Engineering conductive protein films through nanoscale self-assembly and gold nanoparticles doping.

Protein-based materials are usually considered as insulators, although conductivity has been recently shown in proteins. This fact opens the door to develop new biocompatible conductive materials. While there are emerging efforts in this area, there is an open challenge related to the limited conductivity of protein-based systems. This work shows a novel approach to tune the charge transport properties of protein-based materials by using electron-dense AuNPs. Two strategies are combined in a unique way to generate the conductive solid films: (1) the controlled self-assembly of a protein building block; (2) the templating of AuNPs by the engineered building block. This bottom-up approach allows controlling the structure of the films and the distribution of the AuNPs within, leading to enhanced conductivity. This work illustrates a promising strategy for the development of effective hybrid protein-based bioelectrical materials.

Potential use of machine learning methods in assessment of Fusarium culmorum and Fusariumproliferatum growth and mycotoxin production in treatments with antifungal agents.

Fusarium-controlling fungicides are necessary to limit crop loss. Little is known about the effect of antifungal formulations at sub-lethal doses, and their interaction with abiotic factors, on Fusarium culmorum and F. proliferatum development and on zearalenone and fumonisin biosynthesis, respectively. In the present study different treatments based on sulfur, trifloxystrobin and demethylation inhibitor fungicides (cyproconazole, tebuconazole and prothioconazole) under different environmental conditions, in Maize Extract Medium, are assayed in vitro. Several machine learning methods (neural networks, random forest and extreme gradient boosted trees) have been applied for the first time for modeling growth of F. culmorum and F. proliferatum and zearalenone and fumonisin production, respectively. The most effective treatment was prothioconazole, 250 g/L + tebuconazole, 150 g/L. Effective doses of this formulation for reduction or total growth inhibition ranged as follows ED50 0.49-1.70, ED90 2.57-6.02 and ED100 4.0-8.0 µg/mL, depending on the species, water activity and temperature. Overall, the growth rate and mycotoxin levels in cultures decreased when doses increased. Some treatments in combination with certain aw and temperature values significantly induced toxin production. The extreme gradient boosted tree was the model able to predict growth rate and mycotoxin production with minimum error and maximum R2 value.

The Galleria mellonella infection model as a system to investigate the virulence of Candida auris strains.

Candida auris is a multiresistant pathogenic yeast commonly isolated from bloodstream infections in immunocompromised patients. In this work, we infected Galleria mellonella larvae with 105 CFU of a reference strains and two clinical isolates of C. albicans and C. auris and we compared the outcomes of infection between both species. Larvae were evaluated every 24 h for a total of 120 h following the G. mellonella Health Index Scoring System, and survival, activity, melanization and cocoon formation were monitored. Our results showed that clinical isolates were significantly more pathogenic than reference strains independently of the tested species, producing lower survival and activity scores and higher melanization scores and being C. albicans strains more virulent than C. auris strains. We did not find differences in mortality between aggregative and non-aggregative C. auris strains, although non-aggregative strains produced significantly lower activity scores and higher melanization scores than aggregative ones. Survival assays using Galleria mellonella have been previously employed to examine and classify strains of this and other microbial species based on their virulence before scaling the experiments to a mammal model. Taken together, these results show how a more complete evaluation of the model can improve the study of C. auris isolates.

A Biodegradable Antifungal-Loaded Sol-Gel Coating for the Prevention and Local Treatment of Yeast Prosthetic-Joint Infections.

