ABSTRACT
RIPOR2 (previously known as FAM65B) localizes to stereocilia of auditory hair cells and causes deafness when its function is disturbed by mutations. Here, we demonstrate that during the morphogenesis of the hair cell bundle, absence of Ripor2 affects the orientation of this key subcellular structure. We show that Ripor2 interacts with Myh9, a protein encoded by a known deafness gene. Absence of Ripor2 is associated with low Myh9 abundance in the mouse cochlea despite increased amount of Myh9 transcripts. While Myh9 is mainly expressed in stereocilia, a phosphorylated form of Myh9 is particularly enriched in the kinocilium. In Ripor2-deficient mice, kinocilium shows an aberrant localization which associates with a reduced content of phosphorylated Myh9. Acetylated alpha tubulin, another specific kinociliary protein which contributes to microtubule stabilization, is reduced in the absence of Ripor2 as well. We propose that Ripor2 deficiency influences abundance and/or post-translational modifications of proteins expressed in both stereocilia and kinocilia. This effect may have a negative impact on the structure and function of the auditory hair cell bundle.
Subject(s)
Carrier Proteins/physiology , Hair Cells, Auditory/physiology , Membrane Proteins/physiology , Nonmuscle Myosin Type IIA/physiology , Animals , Cell Adhesion Molecules , Cilia/physiology , Ear, Inner/physiology , Epithelium/physiology , HEK293 Cells , Humans , Male , Mice, Inbred C57BL , Mice, Knockout , Myosin Heavy Chains , RNA, Messenger/metabolismABSTRACT
Organic or inorganic stabilizers are often used for coating nanoparticles (NPs) in consumer products. However, upon release of stabilized NPs into the environment, uncertainty exists as to the antimicrobial properties of NPs due to stabilizers and the resultant bioaccumulation in organisms. This study investigates antibacterial effects and subsequent mechanisms of TiO2 NPs on Escherichia coli (E. coli) in the presence and absence of stabilizers (CMC, PVP, and SiO2) commonly used in consumer products. Compared with uncoated TiO2 NPs, the presence of any stabilizers tested in this study increased toxicity of NPs and enhanced growth inhibition in E. coli. While the particle sizes of TiO2 were smaller as the result of coating with PVP or CMC and appeared to contribute to E. coli cell damage, the generation of reactive oxygen species (ROS) was independent of stabilizer type. In fact, coating with PVP and CMC exerted ROS scavenging properties. In contrast, increased ROS production was observed at higher concentrations of TiO2 and upon coating with SiO2. This impact of SiO2 can be related to the formation of a TiOSi chemical bond. The results of the present study emphasize the importance of nanoparticle coating to their anti-bacterial activity and toxicity.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Sunscreening Agents/chemistry , Titanium/chemistry , Escherichia coli/growth & development , Escherichia coli/metabolism , Particle Size , Reactive Oxygen Species/metabolismABSTRACT
Increased manufacture of TiO2 nanoproducts has caused concern about the potential toxicity of these products to the environment and in public health. Identification and confirmation of the presence of TiO2 nanoparticles derived from consumer products as opposed to industrial TiO2 NPs warrant examination in exploring the significance of their release and resultant impacts on the environment. To this end, we examined the significance of the release of these particles and their toxic effect on the marine diatom algae Thalassiosira pseudonana. Our results indicate that nano-TiO2 sunscreen and toothpaste exhibit more toxicity in comparison to industrial TiO2 and inhibited the growth of the marine diatom T. pseudonana. This inhibition was proportional to the exposure time and concentrations of nano-TiO2. Our findings indicate a significant effect, and therefore, further research is warranted in evaluation and assessment of the toxicity of modified nano-TiO2 derived from consumer products and their physicochemical properties.
Subject(s)
Diatoms/drug effects , Metal Nanoparticles/toxicity , Titanium/toxicity , Water Pollutants, Chemical/toxicity , Diatoms/growth & development , Seawater/microbiology , Sunscreening Agents , ToothpastesABSTRACT
BACKGROUND: Amphotericin B (AmB), the most effective drug against leishmaniasis, has serious toxicity. As Leishmania species are obligate intracellular parasites of antigen presenting cells (APC), an immunopotentiating APC-specific AmB nanocarrier would be ideally suited to reduce the drug dosage and regimen requirements in leishmaniasis treatment. Here, we report a nanocarrier that results in effective treatment shortening of cutaneous leishmaniasis in a mouse model, while also enhancing L. major specific T-cell immune responses in the infected host. METHODS: We used a Pan-DR-binding epitope (PADRE)-derivatized-dendrimer (PDD), complexed with liposomal amphotericin B (LAmB) in an L. major mouse model and analyzed the therapeutic efficacy of low-dose PDD/LAmB vs full dose LAmB. RESULTS: PDD was shown to escort LAmB to APCs in vivo, enhanced the drug efficacy by 83% and drug APC targeting by 10-fold and significantly reduced parasite burden and toxicity. Fortuitously, the PDD immunopotentiating effect significantly enhanced parasite-specific T-cell responses in immunocompetent infected mice. CONCLUSIONS: PDD reduced the effective dose and toxicity of LAmB and resulted in elicitation of strong parasite specific T-cell responses. A reduced effective therapeutic dose was achieved by selective LAmB delivery to APC, bypassing bystander cells, reducing toxicity and inducing antiparasite immunity.
Subject(s)
Amphotericin B/administration & dosage , Antiprotozoal Agents/administration & dosage , Dendrimers/administration & dosage , Leishmania major/drug effects , Leishmaniasis, Cutaneous/drug therapy , Malaria Vaccines/administration & dosage , Adaptive Immunity , Amphotericin B/toxicity , Animals , Antigen-Presenting Cells/immunology , Antiprotozoal Agents/toxicity , Disease Models, Animal , Drug Carriers , Epitopes , Female , Injections, Intraperitoneal , Leishmania major/immunology , Leishmaniasis Vaccines , Leishmaniasis, Cutaneous/immunology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microscopy, Electron, Transmission , NanoparticlesABSTRACT
DNA-based vaccines hold promise to outperform conventional antigen-based vaccines by virtue of many unique features. However, DNA vaccines have thus far fallen short of expectations, due in part to poor targeting of professional antigen-presenting cells (APC) and low immunogenicity. In this study, we describe a new platform for effective and selective delivery of DNA to APCs in vivo that offers intrinsic immune-enhancing characteristics. This platform is based on conjugation of fifth generation polyamidoamine (G5-PAMAM) dendrimers, a DNA-loading surface, with MHC class II-targeting peptides that can selectively deliver these dendrimers to APCs under conditions that enhance their immune stimulatory potency. DNA conjugated with this platform efficiently transfected murine and human APCs in vitro. Subcutaneous administration of DNA-peptide-dendrimer complexes in vivo preferentially transfected dendritic cells (DC) in the draining lymph nodes, promoted generation of high affinity T cells, and elicited rejection of established tumors. Taken together, our findings show how PAMAM dendrimer complexes can be used for high transfection efficiency and effective targeting of APCs in vivo, conferring properties essential to generate effective DNA vaccines.
