Silk fibroin increases the elasticity of alginate-gelatin hydrogels and regulates cardiac cell contractile function in cardiac bioinks.

Silk fibroin (SF) is a natural protein extracted fromBombyx morisilkworm thread. From its common use in the textile industry, it emerged as a biomaterial with promising biochemical and mechanical properties for applications in the field of tissue engineering and regenerative medicine. In this study, we evaluate for the first time the effects of SF on cardiac bioink formulations containing cardiac spheroids (CSs). First, we evaluate if the SF addition plays a role in the structural and elastic properties of hydrogels containing alginate (Alg) and gelatin (Gel). Then, we test the printability and durability of bioprinted SF-containing hydrogels. Finally, we evaluate whether the addition of SF controls cell viability and function of CSs in Alg-Gel hydrogels. Our findings show that the addition of 1% (w/v) SF to Alg-Gel hydrogels makes them more elastic without affecting cell viability. However, fractional shortening (FS%) of CSs in SF-Alg-Gel hydrogels increases without affecting their contraction frequency, suggesting an improvement in contractile function in the 3D cultures. Altogether, our findings support a promising pathway to bioengineer bioinks containing SF for cardiac applications, with the ability to control mechanical and cellular features in cardiac bioinks.

High Charge Density in Peptide Dendrimers is Required to Destabilize Membranes: Insights into Endosome Evasion.

Peptide dendrimers are a type of branched, symmetric, and topologically well-defined molecule that have already been used as delivery systems for nucleic acid transfection. Several of the most promising sequences showed high efficiency in many key steps of transfection, namely, binding siRNA, entering cells, and evading the endosome. However, small changes to the peptide dendrimers, such as in the hydrophobic core, the amino acid chirality, or the total available charges, led to significantly different experimental results with unclear mechanistic insights. In this work, we built a computational model of several of those peptide dendrimers (MH18, MH13, and MH47) and some of their variants to study the molecular details of the structure and function of these molecules. We performed CpHMD simulations in the aqueous phase and in interaction with a lipid bilayer to assess how conformation and protonation are affected by pH in different environments. We found that while the different peptide dendrimer sequences lead to no substantial structural differences in the aqueous phase, the total charge and, more importantly, the total charge density are key for the capacity of the dendrimer to interact and destabilize the membrane. These dendrimers become highly charged when the pH changes from 7.5 to 4.5, and the presence of a high charge density, which is decreased for MH47 that has four fewer titratable lysines, is essential to trigger membrane destabilization. These findings are in excellent agreement with the experimental data and help us to understand the high efficiency of some dendrimers and why the dendrimer MH47 is unable to complete the transfection process. This evidence provides further understanding of the mode of action of these peptide dendrimers and will be pivotal for the future design of new sequences with improved transfection capabilities.

Engineering novel phenolic foams with lignin extracted from pine wood residues via a new levulinic-acid assisted process.

Phenolic foams are typically produced from phenolic resins, using phenol and formaldehyde precursors. Therefore, common phenolic foams are non-sustainable, comprising growing environmental, health, and economic concerns. In this work, lignin extracted from pine wood residues using a "green" levulinic acid-based solvent, was used to partially substitute non-sustainable phenol. The novel engineered foams were systematically compared to foams composed of different types of commercially available technical lignins. Different features were analyzed, such as foam density, microstructure (electron microscopy), surface hydrophilicity (contact angle), chemical grafting (infrared spectroscopy) and mechanical and thermal features. Overall, it was observed that up to 30 wt% of phenol can be substituted by the new type of lignin, without compromising the foam properties. This work provides a new insights on the development of novel lignin-based foams as a very promising sustainable and renewable alternative to petrol-based counterparts.

Levulinic Acid-Based "Green" Solvents for Lignocellulose Fractionation: On the Superior Extraction Yield and Selectivity toward Lignin.

