Colorimetric Detection of SARS-CoV-2 Using Plasmonic Biosensors and Smartphones.

Low-cost, instrument-free colorimetric tests were developed to detect SARS-CoV-2 using plasmonic biosensors with Au nanoparticles functionalized with polyclonal antibodies (f-AuNPs). Intense color changes were noted with the naked eye owing to plasmon coupling when f-AuNPs form clusters on the virus, with high sensitivity and a detection limit of 0.28 PFU mL-1 (PFU stands for plaque-forming units) in human saliva. Plasmon coupling was corroborated with computer simulations using the finite-difference time-domain (FDTD) method. The strategies based on preparing plasmonic biosensors with f-AuNPs are robust to permit SARS-CoV-2 detection via dynamic light scattering and UV-vis spectroscopy without interference from other viruses, such as influenza and dengue viruses. The diagnosis was made with a smartphone app after processing the images collected from the smartphone camera, measuring the concentration of SARS-CoV-2. Both image processing and machine learning algorithms were found to provide COVID-19 diagnosis with 100% accuracy for saliva samples. In subsidiary experiments, we observed that the biosensor could be used to detect the virus in river waters without pretreatment. With fast responses and requiring small sample amounts (only 20 µL), these colorimetric tests can be deployed in any location within the point-of-care diagnosis paradigm for epidemiological control.

Engineering an optimized expression operating unit for improved recombinant protein production in Escherichia coli.

Common strategies to improve recombinant protein production in Escherichia coli often involve the test and optimization of several different variables, when using traditional expression vectors that are commercially available. Now, modern synthetic biology-based strategies allow for extensive modifications of these traditional vectors, or even construction of entirely new modular vectors, so as to permit tunable production of the recombinant proteins of interest. Herein, we describe the engineering of a new expression operating unit (EOU; 938 bp) for producing recombinant proteins in E. coli, through the combinatorial assembly of standardized and well-characterized genetic elements required for transcription and translation (promoter, operator site, RBS, junction RBS-CDS, cloning module, transcriptional terminator). We also constructed a novel T7 promoter variant with increased transcriptional activity (1.7-fold higher), when compared to the canonical wild type T7 promoter sequence. This new EOU yielded an improved production of the reporter protein superfolder GFP (sfGFP) in E. coli BL21(DE3) (relative fluorescence units/RFU = 70.62 ± 1.62 A U.) when compared to a high-producing control expression vector (plasmid BBa_I746909; RFU = 59.68 ± 1.82 A U.). The yields of purified soluble recombinant sfGFP were also higher when using the new EOU (188 mg L-1 culture vs. 108 mg L-1 in the control) and it performed similarly well when inserted into different plasmid backbones (pOPT1.0/AmpR and pOPT2.0/CmR).

Rationally designed hypoallergenic mutant variants of the house dust mite allergen Der p 21.

BACKGROUND: Allergic diseases figure among the most common immune-mediated diseases worldwide, affecting more than 25% of the world's population. Allergic reactions can be triggered by house dust mite (HDM) allergens, of which the so-called group 21 of allergens is considered as clinically relevant. METHODS: Herein, we used a structural bioinformatics and immunoinformatics approach to design hypoallergenic mutant variants of the Der p 21 allergen of Dermatophagoides pteronyssinus, which were then recombinantly expressed in bacteria and tested for their IgE-reactivities. For this, we scanned the wild-type Der p 21 protein for all possible single amino acid substitutions in key IgE-binding regions that could render destabilization of the major epitope regions. RESULTS: Four main substitutions (D82P, K110G, E77G, and E87S) were selected to build mutant variants of the Der p 21 allergen, which were produced in their recombinant forms; two of these variants showed reduced reactivity with IgE. Molecular dynamic simulations and immune simulations demonstrated the overall effects of these mutations on the structural stability of the Der p 21 allergen and on the profile of immune response induced through immunotherapy. CONCLUSIONS: When produced in their recombinant forms, two of the Der p 21 mutant variants, namely proteins K110G and E87S, showed significantly reduced IgE reactivities against sera from HDM-allergic individuals (n = 20; p < 0.001). GENERAL SIGNIFICANCE: This study successfully translated a rational in silico mutagenesis design into low IgE-binding mutant variants of the allergen rDer p 21. These novel hypoallergens are promising to compose next-generation allergen-immunotherapy formulations in near future.

Biomechanical analyses of one-piece dental implants composed of titanium, zirconia, PEEK, CFR-PEEK, or GFR-PEEK: Stresses, strains, and bone remodeling prediction by the finite element method.

