Multi-Inlet Spheroid Generator for High-Throughput Combinatorial Drug Screening Based on the Tumor Microenvironment.

Tumor spheroids are powerful tools for drug screening and understanding tumor physiology. Among spheroid formation methods, the hanging drop method is considered most suitable for high-throughput screening (HTS) of anticancer drugs because it does not require surface treatment. However, it still needs to increase the liquid-holding capacity because hanging drops often fall due to the increased pressure caused by the addition of drugs, cells, etc. Here, we report a multi-inlet spheroid generator (MSG) enabling the stable addition of liquid-containing drugs or cells into a spheroid through its side inlet. The MSG was able to load additional solutions through the side inlet without increasing the force applied to the hanging drop. The volume of the additional liquid was easily controlled by varying the diameter of the side inlet. Furthermore, the sequences of the solution injections were manipulated using multiple side inlets. The feasibility of the MSG in clinical application was demonstrated by testing the efficacy of drugs in patient-derived cancer (PDC) cells and controlling the stromal cell ratio in the tumor microenvironment (TME) containing spheroids. Our results suggest that the MSG is a versatile platform for HTS of anticancer drugs and recapitulating the TME.

Inhibitory Effect of Epigallocatechin Gallate on the Virulence of Clostridium difficile PCR Ribotype 027.

Clostridium difficile infection (CDI) is the most prevalent cause of health-care-associated infections. CDI-related health-care costs and deaths are both increasing annually on a global scale. C. difficile have been reported in food products in Canada, Europe, and the United States; however, the systematic transmission of C. difficile between humans and animals is yet to be understood. Because of the limitations of current therapeutic options, there is a need for the development of new patient treatments. Epigallocatechin gallate (EGCG) is a major catechin compound found in green tea extracts and exhibits antioxidant and antimicrobial activities. This study was conducted to investigate the inhibitory effects of EGCG on the expression of virulence genes in C. difficile and in C. difficile-associated diseases by inhibition of quorum sensing. The protein expression of autoinducer-2 (AI-2) was evaluated by AI-2 activity. EGCG at various concentrations had an inhibitory effect on AI-2 production, especially at 10 µg/mL. EGCG also significantly repressed the transcription of virulence genes, including luxS and tcdA, and prolonged the survival of Caenorhabditis elegans infected with C. difficile. Furthermore, treatment with EGCG effectively protected C. difficile-infected mice from C. difficile-induced death. Histological analysis of the colon and cecum of these mice revealed that EGCG protected tissues of the lower intestinal tract from damage. EGCG exerted growth-inhibitory and bactericidal activities on C. difficile in C. difficile-infected mice. Our results suggest that EGCG has significant antipathogenic effects on C. difficile and can be used to prevent or treat C. difficile-associated diseases or C. difficile infections.

AFM probing the mechanism of synergistic effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) with cefotaxime against extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae poses serious challenges to clinicians because of its resistance to many classes of antibiotics. METHODS AND FINDINGS: The mechanism of synergistic activity of a combination of (-)-epigallocatechin-3-gallate (EGCG) and ß-lactam antibiotics cefotaxime was studied on Extended-spectrum ß-lactamase producing Escherichia coli (ESBL-EC), by visualizing the morphological alteration on the cell wall induced by the combination using atomic force microscopy (AFM). Cells at sub-MICs (sub-minimum inhibitory concentrations) of cefotaxime were initially filamentated but recovered to the normal shape later, whereas cells at sub-MICs of EGCG experienced temporal disturbance on the cell wall such as leakage and release of cellular debris and groove formation, but later recovered to the normal shape. In contrast, the combination of cefotaxime and EGCG at their respective sub-MICs induced permanent cellular damages as well as continuous elongation in cells and eventually killed them. Flow cytometry showed that intracellular oxidative stress levels in the cell treated with a combination of EGCG and cefotaxime at sub-MICs were higher than those in the cells treated with either cefotaxime or EGCG at sub-MICs. CONCLUSIONS: These results suggest that the synergistic effect of EGCG between EGCG and cefotaxime against ESBL-EC is related to cooperative activity of exogenous and endogenous reactive oxygen species (ROS) generated by EGCG and cefotaxime, respectively.

