Bar graphs show the relative level of expression of 15 of the differentially expressed genes in paired myometrium (M) and leiomyomas (L) from Caucasians (C) and African Americans (AA) determined by realtime PCR low density array

<p><b>Copyright information:</b></p><p>Taken from "Genomic and proteomic profiling I: Leiomyomas in African Americans and Caucasians"</p><p>http://www.rbej.com/content/5/1/34</p><p>Reproductive biology and endocrinology : RB&E 2007;5():34-34.</p><p>Published online 23 Aug 2007</p><p>PMCID:PMC2063502.</p><p></p> Values on the y-axis represent an arbitrary unit derived from the mean expression of each gene independently with the mean value of myometrium from Caucasian set at 1 for each gene. The asterisks * are statistically different from ** comparing paired myometrium and leiomyomas from African Americans and Caucasians with arrows pointing out the difference between the expression of these genes in leiomyoma and myometrium in each group. A probability level of P < 0.05 was considered significant

The bar graphs show the relative mean expression levels of 12 genes (E2F1, RUNX3, EGR3, TBPIP, ECM-2 ESM1, THBS1, GAS1, ADAM17, CST6, FBLN5, and COL18A1) in leiomyomas (LYM), keloids/incisional scars (Scar) and peritoneal adhesions (P

<p><b>Copyright information:</b></p><p>Taken from "Genomic and proteomic profiling II: Comparative assessment of gene expression profiles in leiomyomas, keloids, and surgically-induced scars"</p><p>http://www.rbej.com/content/5/1/35</p><p>Reproductive biology and endocrinology : RB&E 2007;5():35-35.</p><p>Published online 24 Aug 2007</p><p>PMCID:PMC2039739.</p><p></p> Adhesion) using realtime PCR and LDA as described in materials and methods section. Values on the y-axis represent an arbitrary unit derived from the mean expression level of these genes in each tissue with their mean expression values in leiomyomas set at 1 independently for each gene prior to normalization against their expression levels in myometrium form a Caucasian serving as control. The asterisks * indicate statistical difference between the expression of these genes with arrows pointing the difference between each group. A probability level of P < 0.05 was considered significant

Cluster analysis of 206 differentially expressed genes in leiomyomas from Caucasians (CL1, CL2, and CL3) and peritoneal adhesions (A1, A2, A3) using Affymetrix U95 array

<p><b>Copyright information:</b></p><p>Taken from "Genomic and proteomic profiling II: Comparative assessment of gene expression profiles in leiomyomas, keloids, and surgically-induced scars"</p><p>http://www.rbej.com/content/5/1/35</p><p>Reproductive biology and endocrinology : RB&E 2007;5():35-35.</p><p>Published online 24 Aug 2007</p><p>PMCID:PMC2039739.</p><p></p> The genes were selected based on supervised and unsupervised assessment and p ranking at P < 0.01 followed by 2-fold cutoff change selection. The genes represented by rows were clustered according to their similarities in expression patterns for each tissue and identified as A and B

Combined Adenovirus-Mediated Artificial microRNAs Targeting mfgl2, mFas, and mTNFR1 Protect against Fulminant Hepatic Failure in Mice

