In vitro and in vivo analysis of the assembly of the non-collagenous tectorial membrane matrix

Alpha- and beta-tectorin (Tecta and Tectb) are major non-collagenous components of the tectorial membrane (TM). The presence of a zona pellucida (ZP) domain in both tectorins suggests that Tecta and Tectb can form hetero- or homopolymers. It is unclear, however, how these proteins assemble to form the TM matrix. The mechanisms of apical targeting, secretion and processing of the tectorins are also unexplored. I used fluorescently-tagged tectorin constructs for stable transfection into polarised epithelial MDCK cells or transient expression in mouse cochlear cultures to develop an in vitro model of TM matrix assembly. Significant amounts of matrix were not observed with stable tectorin expression in monolayer cultures of MDCK cells. In contrast, I observed substantial amounts of dense extracellular matrix on the apical surfaces of outgrowth zone cells when cochlear cultures were transiently transfected with either Tecta or Tectb. When ectopically expressed in hair cells, Tecta and Tectb locate to the distal tips of the hair bundle. To study the role of the inner-ear protein Ceacam16 in hearing, we generated a Ceacam16 functional null mouse model. The Ceacam16 gene was inactivated by targeted replacement of exons 2-5 with the bacterial lacZ gene. β-gal staining I performed reveals that Ceacam16 is expressed in the epithelial cells of the spiral limbus and inner sulcus, and in both the pillar cells and Deiter’s cells. I first detected the presence of Ceacam16 in the TM at P12, four days before the defined striated-sheet matrix is observed. Transmission electron microscopy reveals a complete loss of striated-sheet matrix in Ceacam16 null mice in comparison to the wild-type. The results of this thesis suggest neonatal mouse cochlear cultures as a model for studying tectorin-based extracellular matrix production and also reveal that Ceacam16 is required for normal formation and/or maintenance of striated-sheet matrix

Socio-Economic Role of Infrastructure Projects in the Spatial Development of the Volga Federal District

The article is devoted to assessing the role of infrastructure projects in the spatial development of the Volga Federal District. First of all, the regions of the Volga Federal District were ranked by the factor of financing infrastructure projects. Then, regression dependencies of this factor with socio-economic indicators of the constituent entities development were identified. In addition, a map was developed reflecting the spatial differentiation of the Volga Federal District regions on the development of infrastructure projects

Non-Covalent Associates of siRNAs and AuNPs Enveloped with Lipid Layer and Doped with Amphiphilic Peptide for Efficient siRNA Delivery

Elaboration of non-viral vehicles for delivery of therapeutic nucleic acids, in particular siRNA, into a cell is an actively growing field. Gold nanoparticles (AuNPs) occupy a noticeable place in these studies, and various nanoconstructions containing AuNPs are reported. We aimed our work to the rational design of AuNPs-based siRNA delivery vehicle with enhanced transfection efficiency. We optimized the obtaining of non-covalent siRNAs-AuNPs cores: ionic strength, temperature and reaction time were determined. Formation of cores was confirmed using gel electrophoresis. Stable associates were prepared, and then enveloped into a lipid layer composed of phosphatidylcholine, phosphatidylethanolamine and novel pH-sensitive lipidoid. The constructions were modified with [Str-(RL)4G-NH2] peptide (the resulting construction). All intermediate and resulting nanoconstructions were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) to control their physico-chemical properties. To examine the biological effect of the delivery vehicle, green fluorescent protein (GFP)-expressing human embryonic kidney (HEK) Phoenix cells were incubated with the resulting construction containing anti-GFP siRNA, with the siRNA effect being studied by flow cytometry and confocal microscopy. Transfection of the cells with the resulting construction reduced the GFP fluorescence as efficiently as Lipofectamin 3000. Thus, siRNA vehicle based on non-covalently bound siRNA-AuNP core and enveloped into a lipid layer provides efficient delivery of siRNA into a cell followed by specific gene silencing

Wfs1E864K knock-in mice illuminate the fundamental role of Wfs1 in endocochlear potential production.

peer reviewedWolfram syndrome (WS) is a rare neurodegenerative disorder encompassing diabetes mellitus, diabetes insipidus, optic atrophy, hearing loss (HL) as well as neurological disorders. None of the animal models of the pathology are presenting with an early onset HL, impeding the understanding of the role of Wolframin (WFS1), the protein responsible for WS, in the auditory pathway. We generated a knock-in mouse, the Wfs1E864K line, presenting a human mutation leading to severe deafness in affected individuals. The homozygous mice showed a profound post-natal HL and vestibular syndrome, a collapse of the endocochlear potential (EP) and a devastating alteration of the stria vascularis and neurosensory epithelium. The mutant protein prevented the localization to the cell surface of the Na+/K+ATPase β1 subunit, a key protein for the maintenance of the EP. Overall, our data support a key role of WFS1 in the maintenance of the EP and the stria vascularis, via its binding partner, the Na+/K+ATPase β1 subunit

ER-mitochondria cross-talk is regulated by the Ca(2+) sensor NCS1 and is impaired in Wolfram syndrome.

Communication between the endoplasmic reticulum (ER) and mitochondria plays a pivotal role in Ca(2+) signaling, energy metabolism, and cell survival. Dysfunction in this cross-talk leads to metabolic and neurodegenerative diseases. Wolfram syndrome is a fatal neurodegenerative disease caused by mutations in the ER-resident protein WFS1. Here, we showed that WFS1 formed a complex with neuronal calcium sensor 1 (NCS1) and inositol 1,4,5-trisphosphate receptor (IP3R) to promote Ca(2+) transfer between the ER and mitochondria. In addition, we found that NCS1 abundance was reduced in WFS1-null patient fibroblasts, which showed reduced ER-mitochondria interactions and Ca(2+) exchange. Moreover, in WFS1-deficient cells, NCS1 overexpression not only restored ER-mitochondria interactions and Ca(2+) transfer but also rescued mitochondrial dysfunction. Our results describe a key role of NCS1 in ER-mitochondria cross-talk, uncover a pathogenic mechanism for Wolfram syndrome, and potentially reveal insights into the pathogenesis of other neurodegenerative diseases

Identification of the Hair Cell Soma-1 Antigen, HCS-1, as Otoferlin

Hair cells, the mechanosensitive receptor cells of the inner ear, are critical for our senses of hearing and balance. The small number of these receptor cells in the inner ear has impeded the identification and characterization of proteins important for hair cell function. The binding specificity of monoclonal antibodies provides a means for identifying hair cell-specific proteins and isolating them for further study. We have generated a monoclonal antibody, termed hair cell soma-1 (HCS-1), which specifically immunolabels hair cells in at least five vertebrate classes, including sharks and rays, bony fish, amphibians, birds, and mammals. We used HCS-1 to immunoprecipitate the cognate antigen and identified it as otoferlin, a member of the ferlin protein family. Mutations in otoferlin underlie DFNB9, a recessive, nonsyndromic form of prelingual deafness characterized as an auditory neuropathy. Using immunocytochemistry, we find that otoferlin is associated with the entire basolateral membrane of the hair cells and with vesicular structures distributed throughout most of the hair cell cytoplasm. Biochemical assays indicate that otoferlin is tightly associated with membranes, as it is not solubilized by alterations in calcium or salt concentrations. HCS-1 immunolabeling does not co-localize with ribeye, a constituent of synaptic ribbons, suggesting that otoferlin may, in addition to its proposed function in synaptic vesicle release, play additional roles in hair cells