siDirect 2.0: updated software for designing functional siRNA with reduced seed-dependent off-target effect

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi), mediated by 21-nucleotide (nt)-length small interfering RNAs (siRNAs), is a powerful tool not only for studying gene function but also for therapeutic applications. RNAi, requiring perfect complementarity between the siRNA guide strand and the target mRNA, was believed to be extremely specific. However, a recent growing body of evidence has suggested that siRNA could down-regulate unintended genes whose transcripts possess complementarity to the 7-nt siRNA seed region. This off-target gene silencing may often provide incongruous results obtained from knockdown experiments, leading to misinterpretation. Thus, an efficient algorithm for designing functional siRNAs with minimal off-target effect based on the mechanistic features is considered of value.</p> <p>Results</p> <p>We present siDirect 2.0, an update of our web-based software siDirect, which provides functional and off-target minimized siRNA design for mammalian RNAi. The previous version of our software designed functional siRNAs by considering the relationship between siRNA sequence and RNAi activity, and provided them along with the enumeration of potential off-target gene candidates by using a fast and sensitive homology search algorithm. In the new version, the siRNA design algorithm is extensively updated to eliminate off-target effects by reflecting our recent finding that the capability of siRNA to induce off-target effect is highly correlated to the thermodynamic stability, or the melting temperature (Tm), of the seed-target duplex, which is formed between the nucleotides positioned at 2-8 from the 5' end of the siRNA guide strand and its target mRNA. Selection of siRNAs with lower seed-target duplex stabilities (benchmark Tm < 21.5°C) followed by the elimination of unrelated transcripts with nearly perfect match should minimize the off-target effects.</p> <p>Conclusion</p> <p>siDirect 2.0 provides functional, target-specific siRNA design with the updated algorithm which significantly reduces off-target silencing. When the candidate functional siRNAs could form seed-target duplexes with Tm values below 21.5°C, and their 19-nt regions spanning positions 2-20 of both strands have at least two mismatches to any other non-targeted transcripts, siDirect 2.0 can design at least one qualified siRNA for >94% of human mRNA sequences in RefSeq. siDirect 2.0 is available at <url>http://siDirect2.RNAi.jp/</url>.</p

Gi/o Protein-Dependent and -Independent Actions of Pertussis Toxin (PTX)

Pertussis toxin (PTX) is a typical A-B toxin. The A-protomer (S1 subunit) exhibits ADP-ribosyltransferase activity. The B-oligomer consists of four subunits (S2 to S5) and binds extracellular molecules that allow the toxin to enter the cells. The A-protomer ADP-ribosylates the α subunits of heterotrimeric Gi/o proteins, resulting in the receptors being uncoupled from the Gi/o proteins. The B-oligomer binds proteins expressed on the cell surface, such as Toll-like receptor 4, and activates an intracellular signal transduction cascade. Thus, PTX modifies cellular responses by at least two different signaling pathways; ADP-ribosylation of the Gαi/o proteins by the A-protomer (Gi/o protein-dependent action) and the interaction of the B-oligomer with cell surface proteins (Gi/o protein-independent action)

Stress and psychological factors before a migraine attack: A time-based analysis

<p>Abstract</p> <p>Background</p> <p>The objective of this study is to examine the stress and mood changes of Japanese subjects over the 1–3 days before a migraine headache.</p> <p>Methods</p> <p>The study participants were 16 patients with migraines who consented to participate in this study. Each subject kept a headache diary four times a day for two weeks. They evaluated the number of stressful events, daily hassles, domestic and non-domestic stress, anxiety, depressive tendency and irritability by visual analog scales. The days were classified into migraine days, pre-migraine days, buffer days and control days based on the intensity of the headaches and accompanying symptoms, and a comparative study was conducted for each factor on the migraine days, pre-migraine days and control days.</p> <p>Results</p> <p>The stressful event value of pre-migraine days showed no significant difference compared to other days. The daily hassle value of pre-migraine days was the highest and was significantly higher than that of buffer days. In non-domestic stress, values on migraine days were significantly higher than on other days, and there was no significant difference between pre-migraine days and buffer days or between pre-migraine days and control days. There was no significant difference in the values of domestic stress between the categories. In non-domestic stress, values on migraine days were significantly higher than other days, and there was no significant difference between pre-migraine days and buffer days or between pre-migraine days and control days.</p> <p>There was little difference in sleep quality on migraine and pre-migraine days, but other psychological factors were higher on migraine days than on pre-migraine days.</p> <p>Conclusion</p> <p>Psychosocial stress preceding the onset of migraines by several days was suggested to play an important role in the occurrence of migraines. However, stress 2–3 days before a migraine attack was not so high as it has been reported to be in the United States and Europe. There was no significant difference in the values of psychological factors between pre-migraine days and other days.</p

