Infection with a human-derived enteroinvasive Escherichia coli strain altered intestinal barrier function in guinea pigs / La infección con una cepa enteroinvasiva de Escherichia coli de origen humano alteró la función de la barrera intestinal en cobayos

Background/aims: The aim was to characterize a bacterium causing intestinal mucosal barrier damage and to identify the possible invasion mechanism. Materials and methods: The intestinal permeability and tight junction protein levels were detected in guinea pigs infected with Escherichia coli D-09 via immunofluorescence analysis and western blotting. In order to explain this invasion mechanism at the gene level, whole genome sequencing analysis was performed on this bacterium. Results: The results showed an increased intestinal permeability and upregulated expression of the leaky protein claudin-2 in both the colon and liver of the infected animals. In addition, the draft genome of E. coli D-09 comprised 42 scaffolds (size, > 645 bp) with a total size of 4,679,567 bp. A total of 4379 protein coding genes were identified, which contained 45 antibiotic resistance and 86 virulence-related genes and covered 88.0% of the whole genome. Conclusions: This study verified that the human-derived enteroinvasive E. coli strain could destroy intestinal barrier function in guinea pigs. Additionally, our data first characterized the genome features of E. coli O124:K72 D-09, which may provide new insights into the possible invasion mechanism.(AU)

Infection with a human-derived enteroinvasive Escherichia coli strain altered intestinal barrier function in guinea pigs.

BACKGROUND/AIMS: The aim was to characterize a bacterium causing intestinal mucosal barrier damage and to identify the possible invasion mechanism. MATERIALS AND METHODS: The intestinal permeability and tight junction protein levels were detected in guinea pigs infected with Escherichia coli D-09 via immunofluorescence analysis and western blotting. In order to explain this invasion mechanism at the gene level, whole genome sequencing analysis was performed on this bacterium. RESULTS: The results showed an increased intestinal permeability and upregulated expression of the leaky protein claudin-2 in both the colon and liver of the infected animals. In addition, the draft genome of E. coli D-09 comprised 42 scaffolds (size, > 645 bp) with a total size of 4,679,567 bp. A total of 4379 protein coding genes were identified, which contained 45 antibiotic resistance and 86 virulence-related genes and covered 88.0% of the whole genome. CONCLUSIONS: This study verified that the human-derived enteroinvasive E. coli strain could destroy intestinal barrier function in guinea pigs. Additionally, our data first characterized the genome features of E. coli O124:K72 D-09, which may provide new insights into the possible invasion mechanism.

Corrigendum: Sperm Protein Antigen 17 Expression Correlates With Lymph Node Metastasis and Worse Overall Survival in Patients With Breast Cancer.

[This corrects the article DOI: 10.3389/fonc.2019.00710.].

Quantitative analysis for modified Schall's classification by stimulation test with dynamic scintigraphy in Sjögren's syndrome.

OBJECTIVES: To update Schall's classification for Sjögren's syndrome (SS) by the new quantitative stimulation test with dynamic salivary glands scintigraphy (qsDSGS) and to standardize quantitative salivary gland scintigraphy. METHODS: The histopathology, oral, ocular, serological examination and qsDSGS of 268 consecutive patients with suggestive SS were evaluated in this retrospective cohort study. The serological examination included 15 autoantibodies, antinuclear antibodies (ANA) and so on. The diagnostic thresholds of the functional parameters were set by the quantitative method, and the modified Schall's classification is well established and verified. RESULTS: Based on the quantitative analysis of qsDSGS, the peak uptake level (PUL) and stimulation excretion fraction (sEF) of each parotid gland were determined as the key imaging features, which had good diagnostic performance for SS. By the modified Schall's classification, all patients were classified into: Class 1 (normal; n = 44), Class 2 (mild to moderate involvement; n = 130), Class 3 (severe involvement; n = 56) and Class 4 (very severe involvement, non-function; n = 38). Using the threshold PUL ≤ 10 counts per sec/pixel as positivity, the modified Schall's classification could provide better diagnostic performance with 88.4% specificity, 71.3% sensitivity, 96.14% positive predictive value and 43.20% negative predictive value for SS (likelihood ratio 6.15). The trends of serologically positive frequencies against SSA/Ro, anti-SSB/La and ANA were significantly increased with the new classification. CONCLUSION: The modified Schall's classification by the new stimulation test with dynamic scintigraphy is eligible to standardize quantitative salivary gland scintigraphy for SS, and may be more convenient and suitable in daily practice for clinical research and management of SS.

Enhancement of epithelial cell autophagy induced by sinensetin alleviates epithelial barrier dysfunction in colitis.

