A natural inhibitor of diapophytoene desaturase attenuates methicillin-resistant Staphylococcus aureus (MRSA) pathogenicity and overcomes drug-resistance.

BACKGROUND AND PURPOSE: At present, the inhibition of staphyloxanthin biosynthesis has emerged as a prominent strategy in combating methicillin-resistant Staphylococcus aureus (MRSA) infection. Nonetheless, there remains a limited understanding regarding the bio-structural characteristics of staphyloxanthin biosynthetic enzymes, as well as the molecular mechanisms underlying the interaction between inhibitors and proteins. Furthermore, the functional scope of these inhibitors is relatively narrow. EXPERIMENTAL APPROACH: In this study, we address these limitations by harnessing the power of deep learning techniques to construct the 3D structure of diapophytoene desaturase (CrtN). We perform efficient virtual screening and unveil alnustone as a potent inhibitor of CrtN. Further investigations employing molecular modelling, site-directed mutagenesis and biolayer interferometry (BLI) confirmed that alnustone binds to the catalytic active site of CrtN. Transcriptomic analysis reveals that alnustone significantly down-regulates genes associated with staphyloxanthin, histidine and peptidoglycan biosynthesis. KEY RESULTS: Under the effects of alnustone, MRSA strains exhibit enhanced sensitivity to various antibiotics and the host immune system, accompanied by increased cell membrane permeability. In a mouse model of systemic MRSA infection, the combination of alnustone and antibiotics exhibited a significant therapeutic effect, leading to reduced bacterial colony counts and attenuated pathological damage. CONCLUSION AND IMPLICATIONS: Alnustone, as a natural inhibitor targeting CrtN, exhibits outstanding antibacterial properties that are single-targeted yet multifunctional. This finding provides a novel strategy and theoretical basis for the development of drugs targeting staphyloxanthin producing bacteria.

Study transport of hesperidin based on the DPPC lipid model and the BSA transport model.

Hesperidin (HE), a significant flavonoid polyphenolic compound present in citrus plants, exhibits diverse pharmacological effects. Considering the crucial involvement of biological membranes and transporter proteins in the transportation and biological processes of HE, it becomes essential to comprehend the potential mechanisms through which HE interacts with membranes and transporter proteins. In order to simulate the process of active molecule transport, a cell membrane model consisting of 1,2-dipalmitoyl-n-glycero-3-phosphatidylcholine (DPPC) and a transporter protein model of bovine serum albumin (BSA) were employed for investigation. The present study aimed to investigate the mechanism of action of hesperidin (HE) in DPPC and BSA using fluorescence quenching, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The localization and interaction of HE within liposomes were also elucidated. Furthermore, the binding of BSA and HE was analyzed through UV/Vis absorption spectroscopy, fluorescence spectroscopy, infrared spectroscopy, and computational biology techniques. Computational biology analysis revealed that the binding between HE and BSA primarily occurred via hydrogen bonding and hydrophobic interactions. This study aimed to investigate the role and mechanism of HE in the DPPC cell membrane model and the BSA transporter protein model, thereby offering novel insights into the action of HE in DPPC and BSA.

A hydrogen-bonded curdlan-chitosan/polyvinyl alcohol edible dual functional hydrogel bandage against MRSA promotes wound healing.

The most prevalent complication arising from skin injuries is bacterial infection, where pathogenic bacteria proliferate significantly at the wound site, leading to subsequent complications like septic shock and sepsis. Although antibiotics presently effectively manage wound infections caused by common bacteria, the escalating prevalence of antibiotic-resistant strains necessitates urgent novel approaches for addressing such infections. Here, we present CS9P1-RA, a dual functional hydrogel dressing, based on polyvinyl alcohol (PVA) matrix crosslinked through hydrogen bonding. CS9P1-RA combines chitosan (CS), a food-derived antibacterial agent, with the natural compound rosmarinic acid (RA) to specifically target skin injuries caused by MRSA. Computational and molecular biology assays illustrate RA's ability to selectively inhibit the activity of Staphylococcus aureus (S. aureus) serine/threonine phosphatase (Stp1), reducing the S. aureus pathogenicity. CS9P1-RA showcases exceptional antibacterial efficacy (MIC = 1 mg/mL) and demonstrates potency in reducing virulence (IC50 = 7.424 µM on Stp1). Notably, it effectively curbs bacterial growth and accelerates wound healing in the mice model, thereby fulfilling the practical requirements for clinical applications. Moreover, the mechanical properties of CS9P1-RA ensure user comfort during treatment. This work introduces a fresh design paradigm for dressing materials, offering a promising solution for treating skin injuries inflicted by antibiotic-resistant bacterial infections.