Fungal prosthetic-joint infections are rare but devastating complications following arthroplasty. These infections are highly recurrent and expose the patient to the development of candidemia, which has high mortality rates. Patients with this condition are often immunocompromised and present several comorbidities, and thus pose a challenge for diagnosis and treatment. The most frequently isolated organisms in these infections are Candida albicans and Candida parapsilosis, pathogens that initiate the infection by developing a biofilm on the implant surface. In this study, a novel hybrid organo-inorganic sol-gel coating was developed from a mixture of organopolysiloxanes and organophosphite, to which different concentrations of fluconazole or anidulafungin were added. Then, the capacity of these coatings to prevent biofilm formation and treat mature biofilms produced by reference and clinical strains of C. albicans and C. Parapsilosis was evaluated. Anidulafungin-loaded sol-gel coatings were more effective in preventing C. albicans biofilm formation, while fluconazole-loaded sol-gel prevented C. parapsilosis biofilm formation more effectively. Treatment with unloaded sol-gel was sufficient to reduce C. albicans biofilms, and the sol-gels loaded with fluconazole or anidulafungin slightly enhanced this effect. In contrast, unloaded coatings stimulated C. parapsilosis biofilm formation, and loading with fluconazole reduced these biofilms by up to 99%. In conclusion, these coatings represent a novel therapeutic approach with potential clinical use to prevent and treat fungal prosthetic-joint infections.

Sol-Gel-Deposited Ti-Doped ZnO: Toward Cell Fouling Transparent Conductive Oxides.

Ti-doped ZnO thin films were obtained with the aim of tailoring ZnO film bioadhesiveness and making the optoelectronic properties of ZnO materials transferable to biological environments. The films were prepared on silicon substrates by sol-gel spin-coating and subsequent annealing. A Ti-O segregation limits the ZnO crystallite growth and creates a buffer out-layer. Consequently, the Ti-doped ZnO presents slightly increased resistivity, which remains in the order of 10-3 Ω·cm. The strong biochemical interference of Zn2+ ions released from pure ZnO surfaces was evidenced by culturing Staphylococcus epidermidis with and without the Zn2+ coupling agent clioquinol. The Ti-doped ZnO surfaces showed a considerable increase of bacterial viability with respect to pure ZnO. Cell adhesion was assayed with human mesenchymal stem cells (hMSCs). Although hMSCs find difficulties to adhere to the pure ZnO surface, they progressively expand on the surface of ZnO when the Ti doping is increased. A preliminary microdevice has been built on the Si substrate with a ZnO film doped with 5% Ti. A one-dimensional micropattern with a zigzag structure shows the preference of hMSCs for adhesion on Ti-doped ZnO with respect to Si. The induced contrast of surface tension further induces a cell polarization effect on hMSCs. It is suggested that the presence of Ti-O covalent bonding on the doped surfaces provides a much more stable ground for bioadhesion. Such fouling behavior suggests an influence of Ti doping on film bioadhesiveness and sets the starting point for the selection of optimal materials for implantable optoelectronic devices.

Candida auris: a comparison between planktonic and biofilm susceptibility to antifungal drugs.

Introduction. Candida auris is a pathogenic yeast that mainly affects immunosuppressed patients and those with implanted medical devices. This pathogen also displays elevated resistance to common antifungals and high survival and spreading capacities. Since no antifungal breakpoints have yet been defined for this pathogen, the data obtained here can be useful for further research concerning treatment or implementation of a prevention and disinfection protocol. Our aim was to study the antifungal resistance of C. auris to current antifungals in planktonic and sessile states. Using confocal laser scanning microscopy and viable biomass production, we demonstrated the ability of C. auris to develop a mature biofilm. We compared the minimal inhibitory concentration (MIC) and the minimal biofilm eradication concentration (MBEC) for the C. auris DSM 21092 strain plus two clinical isolates, and the results were compared with those obtained for Candida albicans and Candida parapsilosis, two species strongly linked to bloodstream infections and infections associated with biomaterials. We found that the clinical isolates of C. auris were resistant to fluconazole and sensitive to echinocandins and polyenes. The C. auris biofilms did not show susceptibility to any antifungal agent, showing MBECs that were up to 512-fold higher than the MICs. These findings highlight the importance of biofilm formation as a key factor underlying the resistance of this species to antifungals and suggest that the presence of implantable medical devices is one of the major risk factors in immunocompromised patients.

