A 3D-Printed Portable UV and Visible Photoreactor for Water Purification and Disinfection Experiments.

Water scarcity and contamination are urgent issues to be addressed. In this context, different materials, techniques, and devices are being developed to mitigate contemporary and forthcoming water constraints. Photocatalysis-based approaches are suitable strategies to address water contamination by degrading contaminants and eliminating microbes. Photoreactors are usually designed to perform photocatalysis in a scalable and standardised way. Few or none have been developed to combine these characteristics with portability, flexibility, and cost effectiveness. This study reports on designing and producing a portable (490 g), low-cost, and multifunctional photoreactor that includes adjustable radiation intensity and two types of wavelengths (UV-A and visible), including combined agitation in a compact mechanism produced through 3D printing technology. The mechanical, electrical, and optical subsystems were designed and assembled into a robust device. It is shown that it is possible to apply radiations that can reach 65 mW/cm2 and 110 mW/cm2 using the installed visible and UV LEDs and apply mechanical agitation up to 200 rpm, all under a ventilated system. Regarding functionality, the photoreactor proof of concept indicated the ability to degrade ~80% and 30% ciprofloxacin under UV and visible irradiation of TiO2 and Ag/TiO2 nanoparticles. The device also showed the ability to eliminate E. coli bacteria, recurring to radiation set-ups and nanoparticles. Therefore, the originally designed and constructed photoreactor concept was characterised and functionally validated as an exciting and flexible device for lab-scaled or outdoor experiments, assuring standardised and comparable results.

NAGPKin: Nucleation-and-growth parameters from the kinetics of protein phase separation.

The assembly of biomolecular condensate in eukaryotic cells and the accumulation of amyloid deposits in neurons are processes involving the nucleation and growth (NAG) of new protein phases. To therapeutically target protein phase separation, drug candidates are tested in in vitro assays that monitor the increase in the mass or size of the new phase. Limited mechanistic insight is, however, provided if empirical or untestable kinetic models are fitted to these progress curves. Here we present the web server NAGPKin that quantifies NAG rates using mass-based or size-based progress curves as the input data. A report is generated containing the fitted NAG parameters and elucidating the phase separation mechanisms at play. The NAG parameters can be used to predict particle size distributions of, for example, protein droplets formed by liquid-liquid phase separation (LLPS) or amyloid fibrils formed by protein aggregation. Because minimal intervention is required from the user, NAGPKin is a good platform for standardized reporting of LLPS and protein self-assembly data. NAGPKin is useful for drug discovery as well as for fundamental studies on protein phase separation. NAGPKin is freely available (no login required) at https://nagpkin.i3s.up.pt.

Smart and Multifunctional Materials Based on Electroactive Poly(vinylidene fluoride): Recent Advances and Opportunities in Sensors, Actuators, Energy, Environmental, and Biomedical Applications.

From scientific and technological points of view, poly(vinylidene fluoride), PVDF, is one of the most exciting polymers due to its overall physicochemical characteristics. This polymer can crystalize into five crystalline phases and can be processed in the form of films, fibers, membranes, and specific microstructures, being the physical properties controllable over a wide range through appropriate chemical modifications. Moreover, PVDF-based materials are characterized by excellent chemical, mechanical, thermal, and radiation resistance, and for their outstanding electroactive properties, including high dielectric, piezoelectric, pyroelectric, and ferroelectric response, being the best among polymer systems and thus noteworthy for an increasing number of technologies. This review summarizes and critically discusses the latest advances in PVDF and its copolymers, composites, and blends, including their main characteristics and processability, together with their tailorability and implementation in areas including sensors, actuators, energy harvesting and storage devices, environmental membranes, microfluidic, tissue engineering, and antimicrobial applications. The main conclusions, challenges and future trends concerning materials and application areas are also presented.

Drug repurposing of dopaminergic drugs to inhibit ataxin-3 aggregation.

