Multimodality treatment-induced JNK phosphorylation, Bcl-xL phosphorylation and reduction in c-FLIP_L level.

<p>(A) CX-1 and (B) HCT116 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of Mapa and oxaliplatin and incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, the cleavage of caspase 8, caspase 9, caspase 3, or PARP was detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane. (C) CX-1 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of 100 ng/ml Mapa and 10 µg/ml oxaliplatin and then incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, cells were immunoblotted with anti-phospho-JNK/JNK, anti-phospho-Bcl-xL/Bcl-xL and anti-FLIP antibodies. (D) CX-1 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of Mapa (100 ng/ml-1000 ng/ml) and oxaliplatin (10 µg/ml-100 µg/ml) and incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, phospho-JNK/JNK, phospho-Bcl-xL/Bcl-xL and FLIP_L were detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane. (E) HCT116 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of Mapa (10 ng/ml-100 ng/ml) and oxaliplatin (10 µg/ml-100 µg/ml) and then incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, phospho-JNK/JNK, phospho-Bcl-xL/Bcl-xL and FLIP_L were detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane.</p

Effect of oxaliplatin and hyperthermia on Mapa-induced cytotoxicity and apoptosis.

<p>(A, B) CX-1 and HCT116 cells were exposed to normothermic or hyperthermic (42°C) conditions for 1 h in the presence/absence of Mapa and oxaliplatin and then incubated for 23 h at 37°C in the presence/absence of Mapa and oxaliplatin. Cell viability was analyzed by MTS assay. Error bars represent SD from triplicate experiments. Asterisk ** represents a statistically significant difference (P <0.01). (C) CX-1 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of Mapa and oxaliplatin and then incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, cells were stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI). Apoptosis was detected by the flow cytometric assay. (D) After treatment, the cleavage of caspase 8, caspase 9, caspase 3, or PARP was detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane. (E) CX-1 and HCT116 cells were treated with or without 20 µM Z-IETD-FMK (caspase 8 inhibitor), Z-LEHD-FMK (caspase 9 inhibitor), and Z-DEVD-FMK (caspase 3 inhibitor) for µmin followed by oxaliplatin/Mapa/hyperthermia and the cleavage of PARP was detected by immunoblotting. (F) Human colon cancer stem cells, Tu-12, Tu-21 and Tu-22, were exposed to normothermic or hyperthermic (42°C) conditions for 1 h in the presence/absence of Mapa and oxaliplatin at the indicated concentration and then incubated for 23 h at 37°C in the presence/absence of Mapa and oxaliplatin. PARP was detected by immunoblotting. Actin was used as loading control.</p

The kinetics of multimodality treatment in CX-1 and HCT116 cells.

<p>CX-1 (A) and HCT116 (B) cells were exposed to hyperthermic (42°C) conditions for 1 h in the presence/absence of Mapa and oxaliplatin and incubated at 37°C in the presence/absence of Mapa and oxaliplatin for 3 h, 7 h, 11 h and 23 h. After treatment, the cleavage of caspase 8/9/3 and PARP, phospho-JNK/JNK, phospho-Bcl-xL/Bcl-xL and FLIP_L were detected by immunoblotting. Actin was used to confirm the equal amount of proteins.</p

The requirement of phosphorylation of JNK and Bcl-xL in the multimodality treatment-induced apoptosis in CX-1 cells.

<p>(A) Cells were pretreated with JNK inhibitor 25 µM SP6001125 followed by oxaliplatin/Mapa/hyperthermia and immunoblotted with anti-PARP, anti-phospho-Bcl-xL and anti-Bcl-xL antibody. (B) Transfectants with control plasmid (pcDNA), wild-type Bcl-xL (Bcl-xL-WT), Ser62/Ala phospho-defective Bcl-xL mutant (Bcl-xL-S62A), or Ser62/Asp phospho-mimic Bcl-xL mutant (Bcl-xL-S62D) were treated with oxaliplatin/Mapa/hyperthermia and immunoblotted with anti-PARP or anti-Bcl-xL antibody. Actin was used to confirm the equal amount of proteins loaded in each lane.</p

Changes of bacterial community.

(A) PCA analysis between samples base on the OTUs; (B) the structure of bacterial between samples; (C) the variation of bacterial at phylum level between samples. (D) the variation of pathogen at species level between samples.</p

The ARGs changed along the WWTPs process.

(A) venn diagrams analysis between the samples. (B) 53 share genes changed between samples; (C) 18 new generated ARGs changed between samples.</p

The correlation between ARG and their host bacteria.

(A) the analysis base on Spearman’s correlation between ARGs and their host; network analysis revealing the correlations among the fates of ARGs, MGEs, DMF and bacterial in influent (B) and effluent (C).</p

The abundance of ARGs in WWTPs.

(A) PCA analysis between different samples in WWTPs. (B) the relative abundance of ARGs in WWTPs; (C) the absolute abundance of ARGs in WWTPs; (D) Chord diagram analysis between the sample and ARG subtype; (E) the mechanisms of ARGs variation in WWTPs.</p

Graphical abstract.

The excessive use of antibiotics has resulted in the contamination of the environment with antibiotic resistance genes (ARGs), posing a significant threat to public health. Wastewater treatment plants (WWTPs) are known to be reservoirs of ARGs and considered to be hotspots for horizontal gene transfer (HGT) between bacterial communities. However, most studies focused on the distribution and dissemination of ARGs in hospital and urban WWTPs, and little is known about their fate in industrial WWTPs. In this study, collected the 15 wastewater samples containing N,N-dimethylformamide (DMF) from five stages of the anaerobic anoxic aerobic (AAO) process in an industrial WWTPs. The findings revealed a stepwise decrease in DMF and chemical oxygen demand (COD) content with the progression of treatment. However, the number and abundances of ARGs increase in the effluents of biological treatments. Furthermore, the residues of DMF and the treatment process altered the structure of the bacterial community. The correlation analysis indicated that the shift in bacterial community structures might be the main driver for the dynamics change of ARGs. Interestingly, observed that the AAO process may acted as a microbial source and increased the total abundance of ARGs instead of attenuating it. Additionally, found that non-pathogenic bacteria had higher ARGs abundance than pathogenic bacteria in effluents. The study provides insights into the microbial community structure and the mechanisms that drive the variation in ARGs abundance in industrial WWTPs.</div

The change among the different treatment in WWTPs.

(A) Dynamic changes of the content of DMF and (B) COD; (C) Correlation analysis of DMF and COD; (D) The abundance of 16S rRNA; Influent wastewater, IN; Anaerobic process wastewater, AP; Anoxic process wastewater, ANP; Aerobic process wastewater, AEP; Effluent wastewater, EF.</p