Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose.

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

Inhibitory Effects, Fluorescence Studies, and Molecular Docking Analysis of Some Novel Pyridine-Based Compounds on Mushroom Tyrosinase.

Melanin biosynthesis in different organisms is performed by a tyrosinase action. Excessive enzyme activity and pigment accumulation result in different diseases and disorders including skin cancers, blemishes, and darkening. In fruits and vegetables, it causes unwanted browning of these products and reduces their appearance quality and economic value. Inhibiting enzyme activity and finding novel powerful and safe inhibitors are highly important in agriculture, food, medical, and pharmaceutical industries. In this regard, in the present study, some novel synthetic pyridine-based compounds including 2,6-bis (tosyloxymethyl) pyridine (compound 3), 2,6-bis (butylthiomethyl) pyridine (compound 4), and 2,6-bis (phenylthiomethyl) pyridine (compound 5) were synthesized for the first time, and their inhibitory potencies were assessed on mushroom tyrosinase diphenolase activity. The results showed that while all tested compounds significantly decreased the enzyme activity, compounds 4 and 5 had the highest inhibitory effects (respectively, 80 and 89% inhibition with the IC50 values of 17.0 and 9.0 µmol L-1), and the inhibition mechanism was mixed-type for both compounds. Ligand-binding studies were carried out by fluorescence quenching and molecular docking methods to investigate the enzyme-compound interactions. Fluorescence quenching results revealed that the compounds can form nonfluorescent complexes with the enzyme and result in quenching of its intrinsic emission by the static process. Molecular docking analyses predicted the binding positions and the amino acid residues involved in the interactions. These compounds appear to be suitable candidates for more studies on tyrosinase inhibition.

Synergy of histone acetyltransferase inhibitor (HATi) with quercetin inhibits biofilm formation in Candida tropicalis.

Histone acetyltransferase inhibitors (HATi) are mechanism-based inhibitors that show promise in the treatment of several illnesses, including diabetes, hyperlipidemia, cancer, and Alzheimer's disease. The work emphasizes the significance of HATi as a possible treatment strategy against Candida species biofilms. Here, in this study, we found that combining a HATi, anacardic acid (AA), and quercetin, a known flavonoid, significantly prevented biofilm formation by C. tropicalis. We further show that C. tropicalis exhibited a considerable downregulation of drug-resistance gene expression (CDR1 and MDR1) when co-administrated. Additionally, in silico studies revealed that the AA interacts strongly with a histone acetyltransferase, Rtt109, which may account for the observed biofilm inhibitory effect. In conclusion, the study illustrates how HATi may be used to potentiate the inhibitory action of phytoactives or antifungals against drug-resistant yeast infections.

Phytochemical inhibitors from Leucas aspera against the target proteins induced by Trichophyton mentagrophytes using computational techniques.

Dermatophytosis is a cutaneous infection that is able to degrade the keratinized tissues of the animal/human body, like skin, nails, and hair, causing chronic or subacute infection with the contact of some specific fungal strains. Trichophyton mentagrophytes are the most potential fungal pathogen causing dermatophytoses. The present study focuses on computationally based in silico antifungal activity of selected phytocompounds of Leucas aspera (Willd.) Link. against dermatophytic fungus, T. mentagrophytes. Validation and screening of derived phytocompounds is performed using Lipinski rule of five and toxicity test through Protox-II. Five target genes involved in dermatophytosis, induced by T. mentagrophytes are retrieved from the UniProt Database, and the corresponding proteins such as glucan 1,3-beta-glucosidase ARB_02797, Probable class II chitinase ARB_00204, squalene monooxygenase, actin, and ubiquitin are selected for in silico study. Three-dimensional structures of the target protein were computationally determined and validated through modeling tools and techniques due to the lack of validated protein structures in the database. Then, these proteins are used for in silico molecular docking through the AutoDock Vina tool to find out the promising phytocompounds. This study could be utilized in designing more effective drugs against T. mentagrophytes. Based on this work, a plant-based natural alternative can be added to the treatment of dermatophytosis rather than synthetic supplements.

Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Structural features of Cryptococcus neoformans bifunctional GAR/AIR synthetase may present novel antifungal drug targets.

Cryptococcus neoformans is a fungus that causes life-threatening systemic mycoses. During infection of the human host, this pathogen experiences a major change in the availability of purines; the fungus can scavenge the abundant purines in its environmental niche of pigeon excrement, but must employ de novo biosynthesis in the purine-poor human CNS. Eleven sequential enzymatic steps are required to form the first purine base, IMP, an intermediate in the formation of ATP and GTP. Over the course of evolution, several gene fusion events led to the formation of multifunctional purine biosynthetic enzymes in most organisms, particularly the higher eukaryotes. In C. neoformans, phosphoribosyl-glycinamide synthetase (GARs) and phosphoribosyl-aminoimidazole synthetase (AIRs) are fused into a bifunctional enzyme, while the human ortholog is a trifunctional enzyme that also includes GAR transformylase. Here we functionally, biochemically, and structurally characterized C. neoformans GARs and AIRs to identify drug targetable features. GARs/AIRs are essential for de novo purine production and virulence in a murine inhalation infection model. Characterization of GARs enzymatic functional parameters showed that C. neoformans GARs/AIRs have lower affinity for substrates glycine and PRA compared with the trifunctional metazoan enzyme. The crystal structure of C. neoformans GARs revealed differences in the glycine- and ATP-binding sites compared with the Homo sapiens enzyme, while the crystal structure of AIRs shows high structural similarity compared with its H. sapiens ortholog as a monomer but differences as a dimer. The alterations in functional and structural characteristics between fungal and human enzymes could potentially be exploited for antifungal development.

A Novel Tyrosinase from Armillaria ostoyae with Comparable Monophenolase and Diphenolase Activities Suffers Substrate Inhibition.

Tyrosinase is a bifunctional enzyme mediating the o-hydroxylation and two-electron oxidation of monophenols to o-quinones. The monophenolase activity of tyrosinase is much desired for the industrial synthesis of catechols. However, the generally low ratio of monophenolase/diphenolase activity of tyrosinase limited its utilization in the industry. In this study, a novel tyrosinase from Armillaria ostoyae strain C18/9 (AoTyr) was characterized, and the results showed that the enzyme has an optimal temperature of 25°C and an optimal pH of 6. The enzyme has comparable monophenolase and diphenolase activities and exhibits substrate inhibition in both of the activities. In silico analysis and mutagenesis experiments showed that residues 262 and 266 play important roles in modulating the substrate inhibition and enzymatic activities of AoTyr, and the replacement of D262 with asparagine significantly increased the monophenolase/diphenolase catalytic efficiencies (kcat/Km ratios) (1.63-fold) of the enzyme. The results from this study indicated that this novel tyrosinase could be a potential candidate for the industrial biosynthesis of catechols. IMPORTANCE Tyrosinase is able to oxidize various phenolic compounds, and its ability to convert monophenols into diphenols has caught great attention in the research field and industrial applications. However, the utilization of tyrosinase for the industrial synthesis of catechols has been limited due to the fact that the monophenolase activity of most of the known tyrosinases is much lower than the diphenolase activity. In the present study, a novel tyrosinase with comparable monophenolase and diphenolase activities was characterized. The enzyme exhibits substrate inhibition in both monophenolase and diphenolase activities. In silico analysis followed by mutagenesis experiments confirmed the important roles of residues 262 and 266 in the substrate inhibition and activity modulation of the enzyme, and the D262N variant showed an enhanced monophenolase/diphenolase catalytic efficiency ratio compared to the wild-type enzyme.

Unravelling the antifungal mode of action of curcumin by potential inhibition of CYP51B: A computational study validated in vitro on mucormycosis agent, Rhizopus oryzae.

Like human, fungi too are known to share lot of structural similarities amongst their CYPs (Cytochrome P450 super family of enzymes) which allows antifungal 'azole' compounds to interact with CYPs of human. Clotrimazole, an 'azole' antifungal drug, is a known inhibitor of fungal CYP named CYP51B. Curcumin, a phytochemical obtained from Curcuma longa has the ability to interact with several different human CYPs to induce inhibition. The sequence and the structural similarities amongst both human and fungal CYPs suggest a strong possibility for curcumin to interact with fungal CYP51B to behave like an antifungal agent. To test this hypothesis a study was designed involving mucormycosis agent, Rhizopus oryzae. The ability of curcumin to interact with fungal CYP51B was analysed computationally through molecular docking, MM-GBSA and Molecular Dynamics (MD) simulation assessment. Further, interaction profile for fungal CYP51B-curcumin was compared with human CYP3A4-curcumin, as there are published evidence describing curcumin as an inhibitor of human CYPs. Additionally, to validate in silico findings, an in vitro assay was performed to examine the antifungal potentials of curcumin on the R. oryzae. Conclusive results allow us to determine a plausible mode of action of curcumin to act as an antifungal against a mucormycosis agent.