The high potential use of lignin in novel biomaterials and chemicals represents an important opportunity for the valorization of the most abundant natural resource of aromatic molecules. From an environmental perspective, it is highly desirable replacing the hazardous methods currently used to extract lignin from lignocellulosic biomass and develop more sustainable and environmentally friendly approaches. Therefore, in this work, levulinic acid (a "green" solvent obtained from biomass) was successfully used, for the first time, to selectively extract high-quality lignin from pine wood sawdust residues at 200 °C for 6 h (at atmospheric pressure). Moreover, the addition of catalytic concentrations of inorganic acids (i.e., H2SO4 or HCl) was found to substantially reduce the temperature and reaction times needed (i.e., 140 °C, 2 h) for complete lignin extraction without compromising its purity. NMR data suggests that condensed OH structures and acidic groups are present in the lignin following extraction. Levulinic acid can be easily recycled and efficiently reused several times without affecting its performance. Furthermore, excellent solvent reusability and performance of extraction of other wood residues has been successfully demonstrated, thus making the developed levulinic acid-based procedure highly appealing and promising to replace the traditional less sustainable methodologies.

Interfacial Dynamics and Growth Modes of ß2-Microglobulin Dimers.

Protein aggregation is a complex process, strongly dependent on environmental conditions and highly structurally heterogeneous, both at the final level of fibril structure and intermediate level of oligomerization. Since the first step in aggregation is the formation of a dimer, it is important to clarify how certain properties of the latter (e.g., stability or interface geometry) may play a role in self-association. Here, we report a simple model that represents the dimer's interfacial region by two angles and combine it with a simple computational method to investigate how modulations of the interfacial region occurring on the ns-µs time scale change the dimer's growth mode. To illustrate the proposed methodology, we consider 15 different dimer configurations of the ß2m D76N mutant protein equilibrated with long Molecular Dynamics simulations and identify which interfaces lead to limited and unlimited growth modes, having, therefore, different aggregation profiles. We found that despite the highly dynamic nature of the starting configurations, most polymeric growth modes tend to be conserved within the studied time scale. The proposed methodology performs remarkably well taking into consideration the nonspherical morphology of the ß2m dimers, which exhibit unstructured termini detached from the protein's core, and the relatively weak binding affinities of their interfaces, which are stabilized by nonspecific apolar interactions. The proposed methodology is general and can be applied to any protein for which a dimer structure has been experimentally determined or computationally predicted.

Adding knowledge to the design of safer hydrophobically modified poly(acrylic) acids: an ecotoxicological approach.

The architecture of hydrophobically modified polymers can be tailored to produce variants with different levels of functionality. This allows industry to apply rational design methods for the development of more environmentally friendly materials. In the present work, the ecotoxicity of six variants of hydrophobically modified poly(acrylic) acids (HMPAA), obtained by changing the crosslinked conformation, insertion position, and length of the hydrophobic groups, was assessed for the (i) bioluminescence production of Aliivibrio fischeri; (ii) population growth rate of Raphidocelis subcapitata and Chlorella vulgaris; (iii) mortality of Brachionus calyciflorus; (iv) feeding inhibition, somatic growth rate, reproduction, and mortality of Daphnia magna; and (iv) mortality and somatic growth rate of Pelophylax perezi tadpoles. The concentrations causing 50% and 20% of effects (L(E)C50 and 20, respectively) ranged from 9.64 up to > 2000 mg·L-1 for all six HMPAA and species. The bacterium A. fischeri and tadpoles of P. perezi were the most sensitive and most tolerant organisms to the six tested HMPAA, respectively. The computed 5% hazard concentrations (computed on the basis of L(E)C50 s) showed that HMPAA1 (13.0 mg·L-1) and HMPAA2 (26.1 mg·L-1) were the most toxic variants, while HMPAA6 (233 mg·L-1) the least one. These results suggest HMPAA6 (with low crosslink percentage modified by the addition of long and short hydrophobic groups at the surface) to be the most environmentally friendly variant and should be preferentially considered to be used in consumer products, compared to the other five studied variants.