Intestinal epithelial barrier dysfunction is a key pathology of colitis. Autophagy of epithelial cells maintains homeostasis of the intestinal barrier by inhibiting apoptosis and stimulating degradation of the tight junction protein claudin-2. This study investigated the effects and mechanism of activity of sinensetin, a polymethylated flavonoid isolated from tangerine peel and citrus, on intestinal barrier dysfunction in colitis. Animal model of colitis were established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid and oral treatment with dextran sulfate sodium. Epithelial barrier function was evaluated by measuring the serum recovery of fluorescein isothiocyanate-4 kD dextran in vivo and transepithelial electrical resistance in Caco-2 cells, respectively. Epithelial cell autophagy assayed by autophagosome formation and expression of autophagy-related protein. Sinensetin reversed colitis-associated increase in intestinal permeability, significantly promoted epithelial cell autophagy, and further decreased epithelial cell apoptosis, and reduced mucosal claudin-2. Sinenstetin alleviated colitis symptoms rats and mice with colitis. Knockdown of 5' adenosine monophosphate-activated protein kinase (AMPK) reversed the promotion of epithelial autophagy by sinensetin. In conclusion, sinensetin significantly alleviated intestinal barrier dysfunction in colitis by promoting epithelial cell autophagy, and further inhibiting apoptosis and promoting claudin-2 degradation. The results highlighted novel potential benefits of sinensetin in colitis.

Sperm Protein Antigen 17 Expression Correlates With Lymph Node Metastasis and Worse Overall Survival in Patients With Breast Cancer.

Purpose: The expression and role of sperm protein antigen 17 (SPA17), which has been confirmed to be immunogenic, in breast cancer remain unclear. We examined the expression of SPA17 in breast cancer and assessed its effect on patient prognosis and its function in breast cancer development. Methods: SPA17 expression was evaluated by immunohistochemistry and Q-RT-PCR in 120 breast tissue samples. Correlation of SPA17 expression with the patients' clinicopathological parameters and overall survival was assessed. The function of SPA17 was also explored. Results: By reviewing Gene Expression Omnibus datasets, we found that SPA17 expression in ductal breast carcinoma in situ (log2[fold change] = 1.14, p-value = 0.004) and invasive ductal breast cancer (log2[fold change] = 1.03, p-value = 0.016) tissues was 2.20 and 2.05 times higher, respectively, than that in normal breast tissues. Our result also showed that 27% (27/100) of breast cancer samples expressed SPA17 but none of the normal breast (0/20) samples did. Lymph node metastasis (p < 0.001) and molecular subtyping (p = 0.002) were independent factors associated with SPA17 expression. Most importantly, SPA17 expression resulted in poor prognosis. In addition, cell function assay validated that SPA17 increased the migration (p < 0.001) and invasion (p = 0.007) of breast cancer cells, but not affected the proliferation of breast cancer cells. Conclusion: Our results demonstrated the vital role of SPA17 in the development and metastasis of breast cancer and that SPA17 may be a new therapeutic target in improving breast cancer prognosis.

Gut microbial diversity analysis using Illumina sequencing for functional dyspepsia with liver depression-spleen deficiency syndrome and the interventional Xiaoyaosan in a rat model.

AIM: To investigate gut microbial diversity and the interventional effect of Xiaoyaosan (XYS) in a rat model of functional dyspepsia (FD) with liver depression-spleen deficiency syndrome. METHODS: The FD with liver depression-spleen deficiency syndrome rat model was established through classic chronic mild unpredictable stimulation every day. XYS group rats received XYS 1 h before the stimulation. The models were assessed by parameters including state of the rat, weight, sucrose test result and open-field test result. After 3 wk, the stools of rats were collected and genomic DNA was extracted. PCR products of the V4 region of 16S rDNA were sequenced using a barcoded Illumina paired-end sequencing technique. The primary composition of the microbiome in the stool samples was determined and analyzed by cluster analysis. RESULTS: Rat models were successfully established, per data from rat state, weight and open-field test. The microbiomes contained 20 phyla from all samples. Firmicutes, Bacteroidetes, Proteobacteria, Cyanobacteria and Tenericutes were the most abundant taxonomic groups. The relative abundance of Firmicutes, Proteobacteria and Cyanobacteria in the model group was higher than that in the normal group. On the contrary, the relative abundance of Bacteroidetes in the model group was lower than that in the normal group. Upon XYS treatment, the relative abundance of all dysregulated phyla was restored to levels similar to those observed in the normal group. Abundance clustering heat map of phyla corroborated the taxonomic distribution. CONCLUSION: The microbiome relative abundance of FD rats with liver depression-spleen deficiency syndrome was significantly different from the normal cohort. XYS intervention may effectively adjust the gut dysbacteriosis in FD.

