[Impact of Tetracycline Antibiotic on the Transcriptional Expression of Tetracycline Resistance Genes in Shigella flexneri].

The tetracycline (TC) antibiotic has been widely found in different environmental matrices. The tetracycline resistant bacterium (TRB) of Shigella flexneri was screened and purified from activated sludge, and was then used to study the impact of TC stress on the gene abundances and expression levels of TC resistance genes (TC-ARGs), including tetC, tetO, and tetX, which were respectively quantified by quantitative PCR and reverse transcriptional PCR. Correlations between the TC concentration and gene abundances of TC-ARGs and their expression levels were discussed. The results showed that TC stress had an inhibiting effect on the growth of Shigella flexneri during the entire culture cycle (24 h) and that the growth rate of the bacterial concentration decreased with increasing TC concentration. However, less impact on the gene abundance of TC-ARGs was found. TC stress could promote the expression of TC-ARGs in Shigella flexneri, and the expression levels of tetC, tetO, and tetX genes first increased and then decreased. The correlation results indicated that no significant correlation was observed between the TC concentration and gene abundances of TC-ARGs and their expression levels. Nevertheless, the gene abundances of tetC and tetO were significantly correlated with their expression levels, thus indicating that they can be used to evaluate and assess expression levels to a certain extent.

[Effect of Tetracycline Antibiotic on Abundance and Transcriptional Expression Level of Tetracycline Resistance Genes in Activated Sludge].

Tetracycline resistant bacteria (TRB) screened from activated sludge were used to study the effect of tetracycline (TC) antibiotic on the transcriptional expression of tetracycline resistance genes (TC-ARGs). The gene abundances of seven TC-ARGs including tetA, tetC, tetG, tetM, tetO, tetW, and tetX, as well as their expression levels, were quantified by quantitative PCR (qPCR) and reverse transcriptional PCR (RT-PCR). The correlations between TC concentrations and gene abundance of TC-ARGs and their expression levels were discussed. The results showed that the gene abundances of tetA, tetG, and tetW generally increased with increasing TC exposure concentrations during the entire culture cycle, whereas other TC-ARGs fluctuated greatly. The impact of TC stress on the transcriptional expression level of different TC-ARGs varied to a great extent. The gene expression of tetA was relatively stable and exhibited an upregulated trend with increasing TC concentrations. When the TC concentration was 100 mg·L-1, the upregulation of tetA expression was as high as 5.3-fold compared with the control. Under short-term TC stress (one day), the transcriptional expression level was upregulated with increasing TC concentration. The correlation results showed that gene abundances of tetA and tetW correlated significantly with their respective expression levels, indicating that they can evaluate expression levels to a certain extent, which can further mirror functional activities and environmental risks.

[Effect of Zero Valent Iron on the Horizontal Gene Transfer of Tetracycline Resistance Genes During Anaerobic Sludge Digestion Process].

The problem of bacterial resistance has become an important issue in the area of global ecological safety and human health. Waste sludge is an important reservoir and discharge source for antibiotic resistance genes (ARGs). In this study, the quantities of seven tetracycline resistance genes (TC-ARGs), including tetA, tetC, tetG, tetM, tetO, tetW, and tetX, as well as those of class 1 integron (intI1) genes, during anaerobic sludge digestion process were comprehensively quantified by quantitative PCR (qPCR). The effects of different doses of zero valent iron (Fe0) on the decrease and increase in the quantities of TC-ARGs and intI1 genes were investigated. The influence of plasmid conjugation on the horizontal gene transfer (HGT) of the target TC-ARGs was preliminarily analyzed. The correlations between the quantities of TC-ARGs and intI1 gene have been discussed. The results showed that the quantities of TC-ARGs and intI1 genes decreased in different degrees during anaerobic sludge digestion, and the abundance of tetX gene was reduced by 2.4 orders of magnitude. When Fe0 was added, no significant reduction in the quantities of TC-ARGs and intI1 genes was observed. However, as the addition of Fe0 increased, the quantities of TC-ARGs and intI1 genes increased correspondingly, as compared to those in the control group. The results obtained from the quantities of TC-ARGs carried by plasmid DNA showed that plasmid conjugation probably promoted the HGT of TC-ARGs. A positive significant correlation was found between the quantities of tetG and intI1 genes, indicating that intI1 might play an important role in the evolution of tetG during sludge anaerobic digestion process.

Cigarette Smoke Modulates NOD1 Signal Pathway and Human ß Defensins Expression in Human Oral Mucosa.

