Promoter strength determines potency of shRNA-miRs.

<p>EGFP-expressing human immune cell types (Raji B, Jurkat T, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoter. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Relative promoter strength of the polymerase II promoters (mCherry Geo Mean) was measured from control vectors that lacked the miR30-backbone, since processing of shRNAs out of the miR30 backbone destabilized the transcript. Relative promoter strength is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R² values of fitted curves are indicated.</p

Efficacy of shRNA-miRs-guided target knockdown in various cell lines is dependent on the type of polymerase II promoter used.

<p>EGFP-expressing human immune cell types (Raji B cells, Jurkat T cells, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a miR-30 backbone driven by various indicated polymerase-II promoter. Cells were allowed to grow for the indicated number of days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) was assessed by flow cytometry. The presented percentage of EGFP knockdown is calculated by ((Geo-mean of uninfected cells minus Geo-mean of infected cells)/Geo-mean of uninfected cells)*100. The presented data is a representative experiment of 2 experiments performed in triplicate. For all data points, the standard deviation was below 5%.</p

Relative quantity of anti-EGFP siRNAs correlates with target-knockdown.

<p>EGFP-expressing human immune cell types (Raji B, Jurkat T, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoters. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Total RNA was isolated and relative mature anti-EGFP siRNA expression was determined by TaqMan analysis. The lowest quantity of mature anti-EGFP within each cell line was set to 1. Relative mature anti-EGFP siRNA expression is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R² values of fitted curves are indicated.</p

anti-EGFP shRNAs expressed from a mouse U6 promoter is more potent as compared to anti-EGFP shRNA-miRs expressed from an EF1A-promoter.

<p>A) EGFP-expressing human immune cell types (Raji B, Jurkat T, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a mouse U6 promoter (blue histograms) or with anti-EGFP shRNA-miRs expressed from the human EF1A promoter (red histograms). Eight days post infection, infected cells were monitored for EGFP expression by flow cytometry. The presented percentage of EGFP knockdown of the U6-driven shRNAs relative to the EF1A-driven shRNAs is calculated by ((Geo-mean of EF1A infected cells minus Geo-mean of U6 infected cells)/Geo-mean of EF1A infected cells)*100. B) mCherry expression is reduced upon cloning of an shRNA-containing miR-30 cassette in the 3′UTR of the fluorescent protein. Two examples are shown where either the CAGGS or EF1A promoter drives expression of mCherry with/without a functional miR cassette in it's 3′UTR. The percentage of reduced relative mCherry expression caused by the miR-cassette containing UTR is indicated. The presented data are representative experiments of 2 experiments performed in triplicate.</p

anti-EGFP shRNAs expressed from a miRNA backbone are less efficient in target-knockdown in Jurkat T cells than in 293T cells.

<p>A) schematic representation of the miR-context anti-EGFP shRNA construct cloned in the 3′UTR of mCherry which is expressed under the control of the polymerase II CMV promoter (lower panel) and the control anti-EGFP shRNA expressed from a mouse polymerase III U6 promoter (top panel). B) EGFP-expressing Jurkat T cells (top panels) or 293T cells (lower panels) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a mouse U6 promoter (U6 anti-EGFP shRNA) or from a miR-30 backbone expressed from a CMV promoter (miR-context anti-EGFP shRNA) or the relevant control viruses that lack shRNA inserts. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) and mCherry (as marker for infected cells) were assessed by flow cytometry. The indicated percentage of EGFP knockdown is calculated by ((Geo-mean of uninfected cells minus Geo-mean of infected cells)/Geo-mean of uninfected cells)*100. The presented data is a representative experiment of 2 experiments performed in triplicate.</p

shRNA-miR-directed target knockdown is highly dependent on the polymerase II promoter used.

<p>EGFP-expressing Jurkat T cells were infected at an MOI of <0.2 with anti-EGFP shRNA-miRs (left panels) or controls (no shRNA insert, right panels) driven by various indicated polymerase-II promoter. Cells were allowed to grow for 8 days and the expression EGFP (to monitor knockdown) and mCherry (as marker for infected cells) were assessed by flow cytometry. The indicated percentage of EGFP knockdown is calculated by ((Geo-mean of uninfected cells minus Geo-mean of infected cells)/Geo-mean of uninfected cells)*100. The presented data is a representative experiment of 2 experiments performed in triplicate.</p

Overview of the efficacy of shRNA-miRs-guided target knockdown in various human cell lines.