The accumulation of mutant ataxin-3 (Atx3) in neuronal nuclear inclusions is a pathological hallmark of Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia Type 3. Decreasing the protein aggregation burden is a possible disease-modifying strategy to tackle MJD and other neurodegenerative disorders for which only symptomatic treatments are currently available. We performed a drug repurposing screening to identify inhibitors of Atx3 aggregation with known toxicological and pharmacokinetic profiles. Interestingly, dopamine hydrochloride and other catecholamines are among the most potent inhibitors of Atx3 aggregation in vitro. Our results indicate that low micromolar concentrations of dopamine markedly delay the formation of mature amyloid fibrils of mutant Atx3 through the inhibition of the earlier oligomerization steps. Although dopamine itself does not cross the blood-brain barrier, dopamine levels in the brain can be increased by low doses of dopamine precursors and dopamine agonists commonly used to treat Parkinsonian symptoms. In agreement, treatment with levodopa ameliorated motor symptoms in a C. elegans model of MJD. These findings suggest a possible application of dopaminergic drugs to halt or reduce Atx3 accumulation in the brains of MJD patients.

Quantification of Surface Tension Effects and Nucleation-and-Growth Rates during Self-Assembly of Biological Condensates.

Liquid-solid and liquid-liquid phase separation (PS) drives the formation of functional and disease-associated biological assemblies. Principles of phase equilibrium are here employed to derive a general kinetic solution that predicts the evolution of the mass and size of biological assemblies. Thermodynamically, protein PS is determined by two measurable concentration limits: the saturation concentration and the critical solubility. Due to surface tension effects, the critical solubility can be higher than the saturation concentration for small, curved nuclei. Kinetically, PS is characterized by the primary nucleation rate constant and a combined rate constant accounting for growth and secondary nucleation. It is demonstrated that the formation of a limited number of large condensates is possible without active mechanisms of size control and in the absence of coalescence phenomena. The exact analytical solution can be used to interrogate how the elementary steps of PS are affected by candidate drugs.

Hybrid TiO2:Au nanostars based polymeric membranes for photocatalytic degradation of ciprofloxacin in water samples.

Antibiotics represent one increasingly harmful type of contaminant of emerging concern in treated and non-treated water. They cause the generation of antibiotic-multiresistant organisms, one of the major challenges in current medicine. Plasmonic-photocatalysis using solar energy represents a promising solution for their removal with low energy consumption. Its successful application requires the improvement of photocatalysts' efficiency under sunlight and the development of robust, durable, and efficient substrates for photocatalysts immobilisation. In this work, hybrid TiO2:Au nanostars were initially synthesised. Then, two porous membranes were prepared to support this nanocatalyst based on poly (vinylidenefluoride-co-hexafluoropropylene) polymer. Doctor blade and salt leaching casting methods, combined with temperature-induced phase separation, were used to generate membranes with high porosity, 80-90%, which was maintained after nanoparticle incorporation (3, 8 and 10 wt%). The photocatalytic activity of the nanocomposite membranes was tested through the degradation of the antibiotic ciprofloxacin under UV and visible radiation. Salt-leaching membranes containing 10 wt% nanoparticles presented the highest degradation efficiencies, 45% under UV and 35% under visible radiation. In contrast, doctor blade membranes showed 36% and 32% degradation efficiencies, respectively. The reusability of the membranes was assessed in repeated cycles, presenting an average efficiency loss of only 2% after three uses. Finally, the reusability of these membranes was also tested in treated effluent water matrixes, presenting similar, or even better, degradation efficiencies, and a minimum reusability efficiency lost 0-1%. The results demonstrate that these membranes are a promising alternative for the degradation of a wide variety of contaminants under sunlight radiation.

Size Effect in Hybrid TiO2:Au Nanostars for Photocatalytic Water Remediation Applications.

