How deprotonation of cohumulone and colupulone influence their UV/Vis and CD spectrum.

Cohumulone and colupulone are representatives of α- and ß-acids, respectively. These compounds are important antimicrobial hop (Humulus lupulus) constituents, where cohumulone is an important source of the bitter taste of beer. In this study, we examined the pH dependence of UV/Vis spectra of both compounds while CD spectra of cohumulone were also measured at various wavelengths. This facilitated the examination of the protolytic equilibrium of both compounds, where the second pKa value of cohumulone was determined for the first time. Additionally, comparing experimental spectra with spectra calculated using time-dependent density functional theory (TD-DFT) enabled the determination of the most likely deprotonation positions and corresponding species most likely present in the aqueous solution at various pH values. Last but not least, comparing calculated and experimental CD spectra of cohumulone facilitated the determination of the absolute stereoconfiguration of cohumulone.

Osmolality and Tonicity of Isotonic Beverages.

This study aimed to measure and compare the osmolality and tonicity of isotonic beverages that can be bought on the Slovenian market. The main goal was to examine how good is the agreement between the measured osmolalities of the beverages and the requirements for isotonic beverages set up by EFSA. Osmolalities were measured with an osmometer using the freezing point depression method. Afterwards, two complementary methods for the observation of tonicity were developed. Erythrocytes were exposed to standard NaCl solutions of different osmolalities to observe their influence on the volume and shape of cells following the turbidity of the solution and the morphology of erythrocytes. These two methods enabled us to determine whether standard solutions were hypo-, iso-, or hypertonic. In this way, we found that the osmolality of 12 out of the 18 investigated isotonic beverages was in the range of 270-330 mOsm/kg, as required by EFSA. However, six samples did not meet this criterion and should therefore not have the label "isotonic" or be described as such. The measurements of turbidity of solutions indicated that most isotonic beverages exhibit a lower tonicity than standard NaCl solutions of identical osmolality. However, examination of the erythrocytes in isotonic beverages showed that the measurements were additionally complicated by the low pH values of these beverages. Finally, by demonstrating how different components of isotonic beverages pass through the erythrocyte membranes, we found that even isoosmolal beverages are often not isotonic, as the concentration of actively transported sugars in these beverages is relatively high.

The Effect of Microwaves on Protein Structure: Molecular Dynamics Approach.

The impact of microwave (MW) irradiation on protein folding, potentially inciting misfolding, was investigated by employing molecular dynamics (MD) simulations. Twenty-nine proteins were subjected to MD simulations under equilibrium (300 K) and MW conditions, where the rotational temperature was elevated to 700 K. The utilized replacement model captures the microwave effects of δ- and γ-relaxation processes (frequency range of ∼300 MHz to ∼20 GHz). The results disclosed that MW heating incited a shift toward more compact protein conformations, as indicated by decreased root-mean-square deviations, root-mean-square fluctuations, head-to-tail distances, and radii of gyration. This compaction was attributed to the intensification of intramolecular electrostatic interactions and hydrogen bonds within the protein caused by MW-destabilized hydrogen bonds between the protein and solvent. The solvent-accessible surface area (SASA), particularly that of polar amino-acid residues, shrank under MW conditions, corresponding to a reduced polarity of the water solvent. However, MW irradiation produced no significant alterations in protein secondary structures; hence, MW heating was observed to primarily affect the protein tertiary structures.

A Monocarbonyl Curcuminoid Derivative Inhibits the Activity of Human Glutathione Transferase A4-4 and Chemosensitizes Glioblastoma Cells to Temozolomide.

