Analysis of Nitrification Efficiency and Microbial Community in a Membrane Bioreactor Fed with Low COD/N-Ratio Wastewater

<div><p>In this study, an approach using influent COD/N ratio reduction was employed to improve process performance and nitrification efficiency in a membrane bioreactor (MBR). Besides sludge reduction, membrane fouling alleviation was observed during 330 d operation, which was attributed to the decreased production of soluble microbial products (SMP) and efficient carbon metabolism in the autotrophic nitrifying community. 454 high-throughput 16S rRNA gene pyrosequencing revealed that the diversity of microbial sequences was mainly determined by the feed characteristics, and that microbes could derive energy by switching to a more autotrophic metabolism to resist the environmental stress. The enrichment of nitrifiers in an MBR with a low COD/N-ratio demonstrated that this condition stimulated nitrification, and that the community distribution of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) resulted in faster nitrite uptake rates. Further, ammonia oxidation was the rate-limiting step during the full nitrification.</p></div

Characteristics of the raw, influent and treated wastewater (Unit: mg/L)^a.

a<p>Values are given as mean ± standard deviation and <i>n</i> = 18, 11, 13 for Phase I, Phase II and Phase III, respectively.</p>b<p>n.d.: not detectable.</p

Operational conditions of R₀ in three phases.

<p>Operational conditions of R₀ in three phases.</p

AOB and NOB sequences from R₀–R₅ assigned to NCBI taxonomies using BLAST and MEGAN.

<p>AOB and NOB sequences from R₀–R₅ assigned to NCBI taxonomies using BLAST and MEGAN.</p

Hierarchical cluster analysis of R₀–R₅ bacterial communities.

<p>The <i>y</i>-axis is the clustering of the 174 most abundant OTUs (3% distance) in reads. The OTUs were divided into seven zones (Z₁–Z₇). Sample communities were clustered based on complete linkage method. The color intensity of scale indicates relative abundance of each OTU read. Relative abundance was defined as the number of sequences affiliated with that OTU divided by the total number of sequences per sample.</p

JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation

<div><p>Jumonji domain-containing 6 (JMJD6) is a member of the Jumonji C domain-containing family of proteins. Compared to other members of the family, the cellular activity of JMJD6 is still not clearly defined and its biological function is still largely unexplored. Here we report that JMJD6 is physically associated with the tumor suppressor p53. We demonstrated that JMJD6 acts as an α-ketoglutarate– and Fe(II)-dependent lysyl hydroxylase to catalyze p53 hydroxylation. We found that p53 indeed exists as a hydroxylated protein <i>in vivo</i> and that the hydroxylation occurs mainly on lysine 382 of p53. We showed that JMJD6 antagonizes p53 acetylation, promotes the association of p53 with its negative regulator MDMX, and represses transcriptional activity of p53. Depletion of JMJD6 enhances p53 transcriptional activity, arrests cells in the G₁ phase, promotes cell apoptosis, and sensitizes cells to DNA damaging agent-induced cell death. Importantly, knockdown of JMJD6 represses p53-dependent colon cell proliferation and tumorigenesis <i>in vivo</i>, and significantly, the expression of JMJD6 is markedly up-regulated in various types of human cancer especially in colon cancer, and high nuclear JMJD6 protein is strongly correlated with aggressive clinical behaviors of colon adenocarcinomas. Our results reveal a novel posttranslational modification for p53 and support the pursuit of JMJD6 as a potential biomarker for colon cancer aggressiveness and a potential target for colon cancer intervention.</p></div

The negative effect of JMJD6 on p53 pathway depends on its hydroxylase activity.

<p>(A) HCT116 cells were transfected with vector, FLAG-JMJD6, or FLAG-JMJD6(H187A/D189A) (mut-JMJD6). The levels of the indicated proteins were detected by Western blotting. The mRNA levels of p21 and PUMA were detected by real-time RT PCR. (B) HCT116 cells were transfected with p21 promoter-driven luciferase construct together with vector, FLAG-JMJD6, or FLAG-JMJD6(H187A/D189A) (mut-JMJD6) plasmids. Cells were then harvested and luciferase activity was measured and normalized to that of renilla. Each bar represents the mean ± S.D. for triplicate experiments. (C) HCT116 cells transfected with vector or FLAG-JMJD6, or FLAG-JMJD6(H187A/D189A) (mut-JMJD6) were synchronized by double thymidine block and released into the cell cycle. Cells were collected for cell cycle analysis by flow cytometry. Experiments were repeated three times and the data from a representative experiment are shown. (D) HCT116 cells were transfected with vector, FLAG-JMJD6, or FLAG-JMJD6(H187A/D189A) (mut-JMJD6), and challenged with VP-16. Annexin V/PI staining and flow cytometry were performed to assess the effect of JMJD6 on the apoptosis of HCT116 cells.</p

JMJD6 is physically associated with p53 <i>in vivo</i> and <i>in vitro</i>.