This work aimed to assess the biomechanics, using the finite element method (FEM), of traditional titanium Morse taper (MT) dental implants compared to one-piece implants composed of zirconia, polyetheretherketone (PEEK), carbon fiber-reinforced PEEK (CFR-PEEK), or glass fiber-reinforced PEEK (GFR-PEEK). MT and one-piece dental implants were modeled within a mandibular bone section and loaded on an oblique force using FEM. A MT implant system involving a Ti6Al4V abutment and a cp-Ti grade IV implant was compared to one-piece implants composed of cp-Ti grade IV, zirconia (3Y-TZP), PEEK, CFR-PEEK, or GFR-PEEK. Stress on bone and implants was computed and analyzed while bone remodeling prediction was evaluated considering equivalent strain. In comparison to one-piece implants, the traditional MT implant revealed higher stress peak (112 MPa). The maximum stresses on the one-piece implants reached ~80 MPa, regardless their chemical composition. MT implant induced lower bone stimulus, although excessive bone strain was recorded for PEEK implants. Balanced strain levels were noticed for reinforced PEEK implants of which CFR-PEEK one-piece implants showed proper biomechanical behavior. Balanced strain levels might induce bone remodeling at the peri-implant region while maintaining low risks of mechanical failures. However, the strength of the PEEK-based composite materials is still low for long-term clinical performance.

In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli.

Recombinant proteins are generally fused with solubility enhancer tags to improve the folding and solubility of the target protein of interest. However, the fusion protein strategy usually requires expensive proteases to perform in vitro proteolysis and additional chromatographic steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, simplify the recombinant protein purification process, and promote the screening of molecules that fail to remain soluble after tag removal. High yields of soluble target proteins have already been achieved using these protease co-expression systems. Herein, we review approaches for controlled intracellular processing systems tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular processing of recombinant proteins regarding system design features, advantages, and limitations of the various strategies.

Biomechanical behavior of functionally graded S53P4 bioglass-zirconia dental implants: Experimental and finite element analyses.

OBJECTIVES: The aim of this work was to evaluate the biomechanical behavior of one-piece zirconia implants with a functionally graded bioglass (BG) layer as compared to monolithic zirconia and BG-coated implants, using the finite element method (FEM). METHODS: Zirconia disks were infiltrated with bioglass S53P4 and then morphologically inspected by scanning electron microscopy (SEM) followed by mechanical analyses on micro-indentation tests for further biomechanical validation using the finite element method (FEM). On modeling, zirconia dental implants anchored into mandibular bone were simulated on occlusal loading as recorded under mastication. Three types of implants were simulated: i) free of BG coating, ii) with 100 µm or 150 µm thick conventional BG coatings; and iii) with graded BG coatings involving 3 different chemical composition distributions. The stress state at both implant and bone were evaluated using the FEM. The mechanically-induced bone remodelling was analyzed through the bone strain results. RESULTS: Infiltration of BG into a zirconia structure resulted in a ∼100 µm thick layer with an exponential-like gradation of chemical composition and properties. Regarding the FEM calculations, the BG coating induced up to 30% decrease on stress in the implant body when compared to the monolithic zirconia implant. The gradient of chemical composition also improved the stresses' distribution. The stresses distribution towards the BG-coatings were significantly high and could lead to failure. Stresses on the bone were recorded down to its strength threshold, with insignificant influence of the coating layer. The bone strain values on all models indicates further bone remodelling although BG-coated and BG-graded zirconia implants showed the highest strain magnitude that may enhance the mechanical stimulation for bone maintenance. SIGNIFICANCE: Graded BG-zirconia dental implants showed enhanced overall biomechanical behaviour as compared to the BG-coated or monolithic zirconia dental implants. Also, such biomechanical improvements noticed for the BG-graded system should be considered in combination with the well-known osseointegration benefits of bioactive glasses.

Single-Input Regulatory Cascade for in vivo Removal of the Solubility Tag in Fusion Recombinant Proteins Produced by Escherichia coli.

Solubility tags are commonly fused to target recombinant proteins to enhance their solubility and stability. In general, these protein tags must be removed to avoid misfolding of the partner protein and to allow for downstream applications. Nevertheless, in vitro tag removal increases process complexity and costs. Herein, we describe a synthetic biology-based strategy to permit in vivo removal of a solubility tag (EDA, KDPG aldolase), through co-expression of the fusion recombinant protein (EDA-EGFP) and the tag-cleaving protease (TEVp), in a controlled manner. Basically, the system uses three repressor proteins (LacI, cI434, and TetR) to regulate the expressions of EDA-EGFP and TEVp, in a regulatory cascade that culminates with the release of free soluble target protein (EGFP), following a single chemical induction by IPTG. The system worked consistently when all biological parts were cloned in a single plasmid, pSolubility(SOL)A (7.08 Kb, AmpR), and transformed in Escherichia coli Rosetta (DE3) or BL21(DE3) strains. Total soluble recombinant protein yield (EDA-EGFP + free EGFP) was ca. 272.0 ± 60.1 µg/mL of culture, following IMAC purification; free EGFP composed great part (average = 46.5%; maximum = 67.3%) of the total purified protein fraction and was easily separated from remaining fusion EDA-EGFP (53 KDa) through filtration using a 50 KDa cut-off centrifugal filter.