Human glutathione transferase A4-4 (hGSTA4-4) displays high catalytic efficiency towards 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation. Its role as a target for the chemosensitization of cancer cells has not been investigated so far. In this study, the inhibitory potency of twelve selected natural products and ten monocarbonyl curcumin derivatives against hGSTA4-4 was studied. Among natural products, ellagic acid turned out to be the strongest inhibitor with an IC50 value of 0.44 ± 0.01 µM. Kinetic analysis using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as variable substrates showed that ellagic acid behaved as a competitive inhibitor towards both GSH and CDNB, with Ki values of 0.39 ± 0.02 and 0.63 ± 0.03 µM, respectively. Among the curcumin derivatives studied, three proved to be the most potent inhibitors, in the order DM151 > DM101 > DM100, with IC50 values of 2.4 ± 0.1 µM, 12.7 ± 1.1 µΜ and 16.9 ± 0.4 µΜ, respectively. Further kinetic inhibition analysis of the most active derivative, DM151, demonstrated that this compound is a mixed inhibitor towards CDNB with inhibition constants of Ki = 4.1 ± 0.5 µM and Ki' = 0.536 ± 0.034 µM, while it is a competitive inhibitor towards GSH with a Ki = 0.98 ± 0.11 µM. Molecular docking studies were performed to interpret the differences in binding of ellagic acid and curcumin derivatives to hGSTA4-4. The in silico measured docking scores were consistent with the obtained experimental data. Hydrogen bonds appear to be the main contributors to the specific binding of monocarbonyl curcumin derivatives, while π-π stacking interactions play a key role in the enzyme-ellagic acid interaction. In vitro cytotoxicity assessment of the worst (DM148) and the best (DM151) inhibitors was performed against glioblastoma cell lines U-251 MG and U-87 MG. The results revealed that DM151 displays considerably higher cytotoxicity against both glioblastoma cell lines, while the glioblastoma cytotoxicity of DM148 was very limited. Furthermore, low and non-toxic doses of DM151 sensitized U-251 MG cells to the first-line glioblastoma chemotherapeutic temozolomide (TMZ), allowing us to propose for the first time that hGSTA4-4 inhibitors may be attractive therapeutic partners for TMZ to optimize its clinical effect in glioblastoma chemotherapy.

Chelation of iron(II) ions by ellagitannins-Effects of hexahydroxydiphenoyl and nonahydroxytriphenoyl groups.

Tannins represent secondary plant metabolites that are used to control bacterial populations by chelation of essential metal ions. Their presence in food also affects the bioavailability of iron. This study investigates the influence of ellagitannins (vescalin, castalin, vescalagin, castalagin) structure and pH on the stoichiometry and formation constants of ellagitannin-Fe(II) coordination compounds. We demonstrated that ellagitannins are stable for at least one hour at pH values lower than 7.25. The spectra of neutral compounds were measured and explained with the help of TDDFT calculations. Furthermore, the pH-dependence of the ellagitannins UV-Vis spectra was examined to obtain insight into their protolytic equilibrium. Using Job's method in the pH range 3.50-5.51, the stoichiometry of the formed ellagitannin-Fe(II) ions complexes was determined. A model explaining interactions between ellagitannins and Fe(II) ions, that took into account the protolytic equilibrium of ellagitannins, was fitted globally to all four Job plots, whereby the corresponding formation constants were obtained.

Identification of Triazolopyrimidinyl Scaffold SARS-CoV-2 Papain-Like Protease (PLpro) Inhibitor.

The global impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its companion disease, COVID-19, has reminded us of the importance of basic coronaviral research. In this study, a comprehensive approach using molecular docking, in vitro assays, and molecular dynamics simulations was applied to identify potential inhibitors for SARS-CoV-2 papain-like protease (PLpro), a key and underexplored viral enzyme target. A focused protease inhibitor library was initially created and molecular docking was performed using CmDock software (v0.2.0), resulting in the selection of hit compounds for in vitro testing on the isolated enzyme. Among them, compound 372 exhibited promising inhibitory properties against PLpro, with an IC50 value of 82 ± 34 µM. The compound also displayed a new triazolopyrimidinyl scaffold not yet represented within protease inhibitors. Molecular dynamics simulations demonstrated the favorable binding properties of compound 372. Structural analysis highlighted its key interactions with PLpro, and we stress its potential for further optimization. Moreover, besides compound 372 as a candidate for PLpro inhibitor development, this study elaborates on the PLpro binding site dynamics and provides a valuable contribution for further efforts in pan-coronaviral PLpro inhibitor development.

Nanomaterials and Their Recent Applications in Impedimetric Biosensing.

Impedimetric biosensors measure changes in the electrical impedance due to a biochemical process, typically the binding of a biomolecule to a bioreceptor on the sensor surface. Nanomaterials can be employed to modify the biosensor's surface to increase the surface area available for biorecognition events, thereby improving the sensitivity and detection limits of the biosensor. Various nanomaterials, such as carbon nanotubes, carbon nanofibers, quantum dots, metal nanoparticles, and graphene oxide nanoparticles, have been investigated for impedimetric biosensors. These nanomaterials have yielded promising results in improving sensitivity, selectivity, and overall biosensor performance. Hence, they offer a wide range of possibilities for developing advanced biosensing platforms that can be employed in various fields, including healthcare, environmental monitoring, and food safety. This review focuses on the recent developments in nanoparticle-functionalized electrochemical-impedimetric biosensors.

Antioxidant Activity of Different Hop (Humulus lupulus L.) Genotypes.

The antioxidant activity (AA) of hop extracts obtained from different hop genotypes (n = 14) was studied. For comparison, the purified ß-acids-rich fraction and α-acids-with-ß-acids-rich fraction were also used to test the antioxidative potential. The AA of purified hydroacetonic hop extracts was investigated using the Ferric Reducing Ability of Plasma (FRAP), Oxygen Radical Absorption Capacity (ORAC) and Intracellular Antioxidant (IA) methods. The FRAP values in different hop genotypes ranged between 63.5 and 101.6 µmol Trolox equivalent (TE)/g dry weight (DW), the ORAC values ranged between 1069 and 1910 µmol TE/g DW and IA potential values ranged between 52.7 and 118.0 mmol TE/g DW. Significant differences in AA between hop genotypes were observed with all three methods. AAs were determined using three different methods, which did not highly correlate with each other. We also did not find significant correlations between AA and different chemical components, which applies both to AA determined using individual methods as well as the total AA. Based on this fact, we assume that the synergistic or antagonistic effects between hop compounds have a more pronounced effect on AA than the presence and quantity of individual hop compounds.

Naive Prediction of Protein Backbone Phi and Psi Dihedral Angles Using Deep Learning.

Protein structure prediction represents a significant challenge in the field of bioinformatics, with the prediction of protein structures using backbone dihedral angles recently achieving significant progress due to the rise of deep neural network research. However, there is a trend in protein structure prediction research to employ increasingly complex neural networks and contributions from multiple models. This study, on the other hand, explores how a single model transparently behaves using sequence data only and what can be expected from the predicted angles. To this end, the current paper presents data acquisition, deep learning model definition, and training toward the final protein backbone angle prediction. The method applies a simple fully connected neural network (FCNN) model that takes only the primary structure of the protein with a sliding window of size 21 as input to predict protein backbone ϕ and ψ dihedral angles. Despite its simplicity, the model shows surprising accuracy for the ϕ angle prediction and somewhat lower accuracy for the ψ angle prediction. Moreover, this study demonstrates that protein secondary structure prediction is also possible with simple neural networks that take in only the protein amino-acid residue sequence, but more complex models are required for higher accuracies.

The Influence of Chestnut Extract and Its Components on Antibacterial Activity against Staphylococcus aureus.

Increasing antimicrobial resistance has caused a great interest in natural products as alternatives or potentiators of antibiotics. The objective of this study was to isolate individual tannins from crude chestnut extract as well as to determine the influence of both crude extracts (tannic acid extract, chestnut extract) and individual pure tannins (gallic acid, vescalin, vescalagin, castalin, castalagin) on the growth of Gram-positive Staphylococcus aureus bacteria. Their antibacterial activity was monitored by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as well as the duration of the lag phase, growth rate and generation time. The effect of growth medium strength on the MIC of different tannins was also investigated. Bacterial growth was followed spectrophotometrically, and MIC values were determined by the microdilution method. The MIC values of various isolated compounds allowed us to determine the bioactive compounds and their contribution to antimicrobial activity. It was found that MIC values increase with increasing growth medium strength and that the lag phase lengthens with increasing tannin concentrations, while the growth rates decrease. Comparing the results of the two studies, the antimicrobial activity of tannins against S. aureus was not as pronounced as in the case of E. coli, which may indicate that a different mechanism of action is responsible for the antimicrobial effects of tannins on Gram-positive than on Gram-negative bacteria, or that a different mechanism is more pronounced.