Novel metallo-ß-lactamases inhibitors restore the susceptibility of carbapenems to New Delhi metallo-lactamase-1 (NDM-1)-harbouring bacteria.

BACKGROUND AND PURPOSE: The production of metallo-ß-lactamases is a major mechanisms adopted by bacterial pathogens to resist carbapenems. Repurposing approved drugs to restore the efficacy of carbapenems represents an efficient and cost-effective approach to fight infections caused by carbapenem resistant pathogens. EXPERIMENTAL APPROACH: The nitrocefin hydrolysis assay was employed to screen potential New Delhi metallo-lactamase-1 (NDM-1) inhibitors from a commercially available U.S. Food and Drug Administration (FDA) approved drug library. The mechanism of inhibition was clarified by metal restoration, inductively coupled plasma mass spectrometry (ICP-MS) and molecular dynamics simulation. The in vitro synergistic antibacterial effect of the identified inhibitors with meropenem was determined by the checkerboard minimum inhibitory concentration (MIC) assay, time-dependent killing assay and combined disc test. Three mouse infection models were used to further evaluate the in vivo therapeutic efficacy of combined therapy. KEY RESULTS: Twelve FDA-approved compounds were initially screened to inhibit the ability of NDM-1 to hydrolyse nitrocefin. Among these compounds, dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid were demonstrated to inhibit all tested metallo-ß-lactamases and showed an in vitro synergistic bactericidal effect with meropenem against metallo-ß-lactamases-producing bacteria. Dexrazoxane, embelin and candesartan cilexetil are metal ion chelating agents, while the inhibition of NDM-1 by nordihydroguaiaretic acid involves its direct binding to the active region of NDM-1. Furthermore, these four drugs dramatically rescued the treatment efficacy of meropenem in three infection models. CONCLUSIONS AND IMPLICATIONS: Our observations indicated that dexrazoxane, embelin, candesartan cilexetil and nordihydroguaiaretic acid are promising carbapenem adjuvants against metallo-ß-lactamases-positive carbapenem resistant bacterial pathogens.

Dibenzylideneacetone Overcomes Botrytis cinerea Infection in Cherry Tomatoes by Inhibiting Chitinase Activity.

Chitinase, a crucial component of the fungal cell wall and septa, plays an important role in fungal germination by hydrolyzing chitin to provide carbon and energy for fungal growth and reproduction. In this study, we initially screened dibenzylideneacetone (DBA), a small molecule with inhibitory activity against Botrytis cinerea Chitinase, exhibiting an IC50 of 13.10 µg/mL. By constructing a three-dimensional (3D) model of the B. cinerea Chitinase and utilizing computational biology approaches, we found DBA bound to the active site pocket and formed strong π-π interactions and hydrophobic interactions with Chitinase, indicative of its competitive inhibitory mode. Site-directed mutagenesis also revealed that TRP-382, TRP-135, and ALA-215 were key amino acid residues involved in DBA binding. Subsequent antifungal assays showed that DBA had an MIC of 32 µg/mL against B. cinerea and EC50 values of 16.29 and 14.64 µg/mL in inhibiting mycelial growth and spore germination, respectively. Importantly, in vivo experiments demonstrated that DBA treatment significantly extended the shelf life of cherry tomatoes by 2-fold. Therefore, DBA represents a promising antifungal agent for fruit preservation applications.

Electron coupling effect-triggered monatomic copper laccase-mimicking nanozyme for the degradation and detection of guaiacol produced by Alicyclobacillus acidoterrestris.

