Accumulation of endogenous free radicals is required to induce titan-like cell formation in Cryptococcus neoformans

Cryptococcus neoformans is an excellent model to investigate fungal pathogenesis. This yeast can produce "titan cells," which are cells of an abnormally larger size that contribute to the persistence of the yeast in the host. In this work, we have used a new approach to characterize them by identifying drugs that inhibit this process. We have used a repurposing off-patent drug library, combined with an automatic method to image and analyze fungal cell size. In this way, we have identified many compounds that inhibit this transition. Interestingly, several compounds were antioxidants, allowing us to confirm that endogenous ROS and mitochondrial changes are important for titan cell formation. This work provides new evidence of the mechanisms required for titanization. Furthermore, the future characterization of the inhibitory mechanisms of the identified compounds by the scientific community will contribute to better understand the role of titan cells in virulence.Oscar Zaragoza was funded by grant PID2020-114546RB by MCIN/AEI/10.13039/501100011033 and by grant SAF2017-86912-R from the Spanish Ministry for Science and Innovation. Irene García-Barbazán was funded by the Ministry for Science and Innovation (contract FPI PRE2018-083436). Alba Torres-Cano was funded by the Ministry for Science and Innovation (contract FPI PRE2021-099486). Rocío García-Rodas was funded by a “Juan de la Cierva” Contract from the Spanish Ministry for Economics, Industry and Competitivity (reference: IJCI-2015-25683).S

A ceramic electrode of ZrO2-Y2O3 for the generation of oxidant species in anodic oxidation. Assessment of the treatment of Acid Blue 29 dye in sulfate and chloride media

A micron-sized powder of 7% mol Y2O3 stabilized ZrO2 (YSZ) was used to deposit a ceramic coating onto Ti substrate by atmospheric plasma spray. The novel YSZ ceramic presented a dense structure with cubic crystalline structure. The as-synthesized YSZ ceramic as stable anode, coupled to a stainless-steel cathode, was assessed for the anodic oxidation of Acid Blue 29 diazo dye solutions in sulfate and chloride media. The decolorization of these solutions in acidic conditions was clearly faster with chloride as electrolyte, since the generated active chlorine HClO from anodic oxidation of Clwas more powerful oxidant than ¿OH formed from water discharge at the 7YSZ surface in sulfate medium. In alkaline conditions, the loss of color was drastically reduced because of the conversion of HClO into the weaker oxidant ClO, as well as the loss of oxidation power of ¿OH, partially compensated by the increasing oxidation ability of SO4¿formed from anodic oxidation of SO42ion. The effect of other experimental variables such as current density, as well as the concentration of each electrolyte and the dye, was examined. The best experimental conditions at pH 7.0 were found for 0.050 M of electrolyte at 20 and 10 mA cm-2 using sulfate and chloride media, respectively. In contrast, lower mineralization was achieved in chloride medium because of the formation of very recalcitrant and persistent chloro-derivatives that decelerated the mineralization process. In sulfate medium, NH4+, NO3and, to much lesser extent, NO2ions were released during mineralization, whereas tartaric, maleic, acetic and oxalic acids remained in the final solution

Enhancing the bioactivity of polymeric implants by means of cold gas spray coatings

Nanostructured anatase coatings were built-up on biocompatible polyetheretherketone (PEEK) by means of cold gas spray (CGS). Titanium layer was previously desposited, which acted as bond coat between PEEK and metal oxide. Semicrystalline polymer was not degraded during the spraying process and starting composition of titanium dioxide was not affected. TiO2 was homogeneously obtained onto CGS Ti layer and completely covered the piece. Primary human osteoblasts were seeded onto biomaterials and in vitro cell experiments provided evidence to confirm that nanostructured anatase coatings deposited by cold gas spray improve the performance of PEEK implants

Infection of Artemia sp. by Lymphocystis disease virus (LCDV)

In the present work, different developmental stages of Artemia sp. (metanauplius, juvenile and adult) were experimentally infected with LCDV by immersion. Results of viral quantification (both by qPCR and cell-culture viral titer determination) and expression showed that LCDV establishes a productive infection in Artemia, at least under experimental conditions, extending the host range of this virus to crustaceans.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Porous titanium-hydroxyapatite composite coating obtained on titanium by cold gas spray with high bond strength for biomedical applications

The lack of bioactivity of titanium (Ti) is one of the main drawbacks for its application in biomedical implants since it can considerable reduce its osseointegration capacities. One strategy to overcome this limitation is the coating of Ti with hydroxyapatite (HA), which presents similar chemical composition than bone. Nonetheless, most of the strategies currently used generate a non-stable coating and may produce the formation of amorphous phases when high temperatures are used. Herein, we proposed to generate a Ti-HA composite coating on Ti surface to improve the stability of the bioactive coating. The coating was produced by cold gas spraying, which uses relatively low temperatures, and compared to a Ti coating. The coating was thoroughly characterized in terms of morphology, roughness, porosity and phase composition. In addition, the coating was mechanically characterized using a tensile loading machine. Finally, biological response was evaluated after seeding SaOS-2 osteoblasts and measuring cell adhesion, proliferation and differentiation. The novel Ti-HA coating presented high porosity and high adhesion and bond strengths. No change in HA phases was observed after coating formation. Moreover, osteoblast-like cells adhered, proliferated and differentiated on Ti-HA coated surfaces suggesting that the novel coating might be a good candidate for biomedical applications.Peer ReviewedPostprint (author's final draft