Design of Tetra-Peptide Ligands of Antibody Fc Regions Using In Silico Combinatorial Library Screening.

Peptides, or short chains of amino-acid residues, are becoming increasingly important as active ingredients of drugs and as crucial probes and/or tools in medical, biotechnological, and pharmaceutical research. Situated at the interface between small molecules and larger macromolecular systems, they pose a difficult challenge for computational methods. We report an in silico peptide library generation and prioritization workflow using CmDock for identifying tetrapeptide ligands that bind to Fc regions of antibodies that is analogous to known in vitro recombinant peptide libraries' display and expression systems. The results of our in silico study are in accordance with existing scientific literature on in vitro peptides that bind to antibody Fc regions. In addition, we postulate an evolving in silico library design workflow that will help circumvent the combinatorial problem of in vitro comprehensive peptide libraries by focusing on peptide subunits that exhibit favorable interaction profiles in initial in silico peptide generation and testing.

Identifying Metal Binding Sites in Proteins Using Homologous Structures, the MADE Approach.

In order to identify the locations of metal ions in the binding sites of proteins, we have developed a method named the MADE (MAcromolecular DEnsity and Structure Analysis) approach. The MADE approach represents an evolution of our previous toolset, the ProBiS H2O (MD) methodology, for the identification of conserved water molecules. Our method uses experimental structures of proteins homologous to a query, which are subsequently superimposed upon it. Areas with a particular species present in a similar location among many homologous protein structures are identified using a clustering algorithm. Dense clusters likely represent positions containing species important to the query protein structure or function. We analyze well-characterized apo protein structures and show that the MADE approach can identify clusters corresponding to the expected positions of metal ions in their binding sites. The greatest advantage of our method lies in its generality. It can in principle be applied to any species found in protein records; it is not only limited to metal ions. We additionally demonstrate that the MADE approach can be successfully applied to predict the location of cofactors in computer-modeled structures, e.g., via AlphaFold. We also conduct a careful protein superposition method comparison and find our methodology robust and the results largely independent of the selected protein superposition algorithm. We postulate that with increasing structural data availability, additional applications of the MADE approach will be possible such as non-protein systems, water network identification, protein binding site elaboration, and analysis of binding events, all in a dynamic manner. We have implemented the MADE approach as a plugin for the PyMOL molecular visualization tool. The MADE plugin is available free of charge at https://gitlab.com/Jukic/made_software.

Fentanyl and the Fluorinated Fentanyl Derivative NFEPP Elicit Distinct Hydrogen-Bond Dynamics of the Opioid Receptor.

The development of safe therapeutics to manage pain is of central interest for biomedical applications. The fluorinated fentanyl derivative N-(3-fluoro-1-phenethylpiperidin-4-yl)-N-phenylpropionamide (NFEPP) is potentially a safer alternative to fentanyl because unlike fentanyl─which binds to the µ-opioid receptor (MOR) at both physiological and acidic pH─NFEPP might bind to the MOR only at acidic pH typical of inflamed tissue. Knowledge of the protonation-coupled dynamics of the receptor-drug interactions is thus required to understand the molecular mechanism by which receptor activation initiates cell signaling to silence pain. To this end, here we have carried out extensive atomistic simulations of the MOR in different protonation states, in the absence of opioid drugs, and in the presence of fentanyl vs NFEPP. We used graph-based analyses to characterize internal hydrogen-bond networks that could contribute to the activation of the MOR. We find that fentanyl and NFEPP prefer distinct binding poses and that, in their binding poses, fentanyl and NFEPP partake in distinct internal hydrogen-bond networks, leading to the cytoplasmic G-protein-binding region. Moreover, the protonation state of functionally important aspartic and histidine side chains impacts hydrogen-bond networks that extend throughout the receptor, such that the ligand-bound MOR presents at its cytoplasmic G-protein-binding side, a hydrogen-bonding environment where dynamics depend on whether fentanyl or NFEPP is bound, and on the protonation state of specific MOR groups. The exquisite sensitivity of the internal protein-water hydrogen-bond network to the protonation state and to details of the drug binding could enable the MOR to elicit distinct pH- and opioid-dependent responses at its cytoplasmic G-protein-binding site.