The generation of guaiacol by Alicyclobacillus acidoterrestris (A. acidoterrestris) in fruit juices negatively affects public health and causes severe environmental pollution. Therefore, the sensitive detection and efficient degradation of guaiacol in real samples are crucial. Here, we develop an electrochemical sensor utilizing a copper single-atom nanozyme (CuN4-G) to detect and degrade guaiacol at the picomolar level. Density functional theory (DFT) calculations verify that the bonding electron coupling effect in the CuN4-G facilitates rapid electron transfer, enhances electrical conductivity, and provides abundant active sites, thereby leading to exceptional catalytic performance. Moreover, CuN4-G demonstrates a Km value similar to that of natural laccase but a higher Vmax, highlighting its potential as a highly efficient biocatalyst. The CuN4-G-based electrochemical sensor achieves a detection from 5 to 50,000 pM for guaiacol, with a 1.2 pM (S/N = 3) detection limit. Additionally, CuN4-G-modified electrodes display high selectivity and excellent stability. CuN4-G nanozyme can keep its activity in conditions of pH (3-9), temperature (30-90 °C), ionic strength (0-400 mM), and organic solvent (0-50% (v/v)), overcoming the deficiencies of natural enzymes. Furthermore, our electrochemical sensor can not only accurately detect guaiacol, but also degrade it in actual fruit juice samples infected by A. acidoterrestris, demonstrating its potential applications in food and environmental monitoring.

Molecular mechanism of green tea polyphenol epicatechin gallate attenuating Staphylococcus aureus pathogenicity by targeting Ser/Thr phosphatase Stp1.

In this study, through virtual screening and in vitro bioactivity assays, we discovered that (-)-epicatechin gallate (ECG), a polyphenol compound extracted from green tea, demonstrated marked anti-Ser/Thr phosphatase (Stp1) activity towards Staphylococcus aureus (S. aureus) with an IC50 value of 8.35 µM. By targeting S. aureus Stp1, ECG prevented the up-regulation of virulence gene and the formation of antibody membrane and protected the mice from S. aureus infection. Through MD simulation, the allosteric inhibitory mechanism of ECG on Stp1 was determined. The Stp1-ECG complex model underwent a significant change in conformation; its flap subdomain changed from opening to closing, whereas Stp1 activity was lost when bound to ECG. In addition, the MD simulation results of Stp1 and several tea polyphenol compounds showed that gallate groups and fewer adjacent phenolic hydroxyl groups contributed to the binding of Stp1 and inhibitors. As an inhibitor targeting S. aureus Stp1, ECG reduced the pathogenicity of S. aureus without inhibiting S. aureus, which largely reduced the possibility of drug resistance. Our findings demonstrated a novel molecular mechanism of green tea as the usual drink against S. aureus infection and elucidated the future design of allosteric inhibitors targeting Stp1.

Colorimetric sensor array based on Au2Pt nanozymes for antioxidant nutrition quality evaluation in food.

Total antioxidant capacity (TAC) has become an important index to evaluate the food quality. Effective antioxidant detection has been the research hotspot of scientists. In this work, a novel three-channel colorimetric sensor array founded on Au2Pt bimetallic nanozymes for the discrimination of antioxidants in food was constructed. Benefiting from the unique bimetallic doping structure, Au2Pt nanospheres exhibited the excellent peroxidase-like activity with Km of 0.044 mM and Vmax of 19.37 × 10-8 M s-1 toward TMB. The density functional theory (DFT) calculation revealed that Pt atom in the doping system was active sites and there was no energy barrier in catalytic reaction which made Au2Pt nanospheres had excellent catalytic activity. Accordingly, a multifunctional colorimetric sensor array was constructed based on Au2Pt bimetallic nanozymes for rapid and sensitive detection of five antioxidants. Based on the different reduction ability of antioxidants, oxidized TMB could be reduced in different degrees. In the presence of H2O2, the colorimetric sensor array could generate differential colorimetric signals (fingerprints) by using TMB as the chromogenic substrate, which could be accurately discriminated through linear discriminant analysis (LDA) with a detection limit of <0.2 µM. The sensor array was able to the evaluate TAC in three actual samples (milk, green tea and orange juice). Furthermore, we prepared a rapid detection strip to meet the needs of practical application, making a positive contribution to food quality evaluation.

Nanozyme colorimetric sensor array based on monatomic cobalt for the discrimination of sulfur-containing metal salts.