Surface damage of dental implant systems and ions release after exposure to fluoride and hydrogen peroxide.

OBJECTIVE: The aim of this study was to evaluate surface changes on dental implant systems and ions release after immersion in fluoride and hydrogen peroxide. METHODS: Ten implant-abutment assemblies were embedded in acrylic resin and cross-sectioned along the implant vertical axis. Samples were wet ground and polished. Delimited areas of groups of samples were immersed in 1.23% sodium fluoride gel (F) or in 35% hydrogen peroxide (HP) for 16 min. Gels (n = 3) were collected from the implant surfaces and analyzed by inductively coupled plasma mass spectrometry (ICP-MS), to detect the concentration of metallic ions released from the implant systems. Selected areas of the abutment and implant (n = 15) were analyzed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). RESULTS: SEM images revealed surface topographic changes on implant-abutment joints after immersion in fluoride. Implants showed excessive oxidation within loss of material, while abutment surfaces revealed intergranular corrosion after immersion in fluoride. ICP-MS results revealed a high concentration of Ti, Al, V ions in fluoride after contact with the implant systems. Localized corrosion of implant systems could not be detected by SEM after immersion in hydrogen peroxide although the profilometry showed increase in roughness. ICP-MS showed the release of metallic ions in hydrogen peroxide medium after contact with dental implants. CONCLUSION: Therapeutical substances such as fluorides and hydrogen peroxide can promote the degradation of titanium-based dental implant and abutments leading to the release of toxic ions.

Influence of specimens' geometry and materials on the thermal stresses in dental restorative materials during thermal cycling.

OBJECTIVES: Thermal cycling is widely used to simulate the aging of restorative materials corresponding to the changes of temperature in the oral cavity. However, test parameters present in literature vary considerably, which prevents comparison between different reports. The aim of this work is to assess the influence of the specimens' geometry and materials on the thermal stresses developed during thermal cycling tests. MATERIALS AND METHODS: Finite elements method was used to simulate the conditions of thermal cycling tests for three different sample geometries: a three-points bending test sample, a cylinder rod and more complex shape of a restoration crown. Two different restorative systems were considered: all-ceramic (zirconia coupled with porcelain) and metal-ceramic (CoCrMo alloy coupled with porcelain). The stress state of each sample was evaluated throughout the test cycle. RESULTS: The results show that the sample geometry has great influence on the stress state, with difference of up to 230% in the maximum stress between samples of the same composition. The location of maximum stress also changed from the interface between materials to the external wall. CONCLUSIONS: Maximum absolute stress values were found to vary between 2 and 4MPa, which might not be critical even for ceramics. During multi-cycle testing these stresses would cause different fatigue in various locations. The zirconia-based specimens and zirconia-based restoration (crown) exhibited the most similar stress states. Thus it might be recommended to use these geometries for fast screening of the materials for this type of restorations. CLINICAL SIGNIFICANCE: The selection of specimens' geometry and materials should be carefully considered when aging conditions close to clinical ones want to be simulated.

Wear of Morse taper and external hexagon implant joints after abutment removal.

The aim of this in vitro study was to evaluate the removal torque values on abutments and the morphological wear aspects of two different dental implant joints after immersion in a medium containing biofilm from human saliva. Twenty implant-abutment assemblies were divided into four groups in this study: (A) Morse taper free of medium containing biofilm, and (B) after contact with a medium containing biofilm from human saliva; (C) External Hexagon free of medium containing biofilm, and (D) after contact with medium containing biofilm from human saliva. The abutments were firstly torqued to the implants according to the manufacturer´s recommendations, using a handheld torque meter. Groups B and D were immersed into 24 well-plates containing 2 ml BHI medium with microorganisms for 72 h at 37 °C under microaerophilic conditions. After detorque evaluation, the abutments were removed and the implants were analyzed by scanning electron microscopy (SEM) and profilometry. On the detorque evaluation, the torque values decreased for the external hexagon implants and increased for the Morse taper implants. However, the values were lower when both implant-abutment assemblies were in contact with a medium containing biofilm from human saliva. The wear areas of contacting surfaces of the implants were identified by SEM. The highest average roughness values were recorded on the surfaces free of biofilm. The medium containing biofilm from human saliva affected the maintenance of the torque values on Morse taper and external hexagon abutments. Additionally, the removal of abutment altered the inner implant surfaces resulting in an increase of wear of the titanium-based connection.