Helichrysum italicum: From Extraction, Distillation, and Encapsulation Techniques to Beneficial Health Effects.

Helichrysum italicum (family Asteraceae), due to its various beneficial health effects, represents an important plant in the traditional medicine of Mediterranean countries. Currently, there is a renewed interest in this medicinal plant, especially in investigations involving the isolation and identification of its bioactive compounds from extracts and essential oils, as well as in experimental validation of their pharmacological activities. In this paper, we review the current knowledge on the beneficial health effects of Helichrysum italicum extracts, essential oils, and their major bioactive polyphenolic compounds, ranging from antioxidative, anti-inflammatory, and anticarcinogenic activities to their antiviral, antimicrobial, insecticidal, and antiparasitic effects. This review also provides an overview of the most promising extraction and distillation techniques for obtaining high-quality extracts and essential oils from Helichrysum italicum, as well as methods for determining their antioxidative, antimicrobial, anti-inflammatory, and anticarcinogenic activities. Finally, new ideas for in silico studies of molecular mechanisms of bioactive polyphenols from Helichrysum italicum, together with novel suggestions for their improved bioavailability through diverse encapsulation techniques, are introduced.

Mechanistic Insights of Polyphenolic Compounds from Rosemary Bound to Their Protein Targets Obtained by Molecular Dynamics Simulations and Free-Energy Calculations.

Rosemary represents an important medicinal plant that has been attributed with various health-promoting properties, especially antioxidative, anti-inflammatory, and anticarcinogenic activities. Carnosic acid, carnosol, and rosmanol, as well as the phenolic acid ester rosmarinic acid, are the main compounds responsible for these actions. In our earlier research, we carried out an inverse molecular docking at the proteome scale to determine possible protein targets of the mentioned compounds. Here, we subjected the previously identified ligand-protein complexes with HIV-1 protease, K-RAS, and factor X to molecular dynamics simulations coupled with free-energy calculations. We observed that carnosic acid and rosmanol act as viable binders of the HIV-1 protease. In addition, carnosol represents a potential binder of the oncogene protein K-RAS. On the other hand, rosmarinic acid was characterized as a weak binder of factor X. We also emphasized the importance of water-mediated hydrogen-bond networks in stabilizing the binding conformation of the studied polyphenols, as well as in mechanistically explaining their promiscuous nature.

Chemically inducible split protein regulators for mammalian cells.

Chemically inducible systems represent valuable synthetic biology tools that enable the external control of biological processes. However, their translation to therapeutic applications has been limited because of unfavorable ligand characteristics or the immunogenicity of xenogeneic protein domains. To address these issues, we present a strategy for engineering inducible split protein regulators (INSPIRE) in which ligand-binding proteins of human origin are split into two fragments that reassemble in the presence of a cognate physiological ligand or clinically approved drug. We show that the INSPIRE platform can be used for dynamic, orthogonal and multiplex control of gene expression in mammalian cells. Furthermore, we demonstrate the functionality of a glucocorticoid-responsive INSPIRE platform in vivo and apply it for perturbing an endogenous regulatory network. INSPIRE presents a generalizable approach toward designing small-molecule responsive systems that can be implemented for the construction of new sensors, regulatory networks and therapeutic applications.

The Effect of the Ala16Val Mutation on the Secondary Structure of the Manganese Superoxide Dismutase Mitochondrial Targeting Sequence.