Exploring the potential of engineering polygalacturonase-inhibiting protein as an ecological, friendly, and nontoxic pest control agent.

In plants, polygalacturonase-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromises plant cell walls and leaves the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1 and BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. We found that tPvPGIP2_5-8, which contains LRR5 to LRR8 and is only one-third the size of the full length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full length PvPGIP2 via Y2H. The inhibitory activities of tPvPGIP2_5-8 on the growth of A. niger and B. cinerea were then examined and confirmed on pectin agar. On pectin assays, application of both full length PvPGIP2 and tPvPGIP2_5-8 clearly slows down the growth of A. niger and B. cinerea. Investigation on the sequence-function relationships of PGIP utilizing a combination of site directed mutagenesis and a variety of peptide truncations suggests that LRR5 could have the most essential structural feature for the inhibitory activities, and may be a possible target for the future engineering of PGIP with enhanced activity. This study highlights the potential of plant-derived PGIPs as a candidate for future in planta evaluation as a pest control agent.

A binding mode hypothesis for prothioconazole binding to CYP51 derived from first principles quantum chemistry.

In order to assess safety and efficacy of small molecule drugs as well as agrochemicals, it is key to understanding the nature of protein-ligand interaction on an atomistic level. Prothioconazole (PTZ), although commonly considered to be an azole-like inhibitor of sterol 14-α demethylase (CYP51), differs from classical azoles with respect to how it binds its target. The available evidence is only indirect, as crystallographic elucidation of CYP51 complexed with PTZ have not yet been successful. We derive a binding mode hypothesis for PTZ binding its target, compare to DPZ, a triazole-type metabolite of PTZ, and set our findings into context of its biochemistry and spectroscopy. Quantum Theory of Atoms in Molecules (QTAIM) analysis of computed DFT electron densities is used to qualitatively understand the topology of binding, revealing significant differences of how R- and S-enantiomers are binding and, in particular, how the thiozolinthione head of PTZ binds to heme compared to DPZ's triazole head. The difference of binding enthalpy is calculated at coupled cluster (DLPNO-CCSD(T)) level of theory, and we find that DPZ binds stronger to CYP51 than PTZ by more than ΔH ~ 11 kcal/mol.

Structural determinants for subnanomolar inhibition of the secreted aspartic protease Sapp1p from Candida parapsilosis.

Pathogenic Candida albicans yeasts frequently cause infections in hospitals. Antifungal drugs lose effectiveness due to other Candida species and resistance. New medications are thus required. Secreted aspartic protease of C. parapsilosis (Sapp1p) is a promising target. We have thus solved the crystal structures of Sapp1p complexed to four peptidomimetic inhibitors. Three potent inhibitors (Ki: 0.1, 0.4, 6.6 nM) resembled pepstatin A (Ki: 0.3 nM), a general aspartic protease inhibitor, in terms of their interactions with Sapp1p. However, the weaker inhibitor (Ki: 14.6 nM) formed fewer nonpolar contacts with Sapp1p, similarly to the smaller HIV protease inhibitor ritonavir (Ki: 1.9 µM), which, moreover, formed fewer H-bonds. The analyses have revealed the structural determinants of the subnanomolar inhibition of C. parapsilosis aspartic protease. Because of the high similarity between Saps from different Candida species, these results can further be used for the design of potent and specific Sap inhibitor-based antimycotic drugs.

Inhibitory Effects of Epipolythiodioxopiperazine Fungal Metabolites on Isocitrate Lyase in the Glyoxylate Cycle of Candida albicans.

Four epipolythiodioxopiperazine fungal metabolites (1-4) isolated from the sponge-derived Aspergillus quadrilineatus FJJ093 were evaluated for their capacity to inhibit isocitrate lyase (ICL) in the glyoxylate cycle of Candida albicans. The structures of these compounds were elucidated using spectroscopic techniques and comparisons with previously reported data. We found secoemestrin C (1) (an epitetrathiodioxopiperazine derivative) to be a potent ICL inhibitor, with an inhibitory concentration of 4.77 ± 0.08 µM. Phenotypic analyses of ICL-deletion mutants via growth assays with acetate as the sole carbon source demonstrated that secoemestrin C (1) inhibited C. albicans ICL. Semi-quantitative reverse-transcription polymerase chain reaction analyses indicated that secoemestrin C (1) inhibits ICL mRNA expression in C. albicans under C2-assimilating conditions.

Commercial AHAS-inhibiting herbicides are promising drug leads for the treatment of human fungal pathogenic infections.

The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (Ki values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates.

