Physcomitrella patens DCL3 Is Required for 22–24 nt siRNA Accumulation, Suppression of Retrotransposon-Derived Transcripts, and Normal Development

Endogenous 24 nt short interfering RNAs (siRNAs), derived mostly from intergenic and repetitive genomic regions, constitute a major class of endogenous small RNAs in flowering plants. Accumulation of Arabidopsis thaliana 24 nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24 nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21 nt microRNAs (miRNAs) and 21–22 nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci that consistently produced a mixture of 21–24 nt siRNAs with a peak at 23 nt. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5 mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22–24 nt siRNAs, but not 21 nt siRNAs, at Pp23SR loci. The 21 nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants failed to accumulate 22–24 nt small RNAs from repetitive regions while transcripts from two abundant families of long terminal repeat (LTR) retrotransposon-associated reverse transcriptases were up-regulated. Ppdcl3 mutants also displayed an acceleration of leafy gametophore production, suggesting that repetitive siRNAs may play a role in the development of P. patens. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants

Sensitive spectrophotometric assay of simvastatin in pharmaceuticals using permanganate

Two simple, sensitive, selective and inexpensive spectrophotometric methods are described for the determination of simvastatin (SMT) in bulk drug and in tablets using permanganate as the oxidimetric reagent. In method A, SMT is treated with a measured excess of permanganate in acetic acid medium and the unreacted oxidant is measured at 550 nm, whereas in method B the reaction is carried out in alkaline medium and the resulting manganate is measured at 610 nm. In method A, the amount of permanganate reacted corresponds to the SMT content and the absorbance is found to decrease linearly with the concentration; and in method B, the absorbance increases with concentration. The working conditions of assays were optimized, and the methods were validated according to the current ICH guidelines. Under optimum conditions, SMT could be assayed in the concentration ranges, 1.47 - 17.67x10-5 and 2.27 - 27.18 x10-6 mol/L by method A and method B, respectively. The calculated molar absorptivities are 3.2 x 10³ and 2.5 x 10(4) L/mol/cm for method A and method B, respectively with corresponding Sandell sensitivity values of 0.0387 and 0.0178 &#956;g/cm². The limits of detection (LOD) and quantification (LOQ) have also been reported. Accuracy and precision for the assay were determined by calculating the intra-day and inter-day at three concentrations; the intra-day RSD was < 2% and the accuracy was better than 2.15 % (RE). The methods were applied successfully for the determination of SMT in tablet dosage form with a high percentage of recovery, good accuracy and precision, and without measurable interference by the excipients. The accuracy was further ascertained from placebo and synthetic mixture analysis and also from the spike-recovery method.<br>Dois métodos espectrofotométricos simples, sensíveis, seletivos e baratos são descritos para a determinação de sinvastatina (SMT) a granel e em comprimidos, utilizando permanganato como reagente oxidimétrico. No método A, a SMT é tratada com excesso conhecido de permanganato em meio de ácido acético e o oxidante que não reage é medido a 550 nm, enquanto no método B, a reação é efetuada em meio alcalino e o manganato resultante é medido a 610 nm. No método A, a quantidade de permanganato que reage corresponde ao conteúdo de SMT e a absorbância diminui linearmente com o aumento da concentração; no método B, a absorbância aumenta com o aumento da concentração. As condições de trabalho do ensaio foram otimizadas e os métodos, validados de acordo com as normas do ICH. Sob condições ótimas, a SMT pode ser ensaiada nas faixas de concentração de 1,47- 17,67x10-5 e de 2,27-27,18 x10-6 mol/L pelo método A e B, respectivamente. As absortividades molares calculadas são 2 x 10³ e 2,5 x 10(4) L/ mol/cm, respectivamente, para os métodos A e B, com os valores correspondentes de sensibilidade de Sandell de 0,0387 e 0,0178 &#956;g/cm². Os limites de detecção (LOQ) também foram relatados. A exatidão e a precisão do ensaio foram determinadas pelo cálculo de três concentrações intra- e inter-dia; a RSD intra-dia foi <2% e a exatidão foi melhor que 2,15% (RE). Os métodos foram aplicados com sucesso à determinação de SMT em comprimidos com alta porcentagem de recuperação, boa exatidão e precisão e sem interferência mensurável dos excipientes. A exatidão foi posteriormente determinada no placebo e na mistura sintética e, também, pelo método de spike recovery