Intestinal Contents

DNA extractions from intestinal luminal contents were prepared as described previously [14 (link)]. In brief, DNA extracts and plasmids were quantified by using Quant-iT PicoGreen reagent (Invitrogen, UK) and all adjusted to 1 ng DNA/µl.
The abundance of specific intestinal bacterial groups was measured by qPCR with group-specific 16S rRNA gene primers (Tib MolBiol, Germany) as decribed previously [15 (link),16 (link)]. As reference for quantification standard curves with tenfold serial dilutions of plasmids (ranging from 2×10⁸ to 2×10² copies) were generated for each run. The real-time PCR primers were first used to amplify cloned 16S rDNA of reference strains (see Table 1). The number of 16S rRNA gene copies / ng DNA of each sample was determined. Frequencies of the given bacterial groups were calculated proportionally to the eubacterial (V3) amplicon.
Genetic fingerprints were generated by PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) as described previously [14 (link)].

A number of in vitro models have been used to study the toxicity and biokinetics of pharmaceuticals and chemicals in the GIT. The most commonly used model employs Caco-2 cells (immortal human colonic epithelial) cells, which after culture for 2–3 weeks differentiate into cells with markers and morphological characteristics of small intestinal epithelial enterocytes [64 –66 (link)]. While this may be a reasonable choice for many situations, the epithelium of the small intestine is more complex, and in order to more accurately emulate this structure, a variety of modifications have been added. The intestinal mucosa is normally protected by a layer of mucus produced by both goblet cells and submucosal glands (Brunner’s glands, limited mostly to the duodenum) [67 (link)]. It is therefore appropriate to modify the in vitro model to include mucus secreting cells. To this end, HT29-MTX cells, an immortal human cell line that resembles intestinal goblet cells and secretes mucus, is often co-cultured with Caco-2 cells [66 (link)–69 (link)]. Finally, in the Peyer’s patches and other lymphoid-associated epithelium of the small intestine, specialized cells called Microfold- or M-cells are present. These cells engulf and translocate samples of the contents of the intestinal lumen to lymphocytes in the submucosa below, thereby providing continuous antigenic surveillance of the intestinal contents [33 (link)]. It has also recently been shown that M-cells can play an important role in translocation of iENMs in in vitro intestinal epithelial models [33 (link)]. It has previously been shown that differentiated Caco-2 cells can be induced by factors released from another cell line, Raji B (a human B lymphocyte) to differentiate into cells resembling M-cells [70 (link), 71 (link)]. Thus, when Raji B cells are added to the basolateral compartment of a transwell system in which matured caco-2 cells reside on the transwell membrane above, some of the Caco-2 cells are induced to differentiate into M-like cells. The complete hybrid triculture model utilized in our methodology, illustrated in Fig. 5a, has previously been described and characterized and includes cells with morphology and markers consistent with the three primary cells of the intestinal epithelium: enterocytes, goblet cells and M-cells [37 (link)–41 (link)]. Because it represents a reasonably realistic hybrid model of the complete intestinal epithelium, this model was adopted for the proposed integrated methodology. Specifically, we employed the protocol reported by Mahler et al. [37 (link)] for development of our triculture system. Such a physiologically relevant model is well suited to the study of biokinetics and intestinal toxicity of iENMs. However other similar advanced models could also be used.
Details of the methods employed for development, characterization and validation of the triculture model, including protocols for creating the system, measurement of transepithelial electrical resistance (TEER), immunofluorescence staining and imaging for morphological characterization and TEM characterization are provided in Additional file 1.

Two authors (MX and YZ) independently extracted the following data: (1) anastomotic leakage, (2) defecation frequency, (3) anastomotic stricture, (4) reoperation, (5) postoperative mortality within 30 days, (6) fecal urgency, (7) incomplete defecation, (8) use of antidiarrheal medication, and (9) quality of life. We recorded the results of bowel function outcomes at 3, 6, 12, and 24 months following stoma retraction (or without stoma surgery). We considered the most common and concerning anastomotic leakage and defecation frequency as the primary outcome indicators, and the rest were secondary outcome indicators. Anastomotic leakage is defined as a significant crack at the edge of the anastomosis, leakage of bowel contents seen in the pelvis on imaging or endoscopy, or purulent discharge from the pelvic drainage tube. The defecation frequency was determined based on the patient-described average number of daily bowel movements.

Nine intestinal contents (three contents are merged into one pooled sample) from each group were randomly collected from water tanks 1 day after injection for high-throughput sequencing of the 16S rDNA. Total genomic DNA from pooled intestinal content samples of both groups was extracted using the CTAB/SDS method. DNA concentration and purity were monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1 ng/μL using sterile water. 16S rRNA genes of distinct regions (V3-V4 hypervariable region) were amplified using specific primers 515F [5’-GTGCCAGCMGCCGCGGTAA-3′] and 806R [5’-GGACTACHVGGGTWTCTAAT-3′] with the barcode. All PCRs were carried out with 15 μl of Phusion^® High-Fidelity PCR Master Mix (New England Biolabs). The same volume of 1X loading buffer (containing SYBR Green) was mixed with the PCR products, and electrophoresis was performed on a 2% agarose gel for detection. PCR products were mixed in equidensity ratios. Then, the mixed PCR products were purified with a Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using the TruSeq^® DNA PCR-Free Sample Preparation Kit (Illumina, United States) following the manufacturer’s recommendations, and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina NovaSeq platform, and 250 bp paired-end reads were generated. The 16S rRNA sequencing service was completed by Wuhan Metwell Biotechnology Co., Ltd.