TiO2:Au-based photocatalysis represents a promising alternative to remove contaminants of emerging concern (CECs) from wastewater under sunlight irradiation. However, spherical Au nanoparticles, generally used to sensitize TiO2, still limit the photocatalytic spectral band to the 520 nm region, neglecting a high part of sun radiation. Here, a ligand-free synthesis of TiO2:Au nanostars is reported, substantially expanding the light absorption spectral region. TiO2:Au nanostars with different Au component sizes and branching were generated and tested in the degradation of the antibiotic ciprofloxacin. Interestingly, nanoparticles with the smallest branching showed the highest photocatalytic degradation, 83% and 89% under UV and visible radiation, together with a threshold in photocatalytic activity in the red region. The applicability of these multicomponent nanoparticles was further explored with their incorporation into a porous matrix based on PVDF-HFP to open the way for a reusable energy cost-effective system in the photodegradation of polluted waters containing CECs.

A Robust Assay to Monitor Ataxin-3 Amyloid Fibril Assembly.

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine repeat in the protein ataxin-3, which is deposited as intracellular aggregates in affected brain regions. Despite the controversial role of ataxin-3 amyloid structures in SCA3 pathology, the identification of molecules with the capacity to prevent aberrant self-assembly and stabilize functional conformation(s) of ataxin-3 is a key to the development of therapeutic solutions. Amyloid-specific kinetic assays are routinely used to measure rates of protein self-assembly in vitro and are employed during screening for fibrillation inhibitors. The high tendency of ataxin-3 to assemble into oligomeric structures implies that minor changes in experimental conditions can modify ataxin-3 amyloid assembly kinetics. Here, we determine the self-association rates of ataxin-3 and present a detailed study of the aggregation of normal and pathogenic ataxin-3, highlighting the experimental conditions that should be considered when implementing and validating ataxin-3 amyloid progress curves in different settings and in the presence of ataxin-3 interactors. This assay provides a unique and robust platform to screen for modulators of the first steps of ataxin-3 aggregation-a starting point for further studies with cell and animal models of SCA3.

Large-scale aqueous synthesis of Cu(In,Ga)Se2 nanoparticles for photocatalytic degradation of ciprofloxacin.

Environmentally friendly synthesis of Cu(In,Ga)Se2 (CIGS) nanoparticles (NPs) is pivotal for producing sustainable photocatalytic compounds to be applied in the remediation of contaminants of emerging concern from water. To this end, we herein report an aqueous synthesis of CIGS NPs, followed by annealing, to give access to phase-pure CIGS crystals with chalcopyrite structure and no signs of secondary phases. Morphological and compositional characterization revealed NPs with an average size of 10-35 nm and uniform distribution of Cu, In, Ga, and Se elements. In addition, the first aqueous large-scale synthesis of CIGS NPs is developed by up-scaling the synthesis procedure, resulting in 5 g of highly crystalline nanoparticles exhibiting an ideal optical band gap of 1.14 eV. The as-synthesized NPs proved the ability to remove 71 and 83% of a contaminant of emerging concern, ciprofloxacin (CIP), under ultraviolet (UV) and visible (Vis) radiations, respectively.

Propedia: a database for protein-peptide identification based on a hybrid clustering algorithm.

