In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.

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Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2–8 from the 5′end of the guide strand; its complementary sequence; the 5′end of the guide strand and the 3′overhang of the passenger strand. However, most part of the 3′ two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3′end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA–RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA–RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.

Screening of dominant enhancers for sev-Src42A[KR]Enhancers were searched for using P[sev-Src42A[KR]] (Takahashi et al., 1996) inserted into CyO or TM3 balancers. E(7A-1), which is incapable of complementing shg R64a

In Drosophila, Dsrc64 is considered a unique ortholog of the vertebrate c-src; however, we show evidence to the contrary. The closest relative of vertebrate c-src so far found in Drosophila is not Dsrc64, but Dsrc41, a gene identified for the first time here. In contrast to Dsrc64, overexpression of wild-type Dsrc41 caused little or no appreciable phenotypic change in Drosophila. Both gain-of-function and dominant-negative mutations of Dsrc41 caused the formation of supernumerary R7-type neurons, suppressible by one-dose reduction of boss, sev, Rasl, or other genes involved in the Sev pathway. Dominant-negative mutant phenotypes were suppressed and enhanced, respectively, by increasing and decreasing the copy number of wild-type Dsrc41. Colocalization of Dsrc41 protein, actin fibers and DE-cadherin, and Dsrc41-dependent disorganization of actin fibers and putative adherens junctions in precluster cells suggested that Dsrc41 may be involved in the regulation of cytoskeleton organization and cell-cell contacts in developing ommatidia.

Post-hoc analyses of the ALS Functional Rating Scale-Revised (ALSFRS-R) score data, the primary endpoint in the 24-week double-blind placebo-controlled study of edaravone (MCI186-19, NCT01492686), were performed to confirm statistical robustness of the result. The previously reported original analysis had used a last observation carried forward (LOCF) method and also excluded patients with fewer than three completed treatment cycles. The post-hoc sensitivity analyses used different statistical methods as follows: 1) including all patients regardless of treatment cycles received (ALL LOCF); 2) a mixed model for repeated measurements (MMRM) analysis; and 3) the Combined Assessment of Function and Survival (CAFS) endpoint. Findings were consistent with the original primary analysis in showing superiority of edaravone over placebo. We also investigated the distribution of change in ALSFRS-R total score across all patients in the study as well as which ALSFRS-R items and domains may have contributed to the overall efficacy findings. The distribution of changes in ALSFRS-R total score from baseline to the end of cycle 6 (ALL LOCF) shifted in favour of edaravone compared to placebo. Edaravone was descriptively favoured for each ALSFRS-R item and each of the four ALSFRS-R domains at the end of cycle 6 (ALL LOCF), suggesting a generalised effect of edaravone in slowing functional decline across all anatomical regions. The effect of edaravone appeared to be similar in patients with bulbar onset and limb onset. Together, these observations would be consistent with its putative neuroprotective effects against the development of oxidative damage unspecific to anatomical regions.

In the 24-week double-blind study of edaravone in ALS (MCI186-16), edaravone did not show a statistically significant difference versus placebo for the primary efficacy endpoint. For post-hoc analyses, two subpopulations were identified in which edaravone might be expected to show efficacy: the efficacy-expected subpopulation (EESP), defined by scores of ≥2 points on all 12 items of the ALS Functional Rating Scale-Revised (ALSFRS-R) and a percent predicted forced vital capacity (%FVC) ≥80% at baseline; and the definite/probable EESP 2 years (dpEESP2y) subpopulation which, in addition to EESP criteria, had definite or probable ALS diagnosed by El Escorial revised criteria, and disease duration of ≤2 years. In the 36-week extension study of MCI186-16, a 24-week double-blind comparison followed by 12 weeks of open-label edaravone (MCI186-17; NCT00424463), analyses of ALSFRS-R scores of the edaravone-edaravone group and edaravone-placebo group for the full analysis set (FAS) and EESP, as prospectively defined, were reported in a previous article. Here we additionally report results in patients who met dpEESP2y criteria at the baseline of MCI186-16. In the dpEESP2y, the difference in ALSFRS-R changes from 24 to 48 weeks between the edaravone-edaravone and edaravone-placebo groups was 2.79 (p = 0.0719), which was greater than the differences previously reported for the EESP and the FAS. The pattern of adverse events in the dpEESP2y did not show any additional safety findings to those from the earlier prospective study. In conclusion, this post-hoc analysis suggests a potential effect of edaravone between 24 and 48 weeks in patients meeting dpEESP2y criteria at baseline.

OBJECTIVE To evaluate and compare morphological characteristics of the dens in atlantoaxial instability (AAI)-predisposed toy-breed dogs (TBDs) with and without AAI and non-AAI-predisposed healthy Beagles. ANIMALS 80 AAI-affected and 40 nonaffected TBDs and 40 Beagles. PROCEDURES Each dog underwent CT examination of the cervical vertebral column. On median 3-D multiplanar reconstruction images, the dens angle (DA) was measured as were the lengths of the dens and the body of the axis; the dens-to-axis length ratio (ratio of the dens length to the axis body length [DALR]) was calculated. Data were compared among dog groups. RESULTS The DALR in nonaffected TBDs and Beagles did not differ significantly. The mean DALR for AAI-affected TBDs was significantly lower than that for nonaffected TBDs. The mean DA of AAI-affected TBDs was significantly greater than that of Beagles and nonaffected TBDs. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that a low DALR might be associated with a high probability of dens abnormalities in TBDs. Additionally, dens length in AAI-affected TBDs appeared to be smaller than that in non-AAI-affected TBDs, given the low DALR in AAI-affected TBDs. Further investigations to determine reference ranges of the DA and DALR and the potential usefulness of those variables as diagnostic markers for AAI in TBDs are warranted.