Comparing 2-nt 3\u27 Overhangs Against Blunt-Ended siRNAs: A Systems Biology Based Study

In this study, we formulate a computational reaction model following a chemical kinetic theory approach to predict the binding rate constant for the siRNA-RISC complex formation reaction. The model allowed us to study the potency difference between 2-nt 3\u27 overhangs against blunt-ended siRNA molecules in an RNA interference (RNAi) system. The rate constant predicted by this model was fed into a stochastic simulation of the RNAi system (using the Gillespie stochastic simulator) to study the overall potency effect. We observed that the stochasticity in the transcription/translation machinery has no observable effects in the RNAi pathway. Sustained gene silencing using siRNAs can be achieved only if there is a way to replenish the dsRNA molecules in the cell. Initial findings show about 1.5 times more blunt-ended molecules will be required to keep the mRNA at the same reduced level compared to the 2-nt overhang siRNAs. However, the mRNA levels jump back to saturation after a longer time when blunt-ended siRNAs are used. We found that the siRNA-RISC complex formation reaction rate was 2 times slower when blunt-ended molecules were used pointing to the fact that the presence of the 2-nt overhangs has a greater effect on the reaction in which the bound RISC complex cleaves the mRNA

Comparing 2-nt 3' overhangs against blunt-ended siRNAs: a systems biology based study

In this study, we formulate a computational reaction model following a chemical kinetic theory approach to predict the binding rate constant for the siRNA-RISC complex formation reaction. The model allowed us to study the potency difference between 2-nt 3' overhangs against blunt-ended siRNA molecules in an RNA interference (RNAi) system. The rate constant predicted by this model was fed into a stochastic simulation of the RNAi system (using the Gillespie stochastic simulator) to study the overall potency effect. We observed that the stochasticity in the transcription/translation machinery has no observable effects in the RNAi pathway. Sustained gene silencing using siRNAs can be achieved only if there is a way to replenish the dsRNA molecules in the cell. Initial findings show about 1.5 times more blunt-ended molecules will be required to keep the mRNA at the same reduced level compared to the 2-nt overhang siRNAs. However, the mRNA levels jump back to saturation after a longer time when blunt-ended siRNAs are used. We found that the siRNA-RISC complex formation reaction rate was 2 times slower when blunt-ended molecules were used pointing to the fact that the presence of the 2-nt overhangs has a greater effect on the reaction in which the bound RISC complex cleaves the mRNA

Pengaruh Harga Terhadap Peningkatan Penjualan Produk Semen Tiga Roda Pada PT. Robcaga Beo Kabupaten Kepulauan Talaud

The development in business world these days is market by the competition between the business company is getting fierce. Especially in managing the company business unit. It is shown by the appearance of a company that offer a good quality product with a compete price on the market. To handle the fierce competition on the market then one from so many effort that the company do is by apply the strategic price. Which on the way of applying that strategi the company try to set a price that can be compete in the market so the increase sale of the product become maximum. With right price and controlled will result the domino effect to a company to build long term relationship with costumer so it can increase the sales volume. This research is a descriptive quantitative research by using the correlation approach and simple regression. To see relation between variable and to measure the impact to the variable itself. So the purpose of this research is to know how far the price effect and to the increase of PT. ROBCAGA in Talaud. According to the sesult of the research, can be shown as following: price has a correlation and significant determination effort to the increase sale of PT. ROBCAGA Talaud. According to the data analysis, coefficient value moment r = 0,685. That show there is a positive relation, and can be categorize as high and strong, also price coefficient determination to the increase sale is by 46,5% and 53,5% by the rest of it depends on the unknown factors that not been analyze in this research

Correction: Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

[This corrects the article DOI: 10.1371/journal.pbio.2001882.]

Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo

Identification and validation of PF-06446846–sensitive nascent chains.

<p>(A) Outline of the approach to identify PF-06446846–targeted mRNAs. (B) Example readplot and (C) example cumulative fractional read (CFR) plot for proprotein convertase subtilisin/kexin type 9 (PCSK9). A CFR plot depicts at each codon the percentage of reads aligning at or 5ʹ to that codon. In all plots, data from 1.5 μM PF-06446846 treatments are shown in red and vehicle treatments are shown in blue. The major stall and the position of D_max is marked. (D) Scatterplot showing the distribution of D_max values as a function of read counts; red indicates D_max Z-score > 3 (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#sec007" target="_blank">Materials and methods</a> for Z-score calculations) and green indicates 2 < Z-score < 3. (E) Scatterplot of fold change versus expression level when reads mapping 3ʹ to D_max position (for Z-score > 2) or codon 50 (for Z-score < 2) are used. Genes for which D_max Z-score > 2 and DeSeq fasle discovery rate (FDR) < 10% are highlighted in red, in green for Dmax Z-score > 2 but FDR > 10%, and in purple for D_max Z-score < 2 with FDR < 10%. (F–I) Example readplots for PF-06446846–sensitive proteins (F) HSD17B11, (G) RPL27, (H) PCBP1, and (I) cadherin-1 (CDH1). Bars representing the treatment dataset are red and go upwards and bars representing the vehicle datasets are blue and go downwards. All graphs are derived from the 1-h treatment time in the first study. (J) Cell-free translation assays showing inhibition of translation by 50 μM PF-06446846 when the stall sites identified by ribosome profiling are fused to the N-terminus of luciferase. (K) Inhibition of in vitro translation of full-length Midikine- and BCAP31-luciferase fusions in the cell-free translation system. (L) In vitro translation of control constructs not predicted to be inhibited by PF-06446846 from cell-based experiments. (M) In vitro translation of constructs with PF-06446846–induced stalls identified only at the 10-min treatment time. The individual quantitative observations that underlie Fig 5J–M are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.s029" target="_blank">S14 Table</a>.</p

Oral administration of PF-06446846 reduces plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) and total cholesterol levels in rats.

<p>(A–B) Plasma PCSK9 levels following (A) a single and (B) 12 daily oral doses of PF-06446848. Rats were administered the indicated dose of PF-06446846, and plasma concentrations of PCSK9 were measured by commercial ELISA at 1, 3, 6, and 24 h after dosing (A) or the 12th daily dose (B). Symbols represent mean concentration ± standard error and were jittered to provide a clearer graphical representation. Data were analyzed using a mixed model repeated measure (MMRM) with treatment, day, and hour as fixed factors; treatment by day and hour as an interaction term; and animal as a random factor. The significance level was set at a level of 5%. No adjustment for multiple comparisons was used. *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, ***<i>p</i> ≤ 0.001. (C–E) Total plasma (C), low-density lipoprotein (LDL) (D), and high-density lipoprotein (HDL) (E) cholesterol levels in rats measured 24 h following 14 daily oral doses of PF-06446846. Symbols represent individual animal values. The middle horizontal bar represents the group mean ± standard deviation. Difference between group means relative to vehicle was performed by a one-way ANOVA followed by a Dunnett’s multiple comparisons test; * <i>p</i> ≤ 0.05, **** <i>p</i> ≤ 0.0001. The individual quantitative observations that underlie Fig 2 are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001882#pbio.2001882.s029" target="_blank">S14 Table</a>.</p