Functionalized coatings by cold spray: an in vitro study of micro- and nanocrystalline hydroxyapatite compared to porous titanium

Three different surface treatments on a Ti6Al4V alloy have been in vitro tested for possible application in cementless joint prosthesis. All of them involve the novelty of using the Cold Spray technology for their deposition: (i) an as-sprayed highly rough titanium and, followed by the deposition of a thin hydroxyapatite layer with (ii) microcrystalline or (iii) nanocrystalline structure. Primary human osteoblasts were extracted from knee and seeded onto the three different surfaces. Cell viability was tested by MTS and LIVE/DEAD assays, cell differentiation by alkaline phosphatase (ALP) quantification and cell morphology by Phalloidin staining. All tests were carried out at 1, 7 and 14 days of cell culture. Different cell morphologies between titanium and hydroxyapatite surfaces were exhibited. At 1 day of cell culture, cells on the titanium coating were spread and flattened, expanding the filopodia actin filaments in all directions, while cells on the hydroxyapatite coatings showed round like-shape morphology due to slower attachment. Higher cell viability was detected at all times of cell culture on titanium coating due to a better attachment at 1 day. However, from 7 days of cell culture, cells on hydroxyapatite showed good attachment onto surfaces and highly increased their proliferation, mostly on nanocrystalline, achieving similar cell viability levels than titanium coatings. ALP levels were significantly higher in titanium, in part, because of greatest cell number. Overall, the best cell functional results were obtained on titanium coatings whereas microcrystalline hydroxyapatite presented the worst cellular parameters. However, results indicate that nanocrystalline hydroxyapatite coatings may achieve promising results for the faster cell proliferation once cells are attached on the surface

In-vitro comparison of hydroxyapatite coatings obtained by cold spray and conventional thermal spray technologies

Hydroxyapatite (HA) coatings onto Ti6Al4V alloy substrates were obtained by several thermal spray technologies: atmospheric plasma spray (APS) and high velocity oxy fuel (HVOF), together with the cold spray (CS) technique. A characterization study has been performed by means of surface and microstructure analyses, as well as biological performance. In-vitro tests were performed with primary human osteoblasts at 1, 7 and 14 days of cell culture on substrates. Cell viability was tested by MTS and LIVE/DEAD assays, cell differentiation by alkaline phosphatase (ALP) quantification, and cell morphology was analyzed by scanning electron microscopy. The HA coatings showed an increase of HA crystallinity from 62,4% to 89%, but also an increase of hydrophilicity from ∼32° to 0°, with the decrease of the operating temperature of the thermal spray techniques (APS > HVOF > CS). Additionally, APS HA coatings showed more surface micro-features than HVOF and CS HA coatings; cells onto APS HA coatings showed faster attachment by acquiring osteoblastic morphology in comparison with the rounded cell morphology observed onto CS HA coatings at 1 day of cell culture. HVOF HA coatings also showed proper cell adherence but did not show extended filopodia as cells onto APS HA coatings. However, at 14 days of cell culture, higher cell proliferation and differentiation was detected on HA coatings with higher crystallinity (HVOF and CS techniques). Cell attachment is suggested to be favoured by surface micro-features but also moderate surface wettability whereas cell proliferation and differentiation is suggested to be highly influenced by HA crystallinity and crystal size

In-vitro study of hierarchical structures: Anodic oxidation and alkaline treatments onto highly rough titanium cold spray coatings for biomedical applications

Hierarchical structures were obtained applying two different nanotexturing surface treatments onto highly rough commercial pure titanium coatings by cold spray: (i) anodic oxidation and (ii) alkaline treatments. An extended surface characterization in terms of topography, composition, and wettability has been performed to understand how those parameters affect to cell response. Primary human osteoblasts extracted from knee were seeded onto the as-sprayed titanium surface before and after the nanotexturing treatments. Cell viability was tested by using MTS and LIVE/DEAD assays, as well as osteoblasts differentiation by alkaline phosphatase (ALP) quantification at 3 and 10 days of cell culture. The combination of micro-/nano-roughness results in a significantly increase of cell proliferation, as well as cell differentiation after 10 days of cell culture in comparison with the non-treated coatings

Nanoengineered graphene-reinforced coating for leading edge protection of wind turbine blades

Possibilities of the development of new anti-erosion coatings for wind turbine blade surface protection on the basis of nanoengineered polymers are explored. Coatings with graphene and hybrid nanoreinforcements are tested for their anti-erosion performance, using the single point impact fatigue testing (SPIFT) methodology. It is demonstrated that graphene and hybrid (graphene/silica) reinforced polymer coatings can provide better erosion protection with lifetimes up to 13 times longer than non-reinforced polyurethanes. Thermal effects and energy dissipation during the repeated soft impacts on the blade surface are discussed

Functional protein-based nanomaterial produced in GRAS microorganism : a new platform for biotechnology

Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest