M6A modification regulates tumor suppressor DIRAS1 expression in cervical cancer cells.

DIRAS family GTPase 1 (DIRAS1) has been reported as a potential tumor suppressor in other human cancer. However, its expression pattern and role in cervical cancer remain unknown. Knockdown of DIRAS1 significantly promoted the proliferation, growth, migration, and invasion of C33A and SiHa cells cultured in vitro. Overexpression of DIRAS1 significantly inhibited the viability and motility of C33A and SiHa cells. Compared with normal cervical tissues, DIRAS1 mRNA levels were significantly lower in cervical cancer tissues. DIRAS1 protein expression was also significantly reduced in cervical cancer tissues compared with para-cancerous tissues. In addition, DIRAS1 expression level in tumor tissues was significantly negatively correlated with the pathological grades of cervical cancer patients. DNA methylation inhibitor (5-Azacytidine) and histone deacetylation inhibitor (SAHA) resulted in a significant increase in DIRAS1 mRNA levels in C33A and SiHa cells, but did not affect DIRAS1 protein levels. FTO inhibitor (FB23-2) significantly down-regulated intracellular DIRAS1 mRNA levels, but significantly up-regulated DIRAS1 protein levels. Moreover, the down-regulation of METTL3 and METTL14 expression significantly inhibited DIRAS1 protein expression, whereas the down-regulation of FTO and ALKBH5 expression significantly increased DIRAS1 protein expression. In conclusion, DIRAS1 exerts a significant anti-oncogenic function and its expression is significantly downregulated in cervical cancer cells. The m6A modification may be a key mechanism to regulate DIRAS1 mRNA stability and protein translation efficiency in cervical cancer.

Study on the Structure of Candida Albicans-Staphylococcus Epidermidis Mixed Species Biofilm on Polyvinyl Chloride Biomaterial.

The objective of the study was to establish an in vitro model of Candida albicans-Staphylococcus epidermidis mixed species biofilm (BF) on polyvinyl chloride (PVC) material, and to investigate the formation and the structure of mixed species BF formation using a combined approach of confocal laser scanning microscope (CLSM), scanning electron microscope (SEM), and 3D image reconstruction technique. Mixed species BF is achieved by co-incubating Staphylococcus epidermidis bacteria (ATCC35984) and Candida albicans fungal (ATCC10231) with PVC pieces in Tris-buffered saline. BF formation was examined at 2, 6, 12, 24, 48, and 72 h of co-culture. Thickness of these BFs and the number, and percentage of viable cells in BFs were measured. CT scan images of BFs were obtained using CLSM and SEM and reconstructed 3D images of mixed species BF were acquired, in an effort to examine structure of the BF. Staphylococcus epidermidis attached to various forms of candida albicans (spores, pseudohyphae, and hyphae), formed complex and dense mesh arrays. The BF is constituted of a large number of viable and dead pathogens, the surface of mixed species BF is uneven, with living pathogens predominating protrusive portions and dead pathogens aggregating in concaves. Mixed species BF formation on the surface of PVC material was found to be a dynamic process, with rapid growth being at 24 h of co-culture, maximal thickness peaked at 48 h. These mixed species BF matured at 48-72 h. Significant differences were observed in the proportion of viable cells between interior, middle, and outer layers of BFs (p < 0.05). Mixed species BF Candida albicans-Staphylococcus epidermidis is sophisticated in structure. The combined approach involving CLSM, SEM, and 3D image reconstruction technique is ideal for the investigation of mixed species BF on PVC material.

Establishment of rat model of central venous catheter (CVC): associated infection and evaluation of the virulence of bacterial biofilms.

In this study, a central venous catheter (CVC)-associated infection model was established in rats to investigate and evaluate the effect of biofilms on the virulence of the pathogens. Twenty-four adult SD rats were randomly divided into biofilm positive (BF+) and biofilm negative (BF-) groups to be challenged with strains of S.epidermidis. Serum levels of inflammatory cytokines were measured and the infection rate and counts of bacteria cells were studied. Compared to rats of BF- group, the serum level of TNF and IL-6 significantly increased in rats of BF+ group (P < 0.01) and the level of IL-10 and IFN-γ significantly decreased (P < 0.01), striking the balance of pro-inflammatory/anti-inflammatory cytokines. The infection rate and bacterial counts in tissues and blood of rats of BF + group were significantly higher than those of rats of BF- group (P < 0.05).Inflammatory cell infiltration in vital organs (heart, lung, liver and kidneys) was more significant in rats of BF+ group than that of rats of BF- group. CVC-associated infection model can be successfully reproduced in rats by injecting 5 × 10(6) CFU of S.epidermidis. Biofilm formation can significantly enhance the virulence of the bacteria, leading to uncontrolled infection. The serum level of inflammatory cytokines, infection rate and the extent of inflammatory cell infiltration are important markers for evaluating the virulence of biofilm.

The role of the QseC quorum-sensing sensor kinase in epinephrine-enhanced motility and biofilm formation by Escherichia coli.

Biofilms play a pivotal role in infections related to devices. Biofilm formation in Escherichia coli is mediated by the quorum-sensing E. coli regulator C (QseC), the histidine sensor kinase that can sense epinephrine (EPI)/norepinephrine (NE). In this study, we evaluate the role of the QseC quorum-sensing sensor kinase in epinephrine-enhanced motility and biofilm formation by E. coli. An E. coli MC1000 qseC mutant was constructed. We investigated the role of the QseC in the formation of biofilms on the surface of medical-grade polyvinyl chloride using the E. coli K-12 MC1000 strain as well as a corresponding qseC mutant. Addition of EPI/NE increased biofilm formation by wild-type K-12 MC1000 but not by the isogenic qseC mutant. Scanning confocal laser microscopy corroborated these results by showing that EPI/NE addition significantly increased biofilm's thickness. As expected, the addition of EPI/NE to the qseC mutant, which lacks the ability to sense the hormones, failed to stimulate biofilm formation. Since EPI/NE addition increased bacterial motility, we proposed that their stimulatory effects on biofilm formation occur by enhancing bacterial motility and altering biofilm architecture. We also found that EPI/NE regulate motility and the biofilm phenotype via QseC, as motility was diminished and biofilm formation was significantly decreased in a qseC deletion mutant. These results indicate that EPI/NE induce E. coli biofilm formation on the surface of polyvinyl chloride through QseC. Cross-talk between E. coli (quorum sensing) and host hormones may explain the pathogen-caused opportunistic infections that occur in patients with prosthetic devices used during hormone level fluctuations in the host.

In vitro model of bacterial biofilm formation on polyvinyl chloride biomaterial.

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12-18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.