A New Antibiotic-Loaded Sol-Gel Can Prevent Bacterial Prosthetic Joint Infection: From in vitro Studies to an in vivo Model.

The aim of this study was to evaluate the effect of a moxifloxacin-loaded organic-inorganic sol-gel with different antibiotic concentration in the in vitro biofilm development and treatment against Staphylococcus aureus, S. epidermidis, and Escherichia coli, cytotoxicity and cell proliferation of MC3T3-E1 osteoblasts; and its efficacy in preventing the prosthetic joint infection (PJI) caused by clinical strains of S. aureus and E. coli using an in vivo murine model. Three bacterial strains, S. epidermidis ATCC 35984, S. aureus 15981, and, E. coli ATCC 25922, were used for microbiological studies. Biofilm formation was induced using tryptic-soy supplemented with glucose for 24 h, and then, adhered and planktonic bacteria were estimated using drop plate method and absorbance, respectively. A 24-h-mature biofilm of each species growth in a 96-well plate was treated for 24 h using a MBECTM biofilm Incubator lid with pegs coated with the different types of sol-gel, after incubation, biofilm viability was estimated using alamrBlue. MC3T3-E1 cellular cytotoxicity and proliferation were evaluated using CytoTox 96 Non-Radioactive Cytotoxicity Assay and alamarBlue, respectively. The microbiological studies showed that sol-gel coatings inhibited the biofilm development and treated to a mature biofilm of three evaluated bacterial species. The cell studies showed that the sol-gel both with and without moxifloxacin were non-cytotoxic and that cell proliferation was inversely proportional to the antibiotic concentration containing by sol-gel. In the in vivo study, mice weight increased over time, except in the E. coli-infected group without coating. The most frequent symptoms associated with infection were limping and piloerection; these symptoms were more frequent in infected groups with non-coated implants than infected groups with coated implants. The response of moxifloxacin-loaded sol-gel to infection was either total or completely absent. No differences in bone mineral density were observed between groups with coated and non-coated implants and macrophage presence lightly increased in the bone grown directly in contact with the antibiotic-loaded sol-gel. In conclusion, moxifloxacin-loaded sol-gel coating is capable of preventing PJI caused by both Gram-positive and Gram-negative species.

Engineered protein-based functional nanopatterned materials for bio-optical devices.

The development of new active biocompatible materials and devices is a current need for their implementation in multiple fields, including the fabrication of implantable devices for biomedical applications and sustainable devices for bio-optics and bio-optoelectronics. This paper describes a simple strategy to use designed proteins to develop protein-based functional materials. Using simple proteins as self-assembling building blocks as a platform for the fabrication of new optically active materials takes previous work one step further towards the design of materials with defined structures and functions using naturally occurring protein materials, such as silk. The proposed fabrication strategy generates thin and flexible nanopatterned protein films by letting the engineered protein elements self-assemble over the surface of an elastomeric stamp with nanoscale features. These nanopatterned protein films are easily transferred onto 3D objects (flat and curved) by moisture-induced adhesion. Additionally, flexible nanopatterned protein films are prepared by incorporating a thin polymeric layer as a back support. Finally, taking advantage of the tunability of the selected protein scaffold, the flexible protein-based surfaces are endowed with optical functions, achieving efficient lasing features. As such, this work enables the simple and cost-effective production of flexible and nanostructured, protein-based, optically active biomaterials and devices over large areas toward emerging applications.

Determination of multiple mycotoxins in feedstuffs by combined use of UPLC-MS/MS and UPLC-QTOF-MS.