<div><p>Hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) has a poor prognosis with high in-hospital mortality. Hepatic and circulating inflammatory cytokines, such as fibrinogen like protein 2 (fgl2), FasL/Fas, and TNFα/TNFR1, play a significant role in the pathophysiology of ACLF. This study aimed to investigate the therapeutic effect of recombinant adenoviral vectors carrying constructed DNA code for non-native microRNA (miRNA) targeting mouse fgl2 (mfgl2) or both mFas and mTNFR1 on murine hepatitis virus (MHV)-3-induced fulminant hepatitis in BALB/cJ mice. Artificial miRNA eukaryotic expression plasmids against mfgl2, mFas, and mTNFR1 were constructed, and their inhibitory effects on the target genes were confirmed <i>in vitro</i>. pcDNA6.2-mFas-mTNFR1- miRNA，which expresses miRNA against both mFas and mTNFR1 simultaneously，was constructed. To construct a miRNA adenovirus expression vector against mfgl2, pcDNA6.2-mfgl2-miRNA was cloned using Gateway technology. Ad-mFas-mTNFR1- miRNA was also constructed by the same procedure. Adenovirus vectors were delivered by tail-vein injection into MHV-3-infected BALB/cJ mice to evaluate the therapeutic effect. 8 of 18 (44.4%) mice recovered from fulminant viral hepatitis in the combined interference group treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA. But only 4 of 18 (22.2%) mice receiving Ad-mfgl2-miRNA and 3 of 18 (16.7%) mice receiving Ad-mFas-mTNFR1- miRNA survived. These adenovirus vectors significantly ameliorated inflammatory infiltration, fibrin deposition, hepatocyte necrosis and apoptosis, and prolonged survival time. Our data illustrated that combined interference using adenovirus-mediated artificial miRNAs targeting mfgl2, mFas, and mTNFR1 might have significant therapeutic potential for the treatment of fulminant hepatitis.</p> </div

The constructed miR adenovirus did not affect the expression of certain apoptosis-related proteins, but significantly decreased cleavage of caspase-3.

<p>(<b>A</b>) There was significantly decreased cleavage of caspase-3 in Ad-mFas-mTNFR1-miRNA treated mice and combined interference group at 72 h after MHV-3 infection. (<b>B</b>) Both Ad-mFas-mTNFR1-miR and combined interference with the two adenoviral miRNAs did not affect the expression of pro-apoptotic proteins, including Bax and Bad, and anti-apoptotic proteins, including Bcl-2 and c-IAP2 at 72 h after MHV-3 infection. The average protein expression from Negative miRNA control group was designated as 1. *<i>P</i> <0.05.</p

Combined interference increased the survival rate and improved liver function and histopathology in mice.

<p>(<b>A</b>): Combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA had a higher survival rate than that of interference with either construct alone in MHV-3–infected BALB/cJ mice. Ad-mfgl2-miRNA, Ad-mFas-mTNFR1-miRNA, or irrelevant miRNA control adenovirus were introduced into BALB/cJ mice by tail vein injection. The mice then received 20 PFU of MHV-3 intraperitoneally 24 hours later to promote the development of fulminant viral hepatitis. Survival data are presented. Serial serum ALT levels (<b>B</b>) and histopathology (<b>C</b>) (H&E staining; original magnification, ×400) at 24, 48, and 72 h post MHV-3 infection were evaluated in the five groups of mice. (<b>B</b>) Effect of Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA on serum ALT levels. Values represent means and standard error of three independent experiments done in triplicate. *<i>P</i> <0.05, #P <0.01 compared with the negative miRNA control adenovirus group. (<b>C</b>) Effect of Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA on liver histopathology in MHV-3–infected BALB/cJ mice. Livers were collected from Ad-mfgl2-miRNA–treated (<b>g</b>, <b>h</b>, and <b>i</b>), Ad-mFas-mTNFR1-miRNA–treated (<b>j</b>, <b>k</b>, and <b>l</b>), combined interference-treated (<b>m</b>, <b>n</b>, and <b>o</b>), irrelevant miRNA adenovirus-treated (<b>d</b>, <b>e</b>, and <b>f</b>), or PBS-treated (<b>a</b>, <b>b</b>, and <b>c</b>) BALB/cJ mice at 24 h (a, d, g, j, and m), 48 h (b, e, h, k, and n), and 72 h (c, f, i, l, and o) after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic areas with the inflammation. </p

Ad-mfgl2-miRNA and/or Ad-mFas-mTNFR1-miRNA inhibited target genes at both mRNA and protein levels <i>in</i><i>vivo</i>.