Modification of chicha gum: Antibacterial activity, ex vivo mucoadhesion, antioxidant activity and cellular viability.

The aim of the present work was to modify the exuded gum of Sterculia striata tree by an amination reaction. The viscosity and zero potential of the chicha gum varied as a function of pH. The modification was confirmed by X-ray diffraction (XRD), infrared spectroscopy (FTIR), size exclusion chromatography (SEC), zeta potential, thermogravimetric analysis (TG), and differential scanning calorimetry (DSC). Furthermore, the chemical modification changed the molar mass and surface charge of the chicha gum. In addition, the gums were used in tests for ex vivo mucoadhesion strength, antibacterial activity against the standard strain of Staphylococcus aureus (ATCC 25923), inhibitory activity of α-glucosidase, antioxidant capacity, and viability of Caco-2 cells. Through these tests, it was found that amination caused an increase in the mucoadhesive and inhibitory activity of chicha gum against the bacterium Staphylococcus aureus. In addition, the gums (pure and modified) showed antioxidant capacity and an inhibitory effect against the α-glucosidase enzyme and did not show cytotoxic potential.

Extending the Stochastic Titration CpHMD to CHARMM36m.

The impact of pH on proteins is significant but often neglected in molecular dynamics simulations. Constant-pH Molecular Dynamics (CpHMD) is the state-of-the-art methodology to deal with these effects. However, it still lacks widespread adoption by the scientific community. The stochastic titration CpHMD is one of such methods that, until now, only supported the GROMOS force field family. Here, we extend this method's implementation to include the CHARMM36m force field available in the GROMACS software package. We test this new implementation with a diverse group of proteins, namely, lysozyme, Staphylococcal nuclease, and human and E. coli thioredoxins. All proteins were conformationally stable in the simulations, even at extreme pH values. The RMSE values (pKa prediction vs experimental) obtained were very encouraging, in particular for lysozyme and human thioredoxin. We have also identified a few residues that challenged the CpHMD simulations, highlighting scenarios where the method still needs improvement independently of the force field. The CHARMM36m all-atom implementation was more computationally efficient when compared with the GROMOS 54A7, taking advantage of a shorter nonbonded interaction cutoff and a less frequent neighboring list update. The new extension will allow the study of pH effects in many systems for which this force field is particularly suited, i.e., proteins, membrane proteins, lipid bilayers, and nucleic acids.

On the Development of Phenol-Formaldehyde Resins Using a New Type of Lignin Extracted from Pine Wood with a Levulinic-Acid Based Solvent.

Resole resins have many applications, especially for foam production. However, the use of phenol, a key ingredient in resoles, has serious environmental and economic disadvantages. In this work, lignin extracted from pine wood using a "green" solvent, levulinic acid, was used to partially replace the non-sustainable phenol. The physicochemical properties of this novel resin were compared with resins composed of different types of commercial lignins. All resins were optimized to keep their free formaldehyde content below 1 wt%, by carefully adjusting the pH of the mixture. Substitution of phenol with lignin generally increases the viscosity of the resins, which is further increased with the lignin mass fraction. The addition of lignin decreases the kinetics of gelification of the resin. The type and amount of lignin also affect the thermal stability of the resins. It was possible to obtain resins with higher thermal stability than the standard phenol-formaldehyde resins without lignin. This work provides new insights regarding the development of lignin-based resoles as a very promising sustainable alternative to petrol-based resins.

Ecotoxicity of cationic cellulose polymers to aquatic biota: The influence of charge density.

