO), which were partially resistant to AML challenge, up-regulated Tim-3 during AML progression and such Tim-3 ؉ PD-1-KO CD8 ؉ T cells had reduced cytokine production. Galectin-9 KO mice were more resistant to AML, which was associated with reduced T-regulatory cell accumulation and a modest induction of PD-1 and Tim-3 expression on CD8 ؉ T cells. Whereas blocking the PD-1/ PDL1 or Tim-3/galectin-9 pathway alone was insufficient to rescue mice from AML lethality, an additive effect was seen in reducing-albeit not eliminating-both tumor burden and lethality when both pathways were blocked. Therefore, combined PD-1/PDL1 and Tim-3/galectin-9 blockade may be beneficial in preventing CD8 ؉ T-cell exhaustion in patients with hematologic malignancies such as advanced AML. (Blood. 2011;117(17):4501-4510) Introduction T-cell exhaustion, a state of T-cell dysfunction characterized by diminished cytokine production, impaired killing, and hypoproliferation, was first characterized in the settings of chronic lymphocytic choriomeningitis virus (LCMV) infection. 1,2-5 Since its discovery, the process of T-cell exhaustion has been of intense interest and has been the subject of study in viral infections such as hepatitis C virus 2,6 and HIV, 3,7 as well as in tumor models. 8,9,10,11 Cell-surface antigen determinants such as program death-1 (PD-1), CTLA-4, and, in some instances, CD28 (eg, hepatitis C viral infection) can be used to identify antigen-specific T cells that are at an exhaustion stage. 4 T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a type I membrane glycoprotein and its expression can be found on terminally differentiated Th1 cells and innate immune cells. [12][13][14] is its only confirmed Tim-3 ligand to date, 15,16 although it is known that Tim-3 can also bind to certain carbohydrate moieties. 17 Ligation of Tim-3 on T cells and gal-9 inhibits Th1 responses and plays an important role in infection, autoimmunity, peripheral tolerance, and inflammation. 14,[18][19][20][21] In addition to its negative regulatory role in dampening the immune system, a recent report showed a synergistic effect of Tim-3 signaling and lipopolysaccharide in producing proinflammatory cytokines by naive dendritic cells (DCs) and monocytes, 22 indicating a dual role of the Tim-3 signaling pathway at a different phase of immune responses.Studies have demonstrated a strong correlation between PD-1 and Tim-3 coexpressi...

Myeloid-derived suppressor cells (MDSCs

Ultra-deep RNA sequencing has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We develop MATS (multivariate analysis of transcript splicing), a Bayesian statistical framework for flexible hypothesis testing of differential alternative splicing patterns on RNA-Seq data. MATS uses a multivariate uniform prior to model the between-sample correlation in exon splicing patterns, and a Markov chain Monte Carlo (MCMC) method coupled with a simulation-based adaptive sampling procedure to calculate the P-value and false discovery rate (FDR) of differential alternative splicing. Importantly, the MATS approach is applicable to almost any type of null hypotheses of interest, providing the flexibility to identify differential alternative splicing events that match a given user-defined pattern. We evaluated the performance of MATS using simulated and real RNA-Seq data sets. In the RNA-Seq analysis of alternative splicing events regulated by the epithelial-specific splicing factor ESRP1, we obtained a high RT–PCR validation rate of 86% for differential exon skipping events with a MATS FDR of <10%. Additionally, over the full list of RT–PCR tested exons, the MATS FDR estimates matched well with the experimental validation rate. Our results demonstrate that MATS is an effective and flexible approach for detecting differential alternative splicing from RNA-Seq data.

A series of 20 dual amino-functionalised phosphonium ionic liquids, (3-aminopropyl)tributylphosphonium amino acid salts ([aP(4443)][AA], in which [AA](-) = [Ala](-), [Arg](-), [Asn](-), [Asp](-), [Cys](-), [Gln](-), [Glu](-), [Gly](-), [His](-), [Ile](-), [Leu](-), [Lys](-), [Met](-), [Phe](-), [Pro](-), [Ser](-), [Thr](-), [Trp](-), [Tyr](-) and [Val](-)), has been prepared. Their physicochemical properties, such as density, viscosity, glass transition and thermal decomposition temperatures and conductivity, have been determined. In particular, the [aP(4443)][AA] ionic liquids (ILs) have low glass transition temperatures ranging from -69.7 to -29.6 degrees C and high decomposition temperatures (all above 200 degrees C). The effects of the variation of the structure of [AA](-) on the above physicochemical properties are discussed. Furthermore, the CO(2) absorption of [aP(4443)][Gly], [aP(4443)][Ala], [aP(4443)][Val] and [aP(4443)][Leu], taken as examples, was investigated. It was found that the supported absorption of CO(2) by the [aP(4443)][AA] ILs almost reaches equilibrium within 80 min, the chemical absorption of CO(2) by the [aP(4443)][AA] ILs approaches 1 mol CO(2) per mol ionic liquid (twice that reported before) and the [aP(4443)][AA] ILs can be repeatedly recycled for CO(2) uptake.

Tumor-induced immune defects can weaken host immune response and permit tumor cell growth. In a systemic model of murine acute myeloid leukemia (AML), tumor progression resulted in increased regulatory T cells (Treg) and elevation of program death-1 (PD-1) expression on CD8 cytotoxic T cells (CTLs) at the tumor site. PD-1 knockout mice were more resistant to AML despite the presence of similar percentage of Tregs compared with wild type. In vitro, intact Treg suppression of CD8 T-cell responses was dependent on PD-1 expression by T cells and Tregs and PD-L1 expression by anti-gen-presenting cells. In vivo, the function of adoptively transferred AML-reactive CTLs was reduced by AML-associated Tregs. Anti-PD-L1 monoclonal antibody treatment increased the proliferation and function of CTLs at tumor sites, reduced AML tumor burden, and resulted in long-term survivors. Treg depletion followed by PD-1/PD-L1 blockade showed superior efficacy for eradication of established AML. These data demonstrated that interaction between PD-1 and PD-L1 can facilitate Treg-induced suppression of T-effector cells and dampen the antitumor immune response. PD-1/PD-L1 blockade coupled with Treg depletion represents an important new approach that can be readily translated into the clinic to improve the therapeutic efficacy of adoptive AML-reactive CTLs in advanced AML disease. (Blood. 2010;116(14):2484-2493)

Densities and viscosities of water (1) + 1-butyl-3-methylimidazolium tetrafluoroborate (2) were measured over the entire mole fraction range from (303.15 to 353.15) K. From these data, excess molar volumes (V E ), and viscosity deviations (∆η) were calculated. The V E and ∆η were fitted to the Redlich-Kister equation using a multiparametric nonlinear regression method. Estimated coefficients of the Redlich-Kister equation and standard deviation calculated from the Redlich-Kister equation to the experimental data are also presented. The results show that the densities and viscosities are dependent strongly on water content. Comparatively, the viscosity deviation ∆η is more sensitive to temperature than the excess molar volume V E .

Urea/metal salt DESs can catalyze PET degradation into a monomer with high selectivity in a short time under mild conditions.