The identification of sulfur-containing metal salts (SCMs) is of great interest because they play an important role in many biological processes and diseases. Here, we constructed a ternary channel colorimetric sensor array to detect multiple SCMs simultaneously, relying on monatomic Co embedded in nitrogen-doped graphene nanozyme (CoN4-G). Due to the unique structure, CoN4-G exhibits activity similar to native oxidases, capable of catalysing directly the oxidization of 3,3',5,5'-tetramethylbenzidine (TMB) by O2 molecules independent of H2O2. Density functional theory (DFT) calculations suggest that CoN4-G has no potential barrier in the whole reaction route, thus presenting higher oxidase-like catalytic activity. Based on different degrees of TMB oxidation, different colorimetric response changes are obtained as "fingerprints" on the sensor array. The sensor array can discriminate different concentrations of unitary, binary, ternary, and quaternary SCMs and has been successfully applied to detect six real samples (soil, milk, red wine and egg white). To advance the field detection of the above four types of SCMs, we creatively propose a smartphone-based autonomous detection platform with a linear range of 1.6-320 µM and a limit of detection of 0.0778-0.218 µM, which demonstrates the potential use of sensor arrays in the application of disease diagnosis and food and environment monitoring.

Narirutin activates TFEB (transcription factor EB) to protect against Acetaminophen-induced liver injury by targeting PPP3/calcineurin.

Acetaminophen (APAP) overdose is the predominant cause of drug-induced liver injury worldwide. The macroautophagy/autophagy-lysosomal pathway (ALP) is involved in the APAP hepatotoxicity. TFEB (transcription factor EB) promotes the expression of genes related to autophagy and lysosomal biogenesis, thus, pharmacological activation of TFEB-mediated ALP may be an effective therapeutic approach for treating APAP-induced liver injury. We aimed to reveal the effects of narirutin (NR), the main bioactive constituents isolated from citrus peels, on APAP hepatotoxicity and to explore its underlying mechanism. Administration of NR enhanced activities of antioxidant enzymes, improved mitochondrial dysfunction and alleviated liver injury in APAP-treated mice, whereas NR did not affect APAP metabolism and MAPK/JNK activation. NR enhanced TFEB transcriptional activity and activated ALP in an MTOR complex 1 (MTORC1)-independent but PPP3/calcineurin-dependent manner. Moreover, knockout of Tfeb or knockdown of PPP3CB/CNA2 (protein phosphatase 3, catalytic subunit, beta isoform) in the liver abolished the beneficial effects of NR on APAP overdose. Mechanistically, NR bound to PPP3CB via PRO31, LYS61 and PRO347 residues and enhanced PPP3/calcineurin activity, thereby eliciting dephosphorylation of TFEB and promoting ALP, which alleviated APAP-induced oxidative stress and liver injury. Together, NR protects against APAP-induced liver injury by activating a PPP3/calcineurin-TFEB-ALP axis, indicating NR may be a potential agent for treating APAP overdose.Abbreviations: ALP: autophagy-lysosomal pathway; APAP: acetaminophen; APAP-AD: APAP-protein adducts; APAP-Cys: acetaminophen-cysteine adducts; CAT: catalase; CETSA: cellular thermal shift assay; CQ: chloroquine; CYP2E1: cytochrome P450, family 2, subfamily e, polypeptide 1; CYCS/Cyt c: cytochrome c, somatic; DARTS: drug affinity responsive target stability assay; ENGASE/NAG: endo-beta-N-acetylglucosaminidase; GOT1/AST: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALT: glutamic pyruvic transaminase, soluble; GSH: glutathione; GPX/GSH-Px: glutathione peroxidase; KD: dissociation constant; Leu: leupeptin; MCOLN1: mucolipin 1; MTORC1: MTOR complex 1; NAC: N-acetylcysteine; NAPQI: N-acetyl-p-benzoquinoneimine; NFAT: nuclear factor of activated T cells; NR: narirutin; OA: okadaic acid; RRAG: Ras related GTP binding; ROS: reactive oxygen species; PPP3CB/CNA2: protein phosphatase 3, catalytic subunit, beta isoform; PPP3R1/CNB1: protein phosphatase 3, regulatory subunit B, alpha isoform (calcineurin B, type I); SOD: superoxide dismutase; SPR: surface plasmon resonance analysis; TFEB: transcription factor EB.

Preparation of a novel glucose oxidase-N-succinyl chitosan nanospheres and its antifungal mechanism of action against Colletotrichum gloeosporioides.

