Analysis of the Metabolites of Indole Degraded by an Isolated Acinetobacter pittii L1

Indole and its derivatives are typical nitrogen heterocyclic compounds and have been of immense concern since they are known for the risk of their toxic, recalcitrant, and carcinogenic properties for human and ecological environment. In this study, a Gram-negative bacterial strain of eliminating indole was isolated from a coking wastewater. The strain was confirmed as Acinetobacter pittii L1 based on the physiological and biochemical characterization and 16S ribosomal DNA (rDNA) gene sequence homology. 400 mg/L indole could be completely removed within 48 h by the strain on the optimum condition of 37°C, pH 7.4, and 150 rpm. The organic nitrogen was converted to NH3-N and then to NO3- and the organic carbon was partially transferred to CO2 during the indole biodegradation. The metabolic pathways were proposed to explain the indole degradation based on the liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of indigo, 4-(3-Hydroxy-1H-pyrrol-2-yl)-2-oxo-but-3-enoic acid, and isatin. The toxicity of the biodegradation products was evaluated using the Microtox test, which revealed that the metabolites were more toxic than indole. Our research holds promise for the potential application of Acinetobacter pittii L1 for NHCs degradation, production of indigoids, and soil remediation as well as treatment of indole containing wastewater

Correction: MicroRNA-221 Induces Cell Survival and Cisplatin Resistance through PI3K/Akt Pathway in Human Osteosarcoma

BACKGROUND: MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional and/or translational level and are deeply involved in the pathogenesis of several types of cancers. Specifically, microRNA-221 (miR-221) is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. However, the function of miR-221 in human osteosarcoma has not been totally elucidated. In the present study, the effects of miR-221 on osteosarcoma and the possible mechanism by which miR-221 affected the survival, apoptosis, and cisplatin resistance of osteosarcoma were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Real-time quantitative PCR analysis revealed miR-221 was significantly upregulated in osteosarcoma cell lines than in osteoblasts. Both human osteosarcoma cell lines SOSP-9607 and MG63 were transfected with miR-221 mimic or inhibitor to regulate miR-221 expression. The effects of miR-221 were then assessed by cell viability, cell cycle analysis, apoptosis assay, and cisplatin resistance assay. In both cells, upregulation of miR-221 induced cell survival and cisplatin resistance and reduced cell apoptosis. In addition, knockdown of miR-221 inhibited cell growth and cisplatin resistance and induced cell apoptosis. Potential target genes of miR-221 were predicted using bioinformatics. Moreover, luciferase reporter assay and western blot confirmed that PTEN was a direct target of miR-221. Furthermore, introduction of PTEN cDNA lacking 3′-UTR or PI3K inhibitor LY294002 abrogated miR-221-induced cisplatin resistance. Finally, both miR-221 and PTEN expression levels in osteosarcoma samples were examined by using real-time quantitative PCR and immunohistochemistry. High miR-221 expression level and inverse correlation between miR-221 and PTEN levels were revealed in osteosarcoma tissues. CONCLUSIONS/SIGNIFICANCE: These results for the first time demonstrate that upregulation of miR-221 induces the malignant phenotype of human osteosarcoma whereas knockdown of miR-221 reverses this phenotype, suggesting that miR-221 could be a potential target for osteosarcoma treatment

Inverse correlation of expression of miR-221 and PTEN in human osteosarcoma samples.

<p><b>A</b>, expression of miR-221 was analyzed by qRT-PCR in normal adjacent tissues (NAT), primary osteosarcoma tissues (POT) and recurrent osteosarcoma tissues (ROT). U24 was used as internal control. The average expression values of adjacent normal tissues is set at 1. Horizontal bars in the box represent median. *, p<0.05, ***, p<0.001. <b>B</b>, expression of PTEN was analyzed in osteosarcoma tissues with immunohistochemical staining. <b>C</b>, Chisquare test analysis of miR-221 and PTEN expression. The inverse correlation is significant (p<0.001).</p

miR-221 expression and miR-221 oligonucleotides transfection in osteosarcoma cells.