Better performances of cellulose-based polymers can be achieved by adjust their architecture including the density of cationic modifications. In this study, the influence of cationic substitution on the ecotoxicity of four quaternized hydroxyethyl cellulose polymers (SK-H, SK-L, SK-M, SK-MH) was studied, using an aquatic biota acute ecotoxicity classification, and rheological and physicochemical characterization. The ecotoxicity characterization was achieved by performing standard ecotoxicity assays with seven key trophic level species: Vibrio fischeri, Raphidocelis subcapitata, Chlorella vulgaris, Daphnia magna, Brachionus calyciflorus, Heterocypris incongruens, and Danio rerio. Median effective concentrations were used to compute hazard concentrations, through the species sensitive distribution curves method. The microalga C. vulgaris and rotifer B. calyciflorus were the most sensitive species to the studied polymers. The SK-H variant was highly toxic to the rotifer. Overall, variants with intermediate levels of cationic charge (SK-M, SK-MH) presented the lowest toxicity. The SK-M variant showed the lowest value of maximum acceptable concentration (0.00354 mg/L), thus being indicated as the least toxic variant. Therefore, the obtained results suggest that industry could direct the development of this type of polymers by tailoring its cationic substitution to moderate levels, in such a way that both functionality and environmental toxicity could be maximized.

In Silico End-to-End Protein-Ligand Interaction Characterization Pipeline: The Case of SARS-CoV-2.

SARS-CoV-2 triggered a worldwide pandemic disease, COVID-19, for which an effective treatment has not yet been settled. Among the most promising targets to fight this disease is SARS-CoV-2 main protease (Mpro), which has been extensively studied in the last few months. There is an urgency for developing effective computational protocols that can help us tackle these key viral proteins. Hence, we have put together a robust and thorough pipeline of in silico protein-ligand characterization methods to address one of the biggest biological problems currently plaguing our world. These methodologies were used to characterize the interaction of SARS-CoV-2 Mpro with an α-ketoamide inhibitor and include details on how to upload, visualize, and manage the three-dimensional structure of the complex and acquire high-quality figures for scientific publications using PyMOL (Protocol 1); perform homology modeling with MODELLER (Protocol 2); perform protein-ligand docking calculations using HADDOCK (Protocol 3); run a virtual screening protocol of a small compound database of SARS-CoV-2 candidate inhibitors with AutoDock 4 and AutoDock Vina (Protocol 4); and, finally, sample the conformational space at the atomic level between SARS-CoV-2 Mpro and the α-ketoamide inhibitor with Molecular Dynamics simulations using GROMACS (Protocol 5). Guidelines for careful data analysis and interpretation are also provided for each Protocol.

Predicting stable binding modes from simulated dimers of the D76N mutant of ß 2-microglobulin.

The D76N mutant of the ß 2 m protein is a biologically motivated model system to study protein aggregation. There is strong experimental evidence, supported by molecular simulations, that D76N populates a highly dynamic conformation (which we originally named I 2 ) that exposes aggregation-prone patches as a result of the detachment of the two terminal regions. Here, we use Molecular Dynamics simulations to study the stability of an ensemble of dimers of I 2 generated via protein-protein docking. MM-PBSA calculations indicate that within the ensemble of investigated dimers the major contribution to interface stabilization at physiological pH comes from hydrophobic interactions between apolar residues. Our structural analysis also reveals that the interfacial region associated with the most stable binding modes are particularly rich in residues pertaining to both the N- and C-terminus, as well residues from the BC- and DE-loops. On the other hand, the less stable interfaces are stabilized by intermolecular interactions involving residues from the CD- and EF-loops. By focusing on the most stable binding modes, we used a simple geometric rule to propagate the corresponding dimer interfaces. We found that, in the absence of any kind of structural rearrangement occurring at an early stage of the oligomerization pathway, some interfaces drive a self-limited growth process, while others can be propagated indefinitely allowing the formation of long, polymerized chains. In particular, the interfacial region of the most stable binding mode reported here falls in the class of self-limited growth.

Antiviral and antibacterial activity of hand sanitizer and surface disinfectant formulations.

