Relative Abundance of Transcripts ( RATs): Identifying differential isoform abundance from RNA-seq.

The biological importance of changes in RNA expression is reflected by the wide variety of tools available to characterise these changes from RNA-seq data. Several tools exist for detecting differential transcript isoform usage (DTU) from aligned or assembled RNA-seq data, but few exist for DTU detection from alignment-free RNA-seq quantifications. We present the RATs, an R package that identifies DTU transcriptome-wide directly from transcript abundance estimates. RATs is unique in applying bootstrapping to estimate the reliability of detected DTU events and shows good performance at all replication levels (median false positive fraction < 0.05). We compare RATs to two existing DTU tools, DRIM-Seq & SUPPA2, using two publicly available simulated RNA-seq datasets and a published human RNA-seq dataset, in which 248 genes have been previously identified as displaying significant DTU. RATs with default threshold values on the simulated Human data has a sensitivity of 0.55, a Matthews correlation coefficient of 0.71 and a false discovery rate (FDR) of 0.04, outperforming both other tools. Applying the same thresholds for SUPPA2 results in a higher sensitivity (0.61) but poorer FDR performance (0.33). RATs and DRIM-seq use different methods for measuring DTU effect-sizes complicating the comparison of results between these tools, however, for a likelihood-ratio threshold of 30, DRIM-Seq has similar FDR performance to RATs (0.06), but worse sensitivity (0.47). These differences persist for the simulated drosophila dataset. On the published human RNA-seq dataset the greatest agreement between the tools tested is 53%, observed between RATs and SUPPA2. The bootstrapping quality filter in RATs is responsible for removing the majority of DTU events called by SUPPA2 that are not reported by RATs. All methods, including the previously published qRT-PCR of three of the 248 detected DTU events, were found to be sensitive to annotation differences between Ensembl v60 and v87.

PGE(2) induces macrophage IL-10 production and a regulatory-like phenotype via a protein kinase A-SIK-CRTC3 pathway.

The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE(2), in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A-dependent pathway. Both TLR agonists and PGE(2) promote the phosphorylation of the transcription factor CREB on Ser(133). However, although CREB regulates IL-10 transcription, the mutation of Ser(133) to Ala in the endogenous CREB gene did not prevent the ability of PGE(2) to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser(343), inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE(2) on IL-10 production.

A quantitative spatial proteomics analysis of proteome turnover in human cells.

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ~20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.

Temperature effects on emission of quantum dots embedded in polymethylmethacrylate.

We successfully used CdSe/ZnS quantum dots (QDs) as a dopant within a polymethylmethacrylate (PMMA) matrix. This doped material was used in the fabrication of a microstructured polymer optical fiber whose photoluminescence was characterized. A detailed analysis of the emission properties of the QDs as a function of temperature is presented, with the temperature dependence of this emission broken into components to show contributions from the thermo-optic effect of the PMMA and the temperature-dependence of the bandgap of the QDs.

Emission properties of quantum dots in polymer optical fibres.

CdSe/ZnS core-shell quantum dots have been embedded within microstructured polymer optical fibres. The emission properties of quantum dots within fibres have been explored to show that variation in concentration, sample length and pumping regimes effects the emission characteristics of these quantum dots.

Enhanced magneto-optical effect in cobalt nanoparticle-doped optical fiber.

An enhanced magnetic Faraday effect is demonstrated in cobalt nanoparticle-doped polymer optical fiber. Magneto-optically induced rotation of the plane of polarization proportional to both the dopant particle concentration and the magnetic field strength is demonstrated. Potential applications include magnetic field sensors, current sensors, and in-fiber optical isolators.

Quantum dot and silica nanoparticle doped polymer optical fibers.

A novel and highly versatile doping method has been developed to allow active dopants, including materials incompatible with the polymer matrix, to be incorporated into microstructured polymer optical fibers through the use of nanoparticles. The incorporation of quantum dots and silica nanoparticles containing Rhodamine isothiocyanate is demonstrated.

Small-core single-mode microstructured polymer optical fiber with large external diameter.

A preform sleeving technique is demonstrated that allows the fabrication of single-mode polymer microstructured fiber with the smallest core and hole dimensions yet reported to our knowledge. For a fixed triangular hole pattern a range of fibers is produced by adjustment to the operating conditions of the draw tower. Numerical modeling is carried out for one of the fibers produced with a 570-microm external diameter, a core diameter of 2.23 microm, an average hole diameter of 0.53 microm, and an average hole spacing of 1.38 microm. This fiber was shown to be endlessly single mode.