<p>The treatment process was the same as that described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082330#pone-0082330-g002" target="_blank">Figure 2</a>, and livers were collected from treated BALB/cJ mice 0, 24, 48, and 72 h after MHV-3 infection. (<b>a</b>) qRT-PCR showed both Ad-mfgl2-miRNA and combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mfgl2 mRNA expression 48 h and 72 h after MHV-3 infection. Both Ad-mFas-mTNFR1-miRNA and combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mFas (<b>b</b>) and mTNFR1 (<b>c</b>) mRNA expression 48 h and 72 h after MHV-3 infection also. Values represent means and SE of three separate experiments done in triplicate.*<i>P</i> <0.05 compared with Negative miRNA control adenovirus group. (<b>d</b>-<b>f</b>) Western blot analysis showed combined interference with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA inhibited mfgl2 (<b>d</b>), mFas (<b>e</b>), and mTNFR1 (<b>f</b>) protein expression at 72 h after MHV-3 infection. The average protein expression from Negative miRNA control group was designated as 1. *<i>P</i> <0.05.</p

Effects of Targeted Suppression of Glutaryl-CoA Dehydrogenase by Lentivirus-Mediated shRNA and Excessive Intake of Lysine on Apoptosis in Rat Striatal Neurons

<div><p></p><p>In glutaric aciduria type 1 (GA1), glutaryl-CoA dehydrogenase (GCDH) deficiency has been shown to be responsible for the accumulation of glutaric acid and striatal degeneration. However, the mechanisms by which GA1 induces striatal degeneration remain unclear. In this study, we aimed to establish a novel neuronal model of GA1 and to investigate the effects of GCDH deficiency and lysine-related metabolites on the viability of rat striatal neurons. Thus we constructed a lentiviral vector containing short hairpin RNA targeted against the GCDH gene expression (lentivirus-shRNA) in neurons. A virus containing a scrambled short hairpin RNA construct served as a control. Addition of lysine (5 mmol/L) was used to mimic hypermetabolism. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis was assessed using Hoechst33342 staining and Annexin V-PE/7-AAD staining. The mitochondrial membrane potential (MPP) was monitored using tetramethylrhodamine methyl ester. The expression levels of caspases 3, 8, and 9 were determined by Western blotting. We found that lentivirus-shRNA induced apoptosis and decreased MMP levels in neurons, and addition of 5 mmol/L lysine enhanced this effect markedly. Lentivirus-shRNA upregulated the protein levels of caspases 3 and 9 regardless of the presence of 5 mmol/L lysine. The expression level of caspase 8 was higher in neurons co-treated with lentivirus-shRNA and 5 mmol/L lysine than in control. Benzyloxy-carbonyl-Val-Ala-Asp(OMe)-fluoromethylketone, a pan-caspase inhibitor, blocked the apoptosis induced by lentivirus-shRNA and 5 mmol/L lysine to a great extent. These results indicate that the targeted suppression of GCDH by lentivirus-mediated shRNA and excessive intake of lysine may be a useful cell model of GA1. These also suggest that GA1-induced striatal degeneration is partially caspase-dependent.</p></div

OD in the detection of neuron viability by MTT assay.

<p>Viability rate (%)  = (OD_m−OD_blank)/(OD₀−OD_blank); OD_m: The OD of each sample; OD₀: The OD of neurons with 0 mmol/L lysine group. OD_blank: The OD of the blank control (0.169±0.0252).</p>*<p><i>P<</i>0.05 <i>vs.</i> neurons with 0 mmol/L lysine group.</p

Detection of apoptosis using flow cytometry.

<p>Cells were assayed for apoptosis using Annexin V-PE/7-AAD staining with flow cytometry. Cells were grouped and treated as shown to quantify the apoptosis induced by GCDH knockdown and increased lysine. Lentivirus-shRNA#1 induced apoptosis, and 5 mmol/L lysine increased the rate of apoptosis to a significantly greater extent. Z-VAD-FMK, a pan-caspase inhibitor, blocked the apoptosis induced by lentivirus-shRNA and increased lysine to a great extent. *<i>P<</i>0.05.</p