<p>(A) Cellular extracts from HCT116 cells stably expressing vector or FLAG-JMJD6 were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptide. The eluates were resolved by SDS-PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry. (B) HCT116 cell lysates were immunoprecipitated with antibodies against JMJD6 followed by immunoblotting with antibodies against p53 (FL-393), or they were immunoprecipitated with antibodies against p53 (FL-393) followed by immunoblotting with antibodies against JMJD6. (C) Immunofluorescence-stained endogenous JMJD6 (red) and p53 (green) were visualized by confocal microscopy. DAPI staining was included to visualize the cell nucleus (blue). Scale bar, 25 µm. (D) Mapping the domain of p53 that is required for its interaction with JMJD6. GST pull-down experiments were performed with GST-fused JMJD6 and <i>in vitro</i> transcribed/translated Myc-tagged full-length p53 or deletions of p53.</p

Negative regulation of p53 transcriptional activity by JMJD6.

<p>(A) Measurement of mRNA (left panel) and protein (right panel) levels of p21 and PUMA by real-time RT PCR and Western blotting in HCT116 cells that were transfected with JMJD6 siRNAs and/or JMJD6 siRNA-1–resistant JMJD6 form (rJMJD6) followed by treatment with or without VP-16. Each bar represents the mean ± S.D. for triplicate measurements. *<i>p</i><0.05. (B) HCT116 cells were treated with control siRNA or JMJD6 siRNAs and challenged with or without VP-16. Real-time RT PCR was performed using exon–exon junction-specific or intron–exon junction-specific primers to measure spliced and unspliced mRNA levels of <i>p21</i> and <i>PUMA</i> by RT-qPCR analysis to determine splicing efficiency of <i>p21</i> (left panel) and <i>PUMA</i> mRNA (right panel). Each bar represents the mean ± S.D. for triplicate measurements. (C) Reporter assays in HCT116 cells that were transfected with JMJD6 siRNAs and/or rJMJD6 together with p21 promoter-driven luciferase reporter construct and challenged with or without VP-16. *<i>p</i><0.05. (D) qChIP was performed in HCT116 cells treated with control siRNA or JMJD6 siRNA with indicated antibodies. (E) HCT116 cells transfected with JMJD6 siRNAs and/or rJMJD6 were synchronized by double thymidine block and released into the cell cycle. Cells were collected for cell cycle analysis by flow cytometry. Experiments were repeated three times and the data from a representative experiment are shown. *<i>p</i><0.05. (F) HCT116 cells were transfected with control siRNA or JMJD6 siRNAs and challenged with or without VP-16 for 24 h. Annexin V/PI staining and flow cytometry were performed to assess the effect of JMJD6 on the apoptosis of HCT116 cells. Experiments were repeated three times, and the data from a representative experiment are shown. Each bar represents the mean ± S.D. for triplicate experiments. *<i>p</i><0.05.</p

JMJD6 is a potential biomarker for colon cancer aggressiveness.

<p>(A) Immunohistochemical staining of JMJD6 in paired samples of breast ductal carcinoma (1), hepatocellular carcinoma (2), lung adenocarcinoma (3), lung squamous carcinoma (4), suprarenal epithelioma (5), pancreatic ductal carcinoma (6), colon adenocarcinoma (7), esophageal squamous carcinoma (8), rectal adenocarcinoma (9), and gastric adenocarcinoma (10) versus adjacent normal tissues. Representative tumor and adjacent normal sections stained with JMJD6 antibody are shown (magnification, ×25; scale bar, 200 µm). Each type of carcinoma included at least six paired samples and the scores were determined by evaluating the extent and intensity of immunopositivity. (B) Immunohistochemical staining of JMJD6 in 90 samples of colon adenocarcinomas paired with adjacent normal tissues. Representative sections from colon cancer (upper, tubular adenocarcinoma; lower, poorly differentiated adenocarcinoma) or adjacent normal tissue stained with JMJD6 antibody are shown (magnification, ×100; scale bar, 100 µm). The scores were determined by evaluating the extent and intensity of immunopositivity and were analyzed by paired-samples <i>t</i> test (***<i>p</i><0.001). (C) Representative sections of histological grade I, II, and III of colon adenocarcinomas that were stained with JMJD6 antibody are presented (magnification, ×100; scale bar, 100 µm). The scores were determined by evaluating the extent and intensity of immunopositivity and were analyzed by two-tailed unpaired <i>t</i> test (***<i>p</i><0.001). (D) Time-to-event data were plotted using Kaplan–Meier curves, and the 5-year survival rate of different groups was compared using the Mantel–Cox log-rank test (***<i>p</i> = 0.001). The <i>y</i>-axis represents the percentage of patients, and the <i>x</i>-axis represents the survival in months. (E) High expression of JMJD6 protein is found only in the base of intestinal glands (crypt of Lieberkuhn). Representative images of immunohistochemical staining of JMJD6 in normal intestinal glands are shown. Upper—magnification, ×25; scale bar, 200 µm. Lower—magnification, ×400; scale bar, 50 µm.</p