CNP signal pathway up-regulated in rectum of depressed rats and the interventional effect of Xiaoyaosan.

AIM: To investigate the distribution and expression of C-type natriuretic peptide (CNP)/natriuretic peptide receptor B (NPR-B) in the rectum of a rodent depression model and the interventional effect of Xiaoyaosan (XYS). METHODS: Male rats (n = 45) of clean grade (200 ± 20 g) were divided into five groups after one week of adaptive feeding: primary control, depression model, low dose XYS, middle dose XYS, and high dose XYS. The animal experiment continued for 3 wk. Primary controls were fed normally ad libitum. The rats of all other groups were raised in solitary and exposed to classic chronic mild unpredictable stimulation each day. XYS groups were perfused intragastrically with low dose, middle dose, and high dose XYS one hour before stimulation. Primary control and depression model groups were perfused intragastrically with normal saline under similar conditions as the XYS groups. Three weeks later, all rats were sacrificed, and the expression levels of CNP and NPR-B in rectum tissues were analyzed by immunohistochemistry, real-time polymerase chain reaction, and Western blotting. RESULTS: CNP and NPR-B were both expressed in the rectum tissues of all rats. However, the expression levels of CNP and NPR-B at both gene and protein levels in the depression model group were significantly higher when compared to the primary control group (n = 9; P < 0.01). XYS intervention markedly inhibited the expression levels of CNP and NPR-B in depressed rats. The expression levels of CNP and NPR-B in the high dose XYS group did not significantly differ from the expression levels in the primary control group. Additionally, the high and middle dose XYS groups (but not the low dose group) significantly exhibited lower CNP and NPR-B expression levels in the rectum tissues of the respectively treated rats compared to the untreated depression model cohort (n = 9; P < 0.01). CONCLUSION: The CNP/NPR-B pathway is upregulated in the rectum of depressed rats and may be one mechanism for depression-associated digestive disorders. XYS antagonizes this pathway at least partially.

Effect of gingerol on colonic motility via inhibition of calcium channel currents in rats.

AIM: To investigate the effect of gingerol on colonic motility and the action of L-type calcium channel currents in this process. METHODS: The distal colon was cut along the mesenteric border and cleaned with Ca(2+)-free physiological saline solution. Muscle strips were removed and placed in Ca(2+)-free physiological saline solution, which was oxygenated continuously. Longitudinal smooth muscle samples were prepared by cutting along the muscle strips and were then placed in a chamber. Mechanical contractile activities of isolated colonic segments in rats were recorded by a 4-channel physiograph. Colon smooth muscle cells were dissociated by enzymatic digestion. L-type calcium currents were recorded using the conventional whole-cell patch-clamp technique. RESULTS: Gingerol inhibited the spontaneous contraction of colonic longitudinal smooth muscle in a dose-dependent manner with inhibition percentages of 13.3% ± 4.1%, 43.4% ± 3.9%, 78.2% ± 3.6% and 80.5% ± 4.5% at 25 µmol/L, 50 µmol/L, 75 µmol/L and 100 µmol/L, respectively (P < 0.01). Nifedipine, an L-type calcium channel blocker, diminished the inhibition of colonic motility by gingerol. Gingerol inhibited L-type calcium channel currents in colonic longitudinal myocytes of rats. At a 75 µmol/L concentration of gingerol, the percentage of gingerol-induced inhibition was diminished by nifedipine from 77.1% ± 4.2% to 42.6% ± 3.6% (P < 0.01). Gingerol suppressed IBa in a dose-dependent manner, and the inhibition rates were 22.7% ± 2.38%, 35.77% ± 3.14%, 49.78% ± 3.48% and 53.78% ± 4.16% of control at 0 mV, respectively, at concentrations of 25 µmol/L, 50 µmol/L, 75 µmol/L and 100 µmol/L (P < 0.01). The steady-state activation curve was shifted to the right by treatment with gingerol. The value of half activation was -14.23 ± 1.12 mV in the control group and -10.56 ± 1.04 mV in the 75 µmol/L group (P < 0.05) with slope factors, Ks, of 7.16 ± 0.84 and 7.02 ± 0.93 (P < 0.05) in the control and 75 µmol/L groups, respectively. However, the steady-state inactivation curve was not changed, with a half-inactivation voltage, 0.5 V, of -27.43 ± 1.26 mV in the control group and -26.56 ± 1.53 mV in the 75 µmol/L gingerol group (P > 0.05), and a slope factor, K, of 13.24 ± 1.62 in the control group and 13.45 ± 1.68 (P > 0.05) in the 75 µmol/L gingerol group. CONCLUSION: Gingerol inhibits colonic motility by preventing Ca(2+) influx through L-type calcium channels.