Emergent diseases caused by viral and bacterial infections have proven to be a current and future challenge. The occurrence of these diseases is usually accompanied by the lack of vaccines and dedicated therapies leaving prevention as the best strategy to adopt. In that context, and apart from confinement and physical distancing measures, an increase in hygiene actions, namely hand and surface cleaning and disinfection can reduce the infection spread originated from our day-to-day routines. However, during crisis situations the high disinfectants demand can very likely lead to having them running out of stock. This impels many individuals and companies to produce their own disinfectants. Here, we explore the main components of a disinfection formulation, both for hand-rub and surface cleaning. Alcohol and non-alcohol based formulations are described, including the possibility to fine tune the properties of the final product in order to increase public acceptance while maintaining product efficacy. The action mechanisms of the main active principles are also described conjugating information from experimental and theoretical data. Overall, the main aspects to develop a disinfectant formulation are addressed, as well as their function, helping formulation developers to better understand the impact of their choices.

Dancing with oils - the interaction of lipases with different oil/water interfaces.

The use of enzymes as biocatalysts in industrial applications has received much attention during the last few years. Lipases are widely employed in the food and cosmetic industry, for the synthesis of novel biomaterials and as a greener solution for the treatment of waste cooking oils (WCO). The latter topic has been widely explored with the use of enzymes from several origins and types, for the treatment of different used and non-used cooking oils. The experimental conditions of such works are also quite broad, hampering the detailed understanding of the process. In this work we present a detailed characterization of the interaction of several commonly used lipases with different types of vegetal oils and food fats through coarse-grained molecular dynamics simulations. First, the molecular details of the oil/water (O/W) mixtures, namely at the O/W interface, are described. The O/W interface was found to be enriched in triglyceride molecules with higher polarity. Then, the interaction of lipases with oil mixtures is characterized from different perspectives, including the identification of the most important protein residues for this process. The lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML) and Candida antarctica (CALB) were found to bind to the O/W interface in a manner that makes the protein binding site more available for the oil molecules. These enzymes were also found to efficiently bind to the O/W interface of all oil mixtures, which in addition to reactivity factors, may explain the efficient applicability of these enzymes to a large variety of edible oils and WCO.

Computational Analysis of the Interactions between the S100B Extracellular Chaperone and Its Amyloid ß Peptide Client.

S100B is an astrocytic extracellular Ca2+-binding protein implicated in Alzheimer's disease, whose role as a holdase-type chaperone delaying Aß42 aggregation and toxicity was recently uncovered. Here, we employ computational biology approaches to dissect the structural details and dynamics of the interaction between S100B and Aß42. Driven by previous structural data, we used the Aß25-35 segment, which recapitulates key aspects of S100B activity, as a starting guide for the analysis. We used Haddock to establish a preferred binding mode, which was studied with the full length Aß using long (1 µs) molecular dynamics (MD) simulations to investigate the structural dynamics and obtain representative interaction complexes. From the analysis, Aß-Lys28 emerged as a key candidate for stabilizing interactions with the S100B binding cleft, in particular involving a triad composed of Met79, Thr82 and Glu86. Binding constant calculations concluded that coulombic interactions, presumably implicating the Lys28(Aß)/Glu86(S100B) pair, are very relevant for the holdase-type chaperone activity. To confirm this experimentally, we examined the inhibitory effect of S100B over Aß aggregation at high ionic strength. In agreement with the computational predictions, we observed that electrostatic perturbation of the Aß-S100B interaction decreases anti-aggregation activity. Altogether, these findings unveil features relevant in the definition of selectivity of the S100B chaperone, with implications in Alzheimer's disease.

Enhancing Lignin Dissolution and Extraction: The Effect of Surfactants.

The dissolution and extraction of lignin from biomass represents a great challenge due to the complex structure of this natural phenolic biopolymer. In this work, several surfactants (i.e., non-ionic, anionic, and cationic) were used as additives to enhance the dissolution efficiency of model lignin (kraft) and to boost lignin extraction from pine sawdust residues. To the best of our knowledge, cationic surfactants have never been systematically used for lignin dissolution. It was found that ca. 20 wt.% of kraft lignin is completely solubilized using 1 mol L-1 octyltrimethylammonium bromide aqueous solution. A remarkable dissolution efficiency was also obtained using 0.5 mol L-1 polysorbate 20. Furthermore, all surfactants used increased the lignin extraction with formic acid, even at low concentrations, such as 0.01 and 0.1 mol L-1. Higher concentrations of cationic surfactants improve the extraction yield but the purity of extracted lignin decreases.