BACKGROUND: Protein-peptide interactions play a fundamental role in a wide variety of biological processes, such as cell signaling, regulatory networks, immune responses, and enzyme inhibition. Peptides are characterized by low toxicity and small interface areas; therefore, they are good targets for therapeutic strategies, rational drug planning and protein inhibition. Approximately 10% of the ethical pharmaceutical market is protein/peptide-based. Furthermore, it is estimated that 40% of protein interactions are mediated by peptides. Despite the fast increase in the volume of biological data, particularly on sequences and structures, there remains a lack of broad and comprehensive protein-peptide databases and tools that allow the retrieval, characterization and understanding of protein-peptide recognition and consequently support peptide design. RESULTS: We introduce Propedia, a comprehensive and up-to-date database with a web interface that permits clustering, searching and visualizing of protein-peptide complexes according to varied criteria. Propedia comprises over 19,000 high-resolution structures from the Protein Data Bank including structural and sequence information from protein-peptide complexes. The main advantage of Propedia over other peptide databases is that it allows a more comprehensive analysis of similarity and redundancy. It was constructed based on a hybrid clustering algorithm that compares and groups peptides by sequences, interface structures and binding sites. Propedia is available through a graphical, user-friendly and functional interface where users can retrieve, and analyze complexes and download each search data set. We performed case studies and verified that the utility of Propedia scores to rank promissing interacting peptides. In a study involving predicting peptides to inhibit SARS-CoV-2 main protease, we showed that Propedia scores related to similarity between different peptide complexes with SARS-CoV-2 main protease are in agreement with molecular dynamics free energy calculation. CONCLUSIONS: Propedia is a database and tool to support structure-based rational design of peptides for special purposes. Protein-peptide interactions can be useful to predict, classifying and scoring complexes or for designing new molecules as well. Propedia is up-to-date as a ready-to-use webserver with a friendly and resourceful interface and is available at: https://bioinfo.dcc.ufmg.br/propedia.

Can photocatalytic and magnetic nanoparticles be a threat to aquatic detrital food webs?

Freshwaters are likely to serve as reservoirs for engineered nanomaterials (ENMs) due to their accelerated unintentional release, increasing the relevance of assessing their impacts on aquatic biota and the ecosystem processes they drive. Stream-dwelling microbes, particularly fungi, and invertebrate shredders play an essential role in the decomposition of organic matter and transfer of energy to higher trophic levels. We assessed the impacts of two photocatalytic (nano-TiO2 and erbium doped nano-TiO2) and one magnetic (nano-CoFe2O4) ENMs on detrital-based food webs in freshwaters by exposing chestnut leaves, colonized by stream-dwelling microbes, to a series of concentrations (0.25-150 mg L-1) of these ENMs. Microbial decomposition and biomass of fungal communities, associated with leaves, were not affected by the ENMs. However, the activities of antioxidant enzymes of microbial decomposers were significantly (P < 0.05) stimulated by ENMs in a concentration-dependent way, suggesting oxidative stress in stream microbial communities. The stronger responses of these stress biomarkers against nano-TiO2 (increase upto 837.5% for catalase, 1546.8% for glutathione peroxidase and 1154.6% for glutathione S-transferase) suggest a higher toxicity of this ENM comparing to the others. To determine whether the effects could be transferred across trophic levels, the invertebrate shredder Sericostoma sp. was exposed to ENMs (1 and 50 mg L-1) for 5 days either via contaminated water or contaminated food (leaf litter). Leaf consumption rate by shredders decreased significantly (P < 0.05) with increasing concentrations of ENMs via food or water; the effects were more pronounced when exposure occurred via contaminated food (up to 99.3%, 90.7% and 90.3% inhibition by nano-Er:TiO2, nano-CoFe2O4 and nano-TiO2, respectively). Overall, the tested photocatalytic and magnetic ENMs can be harmful to microbial decomposers and invertebrate shredders further compromising detrital-based food webs in streams.

Major Improvements in Robustness and Efficiency during the Screening of Novel Enzyme Effectors by the 3-Point Kinetics Assay.

The throughput level currently reached by automatic liquid handling and assay monitoring techniques is expected to facilitate the discovery of new modulators of enzyme activity. Judicious and dependable ways to interpret vast amounts of information are, however, required to effectively answer this challenge. Here, the 3-point method of kinetic analysis is proposed as a means to significantly increase the hit success rates and decrease the number of falsely identified compounds (false positives). In this post-Michaelis-Menten approach, each screened reaction is probed in three different occasions, none of which necessarily coincide with the initial period of constant velocity. Enzymology principles rather than subjective criteria are applied to identify unwanted outliers such as assay artifacts, and then to accurately distinguish true enzyme modulation effects from false positives. The exclusion and selection criteria are defined based on the 3-point reaction coordinates, whose relative positions along the time-courses may change from well to well or from plate to plate, if necessary. The robustness and efficiency of the new method is illustrated during a small drug repurposing screening of potential modulators of the deubiquinating activity of ataxin-3, a protein implicated in Machado-Joseph disease. Apparently, intractable Z factors are drastically enhanced after (1) eliminating spurious results, (2) improving the normalization method, and (3) increasing the assay resilience to systematic and random variability. Numerical simulations further demonstrate that the 3-point analysis is highly sensitive to specific, catalytic, and slow-onset modulation effects that are particularly difficult to detect by typical endpoint assays.