BACKGROUND/AIMS: Nucleotide binding oligomerization domain 1 (NOD1) signal pathway and human ß defensins (hBDs) play crucial roles in innate immune. Cigarette smoke has been confirmed to dampen innate immune in some human tissues, such as oral mucosa. The aim of this study was to evaluate potential effects of smoking on NOD1 signaling and hBDs expression in oral mucosa. METHODS: Tissue specimens of normal oral mucosa were collected from donors undergoing routine surgical treatment. All 20 participants were classified equally as two groups: non-smokers and smokers. By using Western blotting and immunohistochemistry, we investigated differential expression of crucial molecules in NOD1 signal pathway, hBD-1, -2, and -3 in oral mucosa tissues between non-smokers and smokers. Immortalized human oral mucosal epithelial (Leuk-1) cells were treated with various concentrations of cigarette smoke extract (CSE) for 24h. Western blotting and immunofluorescence assays were performed to study CSE-induced alteration of protein expression. Leuk-1 cells were treated with 4% CSE, iE-DAP (NOD1 agonist), CSE + iE-DAP, BAY 11-7082 (NF-κB inhibitor), 4% CSE + BAY 11-7082, respectively. Real-time PCR and ELISA were performed to detect the mRNA levels and secretion of hBD-1, -2, and -3, respectively. RESULTS: The levels of NOD1, NF-κB, hBD-1 and hBD-3 significantly reduced in oral mucosa tissues of smokers compared with non-smokers. The levels of RIP2 (receptor-interacting protein 2), phospho-NF-κB (P-NF-κB) and hBD-2 remarkably enhanced in oral mucosal tissues of smokers. CSE treatment suppressed NOD1 and NF-κB expression and activated RIP2 and P-NF-κB expression in Leuk-1 cells. The mRNA and secretory levels of hBD-1 and -3 were down-regulated by CSE, while the mRNA and secretory level of hBD-2 were up-regulated by CSE. The iE-DAP or BAY 11-7082 treatment reversed the regulatory effects of CSE on levels of hBDs. CONCLUSION: The present study indicated that cigarette smoke could potentially modulate the expression of crucial molecules of NOD1 signal pathway and hBDs in human oral mucosal epithelium. NOD1 signal pathway could play an important role in the regulatory effects of CSE on hBDs levels in oral mucosal epithelial cells.

Effects of whole cigarette smoke on human beta defensins expression and secretion by oral mucosal epithelial cells.

BACKGROUND: Cigarette smoke a recognized risk factor for many systemic diseases and also oral diseases. Human beta defensins (HBDs), a group of important antimicrobial peptides expressed by the epithelium, are crucial for local defense and tissue homeostasis of oral cavity. The aim of this study was to evaluate potential effects of whole cigarette smoke (WCS) exposure on the expression and secretion of HBDs by oral mucosal epithelial cells. METHODS: Immortalized human oral mucosal epithelial (Leuk-1) cells were exposed to WCS for various time periods. HBD-1, -2 and -3 expression and subcellular localization were detected by real time qPCR, immunofluorescence assay and confocal microscopy. According to the relative fluorescent intensity, the expression levels of HBD-1, -2 and -3 were evaluated by digital image analysis system. The alteration of HBD-1, -2 and -3 secretion levels was measured by the Enzyme-Linked Immunosorbent Assay. RESULTS: WCS exposure remarkably attenuated HBD-1 expression and secretion while clearly enhanced HBD-2, -3 expression levels and HBD-2 secretion by Leuk-l cells. It appeared that there was no significant effect of WCS exposure on HBD-3 secretion. CONCLUSIONS: WCS exposure could modulate expression and secretion of HBDs by oral mucosal epithelial cells, establishing a link between cigarette smoke and abnormal levels of antimicrobial peptides. The present results may give a new perspective to investigate smoking-related local defense suppression and oral disease occurrence.

Caspase-12 silencing attenuates inhibitory effects of cigarette smoke extract on NOD1 signaling and hBDs expression in human oral mucosal epithelial cells.

Cigarette smoke exposure is associated with increased risk of various diseases. Epithelial cells-mediated innate immune responses to infectious pathogens are compromised by cigarette smoke. Although many studies have established that cigarette smoke exposure affects the expression of Toll-liked receptor (TLR), it remains unknown whether the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) expression is affected by cigarette smoke exposure. In the study, we investigated effects of cigarette smoke extract (CSE) on NOD1 signaling in an immortalized human oral mucosal epithelial (Leuk-1) cell line. We first found that CSE inhibited NOD1 expression in a dose-dependent manner. Moreover, CSE modulated the expression of other crucial molecules in NOD1 signaling and human ß defensin (hBD) 1, 2 and 3. We found that RNA interference-induced Caspase-12 silencing increased NOD1 and phospho-NF-κB (p-NF-κB) expression and down-regulated RIP2 expression. The inhibitory effects of CSE on NOD1 signaling can be attenuated partially through Caspase-12 silencing. Intriguingly, Caspase-12 silencing abrogated inhibitory effects of CSE on hBD1, 3 expression and augmented induced effect of CSE on hBD2 expression. Caspase-12 could play a vital role in the inhibitory effects of cigarette smoke on NOD1 signaling and hBDs expression in oral mucosal epithelial cells.

[Inhibitory effect of miRNA silencing hypoxia-inducible factor alpha subunit gene on the proliferation of HepG2 cells].

OBJECTIVE: To investigate the effect of miRNA silencing HIF-1α gene on the proliferation of HepG2 cells. METHODS: The eukaryotic expression plasmids of HIF-1α miRNA and report gene containing hypoxia-reponse element were constructed and transfected into HepG2 cells. The expressions of HIF-1α gene and protein were determined by real time-PCR and Western blotting. The expressions of HIF-1α, vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) were quantitatively detected by ELISA. The alterations of cell cycles and apoptosis rate were quantitatively measured by flow cytometry and Annexin V-FITC/PI double dyeing assay. RESULTS: 72 h after transfection the down regulations of HIF-1α mRNA and protein were 87% and 56% respectively, and the decrease of target gene was 46% in the report gene, 54% in VEGF and 36% in Ang-2, respectively. The apoptotic ratio of HepG2 cells was 22.46+/-0.61% (P < 0.01). The cell cycle changed greatly at the ratio of G1 (61.49+/-1.12%) and S (22.40+/-0.58%, P < 0.01). After being combined with doxorubicin, the apoptotic ratio increased to 36.99+/-0.88% and the ratios of G1 and S phases were upregulated to 65.68+/-0.91% and 19.47+/-1.34% respectively. CONCLUSIONS: HIF-1α miRNA or / and doxorubicin can regulate the growth cycles of HepG2 cells, promote the cell apoptosis and inhibit the cell proliferation.