YkgM and ZinT proteins are required for maintaining intracellular zinc concentration and producing curli in enterohemorrhagic Escherichia coli (EHEC) O157:H7 under zinc deficient conditions.

Zn(2+) uptake systems are required for many enteric pathogens to survive and form biofilm in zinc-deficient conditions. ykgM and zinT (formerly yodA), regulated by Zur (zinc uptake regulator), have been reported as being highly induced during zinc shortage. This work reports that ykgM and zinT in enterohemorrhagic Escherichia coli (EHEC) O157:H7 biofilms under fluidic conditions were highly expressed compared to those in stationary-phase planktonic cells and a mutation of either ykgM or zinT genes led to the inhibition of curli biosynthesis. Inductively coupled plasma mass spectroscopy showed that the ykgM and zinT mutants contained lower concentrations of Zn(2+) than the wild type. Both mutants were less attached to both the glass surface of a microchannel and epithelial cells than the wild type. Quantitative reverse-transcription PCR data indicated that the expression of csgA, which encodes the major curli subunit, was inhibited in both mutants with a zinc deficiency. Scanning electron microscopy showed that the mutants grown under zinc-deficient condition were covered with a lower amount of curli compared to the wild type and often became filamentous. Zn(2+) supplementation restored curli production and prevented filamentation in the mutants. Overall, under zinc-deficient conditions, YkgM and ZinT proteins are required for maintaining optimal zinc concentration in EHEC and intracellular zinc deficiency inhibits curli production.

Inhibitory effects of broccoli extract on Escherichia coli O157:H7 quorum sensing and in vivo virulence.

Broccoli extract (BE) has numerous beneficial effects on human health including anticancer activity. Quorum sensing (QS), mediated by self-produced autoinducer (AI) molecules, is a key process for the production of virulence determinants in pathogenic bacteria. BE suppressed AI-2 synthesis and AI-2-mediated bacterial motility in a dose-dependent manner in Escherichia coli O157:H7. In addition, expression of the ler gene that regulates AI-3 QS system was also diminished in response to treatment with BE. Furthermore, in an in vivo efficacy test using Caenorhabditis elegans as a host organism, C. elegans fed on E. coli O157:H7 in the presence of BE survived longer than those fed solely on the pathogenic bacteria. Quantitative real-time PCR analysis indicated that quercetin was the most active among the tested broccoli-derived compounds in downregulating virulence gene expression, while treatment with myricetin significantly suppressed the expression of the eae gene involved in type III secretion system. These data suggest that BE and its flavonoid constituents can inhibit expression of QS-associated genes, thereby downregulating the virulence attributes of E. coli O157:H7 both in vitro and in vivo. This study clearly elucidates BE's QS-inhibitory activity and suggests that BE has the potential to be developed as an anti-infective agent.

Antipathogenic properties of green tea polyphenol epigallocatechin gallate at concentrations below the MIC against enterohemorrhagic Escherichia coli O157:H7.

The inhibitory effects of green tea polyphenol epigallocatechin gallate (EGCG) on virulence phenotypes and gene expression regulated by quorum sensing (QS) in Escherichia coli O157:H7 were demonstrated at concentrations of 1 to 100 microg/ml, which are lower than the MIC (539 +/- 22 microg/ml). At 25 microg/ml, the growth rate was not affected, but autoinducer 2 concentration, biofilm formation, and swarm motility decreased to 13.2, 11.8, and 50%, respectively. Survival at 5 days of nematodes (Caenorhabditis elegans) that were fed the pathogen without and with EGCG were 47.1 and 76%, respectively. Real-time PCR data indicated decreased transcriptional level in many quorum sensing-regulated virulence genes at 25 microg/ml. Our results suggest that EGCG at concentrations below itsMIC has significant antipathogenic effects against E. coli O157:H7.