THERMAL RESIDUAL STRESSES IN BILAYERED, TRILAYERED AND GRADED DENTAL CERAMICS.

Layered ceramic systems are usually hit by residual thermal stresses created during cooling from high processing temperature. The purpose of this study was to determine the thermal residual stresses at different ceramic multi-layered systems and evaluate their influence on the bending stress distribution. Finite elements method was used to evaluate the residual stresses in zirconia-porcelain and alumina-porcelain multi-layered discs and to simulate the 'piston-on-ring' test. Temperature-dependent material properties were used. Three different multi-layered designs were simulated: a conventional bilayered design; a trilayered design, with an intermediate composite layer with constant composition; and a graded design, with an intermediate layer with gradation of properties. Parameters such as the interlayer thickness and composition profiles were varied in the study. Alumina-porcelain discs present smaller residual stress than the zirconia-porcelain discs, regardless of the type of design. The homogeneous interlayer can yield a reduction of ~40% in thermal stress relative to bilayered systems. Thinner interlayers favoured the formation of lower thermal stresses. The graded discs showed the lowest thermal stresses for a gradation profile given by power law function with p=2. The bending stresses were significantly affected by the thermal stresses in the discs. The risk of failure for all-ceramic dental restorative systems can be significantly reduced by using trilayered systems (homogenous or graded interlayer) with the proper design.

Effect of Zirconia and Alumina Fillers on the Microstructure and Mechanical Strength of Dental Glass Ionomer Cements.

BACKGROUND: Glass-ionomer cements perform a protective effect on the dentin-pulp complex considering the F ions release and chemical bonding to the dental structures. On the other hand, those materials have poor physic-mechanical properties in comparison with the restorative resin composite. The main aim of this work was to evaluate the influence of zirconia and/or alumina fillers on the microstructure and strength of a resin modified glass-ionomer cement after thermal cycling. METHODS: An in vitro experimental study was carried out on 9 groups (n = 10) of cylindrical samples (6 x 4 mm) made from resin modified glass-ionomer (Vitremer, 3M, USA) with different contents of alumina and/or zirconia fillers. A nano-hybrid resin composite was tested as a control group. Samples were mechanically characterized by axial compressive tests and electron scanning microscopy (SEM) coupled to energy dispersive X-ray spectrophotometry (EDS), before and after thermal cycling. Thermal cycling procedures were performed at 3000, 6000 and 10000 cycles in Fusayama´s artificial saliva at 5 and 60 (o)C. RESULTS: An improvement of compressive strength was noticed on glass-ionomer reinforced with alumina fillers in comparison with the commercial glass ionomer. SEM images revealed the morphology and distribution of alumina or zirconia in the microstructure of glass-ionomers. Also, defects such as cracks and pores were detected on the glass-ionomer cements. The materials tested were not affected by thermal cycling in artificial saliva. CONCLUSION: Addition of inorganic particles at nano-scale such as alumina can increase the mechanical properties of glass-ionomer cements. However, the presence of cracks and pores present in glass-ionomer can negatively affect the mechanical properties of the material because they are areas of stress concentration.

The bending stress distribution in bilayered and graded zirconia-based dental ceramics.

The purpose of this study was to evaluate the biaxial flexural stresses in classic bilayered and in graded zirconia-feldspathic porcelain composites. A finite element method and an analytical model were used to simulate the piston-on-ring test and to predict the biaxial stress distributions across the thickness of the bilayer and graded zirconia-feldspathic porcelain discs. An axisymmetric model and a flexure formula of Hsueh et al. were used in the FEM and analytical analysis, respectively. Four porcelain thicknesses were tested in the bilayered discs. In graded discs, continuous and stepwise transitions from the bottom zirconia layer to the top porcelain layer were studied. The resulting stresses across the thickness, measured along the central axis of the disc, for the bilayered and graded discs were compared. In bilayered discs, the maximum tensile stress decreased while the stress mismatch (at the interface) increased with the porcelain layer thickness. The optimized balance between both variables is achieved for a porcelain thickness ratio in the range of 0.30-0.35. In graded discs, the highest tensile stresses were registered for porcelain rich interlayers (p=0.25) whereas the zirconia rich ones (p=8) yield the lowest tensile stresses. In addition, the maximum stresses in a graded structure can be tailored by altering compositional gradients. A decrease in maximum stresses with increasing values of p (a scaling exponent in the power law function) was observed. Our findings showed a good agreement between the analytical and simulated models, particularly in the tensile region of the disc. Graded zirconia-feldspathic porcelain composites exhibited a more favourable stress distribution relative to conventional bilayered systems. This fact can significantly impact the clinical performance of zirconia-feldspathic porcelain prostheses, namely reducing the fracture incidence of zirconia and the chipping and delamination of porcelain.