Progressive Degradation of Crude Oil <i>n</i>-Alkanes Coupled to Methane Production under Mesophilic and Thermophilic Conditions

<div><p>Although methanogenic degradation of hydrocarbons has become a well-known process, little is known about which crude oil tend to be degraded at different temperatures and how the microbial community is responded. In this study, we assessed the methanogenic crude oil degradation capacity of oily sludge microbes enriched from the Shengli oilfield under mesophilic and thermophilic conditions. The microbial communities were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes combined with cloning and sequencing. Enrichment incubation demonstrated the microbial oxidation of crude oil coupled to methane production at 35 and 55°C, which generated 3.7±0.3 and 2.8±0.3 mmol of methane per gram oil, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that crude oil <i>n</i>-alkanes were obviously degraded, and high molecular weight <i>n</i>-alkanes were preferentially removed over relatively shorter-chain <i>n</i>-alkanes. Phylogenetic analysis revealed the concurrence of acetoclastic <i>Methanosaeta</i> and hydrogenotrophic methanogens but different methanogenic community structures under the two temperature conditions. Candidate divisions of JS1 and WWE 1, <i>Proteobacteria</i> (mainly consisting of <i>Syntrophaceae</i>, <i>Desulfobacteraceae</i> and <i>Syntrophorhabdus</i>) and <i>Firmicutes</i> (mainly consisting of <i>Desulfotomaculum</i>) were supposed to be involved with <i>n</i>-alkane degradation in the mesophilic conditions. By contrast, the different bacterial phylotypes affiliated with <i>Caldisericales</i>, “Shengli Cluster” and <i>Synergistetes</i> dominated the thermophilic consortium, which was most likely to be associated with thermophilic crude oil degradation. This study revealed that the oily sludge in Shengli oilfield harbors diverse uncultured microbes with great potential in methanogenic crude oil degradation over a wide temperature range, which extend our previous understanding of methanogenic degradation of crude oil alkanes.</p></div

Involvement of miR-125b in chemotherapeutic resistance in breast cancer cells.

<p>(A) Relative expression levels of endogenous miR-125b were measured in breast cancer cell lines by quantitative RT-PCR. Data represented mean ± SD. *<i>p</i><0.05 compared to MCF-7. (B) Breast cancer cells, MCF-7, T47D, BT20 and MDA-MB-231, were transfected with 100 nM pre-miR-125b and treated with the indicated concentrations of 5-FU for 48 h. A scramble pre-miRNA sequence served as a negative control (Ctrl). MCF-7 cells were transfected with 0, 25, 50, and 100 nM pre-miR-125b and then incubated with 8 µM 5-FU (C) or were treated with 0, 4, 8, 16, and 32 µM 5-FU for 48 h after transfection of 100 nM miRNA mimics (D). Cell variability was determined by trypan blue dye exclusion assays. Data represented mean ± SD of triplicate experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 compared to Ctrl.</p

Circulating miR-125b expression correlated to chemotherapeutic response in breast cancer patients.

<p>Surgically removed tumor samples were obtained after neoadjuvant chemotherapy. (A) Percentage of PCNA positive cells was determined by immunohistochemical staining. (B) Apoptotic index (AI) was expressed as the percentage of TUNEL-positive tumor cells. Circulating miR-125b levels were determined by quantitative real-time PCR.</p

Levels of circulating miRNAs in breast cancer patients and healthy controls.

<p>Relative levels of circulating miR-10b, miR-34a, miR-125b and miR-155 were measured in healthy individuals (n = 10) and clinical stage II patients (n = 35) and stage III patients (n = 21) by quantitative RT-PCR. Data represented mean ± SD. *<i>p</i><0.05, **<i>p</i><0.01 compared to healthy controls; #<i>p</i><0.05 compared to clinical stage II patients.</p

Knockdown of miR-125b sensitized breast cancer cells to chemotherapy.

<p>MDA-MB-231 (A), MCF-7 and 5-FU resistant FuR/MCF-7 (B) cells were transfected with 100 nM anti-miR-125b or anti-scramble control (Ctrl). Then the cells were treated with the indicated concentrations of 5-FU for 48 h. Data represented mean ± SD of triplicate experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 compared to Ctrl.</p

Immunohistochemical and Western blot analysis of HIF-1α and VEGF expression in OVA-challenged mice.

<p>(A and B) Immunostaining for HIF-1α and VEGF in nasal mucosa following the last challenge. Positive HIF-1α signals are brown and are predominantly nuclear and VEGF is predominantly cytoplasmic (magnification 400×, scale bar = 20 µm). (C and D) Representative Western blot analysis showing HIF-1α and VEGF (with β-actin as a loading control) expression in the nasal mucosa 24 h after the last challenge. All densitometric analyses are presented as the relative ratio of each molecule to β-actin and the ratio in negative control mice was set to 100. Values represent means±SEM (n = 6 mice). *Significantly different from negative control, p<0.05; ^#significantly different from positive control, p<0.05.</p

Changes in group composition and biomarker ratios of crude oil during methanogenic degradation.

<p>Pr/<i>n</i>C₁₇: pristane/<i>n</i>-heptadecane; three replicates in each time points with exception of original day 0. a <i>n</i>-heptadecane was only detected in one of the three replicates; b: <i>n</i>-heptadecane was not detected above the detection limit; Sterilized-330: crude oil sampled from the sterilized control group after 330 days of incubation at 35°C, Sterilized-303: crude oil sampled from the sterilized control group after 303 days of incubation at 55°C.</p><p>Changes in group composition and biomarker ratios of crude oil during methanogenic degradation.</p

Real-time RT-PCR analysis of HIF-1α and VEGF mRNA expression in nasal mucosa.

<p>Real-time RT-PCR was performed to detect HIF-1α (A) and VEGF (B) mRNA in nasal mucosa. Relative target gene expression was normalized to GADPH, an internal control. Values represent the ratios of various treatments to the negative control group. Values represent means±SEM of four mice in each group. *Significantly different from negative control, p<0.05; ^#significantly different from positive control, p<0.05.</p

MiR-125b expression correlated with chemotherapeutic resistance in primary breast cancer cells.

<p>(A) MiR-125b expression was determined by quantitative RT-PCR. (B) Primary breast cancer cells were treated with 5-FU for 48 h with or without miRNA antisense transfection (100 nM). IC50 was calculated as concentration of 5-FU to produce 50% cell inhibition (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034210#s4" target="_blank">Materials and Methods</a>). Data represented mean ± SD of primary breast cancer cell cultures from 5 patients with partial or complete response (PR + CR) and from 4 patients with disease progression or stabilization (PD + SD), *<i>p</i> = 0.01, **<i>p</i> = 0.002, ***<i>p</i><0.001.</p

E2F3 was a direct target of miR-125b in breast cancer cells.

<p>(A) MCF-7 cells were transfected with 100 nM pre-miR-125b or pre-scramble control (Ctrl). (B) MDA-MB-231 cells were transfected with 100 nM anti-miR-125b or anti-scramble control (Ctrl). Twenty-four hours after transfection, nuclear lysates were prepared for Western blotting with an antibody against E2F3, and TBP was used as a loading control. (C) MCF-7 cells were co-transfected with luciferase reporter plasmids with intact or mutant 3′UTR of E2F3, pre-miR-125 or pre-scramble control (Ctrl). Luciferase activity was measured at 48 h post-transfection using a dual luciferase reporter assay. The results were expressed as relative luciferase activity (firefly/renilla luciferase). Data represented mean ± SD of triplicate experiments. *<i>p</i> = 0.002.</p