Visualizing Sweetness: Increasingly Diverse Applications for Fluorescent-Tagged Glucose Bioprobes and Their Recent Structural Modifications

Glucose homeostasis is a fundamental aspect of life and its dysregulation is associated with important diseases, such as cancer and diabetes. Traditionally, glucose radioisotopes have been used to monitor glucose utilization in biological systems. Fluorescent-tagged glucose analogues were initially developed in the 1980s, but it is only in the past decade that their use as a glucose sensor has increased significantly. These analogues were developed for monitoring glucose uptake in blood cells, but their recent applications include tracking glucose uptake by tumor cells and imaging brain cell metabolism. This review outlines the development of fluorescent-tagged glucose analogues, describes their recent structural modifications and discusses their increasingly diverse biological applications

Enhancement of Allele Discrimination by Introduction of Nucleotide Mismatches into siRNA in Allele-Specific Gene Silencing by RNAi

Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs) against mutant alleles of the human Prion Protein (PRNP) gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the ‘seed region’ of microRNAs. Due to the essential role of the ‘seed region’ of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs), of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3′-ends of sense-strand siRNA elements, which possibly increase the assembly of antisense-strand (guide) siRNAs into RNA-induced silencing complexes (RISCs), may enhance ASP-RNAi in the case of inert siRNA duplexes. Therefore, the data presented here suggest that structural modification of functional portions of an siRNA duplex by base substitution could greatly influence allele discrimination and gene silencing, thereby contributing to enhancement of ASP-RNAi

Design Safety Considerations for Water Cooled Small Modular Reactors Incorporating Lessons Learned from the Fukushima Daiichi Accident

The global future deployment of advanced nuclear reactors for electricity generation depends primarily on the ability of nuclear industries, utilities and regulatory authorities to further enhance their reliability and economic competitiveness while satisfying stringent safety requirements. The IAEA has a project to help coordinate Member State efforts in the development and deployment of small and medium sized or small modular reactor (SMR) technology. This project aims simultaneously to facilitate SMR technology developers and potential SMR users, particularly States embarking on a nuclear power programme, in identifying key enabling technologies and enhancing capacity building by resolving issues relevant to deployment, including nuclear reactor safety. The objective of this publication is to explore common practices for Member States, which will be an essential resource for future development and deployment of SMR technology. The accident at the Fukushima Daiichi nuclear power plant was caused by an unprecedented combination of natural events: a strong earthquake, beyond th e design basis, followed by a series of tsunamis of heights exceeding the design basis tsunami considered in the flood analysis for the site. Consequently, all the operating nuclear power plants and advanced reactors under development, including SMRs, have been incorporating lessons learned from the accident to assure and enhance the performance of the engineered safety features in coping with such external events. In response to the Fukushima Daiichi accident, the IAEA established an Action Plan on Nuclear Safety. The preparation of this publication was carried out within the framework of the IAEA Action Plan on effectively utilizing research and development. The main objective of this publication is to present technology developers and user s with common considerations, approaches and measures for enhancing the defence in depth and operability of water cooled SMR design concepts to cope with extreme natural hazards. Indicative requirements to prevent such an accident from recurring are also provided for States planning to adopt water cooled SMR designs and technologies. The IAEA gratefully acknowledges the information on technology and safety aspects provided by SMR design organizations and information regarding technical requirements provided by several Member States. The IAEA officers responsible for this publication were M.H. Subki of the Division of Nuclear Power and M. Kim of the Division of Nuclear Installation Safety