In this report, a UPLC-ESI-MS/MS method for the simultaneous determination of aflatoxins, ochratoxin A, zearalenone, deoxynivalenol, fumonisins, T-2 and HT-2 toxins, fusarenone X, diacetoxyscirpenol, and 3- and 15-acetyldeoxynivalenol in feedstuffs was developed. A quadrupole-time-of-flight mass spectrometer detector (QTOF-MS) operating in full scan mode was combined with the UPLC-ESI-MS/MS system to confirm the identity of detected mycotoxins and to identify other possible microbial metabolites occurring in samples. Sixty-two feed samples from the Spanish market were analyzed. Extraction of metabolites was carried out with acetonitrile-water-formic acid (80:19:1, v/v/v). Method detection and quantification limits and performance criteria set by Commission Regulation (EC) No 401/2006 were fulfilled. Relatively high levels of the main regulated mycotoxins and presence of non-regulated mycotoxins in feed samples were found. This is the first study in which mycotoxins and other microbial metabolites occurring in feed are studied using a UPLC-QTOF-MS system being therefore a reference report.

Assessment of azole fungicides as a tool to control growth of Aspergillus flavus and aflatoxin B1 and B2 production in maize.

Aspergillus flavus is a highly aflatoxin (AF)-producing species infecting maize and other crops. It is dominant in tropical regions, but it is also considered an emerging problem associated with climate change in Europe. The aim of this study was to assess the efficacy of azole fungicides (prochloraz, tebuconazole and a 2:1 (w/w) mixture of prochloraz plus tebuconazole) to control the growth of A. flavus and AF production in yeast-extract-sucrose (YES) agar and in maize kernels under different water activities (aw) and temperatures. Aflatoxins B1 and B2 were determined by LC with fluorescence detection and post-column derivatisation of AFB1. In YES medium and maize grains inoculated with conidia of A. flavus, the growth rate (GR) of the fungus and AFB1 and AFB2 production were significantly influenced by temperature and treatment. In YES medium and maize kernels, optimal temperatures for GR and AF production were 37 and 25°C, respectively. In maize kernels, spore germination was not detected at the combination 37ºC/0.95 aw; however, under these conditions germination was found in YES medium. All fungicides were more effective at 0.99 than 0.95 aw, and at 37 than 25ºC. Fungicides effectiveness was prochloraz > prochloraz plus tebuconazole (2:1) > tebuconazole. AFs were not detected in cultures containing the highest fungicide doses, and only very low AF levels were found in cultures containing 0.1 mg l-1 prochloraz or 5.0 mg l-1 tebuconazole. Azoles proved to be highly efficient in reducing A. flavus growth and AF production, although stimulation of AF production was found under particular conditions and low-dosage treatments. Maize kernels were a more favourable substrate for AF biosynthesis than YES medium. This paper is the first comparative study on the effects of different azole formulations against A. flavus and AF production in a semi-synthetic medium and in maize grain under different environmental conditions.

Biomolecular templating of functional hybrid nanostructures using repeat protein scaffolds.

The precise synthesis of materials and devices with tailored complex structures and properties is a requisite for the development of the next generation of products based on nanotechnology. Nowadays, the technology for the generation of this type of devices lacks the precision to determine their properties and is accomplished mostly by 'trial and error' experimental approaches. The use of bottom-up approaches that rely on highly specific biomolecular interactions of small and simple components is an attractive approach for the templating of nanoscale elements. In nature, protein assemblies define complex structures and functions. Engineering novel bio-inspired assemblies by exploiting the same rules and interactions that encode the natural diversity is an emerging field that opens the door to create nanostructures with numerous potential applications in synthetic biology and nanotechnology. Self-assembly of biological molecules into defined functional structures has a tremendous potential in nano-patterning and the design of novel materials and functional devices. Molecular self-assembly is a process by which complex 3D structures with specified functions are constructed from simple molecular building blocks. Here we discuss the basis of biomolecular templating, the great potential of repeat proteins as building blocks for biomolecular templating and nano-patterning. In particular, we focus on the designed consensus tetratricopeptide repeats (CTPRs), the control on the assembly of these proteins into higher order structures and their potential as building blocks in order to generate functional nanostructures and materials.