Manganese Superoxide Dismutase (MnSOD) represents a mitochondrial protein that scavenges reactive oxygen species (ROS) responsible for oxidative stress. A known single nucleotide polymorphism (SNP) rs4880 on the SOD2 gene, causing a mutation from alanine to valine (Ala16Val) in the primary structure of immature MnSOD, has been associated with several types of cancer and other autoimmune diseases. However, no conclusive correlation has been established yet. This study aims to determine the effect of the alanine to valine mutation on the secondary structure of the MnSOD mitochondrial targeting sequence (MTS). A model for each variant of the MTS was prepared and extensively simulated with molecular dynamics simulations using the CHARMM36m force field. The results indicate that the alanine variant of the MTS preserves a uniform α-helical secondary structure favorable for the protein transport into mitochondria, whereas the valine variant quickly breaks down its α-helix. Thus, the alanine MTS represents the more active MnSOD variant, the benefits of which have yet to be determined experimentally.

Recent Developments in Electrochemical-Impedimetric Biosensors for Virus Detection.

Viruses, including influenza viruses, MERS-CoV (Middle East respiratory syndrome coronavirus), SARS-CoV (severe acute respiratory syndrome coronavirus), HAV (Hepatitis A virus), HBV (Hepatitis B virus), HCV (Hepatitis C virus), HIV (human immunodeficiency virus), EBOV (Ebola virus), ZIKV (Zika virus), and most recently SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), are responsible for many diseases that result in hundreds of thousands of deaths yearly. The ongoing outbreak of the COVID-19 disease has raised a global concern and intensified research on the detection of viruses and virus-related diseases. Novel methods for the sensitive, rapid, and on-site detection of pathogens, such as the recent SARS-CoV-2, are critical for diagnosing and treating infectious diseases before they spread and affect human health worldwide. In this sense, electrochemical impedimetric biosensors could be applied for virus detection on a large scale. This review focuses on the recent developments in electrochemical-impedimetric biosensors for the detection of viruses.

Conserved Water Networks Identification for Drug Design Using Density Clustering Approaches on Positional and Orientational Data.

This work describes the development and testing of a method for the identification and classification of conserved water molecules and their networks from molecular dynamics (MD) simulations. The conserved waters in the active sites of proteins influence protein-ligand binding. Recently, several groups have argued that a water network formed from conserved waters can be used to interpret the thermodynamic signature of the binding site. We implemented a novel methodology in which we apply the complex approach to categorize water molecules extracted from the MD simulation trajectories using clustering approaches. The main advantage of our methodology as compared to current state of the art approaches is the inclusion of the information on the orientation of hydrogen atoms to further inform the clustering algorithm and to classify the conserved waters into different subtypes depending on how strongly certain orientations are preferred. This information is vital for assessing the stability of water networks. The newly developed approach is described in detail as well as validated against known results from the scientific literature including comparisons with the experimental data on thermolysin, thrombin, and Haemophilus influenzae virulence protein SiaP as well as with the previous computational results on thermolysin. We observed excellent agreement with the literature and were also able to provide additional insights into the orientations of the conserved water molecules, highlighting the key interactions which stabilize them. The source code of our approach, as well as the utility tools used for visualization, are freely available on GitHub.

Electrochromic Device Demonstrator from Household Materials.

Electrochromism encompasses reversible changes of material's optical properties (color, opacity) under the influence of an external electric current or applied voltage. The effect has been known for decades, but its importance continues to grow due to the rapid development of smart systems and the accompanying demand to build devices that consume less power. Most commercial electrochromic devices (ECDs) require sophisticated chemicals and advanced material preparation techniques. Also, the demonstration of electrochromism in chemistry classes mainly uses expensive WO3 films, intrinsically conductive polymers, and/or optically transparent electrodes (OTEs). The aim of this article is to present a simple and fast educational method to build ECDs from household materials without the need for OTEs: unsharpened kitchen knives are used as electrodes, curcumin from turmeric is used as the electrochromic dye, and baking soda is used as the electrolyte. The laboratory experiments presented will help students gain a deeper understanding of the fundamentals of electrochemistry (electrolysis, pH change) and electrochromism (in our case, color changes due to pH-induced keto-enol tautomerism of curcumin).