<p><b>A</b>, the expression of miR-221 was analyzed by qRT-PCR and normalized to RNU24 in five osteosarcoma cell lines. The expression of miR-221 in hFOB1.19 osteoblasts was used as a control. For <b>B</b>–<b>D</b>, SOSP-9607 and MG 63 cells were transfected with miR-221 mimic (221 Mi), miR-221 inhibitor (221 In), or scramble oligonucleotides (Scr). The blank (not transfected) cells were used as control. 24 h after transfection, the cells were observed and miR-221 expression levels were evaluated by qRT-PCR analysis. <b>B</b>, SOSP-9607 cells (<b>top</b>) and MG 63 cells (<b>bottom</b>) were observed with white bright (<b>left</b>) and green fluorescence assay (<b>right</b>) in the same vision using fluorescence microscopy (200×). <b>C</b>, miR-221 expression levels were evaluated by qRT-PCR analysis in transfected SOSP-9607 cells. <b>D</b>, miR-221 expression levels were assayed in transfected MG63 cells. RNU24 was used as an internal loading control to normalize the results. For <b>C</b> and <b>D</b>, data was presented as means ± SD. Columns, mean of three independent experiments; bars, SD; ***, p<0.001.</p

Involvement of miR-221 in cisplatin resistance in both SOSP-9607 and MG63 cells.

<p>Cells were transfected with miR-221 mimic (221 Mi), miR-221 inhibitor (221 In) or scramble oligonucleotide (Scr). The blank cells were used as control. Twenty four hours after transfection, treated cells were seeded into 96-well plates at the density of 5×10³ per well. <b>A</b> and <b>B</b>, twenty four hours later, SOSP-9607 and MG 63 cells were treated with or without 10 uM cisplatin for 24 h, 48 h or 72 h. Viability evaluation were examined using MTT assay. Then viability inhibition was calculated. <b>C</b> and <b>D</b>, twenty four hours later, SOSP-9607 and MG 63 cells were treated with or without the indicated concentrations of cisplatin for 48 h. Then, viability evaluation were examined using MTT assay. <b>E</b> and <b>F</b>, twenty four hours later, transfected SOSP-9607 and MG 63 cells were treated with or without 10 uM cisplatin for 24 h. Caspase3 activity was measured using Caspase-Glo® 3/7 assay kit (Promega). The data were presented as the means ± SD. Columns, mean of three independent experiments; bars, SD. **, p<0.01, ***, p<0.001.</p

miR-221 targets PTEN leading to activation of the Akt pathway.

<p><b>A</b>, putative miR-221-binding sites (red) in the 3′-UTRs of PTEN (<b>top</b>). The <b>middle</b> panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The <b>bottom</b> shows a mutant of PTEN 3′-UTR for pMIR-REPORT. <b>B</b>, SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. <b>C</b>, SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). <b>D</b>, similar experiments were performed in MG63 cells as described in panel <b>C</b> except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p<0.01.</p

Performance of the first beam on Shenguang III technical Integration Line

Technical Integration Line (TIL), which is being constructed at CAEP, is the prototype of Shenguang III fusion laser facility. Experimental results during the first integrated tests on the first line demonstrate that the laser system architecture from master oscillator through the laser amplifiers can meet the requirement of TIL engineering design. In this paper, brief introduction of TIL and main experiment results are presented

miR-221 regulates apoptosis in both SOSP-9607 and MG63 cells.

<p><b>A, C</b> SOSP-9607 cells were transfected with 100 nM miR-221 mimic (221 Mi), miR-221 inhibitor (221 In) or scramble oligonucleotide (Scr) for 48 h. The blank cells were used as control. Apoptosis was measured by FACS with Annexin V and propidium iodide staining. <b>B, D</b> Apoptosis of MG 63 was measured by FACS with Annexin V and propidium iodide staining. The data were presented as the means ± SD in panel <b>C</b> and panel <b>D</b>. Columns, mean of three independent experiments; bars, SD; **, p<0.01, ***, p<0.001.</p