Food-Grade Expression and Characterization of a Dextranase from Chaetomium gracile Suitable for Sugarcane Juice Clarification.

The microbial production of dextranase using cheap carbon sources is beneficial to solve the economic loss caused by the accumulation of dextran in syrup. A food-grade microbial cell factory was constructed by introducing the dextranase encoding gene DEX from Chaetomium gracile to the chromosome of Bacillus subtilis, and the antibiotic resistance marker gene was subsequently deleted via the Cre/loxP strategy. The dual-promoter system with a sequentially arranged constitutive P43 promoter resulted in an 85 % increase in DEX expression. Under the optimal fermentation conditions of 10 g/L maltose, 15 g/L casein, 1 g/L Na2 HPO4 , 1 g/L FeSO4 and 8 g/L NaCl, DEX activity was increased from 2.625 to 64.34 U/mL. Recombinant DEX was purified 5.98-fold with a recovery ratio of 26.67 % and specific activity of 3935.02 U/mg. Enzyme activity was optimal at 55 °C and pH 5.0 and remained 80.34 % and 71.36 % of the initial activity at 55 °C and pH 4.0 after 60 min, respectively. The enzyme possessed high activity in the presence of Co2+ , while Ag+ showed the strongest inhibition ability. The optimal substrate was 20 g/L dextran T-2000. The findings could facilitate the low-cost, large-scale production of food-grade DEX for use in the sugar industry.

Urolithin and Reduced Urolithin Derivatives as Potent Inhibitors of Tyrosinase and Melanogenesis: Importance of the 4-Substituted Resorcinol Moiety.

We previously reported (E)-ß-phenyl-α,ß-unsaturated carbonyl scaffold ((E)-PUSC) played an important role in showing high tyrosinase inhibitory activity and that derivatives with a 4-substituted resorcinol moiety as the ß-phenyl group of the scaffold resulted in the greatest tyrosinase inhibitory activity. To examine whether the 4-substituted resorcinol moiety could impart tyrosinase inhibitory activity in the absence of the α,ß-unsaturated carbonyl moiety of the (E)-PUSC scaffold, 10 urolithin derivatives were synthesized. To obtain more candidate samples, the lactone ring in synthesized urolithins was reduced to produce nine reduced urolithins. Compounds 1c (IC50 = 18.09 ± 0.25 µM), 1h (IC50 = 4.14 ± 0.10 µM), and 2a (IC50 = 15.69 ± 0.40 µM) had greater mushroom tyrosinase-inhibitory activities than kojic acid (KA) (IC50 = 48.62 ± 3.38 µM). The SAR results suggest that the 4-substituted resorcinol motif makes an important contribution to tyrosinase inhibition. To investigate whether these compounds bind to human tyrosinase, a human tyrosinase homology model was developed. Docking simulations with mushroom and human tyrosinases showed that 1c, 1h, and 2a bind to the active site of both tyrosinases with higher binding affinities than KA. Pharmacophore analyses showed that two hydroxyl groups of the 4-substituted resorcinol entity act as hydrogen bond donors in both mushroom and human tyrosinases. Kinetic analyses indicated that these compounds were all competitive inhibitors. Compound 2a inhibited cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells more strongly than KA. These results suggest that 2a is a promising candidate for the treatment of skin pigment disorders, and show the 4-substituted resorcinol entity importantly contributes to tyrosinase inhibition.

Inhibitory Potential of New Phenolic Hydrazide-Hydrazones with a Decoy Substrate Fragment towards Laccase from a Phytopathogenic Fungus: SAR and Molecular Docking Studies.

Laccase from pathogenic fungi participates in both the delignification and neutralization of phytoantibiotics. Furthermore, it interferes with the hormone signaling in plants and catalyzes melanization. Infections of these pathogens contribute to loss in forestry, agriculture, and horticulture. As there is still a need to expand knowledge on efficient defense strategies against phytopathogenic fungi, the present study aimed to reveal more information on the molecular mechanisms of laccase inhibition with natural and natural-like carboxylic acid semi-synthetic derivatives. A set of hydrazide-hydrazones derived from carboxylic acids, generally including electron-rich arene units that serve as a decoy substrate, was synthesized and tested with laccase from Trametes versicolor. The classic synthesis of the title inhibitors proceeded with good to almost quantitative yield. Ninety percent of the tested molecules were active in the range of KI = 8-233 µM and showed different types of action. Such magnitude of inhibition constants qualified the hydrazide-hydrazones as strong laccase inhibitors. Molecular docking studies supporting the experimental data explained the selected derivatives' interactions with the enzyme. The results are promising in developing new potential antifungal agents mitigating the damage scale in the plant cultivation, gardening, and horticulture sectors.