In this work, a new glucose oxidase-N-succinyl chitosan (GOD-NSCS) nanospheres was prepared through the immobilization of glucose oxidase (GOD) on N-succinyl chitosan (NSCS) nanospheres. Compared to the free GOD, GOD-NSCS nanospheres demonstrated the excellent anti-Colletotrichum gloeosporioides activity with the EC50 values of 211.2 and 10.7 µg/mL against mycelial growth and spores germination. The computational biology analysis demonstrated that the substrate presented the similar binding free energy with GOD-NSCS nanospheres (-27.64 kcal/mol) compared with the free GOD (-24.04 kcal/mol), indicating that GOD-NSCS nanospheres had the same oxidation efficiency and produced more H2O2. Moreover, the enzyme activity stability of GOD-NSCS nanospheres could be prolonged to 10 d. The cell membrane was destructed by the treatment of H2O2 produced by GOD, leading to the cell death. In vivo test, GOD-NSCS nanospheres treatment significantly prolonged the preservation period of mangoes 2-fold. Collectively, these results suggested that GOD-NSCS nanospheres suppresses anthracnose in postharvest mangoes by inhibiting the growth of C. gloeosporioides and might become a potential natural preservative for fruits and vegetables.

Unveiling the role of defects in iron oxyhydroxide for oxygen evolution.

Development of earth-abundant and robust oxygen evolution reaction (OER) catalysts is imperative for cost-effective hydrogen production via water electrolysis. Herein, we report ultrafine iron (oxy)hydroxide nanoparticles with average particle size of 2.6 nm and abundant surface defects homogeneously supported on oleum-treated graphite (FeOx(n)@HG-T), providing abundant active sites for the OER. The optimal FeOx(0.03)@HG-110 exhibits high electrocatalytic OER activity and excellent stability. Electrochemical testing results and theoretical calculations reveal that the outstanding OER activity of FeOx(0.03)@HG-110 is due to its stronger charge transfer ability and lower OER energy barrier than defect-free FeOx nanoparticles. This work demonstrates that the OER performance of oxyhydroxide-based electrocatalysts can be improved by surface defect engineering.

Electrochemical sensor for detecting streptomycin in milk based on label-free aptamer chain and magnetic adsorption.

Exploiting a simple and sensitive sensor to efficiently detect streptomycin (STR) in milk is critical for resolving the harm caused to humans by STR residues. This study reports an electrochemical sensor using magnetic mesoporous carbon materials (MMCM) as a loaded material through magnetic adsorption immobilized on magnetic glassy carbon electrode (MGCE) and adsorbing unlabeled streptomycin aptamer (STP) as the identification element. The sensor can detect STR sensitively with a wide detection range (0.172-17.2 × 103 and 17.2 × 103 -17.2 × 105 nM) and a low detection limit of 0.015 nM. Experimental results revealed that the specific binding of STP with STR on the electrode changed the configuration of STP, thereby causing current change of differential pulse voltammetry curve. Compared with HPLC, this study provides a new method for rapid and sensitive detection of STR in milk (n = 5, 95 % confidence level, RSD＜5%).

Piceatannol Alleviates Clostridium perfringens Virulence by Inhibiting Perfringolysin O.

Clostridium perfringens (C. perfringens) is an important foodborne pathogen that can cause diseases such as gas gangrene and necrotizing enteritis in a variety of economic animals, seriously affecting public health and the economic benefits and healthy development of the livestock and poultry breeding industry. Perfringolysin O (PFO) is an important virulence factor of C. perfringens and plays critical roles in necrotic enteritis and gas gangrene, rendering it an ideal target for developing new drugs against infections caused by this pathogen. In this study, based on biological activity inhibition assays, oligomerization tests and computational biology assays, we found that the foodborne natural component piceatannol reduced pore-forming activity with an inhibitory ratio of 83.84% in the concentration of 16 µg/mL (IC50 = 7.83 µg/mL) by binding with PFO directly and changing some of its secondary structures, including 3-Helix, A-helix, bend, and in turn, ultimately affecting oligomer formation. Furthermore, we confirmed that piceatannol protected human intestinal epithelial cells from the damage induced by PFO with LDH release reduced by 38.44% at 16 µg/mL, based on a cytotoxicity test. By performing an animal experiment, we found the C. perfringens clones showed an approximate 10-fold reduction in infected mice. These results suggest that piceatannol may be a candidate for anti-C. perfringens drug development.

