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With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes. The survey employed models consisting of DNA, RNA, and corresponding protein complexes to mimic as close as possible typical applications. Denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometric (MS) analyses were implemented in concert to monitor the formation of the desired conjugates. In particular, the former was used as a rapid and inexpensive tool for the efficient evaluation of cross-linker activity under a broad range of experimental conditions. The latter was applied after preliminary rounds of reaction optimization to enable full-fledged product characterization and, more significantly, differentiation between mono-functional and intra- versus inter-molecular conjugates. This information provided the feedback necessary to further optimize reaction conditions and explain possible outcomes. Among the reagents tested in the study, platinum complexes and nitrogen mustards manifested the most favorable characteristics for practical cross-linking applications, whereas other compounds provided inferior yields, or produced rather unstable conjugates that did not survive the selected analytical conditions. The observed outcomes will help guide the selection of the most appropriate cross-linking reagent for a specific task, whereas the experimental conditions described here will provide an excellent starting point for approaching these types of applications. As a whole, the results of the survey clearly emphasize that finding a universal reagent, which may afford excellent performance with all types of nucleic acid substrates, will require extending the exploration beyond the traditional chemistries employed to modify the constitutive functional groups of these vital biopolymers. [ABSTRACT FROM AUTHOR]

In eukaryotes, cap-dependent translation initiation is a sophisticated process that requires numerous trans -acting factors, the eukaryotic Initiation Factors (eIFs). Their main function is to assist the ribosome for accurate AUG start codon recognition. The whole process requires a 5′-3′ scanning step and is therefore highly dynamic. Therefore translation requires a complex interplay between eIFs through assembly/release cycles. Here, we describe an original approach to assess the dynamic features of translation initiation. The principle is to use the m 7 Gcap located at the 5′ extremity of mRNAs as a tracker to monitor RNA and protein components that are in its vicinity. Cap-binding molecules are trapped by chemical and UV crosslinking. The combination of cap crosslinking methods in cell-free translation systems with the use of specific translation inhibitors for different steps such as edeine, GMP-PNP or cycloheximide allowed assessing the cap fate during eukaryotic translation. Here, we followed the position of the cap in the histone H4 mRNA and the cap binding proteins during H4 mRNA translation. [ABSTRACT FROM AUTHOR]

Parkinson's disease has long been associated with redox imbalance and oxidative stress in dopaminergic neurons. The catecholaldehyde hypothesis proposes that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate product of dopamine catabolism, is a central nexus in a network of pathways leading to disease-state neurodegeneration, owing to its toxicity and potent ability to oligomerize α-synuclein, the main component of protein aggregates in Lewy bodies. In this work we examine the connection between reactive oxygen species and DOPAL autoxidation. We show that superoxide propagates a chain reaction oxidation, and that this reaction is dramatically inhibited by superoxide dismutase. Moreover, superoxide dismutase prevents DOPAL from forming dicatechol pyrrole adducts with lysine and from covalently crosslinking α-synuclein. Given that superoxide is a major radical byproduct of impaired cellular respiration, our results provide a possible mechanistic link between mitochondrial dysfunction and synuclein aggregation in dopaminergic neurons. [ABSTRACT FROM AUTHOR]

Photographic methods of environmental monitoring have grown in popularity and now represent one of the main ways in which habitat and biodiversity are monitored for change through time. However, efficacy and efficiency of this technique compared with traditional approaches to environmental monitoring (direct count or observation) are lacking. This study compares the results and time-efficiency of manual versus photographic monitoring of floral abundance in low-growing flowering plants in a relatively open herbfield. Specifically, we compared 1) manual flower counting of individual plants for four species, followed by data entry in the laboratory, with 2) taking photographic images of each plant and quantifying flower counts in the laboratory. Photographic monitoring underestimated flower counts by an average of 7.5%. Manual counting was more time consuming in the field, but less time consuming in post-processing than photographic monitoring. Overall, photographic monitoring took almost twice as long as manual counting (81.5% longer in duration), which was attributed to the much longer post-processing associated with photographic monitoring. This suggests that perhaps the main benefit of photographic monitoring is a permanent record of the sampling frame rather than any cost savings or enhanced data accuracy, at least in the systems investigated in this study. [ABSTRACT FROM AUTHOR]

Highlights: [•] RIPiT-Seq is a novel RNP footprinting approach based on two sequential purifications. [•] Unveils transcriptome-wide binding sites of a single RBP or multi-subunit RNPs. [•] Distinguishes between footprints of RNPs with overlapping yet distinct composition. [•] Suitable for all RBPs irrespective of their RNA recognition mode. [ABSTRACT FROM AUTHOR]

Abstract: Associations between proteins and RNA–RNA duplexes are important in post-transcriptional regulation of gene expression. The CLASH (Cross-linking, Ligation and Sequencing of Hybrids) technique captures RNA–RNA interactions by physically joining two RNA molecules associated with a protein complex into a single chimeric RNA molecule. These events are relatively rare and considerable effort is needed to detect a small number of chimeric sequences amongst millions of non-chimeric cDNA reads resulting from a CLASH experiment. We present the “hyb” bioinformatics pipeline, which we developed to analyse high-throughput cDNA sequencing data from CLASH experiments. Although primarily designed for use with AGO CLASH data, hyb can also be used for the detection and annotation of chimeric reads in other high-throughput sequencing datasets. We examined the sensitivity and specificity of chimera detection in a test dataset using the BLAST, BLAST+, BLAT, pBLAT and Bowtie2 read alignment programs. We obtained the most reliable results in the shortest time using a combination of preprocessing with Flexbar and subsequent read-mapping using Bowtie2. The “hyb” software is distributed under the GNU GPL (General Public License) and can be downloaded from https://github.com/gkudla/hyb. [Copyright &y& Elsevier]