The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail-anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophanrich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca 2+ channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.

Due to the emergence of multi-drug resistant strains, development of novel antibiotics has become a critical issue. One promising approach is the use of transition metals, since they exhibit rapid and significant toxicity, at low concentrations, in prokaryotic cells. Nevertheless, one main drawback of transition metals is their toxicity in eukaryotic cells. Here, we show that the barriers to use them as therapeutic agents could be mitigated by combining them with silver. We demonstrate that synergism of combinatorial treatments (Silver/transition metals, including Zn, Co, Cd, Ni, and Cu) increases up to 8-fold their antimicrobial effect, when compared to their individual effects, against E. coli and B. subtilis. We find that most combinatorial treatments exhibit synergistic antimicrobial effects at low/ non-toxic concentrations to human keratinocyte cells, blast and melanoma rat cell lines. Moreover, we show that silver/(Cu, Ni, and Zn) increase prokaryotic cell permeability at sub-inhibitory concentrations, demonstrating this to be a possible mechanism of the synergistic behavior. Together, these results suggest that these combinatorial treatments will play an important role in the future development of antimicrobial agents and treatments against infections. In specific, the cytotoxicity experiments show that the combinations have great potential in the treatment of topical infections.

Key pointsr The zebrafish pinball wizard (pwi) mutant is deaf and blind. r The pwi phenotype includes a reduced auditory startle response and reduced visual evoked potentials, suggesting fatigue of synaptic release at ribbon synapses in hair cells and photoreceptors.r The gene defective in the pwi mutant is WRB, a protein homologous to the yeast protein Get1, which is involved in the insertion of 'tail-anchored' membrane proteins.r Many tail-anchored proteins are associated with synaptic vesicles, and both vesicles and synaptic ribbons are reduced in synaptic regions of hair cells in pwi.r Abnormal processing of synaptic vesicle proteins important for ribbon synapses can explain the pwi phenotype.Abstract In a large-scale zebrafish insertional mutagenesis screen, we identified the pinball wizard (pwi) line, which displays a deafness and blindness phenotype. Although the gross morphology and structure of the pwi larval inner ear was near normal, acoustic startle stimuli evoked smaller postsynaptic responses in afferent neurons, which rapidly fatigued. In the retina, similarly, an abnormal electroretinogram suggested reduced transmission at the photoreceptor ribbon synapse. A functional deficit in these specialized synapses was further supported by a reduction of synaptic marker proteins Rab3 and cysteine-string protein (CSP/Dnajc5) in hair cells and photoreceptors, as well as by a reduction of the number of both ribbons and vesicles surrounding the ribbons in hair cells. The pwi gene encodes a homologue of the yeast Get1 and human tryptophan-rich basic (WRB) proteins, which are receptors for membrane insertion of tail-anchored (TA) proteins. We identified more than 100 TA proteins expressed in hair cells, including many synaptic proteins. The expression of synaptobrevin and syntaxin 3, TA proteins essential for vesicle fusion, was reduced in the synaptic layers of mutant retina, consistent with a role for the pwi/WRB protein in TA-protein processing. The WRB protein was located near the apical domain and the ribbons in hair cells, and in the inner segment and the axon of the photoreceptor, consistent with a role in vesicle biogenesis or trafficking. Taken together, our results suggest that WRB plays a critical role in synaptic functions in these two sensory cells, and that disrupted processing of synaptic vesicle TA proteins explains much of the mutant phenotype.

Finding novel antibiotics and antimicrobial materials has become of great importance to modern society due to the alarming increase in the development of multidrug resistance in various bacterial strains. This problem is even more complex when infections involve bacterial strains in stationary metabolic states, since most of the antibiotics found in the market do not have an effect on bacteria in dormant metabolic states. A promising field to aid in the solution of this problem is nanotechnology, since it offers a wide avenue for the development of potential therapeutics, specifically the use of silver metal nanoparticles. Silver nanoparticles have proven to be highly effective antimicrobial agents and excellent candidates to be engineered and designed into clever delivery systems, taking advantage of their rapid and potent toxicity on prokaryotic cells at low concentrations. Metal nanoparticles are most commonly synthetized through one or a series of redox chemical reactions using powerful but environmentally toxic-reducing agents. Therefore, in this work, we propose a biosynthesis method that allows the production of nanoparticles, with homogenous shapes and narrow size distributions, through an environmentally friendly technique that does not produce toxic residues. Here, silver nanoparticles were produced from silver salt (AgNO3) using three different growth culture media residues from E. coli top 10. The three different culture media residues used included LB, LBN, and LBE; all of them displaying a different chemical and nutrient composition. Here, after characterization of the different silver nanoparticles produced with the different media, we demonstrated that the LB culture-conditioned media was the most suitable to produce them since they displayed the most narrow size distribution, with an average 10.6 nm in diameter, a relatively low standard deviation of 5.5 nm, and a narrow UV-vis spectrum absorption peak at 420 nm. The other methods presented larger nanoparticle sizes and broader size distributions. Furthermore, nanoparticles produced with LB Lennox were found to be, at very low concentrations, effective antimicrobial agent against E. coli top 10 at stationary phase. Therefore, these results seem to contribute knowledge linked to the production of antimicrobial nanoparticles (Ag-NPs) through green synthesis and represent a platform to treat infections caused by nongrowing bacteria.

Calcium hydroxide (Ca(OH) 2) is recognized as an efficient bactericide and is widely applied as a root canal filler in endodontic treatment. Ca(OH) 2 is mainly produced by hydration of calcium oxide (CaO), a product of the thermal decomposition of calcium carbonate (CaCO 3) from sources such as limestone. In this work, calcium hydroxide particles were synthetized by the thermochemical transformation of waste biomass from the tequila industry. Agave biomass processed at 600 °C was composed mostly of calcium carbonate (CaCO 3), while calcination at 900 °C followed by hydration produced Ca(OH) 2. The morphology and crystalline nature of the Ca(OH) 2 particles were characterized by micro-Raman spectroscopy, scanning electron microscopy and X-ray diffraction analysis. Bactericidal activity of synthesized calcium hydroxide was evaluated with the agar diffusion assay. Our results provide evidence that Ca(OH) 2 obtained from agave biomass is an effective bactericidal against Escherichia coli and Enterococcus faecalis. Biomass from agave is available in Mexico and the rest of the American continent, the use of processed bagasse for medical applications could provide a venue for the useful disposition of industrial waste. Palabras clave: recursos renovables, ceniza, bactericida, Ca(OH) 2 RESUMEN El hidróxido de calcio (Ca(OH) 2) es reconocido como un eficiente bactericida y es ampliamente utilizado como relleno de la raíz dental en tratamientos de endodoncia. El Ca(OH) 2 es producido por la hidratación del óxido de calcio (CaO), un producto de la descomposición térmica del carbonato de calcio (CaCO 3), obtenido principalmente de piedra caliza. En el presente trabajo, se sintetizaron partículas de hidróxido de calcio SHORT COMMUNICATION / COMUNICACIÓN BREVE