Roles of epidermal growth factor receptor, claudin-1 and occludin in multi-step entry of hepatitis C virus into polarized hepatoma spheroids.

The multi-step process of hepatitis C virus (HCV) entry is facilitated by various host factors, including epidermal growth factor receptor (EGFR) and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), which are thought to function at later stages of the HCV entry process. Using single particle imaging of HCV infection of polarized hepatoma spheroids, we observed that EGFR performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. We previously observed, and in this study confirmed, that EGFR is not required for HCV migration to the tight junction. EGFR is required for the recruitment of clathrin to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization at a stage following the recruitment of clathrin. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into APPL1- and EEA1-associated early endosomes, which form the site of virion uncoating. The tight junction proteins, CLDN1 and OCLN, function at two distinct stages of HCV entry. Despite its appreciated function as a "late receptor" in HCV entry, CLDN1 is required for efficient HCV virion accumulation at the tight junction. Huh-7.5 cells lacking CLDN1 accumulate HCV virions primarily at the initial basolateral surface. OCLN is required for the late stages of virion internalization. This study produced further insight into the unusually complex HCV endocytic process.

Zinc oxide nanoparticles provide anti-cholera activity by disrupting the interaction of cholera toxin with the human GM1 receptor.

Vibrio cholerae causes cholera and is the leading cause of diarrhea in developing countries, highlighting the need for the development of new treatment strategies to combat this disease agent. While exploring the possibility of using zinc oxide (ZnO) nanoparticles (NPs) in cholera treatment, we previously found that ZnO NPs reduce fluid accumulation in mouse ileum induced by the cholera toxin (CT) protein. To uncover the mechanism of action of ZnO NPs on CT activity, here we used classical (O395) and El Tor (C6706) V. cholerae biotypes in growth and biochemical assays. We found that a ZnO NP concentration of 10 µg/ml did not affect the growth rates of these two strains, nor did we observe that ZnO NPs reduce the expression levels of CT mRNA and protein. It was observed that ZnO NPs form a complex with CT, appear to disrupt the CT secondary structure, and block its interaction with the GM1 ganglioside receptor in the outer leaflet of the plasma membrane in intestinal (HT-29) cells and thereby reduce CT uptake into the cells. In the range of 2.5-10 µg/ml, ZnO NPs exhibited no cytotoxicity on kidney (HEK293) and HT-29 cells. We conclude that ZnO NPs prevent the first step in the translocation of cholera toxin into intestinal epithelial cells without exerting measurable toxic effects on HEK293 and HT-29 cells.

The antimicrobial activity of ZnO nanoparticles against Vibrio cholerae: Variation in response depends on biotype.

The potency of zinc oxide nanoparticles (NPs), with a core size of ~7-10nm, to inhibit cholera disease was investigated by demonstrating the effect on two biotypes (classical and El Tor) of O1 serogroup of Vibrio cholerae-El Tor was more susceptible both in planktonic and in biofilm forms. Interaction with ZnO NP results in deformed cellular architecture. Increased fluidity and depolarization of membrane, and protein leakage further confirmed the damages inflicted on Vibrio by NP. NP was shown to produce reactive oxygen species (ROS) and induce DNA damage. These results suggest that the antibacterial mechanism of ZnO action is most likely due to generation of ROS and disruption of bacterial membrane. The antimicrobial efficacy of NP has been validated in animal model. The synergistic action of NP and antibiotic suggests an alternative for the treatment of cholera.

PEG-functionalized zinc oxide nanoparticles induce apoptosis in breast cancer cells through reactive oxygen species-dependent impairment of DNA damage repair enzyme NEIL2.

We find that PEG functionalized ZnO nanoparticles (NP) have anticancer properties primarily because of ROS generation. Detailed investigation revealed two consequences depending on the level of ROS - either DNA damage repair or apoptosis - in a time-dependent manner. At early hours of treatment, NP promote NEIL2-mediated DNA repair process to counteract low ROS-induced DNA damage. However, at late hours these NP produce high level of ROS that inhibits DNA repair process, thereby directing the cell towards apoptosis. Mechanistically at low ROS conditions, transcription factor Sp1 binds to the NEIL2 promoter and facilitates its transcription for triggering a 'fight-back mechanism' thereby resisting cancer cell apoptosis. In contrast, as ROS increase during later hours, Sp1 undergoes oxidative degradation that decreases its availability for binding to the promoter thereby down-regulating NEIL2 and impairing the repair mechanism. Under such conditions, the cells strategically switch to the p53-dependent apoptosis.

Bactericidal effect of polyethyleneimine capped ZnO nanoparticles on multiple antibiotic resistant bacteria harboring genes of high-pathogenicity island.

Zinc oxide nanoparticles (ZnO-NP) were synthesized by alcoholic route using zinc acetate as the precursor material and lithium hydroxide as hydrolyzing agent. Further ZnO-PEI NP (derivative of ZnO-NP) was made in aqueous medium using the capping agent polyethyleneimine (PEI). The nanoparticles were characterized by XRD measurements, TEM and other techniques; the weight % of coating shell in the polymer-capped particles was determined by TGA. ZnO-PEI NP is more soluble in water than the uncapped ZnO-NP, and forms a colloidal suspension in water. PEI-capped ZnO-NP exhibited better antibacterial activity when compared with that of uncapped ZnO-NP against a range of multiple-antibiotic-resistant (MAR) Gram-negative bacterial strains harboring genes of high-pathogenicity island. ZnO-NP effectively killed these microorganisms by generating reactive oxygen species (ROS) and damaging bacterial membrane. ZnO-PEI NP at LD50 dose in combination with tetracycline showed synergistic effect to inhibit tetracycline-resistant Escherichia coli MREC33 growth by 80%. These results open up a new vista in therapeutics to use antibiotics (which have otherwise been rendered useless against MAR bacteria) in combination with minimized dosage of nanoparticles for the more effective control of MAR pathogenic bacteria.

The effect of the binding of ZnO nanoparticle on the structure and stability of α-lactalbumin: a comparative study.

Nanoparticles (NPs), when exposed to biofluids, become coated with proteins. As the protein is adsorbed on the surface, the extent of adsorption and the consequent effect on protein conformation and activity depend on the chemical nature, shape, and size of the nanoparticle. We have carried out a detailed study on the interaction of α-lactalbumin (a protein which forms the regulatory subunit of lactose synthase) with zinc oxide nanoparticles. The NPs were prepared by the sol-gel route and characterized by transmission electron microscopy, X-ray diffraction, UV-visible, and photoluminescence spectroscopy. ZnO particles were found to have a size of 4-7 nm with hexagonal structure. The interaction of protein with NP was examined using a combination of spectroscopic and computational methods. The binding was studied by ITC (isothermal calorimetry), and the result revealed that the complexation is mostly entropy driven and involves hydrophobic interaction. There is alteration in secondary structures in protein on binding ZnO nanoparticle, as revealed by circular dichroism (CD) and Fourier transform infrared spectroscopy (FITR). Finally, a comparison of structure, function, and stability of the α-lactalbumin-NP complex has been made by binding ZnO to other model proteins to get a better insight into the process of protein nanoparticle interaction. The present study thus provides useful insights into issues such as protein-nanoparticle recognition.