<p>EGFP-expressing human cell lines (see top row) were infected at an MOI of <0.2 with anti-EGFP shRNAs expressed from a miR-30 backbone driven by various indicated polymerase-II promoters (left column). Cells were allowed to grow for 8 days and the percentage of EGFP target knockdown was assessed by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026213#pone-0026213-g003" target="_blank">Figure 3</a> for details). The presented data is a representative experiment of 2 experiments performed in triplicate.</p

Determination of 55 Veterinary Antibiotics in Chicken Manure Using Ultra-High-Performance Liquid Chromatography – Tandem Mass Spectrometry (UPLC-MS/MS)

A highly efficient method for the detection and quantification of 55 veterinary antibiotics (VAs) in chicken manure belonging to six drug classes using solid-phase extraction (SPE) and ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed. The mixture of EDTA–McIlvaine buffer (pH 3.0) and the organic extractant (methanol–acetonitrile, 1:3, v/v) was used to isolate the target VAs from freeze-dried chicken manure. The extract was purified by a hydrophile–lipophile balance cartridge. All antibiotics showed excellent linear relationships in the range between 1 and 100 µg/kg with the coefficients of determination of the standard curves above 0.990. The recoveries of antibiotics were between 35.66% and 103.54% using matrix-matched calibration for quantification. The limits of detection and quantification were from 0.01 to 1.59 µg/kg and 0.04 to 4.76 µg/kg, respectively. The method was demployed to determine VAs in chicken manure from 12 farms, revealing contamination in all samples. A total of 13 VAs belonging to six classes were detected with concentrations up to 41.47 mg/kg. The presence of high concentrations of antibiotic residues in poultry manure poses a potential risk for environmental contamination. This work has identified significant differences in the types of VAs compared to literature reports. Additionally, it detected pleuromutilin antibiotics in chicken manure for the first time.</p

Table2_Protective role of curcumin in disease progression from non-alcoholic fatty liver disease to hepatocellular carcinoma: a meta-analysis.DOCX

Background: Pathological progression from non-alcoholic fatty liver disease (NAFLD) to liver fibrosis (LF) to hepatocellular carcinoma (HCC) is a common dynamic state in many patients. Curcumin, a dietary supplement derived from the turmeric family, is expected to specifically inhibit the development of this progression. However, there is a lack of convincing evidence.Methods: The studies published until June 2023 were searched in PubMed, Web of Science, Embase, and the Cochrane Library databases. The SYstematic Review Center for Laboratory animal Experimentation (SYRCLE) approach was used to evaluate the certainty of evidence. StataSE (version 15.1) and Origin 2021 software programs were used to analyze the critical indicators.Results: Fifty-two studies involving 792 animals were included, and three disease models were reported. Curcumin demonstrates a significant improvement in key indicators across the stages of NAFLD, liver fibrosis, and HCC. We conducted a detailed analysis of common inflammatory markers IL-1β, IL-6, and TNF-α, which traverse the entire disease process. The research results reveal that curcumin effectively hinders disease progression at each stage by suppressing inflammation. Curcumin exerted hepatoprotective effects in the dose range from 100 to 400 mg/kg and treatment duration from 4 to 10 weeks. The mechanistic analysis reveals that curcumin primarily exerts its hepatoprotective effects by modulating multiple signaling pathways, including TLR4/NF-κB, Keap1/Nrf2, Bax/Bcl-2/Caspase 3, and TGF-β/Smad3.Conclusion: In summary, curcumin has shown promising therapeutic effects during the overall progression of NAFLD–LF–HCC. It inhibited the pathological progression by synergistic mechanisms related to multiple pathways, including anti-inflammatory, antioxidant, and apoptosis regulation.</p

Table1_Protective role of curcumin in disease progression from non-alcoholic fatty liver disease to hepatocellular carcinoma: a meta-analysis.DOCX

Background: Pathological progression from non-alcoholic fatty liver disease (NAFLD) to liver fibrosis (LF) to hepatocellular carcinoma (HCC) is a common dynamic state in many patients. Curcumin, a dietary supplement derived from the turmeric family, is expected to specifically inhibit the development of this progression. However, there is a lack of convincing evidence.Methods: The studies published until June 2023 were searched in PubMed, Web of Science, Embase, and the Cochrane Library databases. The SYstematic Review Center for Laboratory animal Experimentation (SYRCLE) approach was used to evaluate the certainty of evidence. StataSE (version 15.1) and Origin 2021 software programs were used to analyze the critical indicators.Results: Fifty-two studies involving 792 animals were included, and three disease models were reported. Curcumin demonstrates a significant improvement in key indicators across the stages of NAFLD, liver fibrosis, and HCC. We conducted a detailed analysis of common inflammatory markers IL-1β, IL-6, and TNF-α, which traverse the entire disease process. The research results reveal that curcumin effectively hinders disease progression at each stage by suppressing inflammation. Curcumin exerted hepatoprotective effects in the dose range from 100 to 400 mg/kg and treatment duration from 4 to 10 weeks. The mechanistic analysis reveals that curcumin primarily exerts its hepatoprotective effects by modulating multiple signaling pathways, including TLR4/NF-κB, Keap1/Nrf2, Bax/Bcl-2/Caspase 3, and TGF-β/Smad3.Conclusion: In summary, curcumin has shown promising therapeutic effects during the overall progression of NAFLD–LF–HCC. It inhibited the pathological progression by synergistic mechanisms related to multiple pathways, including anti-inflammatory, antioxidant, and apoptosis regulation.</p