Predicting the intention of distracted pedestrians at road crossings.

Despite improvements to road safety, accidents involving pedestrians are still numerous, for example in the UK there were over 20,000 pedestrian casualties on public roads in 2019. One of the potential causal factors is pedestrian distraction. Therefore, this study aimed to predict pedestrian intention to cross the road under conditions of distraction (using phone maps, talking to another pedestrian, listening to music through headphones), by applying the Theory of Planned Behaviour (TPB) using an online survey. This also involved investigation of the impact of selected traffic characteristics (traffic density, vehicle speed) and crossing type (pelican, zebra, unmarked). The survey consisted of 72 questions and took approximately 15 min to complete. The results (N = 81) revealed that the TPB construct of perceived behavioural control (PBC) was a significant predictor of intention to cross the road while distracted across all scenarios. Furthermore, crossing type was a significant predictor of PBC across all scenarios, with marked crossings facilitating feelings of PBC. Findings suggest that high feelings of PBC, as measured through ease and confidence, are linked with stronger intention to cross the road while distracted. This understanding of pedestrian motivation can be used to help design interventions (such as auditory and visual pedestrian warnings) that prevent conflict between distracted pedestrians and vehicles. These interventions should target marked crossing types, whereby pedestrians are more likely to cross while distracted.

CE: Managing Stable COPD: An Evidence-Based Approach.

: Chronic obstructive pulmonary disease (COPD) affects as many as 16 million Americans and is expected to be the third leading cause of death worldwide by 2020. To increase awareness of COPD, encourage related research, and improve care of patients with this chronic disease, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) was launched in 1998 and published an evidence-based report on COPD prevention and management strategies in 2001 that has been revised regularly. The fourth major revision, which was published in 2017 and revised in 2018, includes significant changes related to COPD classification, as well as to pharmacologic, nonpharmacologic, and comorbidity management. The authors discuss the changes to the GOLD recommendations and, using a patient scenario, explain their application to clinical practice.

Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS.

A label-free method for determining the 5'-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quantitative and qualitative information on the 5' cap was determined from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA preparations was determined to be between 88 and 98 % depending on the modification type and length of the mRNA. mRNA of 2.2K and 9K nucleotides in length and containing the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping. Graphical abstract mRNA 5' end analysis with RNAse H cleavage and capture probe.

Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA.

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5'-triphosphate and 5'-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5'-triphosphates and 5'-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5'-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5'-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5'-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.

Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo.

Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed "Dicer-substrate siRNA" (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as duration of silencing in vivo. Duplexes from both structural classes tolerated 2'-OMe chemical modifications well with respect to target silencing, although some modified dsiRNAs demonstrated reduced activity. On the other hand, dsiRNAs were more immunostimulatory as compared with the shorter siRNAs, both in vitro and in vivo. Because the dsiRNA structure does not confer any appreciable benefits in vitro or in vivo while demonstrating specific liabilities, further studies are required to support their applications in RNAi therapeutics.