New deep eutectic solvent assisted extraction of highly pure lignin from maritime pine sawdust (Pinus pinaster Ait.).

Lignocellulosic biomass is a renewable and sustainable feedstock, mainly composed of cellulose, hemicellulose, and lignin. Lignin, as the most abundant natural aromatic polymer occurring on Earth, has great potential to produce value-added products. However, the isolation of highly pure lignin from biomass requires the use of efficient methods during lignocellulose fractionation. Therefore, in this work, novel acidic deep eutectic solvents (DESs) were prepared, characterized and screened for lignin extraction from maritime pine wood (Pinus pinaster Ait.) sawdust. The use of cosolvents and the development of new DES were also evaluated regarding their extraction and selectivity performance. The results show that an 1 h extraction process at 175 °C, using a novel DES composed of lactic acid, tartaric acid and choline chloride, named Lact:Tart:ChCl, in a molar ratio of 4:1:1, allows the recovery of 95 wt% of the total lignin present in pine biomass with a purity of 89 wt%. Such superior extraction of lignin with remarkable purity using a "green" solvent system makes this process highly appealing for future large-scale applications.

Hydrophobic modifications of hydroxyethyl cellulose polymers: Their influence on the acute toxicity to aquatic biota.

The hydrophobic substitution (HS) of cationic cellulose derivatives may be tuned, promoting their efficiency. This work studied the influence of HS on the acute ecotoxicity of quaternized hydroxyethyl cellulose polymers (SL) to aquatic biota. The ecotoxicity of four SL with different HS (SL-5, SL-30, SL-60, SL-100) was assessed for seven species: Vibrio fischeri, Raphidocelis subcapitata, Chlorella vulgaris, Daphnia magna, Brachionus calyciflorus, Heterocypris incongruens, and Danio rerio. The computed median effective concentrations were used to derive hazard concentrations, by using species sensitive distribution curves. All SL suspensions were characterized for particle size, zeta potential and rheological properties. Results indicated instability of the SL in suspension due to their relatively low zeta potential. Raphidocelis subcapitata, C. vulgaris and B. calyciflorus were the most sensitive to the four SL, suggesting that exposure to these compounds may imbalance the lowest trophic levels. Also, HS influenced the toxicity of SL, with the lowest HS (SL-5) revealing lower ecotoxicity. The maximum acceptable concentrations were 14.0, 2.9, 3.9 and 1.4 mg L-1 for SL-5, SL-30, SL-60, and SL-100, respectively. Accordingly, SL-5 is suggested as the eco-friendliest and is recommended to be used in the production of care products, in detriment of the other three tested variants.

Cu2+-binding to S100B triggers polymerization of disulfide cross-linked tetramers with enhanced chaperone activity against amyloid-ß aggregation.

S100B is an extracellular protein implicated in Alzheimer's Disease and a suppressor of amyloid-ß aggregation. Herein we report a mechanism tying Cu2+ binding to a change in assembly state yielding disulfide cross-linked oligomers with higher anti-aggregation activity. This chemical control of chaperone function illustrates a regulatory process relevant under metal and proteostasis dysfunction as in neurodegeneration.

A Simple Method of Transoperative Maxillomandibular Fixation in Pediatric Facial Fractures.

ABSTRACT: Maxillomandibular fixation in pediatric facial fractures is quite challenging to be achieved, especially in mid-mixed dentition. Traditional well established intermaxillary devices have a lot of limitations in these patients. This article presents a case of a 9-year-old female patient with a displaced mandibular fracture in which orthodontic buttons were used for transoperative maxillomandibular fixation followed by internal fixation after adequate occlusion reestablishment and fracture reduction. The method showed high efficacy and celerity, allowing the success of treatment, with satisfactory evolution of the patient.