Analysis of wheat SAGE tags reveals evidence for widespread antisense transcription

BACKGROUND: Serial Analysis of Gene Expression (SAGE) is a powerful tool for genome-wide transcription studies. Unlike microarrays, it has the ability to detect novel forms of RNA such as alternatively spliced and antisense transcripts, without the need for prior knowledge of their existence. One limitation of using SAGE on an organism with a complex genome and lacking detailed sequence information, such as the hexaploid bread wheat Triticum aestivum, is accurate annotation of the tags generated. Without accurate annotation it is impossible to fully understand the dynamic processes involved in such complex polyploid organisms. Hence we have developed and utilised novel procedures to characterise, in detail, SAGE tags generated from the whole grain transcriptome of hexaploid wheat. RESULTS: Examination of 71,930 Long SAGE tags generated from six libraries derived from two wheat genotypes grown under two different conditions suggested that SAGE is a reliable and reproducible technique for use in studying the hexaploid wheat transcriptome. However, our results also showed that in poorly annotated and/or poorly sequenced genomes, such as hexaploid wheat, considerably more information can be extracted from SAGE data by carrying out a systematic analysis of both perfect and "fuzzy" (partially matched) tags. This detailed analysis of the SAGE data shows first that while there is evidence of alternative polyadenylation this appears to occur exclusively within the 3' untranslated regions. Secondly, we found no strong evidence for widespread alternative splicing in the developing wheat grain transcriptome. However, analysis of our SAGE data shows that antisense transcripts are probably widespread within the transcriptome and appear to be derived from numerous locations within the genome. Examination of antisense transcripts showing sequence similarity to the Puroindoline a and Puroindoline b genes suggests that such antisense transcripts might have a role in the regulation of gene expression. CONCLUSION: Our results indicate that the detailed analysis of transcriptome data, such as SAGE tags, is essential to understand fully the factors that regulate gene expression and that such analysis of the wheat grain transcriptome reveals that antisense transcripts maybe widespread and hence probably play a significant role in the regulation of gene expression during grain development

New process to describe radiation damage at the molecular level. Application to I-125 seeds in water

ICPEAC 2015, Toledo, Spain on 22 –28 July 2015; http://www.icpeac2015.com/We present the first step of a general study on induced damage by 125I in water at the molecular level. Requirements for such a study are introduced and preliminary results on the emission spectra of 125I seeds are presented.This work was supported by MINECO (FIS2012-31230) and FCT/MEC, RaBBiT, PD-F,PD/00193/2012); UID/Multi/04378/2013 (UCIBIO); UID/FIS/00068/2013 (CEFITEC); and grant number SFRH/BD/52536/2014.Peer Reviewe

Tissue-specific gene silencing monitored in circulating RNA

Pharmacologic target gene modulation is the primary objective for RNA antagonist strategies and gene therapy. Here we show that mRNAs encoding tissue-specific gene transcripts can be detected in biological fluids and that RNAi-mediated target gene silencing in the liver and brain results in quantitative reductions in serum and cerebrospinal fluid mRNA levels, respectively. Further, administration of an anti-miRNA oligonucleotide resulted in decreased levels of the miRNA in circulation. Moreover, ectopic expression of an adenoviral transgene in the liver was quantified based on measurement of serum mRNA levels. This noninvasive method for monitoring tissue-specific RNA modulation could greatly advance the clinical development of RNA-based therapeutics

CD8α is expressed by human monocytes and enhances FcγR-dependent responses

Abstract Background CD8α enhances the responses of antigen-specific CTL activated through TCR through binding MHC class I, favoring lipid raft partitioning of TCR, and inducing intracellular signaling. CD8α is also found on dendritic cells and rat macrophages, but whether CD8α enhances responses of a partner receptor, like TCR, to activate these cells is not known. TCR and FcR, use analogous or occasionally interchangeable signaling mechanisms suggesting the possibility that CD8α co-activates FcR responses. Interestingly, CD8α+ monocytes are often associated with rat models of disease involving immune-complex deposition and FcR-mediated pathology, such as arthritis, glomerulonephritis, ischaemia, and tumors. While rat macrophages have been shown to express CD8α evidence for CD8α expression by mouse or human monocytes or macrophages was incomplete. Results We detected CD8α, but not CD8β on human monocytes and the monocytic cell line THP-1 by flow cytometry. Reactivity of anti-CD8α mAb with monocytes is at least partly independent of FcR as anti-CD8α mAb detect CD8α by western blot and inhibit binding of MHC class I tetramers. CD8α mRNA is also found in monocytes and THP-1 suggesting CD8α is synthesized by monocytes and not acquired from other CD8α+ cell types. Interestingly, CD8α from monocytes and blood T cells presented distinguishable patterns by 2-D electrophoresis. Anti-CD8α mAb alone did not activate monocyte TNF release. In comparison, TNF release by human monocytes stimulated in a FcR-dependent manner with immune-complexes was enhanced by inclusion of anti-CD8α mAb in immune-complexes. Conclusion Human monocytes express CD8α. Co-engagement of CD8α and FcR enhances monocyte TNF release, suggesting FcR may be a novel partner receptor for CD8α on innate immune cells.</p