Nuciferine protects against high-fat diet-induced hepatic steatosis and insulin resistance via activating TFEB-mediated autophagy-lysosomal pathway.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment. Hyperactivation of mTOR complex 1 (mTORC1) and subsequent impairment of the transcription factor EB (TFEB)-mediated autophagy-lysosomal pathway (ALP) are implicated in the development of NAFLD. Accordingly, agents that augment hepatic TFEB transcriptional activity may have therapeutic potential against NAFLD. The objective of this study was to investigate the effects of nuciferine, a major active component from lotus leaf, on NAFLD and its underlying mechanism of action. Here we show that nuciferine activated ALP and alleviated steatosis, insulin resistance in the livers of NAFLD mice and palmitic acid-challenged hepatocytes in a TFEB-dependent manner. Mechanistic investigation revealed that nuciferine interacts with the Ragulator subunit hepatitis B X-interacting protein and impairs the interaction of the Ragulator complex with Rag GTPases, thereby suppressing lysosomal localization and activity of mTORC1, which activates TFEB-mediated ALP and further ameliorates hepatic steatosis and insulin resistance. Our present results indicate that nuciferine may be a potential agent for treating NAFLD and that regulation of the mTORC1-TFEB-ALP axis could represent a novel pharmacological strategy to combat NAFLD.

Antifungal activity and molecular mechanisms of mulberrin derivatives against Colletotrichum gloeosporioides for mango storage.

In this work, by using high throughput virtual screening and bioactivity assays, this work revealed that three natural compounds, mulberrin (Mul) exhibiting the highest anti-CYP51 activity, isoxanthohumol and (s)-isopsoralen markedly inhibited 14α-demethylase (a pivotal biosynthetic enzyme involved in the biosynthesis of ergosterol) in Colletotrichum gloeosporioides. Results of computational biology analysis demonstrated that, among the three inhibitors bound to the catalytic pocket of CYP51, Mul showed a closer distance with heme in CYP51 and a stronger binding free energy with CYP51. In vitro tests, Mul demonstrated excellent anti-Colletotrichum gloeosporioides activity by inhibiting CYP51 activity. Notably, Mul treatment decreased the bioactivity of CYP51, thereby increasing cell membrane permeability and cell death. Moreover, Mul treatment significantly prolonged the preservation period of fruits. These results suggest that Mul suppresses anthracnose in postharvest mango by inhibiting the growth of Colletotrichum gloeosporioides and can be used as a potential natural preserving agent.

Luteolin binds Src, promotes STAT3 protein ubiquitination and exerts anti-melanoma effects in cell and mouse models.

Signal transducer and activator of transcription 3 (STAT3) has been proposed as a target for melanoma prevention. Luteolin, a bioactive flavonoid abundant inmedicinal herbs, has been reported to have anti-melanoma activity in vitro. However, its in vivo anti-melanoma effects and underlying mechanisms have not been fully elucidated. In this study, ten cell lines and two mouse models (B16F10 allograft and A375 xenograft models) were used for assessing the in vitro and in vivo anti-melanoma effects of luteolin. A STAT3 over-activated stable A375 cell line was used to determine the contribution of STAT3 signaling in luteolin's anti-melanoma effects. Results showed that luteolin dose-dependently reduced viability of melanoma cells. Luteolin also induced apoptosis in, and suppressed migration and invasion of, A375 and B16F10 melanoma cells. Mechanistically, luteolin inhibited phosphorylation of STAT3 and Src (an upstream kinase of STAT3), accelerated ubiquitin-proteasome pathway-mediated STAT3 degradation, and downregulated the expression of STAT3-targeted genes involved in cell survival and invasion in melanoma cells. Molecular modelling and surface plasmon resonance imaging showed that luteolin stably bound to the protein kinase domain of Src. Animal studies demonstrated that prophylactic administration of luteolin restrained melanoma growth and Src/STAT3 signaling in both A375 and B16F10 melanoma-bearing mice. Moreover, luteolin's anti-melanoma effects were diminished by STAT3 over-activation in A375 cells. Our findings indicate that luteolin inhibits STAT3 signaling by suppressing STAT3 activation and promoting STAT3 protein degradation in melanoma cells, thereby exhibiting anti-melanoma effects. This study provides further pharmacological groundwork for developing luteolin as a chemopreventive agent against melanoma.

Mulberrin inhibits Botrytis cinerea for strawberry storage by interfering with the bioactivity of 14α-demethylase (CYP51).