Magnetic Nanoparticles for Biomedical Applications: From the Soul of the Earth to the Deep History of Ourselves.

Precisely engineered magnetic nanoparticles (MNPs) have been widely explored for applications including theragnostic platforms, drug delivery systems, biomaterial/device coatings, tissue engineering scaffolds, performance-enhanced therapeutic alternatives, and even in SARS-CoV-2 detection strips. Such popularity is due to their unique, challenging, and tailorable physicochemical/magnetic properties. Given the wide biomedical-related potential applications of MNPs, significant achievements have been reached and published (exponentially) in the last five years, both in synthesis and application tailoring. Within this review, and in addition to essential works in this field, we have focused on the latest representative reports regarding the biomedical use of MNPs including characteristics related to their oriented synthesis, tailored geometry, and designed multibiofunctionality. Further, actual trends, needs, and limitations of magnetic-based nanostructures for biomedical applications will also be discussed.

interferENZY: A Web-Based Tool for Enzymatic Assay Validation and Standardized Kinetic Analysis.

Enzymatic assays are widely employed to characterize important allosteric and enzyme modulation effects. The high sensitivity of these assays can represent a serious problem if the occurrence of experimental errors surreptitiously affects the reliability of enzyme kinetics results. We have addressed this problem and found that hidden assay interferences can be unveiled by the graphical representation of progress curves in modified reaction coordinates. To render this analysis accessible to users across all levels of expertise, we have developed a webserver, interferENZY, that allows (i) an unprecedented tight quality control of experimental data, (ii) the automated identification of small and major assay interferences, and (iii) the estimation of bias-free kinetic parameters. By eliminating the subjectivity factor in kinetic data reporting, interferENZY will contribute to solving the "reproducibility crisis" that currently challenges experimental molecular biology. The interferENZY webserver is freely available (no login required) at https://interferenzy.i3s.up.pt.

MIRRAGGE - Minimum Information Required for Reproducible AGGregation Experiments.

Reports on phase separation and amyloid formation for multiple proteins and aggregation-prone peptides are recurrently used to explore the molecular mechanisms associated with several human diseases. The information conveyed by these reports can be used directly in translational investigation, e.g., for the design of better drug screening strategies, or be compiled in databases for benchmarking novel aggregation-predicting algorithms. Given that minute protocol variations determine different outcomes of protein aggregation assays, there is a strong urge for standardized descriptions of the different types of aggregates and the detailed methods used in their production. In an attempt to address this need, we assembled the Minimum Information Required for Reproducible Aggregation Experiments (MIRRAGGE) guidelines, considering first-principles and the established literature on protein self-assembly and aggregation. This consensus information aims to cover the major and subtle determinants of experimental reproducibility while avoiding excessive technical details that are of limited practical interest for non-specialized users. The MIRRAGGE table (template available in Supplementary Information) is useful as a guide for the design of new studies and as a checklist during submission of experimental reports for publication. Full disclosure of relevant information also enables other researchers to reproduce results correctly and facilitates systematic data deposition into curated databases.

Morphology Dependence Degradation of Electro- and Magnetoactive Poly(3-hydroxybutyrate-co-hydroxyvalerate) for Tissue Engineering Applications.

Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) is a piezoelectric biodegradable and biocompatible polymer suitable for tissue engineering applications. The incorporation of magnetostrictive cobalt ferrites (CFO) into PHBV matrix enables the production of magnetically responsive composites, which proved to be effective in the differentiation of a variety of cells and tissues. In this work, PHBV and PHBV with CFO nanoparticles were produced in the form of films, fibers and porous scaffolds and subjected to an experimental program allowing to evaluate the degradation process under biological conditions for a period up to 8 weeks. The morphology, physical, chemical and thermal properties were evaluated, together with the weight loss of the samples during the in vitro degradation assays. No major changes in the mentioned properties were found, thus proving its applicability for tissue engineering applications. Degradation was apparent from week 4 and onwards, leading to the conclusion that the degradation ratio of the material is suitable for a large range of tissue engineering applications. Further, it was found that the degradation of the samples maintain the biocompatibility of the materials for the pristine polymer, but can lead to cytotoxic effects when the magnetic CFO nanoparticles are exposed, being therefore needed, for magnetoactive applications, to substitute them by biocompatible ferrites, such as an iron oxide (Fe3O4).

Rapid assembly and profiling of an anticoagulant sulfoprotein library.

Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

A simple linearization method unveils hidden enzymatic assay interferences.

Enzymes are among the most important drug targets in the pharmaceutical industry. The bioassays used to screen enzyme modulators can be affected by unaccounted interferences such as time-dependent inactivation and inhibition effects. Using procaspase-3, caspase-3, and α-thrombin as model enzymes, we show that some of these effects are not eliminated by merely ignoring the reaction phases that follow initial-rate measurements. We thus propose a linearization method (LM) for detecting spurious changes of enzymatic activity based on the representation of progress curves in modified coordinates. This method is highly sensitive to signal readout distortions, thereby allowing rigorous selection of valid kinetic data. The method allows the detection of assay interferences even when their occurrence is not suspected a priori. By knowing the assets and liabilities of the bioassay, enzymology results can be reported with enhanced reproducibility and accuracy. Critical analysis of full progress curves is expected to help discriminating experimental artifacts from true mechanisms of enzymatic inhibition.

Photocatalytic Microporous Membrane against the Increasing Problem of Water Emerging Pollutants.

Emerging pollutants are an essential class of recalcitrant contaminants that are not eliminated from water after conventional treatment. Here, a photocatalytic microporous membrane based on polyvinylidene difluoride-co-trifluoroethylene (PVDF-TrFE) with immobilised TiO2 nanoparticles, prepared by solvent casting, was tested against representative emerging pollutants. The structure and composition of these polymeric membranes were characterized by scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, porosimetry, and contact angle goniometry. The nanocomposites exhibited a porous structure with a uniform distribution of TiO2 nanoparticles. The addition of TiO2 did not change the structure of the polymeric matrix; however, it increased the wettability of the nanocomposite. The nanocomposites degraded 99% of methylene blue (MB), 95% of ciprofloxacin (CIP), and 48% of ibuprofen (IBP). The microporous nanocomposite exhibited no photocatalytic efficiency loss after four use cycles, corresponding to 20 h of UV irradiation. The reusability of this system confirms the promising nature of polymer nanocomposites as the basis for cost-effective and scalable treatments of emerging pollutants.

Chemical Kinetic Strategies for High-Throughput Screening of Protein Aggregation Modulators.

Insoluble aggregates staining positive to amyloid dyes are known histological hallmarks of different neurodegenerative disorders and of type II diabetes. Soluble oligomers are smaller assemblies whose formation prior to or concomitant with amyloid deposition has been associated to the processes of disease propagation and cell death. While the pathogenic mechanisms are complex and differ from disease to disease, both types of aggregates are important biological targets subject to intense investigation in academia and industry. Here we review recent advances in the fundamental understanding of protein aggregation that can be used on the development of anti-amyloid and anti-oligomerization drugs. Specifically, we pinpoint the chemical kinetic aspects that should be attended during the development of high-throughput screening assays and in the hit validation phase. The strategies here devised are expected to establish a connection between basic research and pharmaceutical innovation.