Induced molecular dissociations as a radiation damage descriptor: nanodosimetry

Conferencia invitada; IBER 2015, 6-9th September, Aveiro – Portugal; http://iber2015.web.ua.pt/Traditional dosimetry is based on the proportionality between the energy absorbed by the medium (absorbed dose) and the induced damage. This assumption applies for relatively high irradiated volumes and requires some equilibrium conditions. However, for small volumes being relatively far from the central irradiated areas these conditions are not observed and radiation damage is mainly driven by low energy secondary species (electrons and radicals) which induce molecular dissociations via electronic and vibrational excitations, electron attachment and chemical reactions. We will present here an integrated modelling procedure to simulate particle radiation tracks including those of all generated secondary species and their further interactions with the molecular constituent of the medium. For any selected volume of interest, this model provides not only the total energy transferred to that area but also the number and type of interactions taking place in it [1].Peer Reviewe

The role of pyrimidine and water as underlying molecular constituents for describing radiation damage in living tissue: A comparative study

8 págs.; 7 figs.; 2 tabs.© 2015 AIP Publishing LLC. Water is often used as the medium for characterizing the effects of radiation on living tissue. However, in this study, charged-particle track simulations are employed to quantify the induced physicochemical and potential biological implications when a primary ionising particle with energy 10 keV strikes a medium made up entirely of water or pyrimidine. Note that pyrimidine was chosen as the DNA/RNA bases cytosine, thymine, and uracil can be considered pyrimidine derivatives. This study aims to assess the influence of the choice of medium on the charged-particle transport, and identify how appropriate it is to use water as the default medium to describe the effects of ionising radiation on living tissue. Based on the respective electron interaction cross sections, we provide a model, which allows the study of radiation effects not only in terms of energy deposition (absorbed dose and stopping power) but also in terms of the number of induced molecular processes. Results of these parameters for water and pyrimidine are presented and compared.This research was supported by the Australian Research Council (ARC) through its Centres of Excellence Program. D.B.J. thanks the ARC for provision of a Discovery Early Career Researcher Award. We also acknowledge the support of the Spanish Ministerio de Economia y Competitivad under Project No. FIS 2012-31230 and the European Union COST Actions (MP1002 and CM1301). P.L.V. acknowledges the Portuguese Foundation for Science and Technology (FCTMEC) through research grants PTDC/FIS-ATO/1832/2012, UID/FIS/00068/2013, and SFRH/BSAB/105792/2014. P.L.V. also acknowledges his Visiting Professor position at Flinders University, Adelaide, South Australia.Peer Reviewe

Modeling secondary particle tracks generated by high-energy protons in water

ICPEAC 2015, Toledo, Spain on 22 –28 July 2015; http://www.icpeac2015.com/We present interaction probability data of low-energy secondary electrons and positrons produced due to the proton impact. The probability distribution functions serve as input data for the Low Energy Particle Track Simulation (LEPTS) approach which allows one to include the effect of low-energy species in medical applications of radiation and in ion-beam cancer therapy, in particular.his work has been supported by the Spanish Ministry of Economía y Productividad (Project FIS2012-31320). We also acknowledge partial funding from the Portuguese Foundation for Science and Technology (FCT-MCTES), research grant PEstOE/FIS/UI0068/2011,the EU/ESF COST Actions Nano-IBCT - MP1002 and CELINA CM- 1301, and from the FP7 Multi-ITN Project ”Advanced Radiotherapy, Generated by Exploiting Nanoprocesses and Technologies” (ARGENT) (Grant Agreement n◦608163).Peer Reviewe

Modelling secondary particle tracks generated by high-energy protons in water

1 pág.; XXIX International Conference on Photonic, Electronic, and Atomic Collisions (ICPEAC2015); Open Access funded by Creative Commons Atribution Licence 3.0We present interaction probability data of low-energy secondary electrons and positrons produced due to the proton impact. The probability distribution functions serve as input data for the Low Energy Particle Track Simulation (LEPTS) approach which allows one to include the effect of low-energy species in medical applications of radiation and in ion-beam cancer therapy, in particular.This work has been supported by the Spanish Ministry of Economía y Productividad (Project FIS2012-31320). We also acknowledge partial funding from the Portuguese Foundation for Science and Technology (FCT-MCTES), research grant PEstOE/FIS/UI0068/2011,the EU/ESF COST Actions Nano-IBCT - MP1002 and CELINA CM- 1301, and from the FP7 Multi-ITN Project ”Advanced Radiotherapy, Generated by Exploiting Nanoprocesses and Technologies” (ARGENT) (Grant Agreement n◦608163).Peer Reviewe

Intermediate energy cross sections for electron-impact vibrational-excitation of pyrimidine

We report differential cross sections (DCSs) and integral cross sections (ICSs) for electron-impact vibrational-excitation of pyrimidine, at incident electron energies in the range 15–50 eV. The scattered electron angular range for the DCS measurements was 15°–90°. The measurements at the DCS-level are the first to be reported for vibrational-excitation in pyrimidine via electron impact, while for the ICS we extend the results from the only previous condensed-phase study [P. L. Levesque, M. Michaud, and L. Sanche, J. Chem. Phys. 122, 094701 (2005)], for electron energies ⩽12 eV, to higher energies. Interestingly, the trend in the magnitude of the lower energy condensed-phase ICSs is much smaller when compared to the corresponding gas phase results. As there is no evidence for the existence of any shape-resonances, in the available pyrimidine total cross sections [Baek et al., Phys. Rev. A 88, 032702 (2013); Fuss et al., ibid. 88, 042702 (2013)], between 10 and 20 eV, this mismatch in absolute magnitude between the condensed-phase and gas-phase ICSs might be indicative for collective-behaviour effects in the condensed-phase results

The HIV-1 Nef protein binds argonaute-2 and functions as a viral suppressor of RNA interference

The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR)