Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid–inducible gene-I and melanoma differentiation–associated gene 5

The ribonucleic acid (RNA) helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation–associated gene 5 (MDA5) recognize distinct viral and synthetic RNAs, leading to the production of interferons. Although 5′-triphosphate single-stranded RNA is a RIG-I ligand, the role of RIG-I and MDA5 in double-stranded (ds) RNA recognition remains to be characterized. In this study, we show that the length of dsRNA is important for differential recognition by RIG-I and MDA5. The MDA5 ligand, polyinosinic-polycytidylic acid, was converted to a RIG-I ligand after shortening of the dsRNA length. In addition, viral dsRNAs differentially activated RIG-I and MDA5, depending on their length. Vesicular stomatitis virus infection generated dsRNA, which is responsible for RIG-I–mediated recognition. Collectively, RIG-I detects dsRNAs without a 5′-triphosphate end, and RIG-I and MDA5 selectively recognize short and long dsRNAs, respectively

The correlation between the number of eligible patients in routine clinical practice and the low recruitment level in clinical trials: a retrospective study using electronic medical records.

[Background]A number of clinical trials have encountered difficulties enrolling a sufficient number of patients upon initiating the trial. Recently, many screening systems that search clinical data warehouses for patients who are eligible for clinical trials have been developed. We aimed to estimate the number of eligible patients using routine electronic medical records (EMRs) and to predict the difficulty of enrolling sufficient patients prior to beginning a trial. [Methods]Investigator-initiated clinical trials that were conducted at Kyoto University Hospital between July 2004 and January 2011 were included in this study. We searched the EMRs for eligible patients and calculated the eligible EMR patient index by dividing the number of eligible patients in the EMRs by the target sample size. Additionally, we divided the trial eligibility criteria into corresponding data elements in the EMRs to evaluate the completeness of mapping clinical manifestation in trial eligibility criteria into structured data elements in the EMRs. We evaluated the correlation between the index and the accrual achievement with Spearman's rank correlation coefficient. [Results]Thirteen of 19 trials did not achieve their original target sample size. Overall, 55% of the trial eligibility criteria were mapped into data elements in EMRs. The accrual achievement demonstrated a significant positive correlation with the eligible EMR patient index (r = 0.67, 95% confidence interval (CI), 0.42 to 0.92). The receiver operating characteristic analysis revealed an eligible EMR patient index cut-off value of 1.7, with a sensitivity of 69.2% and a specificity of 100.0%. [Conclusions]Our study suggests that the eligible EMR patient index remains exploratory but could be a useful component of the feasibility study when planning a clinical trial. Establishing a step to check whether there are likely to be a sufficient number of eligible patients enables sponsors and investigators to concentrate their resources and efforts on more achievable trials

The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus

The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.

Patients' attitudes towards generic drug substitution in Japan

Objective The aim of this study was to evaluate the understanding and attitude of Japanese patients towards generic drug substitutions.Method The subjects were male and female patients, who purchased their prescription medications at a pharmacy. A questionnaire was created to assess their attitudes towards generic drugs.Results Of 1215 respondents, 68.4% knew the term "generic drugs." The majority of them had the correct understanding only on the following two points: generic drugs are less expensive than the brand name drugs (86.0%) and generic drugs contain the same active ingredients as brand name drugs (71.1%). However, their understanding was poor in other aspects of generic substitution: the availability and accessibility of generic drugs, etc. Only the experience of a previous generic drug substitution was significantly associated with the increased willingness for generic substitution (OR = 2.93, CI 1.93-4.44). The main reasons for accepting generic substitutions were recommendations by physicians (48.6%) and by pharmacists (33.1%).Conclusion The public awareness program on generic drugs should be expanded to include more detailed information so that patients obtain the correct understanding of generic substitution. It is critical that physicians and pharmacists have the proper understanding of generic drug substitution and provide the correct information to patients.Patients' attitudes Generic drug substitution Japan

Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from <i>Chenopodium ficifolium</i>

<div><p>Photoconvertible water-soluble chlorophyll-binding proteins, called Class I WSCPs, have been detected in Chenopodiaceae, Amaranthaceae and Polygonaceae plant species. To date, <i>Chenopodium album WSCP</i> (<i>CaWSCP</i>) is the only cloned gene encoding a Class I WSCP. In this study, we identified two cDNAs encoding <i>Chenopodium ficifolium</i> Class I WSCPs, <i>CfWSCP1</i>, and <i>CfWSCP2</i>. Sequence analyses revealed that the open reading frames of <i>CfWSCP1</i> and <i>CfWSCP2</i> were 585 and 588 bp, respectively. Furthermore, both CfWSCPs contain cystein2 and cystein30, which are essential for the chlorophyll-binding ability of CaWSCP. Recombinant CfWSCP1 and CfWSCP2, expressed in <i>Escherichia coli</i> as hexa-histidine fusion proteins (CfWSCP1-His and CfWSCP2-His), formed inclusion bodies; however, we were able to solubilize these using a buffer containing 8 M urea and then refold them by dialysis. The refolded CfWSCP1-His and CfWSCP2-His could bind chlorophylls and exhibited photoconvertibility, confirming that the cloned CfWSCPs are further examples of Class I WSCPs.</p></div

Amplification efficiency of target genes.

<p>*Dynamic range represents the range of Cq values between the highest and the lowest concentration of generated standard curves.</p><p>Amplification efficiency of target genes.</p

Detection of smRNAs in frozen tissues.

<p>Average Cq values (Y-axis) of 8 smRNAs with various postmortem intervals (X-axis) are shown. Cq values for the miRNAs (dashed lines) appear to be more stable than those of other classes of smRNAs (solid lines). The mean value and standard deviation for each candidate smRNA are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129338#pone.0129338.t003" target="_blank">Table 3</a>.</p

Amplification efficiency of target genes.

<p>*Dynamic range represents the range of Cq values between the highest and the lowest concentration of generated standard curves.</p><p>Amplification efficiency of target genes.</p

Integrity analysis of smRNAs in autoptic tissues.

<p>Representative electropherograms of frozen tissues (A–C) and FFPE tissues (E–G). Green solid lines indicate the area (10–40 nt) containing miRNA peaks, and green dashed lines indicate the area containing smRNA peaks (0–200 nt). The ratio of smRNAs to miRNAs was evaluated using 11 frozen samples (D), and 17 FFPE samples (H). PMI, postmortem interval; RT, room temperature; FF, formalin-fixed period.</p

Control and biomarker candidate specifications.

<p>Control and biomarker candidate specifications.</p