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文字:[大][中][小] 2024-6-4    瀏覽次數:645    

Determination of cadmium contents in the colon and liver
Colon and liver tissue samples (0.05-0.2 g) were dissolved using a mixture ofHNO3 and HClO4 (300 μL and 100 μL, respectively) and clarified by heating in a95℃ water bath. The volume was then fixed to 5 mL, filtered using inorganicmembranes, and the concentration of Cd was determined using ICP-MS (NexION2000, PerkinElmer, USA). 

Method S7. Detection of the ultrastructure of colonColon 

tissues were fixed in 2.5% glutaraldehyde for 12 hours at 4℃. Next, thesamples were washed three times for 15 minutes each with phosphate buffer (pH=7)and then post-fixed in 1% osmium tetroxide, dehydrated with ethanol, and embeddedin epoxy resin. Afterward, the samples were sliced, stained with lead citrate anduranyl acetate solution. Finally, the ultrastructure was observed using a transmissionelectron microscope (TEM, HT-7800, Hitachi, Japan). 

 ROS detection

The fresh colon tissues were immediately transferred to a -20℃ environment, inorder to make frozen sections. Dihydroethidium (DHE) was added to the tissues andincubated at 37℃ in a light-proof incubator for 30 minutes. Subsequently, the slideswere washed 3 times using PBS. Following this, the 4’,6-diamidino-2-phenylindole(DAPI) staining solution was added and then incubated for 10 minutes in the dark. Finally, the slides were sealed with an anti-fluorescence quenching sealant andobserved using a Nikon Eclipse C1 fluorescence microscope (Tokyo, Japan).

  

High-throughput sequencingGut 

microbiota profiling analysis was performed by Majorbio Bio-PharmTechnology Co., Ltd (Shanghai, China). Total bacterial DNA was isolated from micestool samples by using the fecal genomic DNA extraction kit (DP328, TIANGEN, Beijing, China) according to the manufacturer’s guidelines and optimized forhigh-throughput processing. The 16S V3-V4 region was amplified on ABIGeneAmp®9700 PCR System (AppliedBiosystems, USA) using DNA Polymerase
and fusion primers designed against the surrounding conserved regions, and theseconserved regions are tailed with sequences to incorporate Illumina (San Diego, CA)adapters and index barcodes. The PCR products were concentrated using asolid-phase reversible immobilization method for the purification and quantified by
QuantiFluor? -ST Blue Fluorescence Quantification System (Promega) with anaverage number of reads sampled of 50,000. A pool containing 16S V3-V4 enriched, amplified, barcoded samples will then be loaded into the MiSeq® kit and theamplicons will be sequenced for 250 cycles using custom primers. The resulting sequences were mapped to the speciestaxonomydatabase(silva138/16s_bacteria) using the USEARCH7-UPRASE clustering method tocalculate strain abundance, and all sequences hitting a unique strain with ≥99%identity were assigned a strain Operational Taxonomic Units (OTUs). Alpha andbeta-diversity, linear discriminant analysis (LDA), and linear discriminant analysisEffect Size (LEfSe) were performed on the cloud platform of Majorbio.

感謝食品科學與資源國家重點實驗室,南昌大學南昌330047公關中國b國際食品創新研究所有限公司南昌大學南昌330200公關中國強調圖形抽象鎘吸附改變PS-MPs的物理特性。引用文獻!

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