Discovery of Octahydroisoindolone as a Scaffold for the Selective Inhibition of Chitinase B1 from Aspergillus fumigatus: In Silico Drug Design Studies.

Chitinases represent an alternative therapeutic target for opportunistic invasive mycosis since they are necessary for fungal cell wall remodeling. This study presents the design of new chitinase inhibitors from a known hydrolysis intermediate. Firstly, a bioinformatic analysis of Aspergillus fumigatus chitinase B1 (AfChiB1) and chitotriosidase (CHIT1) by length and conservation was done to obtain consensus sequences, and molecular homology models of fungi and human chitinases were built to determine their structural differences. We explored the octahydroisoindolone scaffold as a potential new antifungal series by means of its structural and electronic features. Therefore, we evaluated several synthesis-safe octahydroisoindolone derivatives by molecular docking and evaluated their AfChiB1 interaction profile. Additionally, compounds with the best interaction profile (1-5) were docked within the CHIT1 catalytic site to evaluate their selectivity over AfChiB1. Furthermore, we considered the interaction energy (MolDock score) and a lipophilic parameter (aLogP) for the selection of the best candidates. Based on these descriptors, we constructed a mathematical model for the IC50 prediction of our candidates (60-200 µM), using experimental known inhibitors of AfChiB1. As a final step, ADME characteristics were obtained for all the candidates, showing that 5 is our best designed hit, which possesses the best pharmacodynamic and pharmacokinetic character.

Health Potential of Clery Strawberries: Enzymatic Inhibition and Anti-Candida Activity Evaluation.

Strawberries, belonging to cultivar Clery (Fragaria × ananassa Duchesne ex Weston) and to a graft obtained by crossing Clery and Fragaria vesca L., were chosen for a study on their health potential, with regard to the prevention of chronic and degenerative diseases. Selected samples, coming from fresh and defrosted berries, submitted to different homogenization techniques combined with thermal and microwave treatments, had been previously analyzed in their polyphenolic content and antioxidant capacity. In the present work, these homogenates were evaluated in relation to their enzymatic inhibition activity towards acetylcholinesterase and butyrylcholinesterase, α-amylase, α-glucosidase and tyrosinase. All these enzymes, involved in the onset of diabetes, and neurodegenerative and other chronic diseases, were modulated by the tested samples. The inhibitory effect on tyrosinase and cholinesterase was the most valuable. Antifungal activity against Candida albicans, recently shown to play a crucial role in human gut diseases as well as diabetes, rheumatoid arthritis and Alzheimer's disease, was also shown in vitro and confirmed by the in vivo text on Galleria mellonella. Overall, the obtained results confirm once again the health potential of strawberries; however, the efficacy is dependent on high quality products submitted to correct processing flow charts.

The Short-Term Exposure to SDHI Fungicides Boscalid and Bixafen Induces a Mitochondrial Dysfunction in Selective Human Cell Lines.

Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.

Inhibitory Activity and Mechanism Investigation of Hypericin as a Novel α-Glucosidase Inhibitor.

α-glucosidase is a major enzyme that is involved in starch digestion and type 2 diabetes mellitus. In this study, the inhibition of hypericin by α-glucosidase and its mechanism were firstly investigated using enzyme kinetics analysis, real-time interaction analysis between hypericin and α-glucosidase by surface plasmon resonance (SPR), and molecular docking simulation. The results showed that hypericin was a high potential reversible and competitive α-glucosidase inhibitor, with a maximum half inhibitory concentration (IC50) of 4.66 ± 0.27 mg/L. The binding affinities of hypericin with α-glucosidase were assessed using an SPR detection system, which indicated that these were strong and fast, with balances dissociation constant (KD) values of 6.56 × 10-5 M and exhibited a slow dissociation reaction. Analysis by molecular docking further revealed that hydrophobic forces are generated by interactions between hypericin and amino acid residues Arg-315 and Tyr-316. In addition, hydrogen bonding occurred between hypericin and α-glucosidase amino acid residues Lys-156, Ser-157, Gly-160, Ser-240, His-280, Asp-242, and Asp-307. The structure and micro-environment of α-glucosidase enzymes were altered, which led to a decrease in α-glucosidase activity. This research identified that hypericin, an anthracene ketone compound, could be a novel α-glucosidase inhibitor and further applied to the development of potential anti-diabetic drugs.