Currently, chemical agents hold great promise in preventing and combating Botrytis cinerea. However, the antifungal mechanism of some agents for B. cinerea remains rather vague, imposing restrictions on the research and development of novel antifungal inhibitors. In this work, we discovered that mulberrin (MBN), a natural compound from the root bark of Ramulus Mori, with an IC50 of 1.38 µM together, demonstrated marked anti-14α-demethylase (CYP51) activity through high throughput virtual screening and in vitro bioactivity assay. The computational biology results demonstrated that MBN and its derivatives were bound to the catalytic activity region of CYP51, but only MBN could form a strong π-cation interaction with the Fe ion of heme in CYP51 via the 2-methylpent-2-ene moiety at atom C9. MBN had a stronger binding free energy than the other three compounds with CYP51, implying that the 2-methylpent-2-ene moiety at atom C9 is a critical pharmacophore for CYP51 inhibitors. Subsequently, through an antifungal test, MBN demonstrated excellent anti-B. cinerea activity by inhibiting CYP51 activity. The EC50 values of MBN toward hyphal growth and spore germination in B. cinerea were 17.27 and 9.56 µg mL-1, respectively. The bioactivity loss of CYP51 by direct interaction with MBN induced the increase of cell membrane permeability, membrane destruction, and cell death. Meanwhile, in the B. cinerea infection model, MBN significantly prolonged the preservation of strawberries by preventing B. cinerea from infecting strawberries and could be used as a potential natural preserving agent for storing fruits.

A novel inhibitor of monooxygenase reversed the activity of tetracyclines against tet(X3)/tet(X4)-positive bacteria.

BACKGROUND: Tigecycline is one of the few last-resort antibiotics for the treatment of carbapenem-resistant Enterobacteriaceae infection, the incidence of which has been rapidly increasing. However, the emergence and spread of tigecycline resistance genes tet(X) (including tet(X3) and tet(X4)) has largely compromised the efficient usage of tetracyclines in the clinical settings. METHODS: The synergistic effect was determined by a checkerboard minimum inhibitory concentration (MIC) assay, a time-killing assay and scanning electron microscopy (SEM) analysis. In-depth mechanisms were defined using an enzyme inhibition assay, western blotting, RT-PCR analysis, molecular dynamics (MD) simulations, biolayer interferometry (BLI) assay and metabolomics analysis. FINDINGS: Herein, our work identified a natural compound, plumbagin, as an effective broad-spectrum inhibitor of Tet(X) (also known as monooxygenase) by simultaneously inhibiting the activity and the production of Tet(X3)/Tet(X4). Plumbagin in combination with tetracyclines showed a synergistic bactericidal effect against Tet(X3)/Tet(X4)-producing bacteria. Mechanistic studies revealed that direct engagement of plumbagin with the catalytic pocket of Tet(X3)/Tet(X4) induced an alternation in its secondary structure to inhibit the activity of these monooxygenases. As a consequence, monotherapy or combination therapy with plumbagin increases the oxidative stress and metabolism in bacteria. Moreover, in a mouse systemic infection model of tet(X4)-positive E. coli, the combination of plumbagin and methacycline exhibited remarkable treatment benefits, as shown by a reduced bacterial load and the alleviation of pathological injury. INTERPRETATION: Plumbagin, as an inhibitor of Tet(X3)/Tet(X4), represents a promising lead drug, as well as an adjunct with tetracyclines to treat bacterial infections, especially for extensively drug-resistant bacteria harbouring Tet(X3)/Tet(X4). FUNDING: The National Natural Science Foundation of China.

Egg White Peptides Increased the Membrane Liquid-Ordered Phase of Giant Unilamellar Vesicles: Visualization, Localization, and Phase Regulation Mechanism.

Cell membranes are heterogeneous and consist of liquid-ordered (Lo) and liquid-disordered (Ld) phases due to phase separation. Membrane regulation of egg white peptides (LCAY and QVPLW) was confirmed in our previous study. However, the underlying mechanism of phase regulation by the peptides has not been elucidated. This study aimed to explore the effect of LCAY and QVPLW on the membrane phase separation and illustrate their mechanism by giant unilamellar vesicles (GUVs). Based on phase separation visualization, LCAY and QVPLW were found to increase the Lo phase by rearranging lipids and ordering the Ld phase. LCAY and QVPLW can bind to the GUVs and localize in the amphiphilic region of the membrane. By hydrogen bonds and hydrophobic interactions, LCAY and QVPLW may play a cholesterol-like role in regulating phase separation.