Mutation of Pseudomonas aeruginosa lasI/rhlI diminishes its cytotoxicity, oxidative stress, inflammation, and apoptosis on THP-1 macrophages.

The management of Pseudomonas aeruginosa (P. aeruginosa) infections presents a substantial challenge to clinics and public health, emphasizing the urgent need for innovative strategies to address this issue. Quorum sensing (QS) is an intercellular communication mechanism that coordinates bacterial activities involved in various virulence mechanisms, such as acquiring host nutrients, facilitating biofilm formation, enhancing motility, secreting virulence factors, and evading host immune responses, all of which play a crucial role in the colonization and infection of P. aeruginosa. The LasI/R and RhlI/R sub-systems dominate in the QS system of P. aeruginosa. Macrophages play a pivotal role in the host's innate immune response to P. aeruginosa invasion, particularly through phagocytosis as the initial host defense mechanism. This study investigated the effects of P. aeruginosa's QS system on THP-1 macrophages. Mutants of PAO1 with lasI/rhlI deletion, as well as their corresponding complemented strains, were obtained, and significant downregulation of QS-related genes was observed in the mutants. Furthermore, the ΔlasI and ΔlasIΔrhlI mutants exhibited significantly attenuated virulence in terms of biofilm formation, extracellular polymeric substances synthesis, bacterial adhesion, motility, and virulence factors production. When infected with ΔlasI and ΔlasIΔrhlI mutants, THP-1 macrophages exhibited enhanced scavenging ability against the mutants and demonstrated resistance to cytotoxicity, oxidative stress, inflammatory response, and apoptosis induced by the culture supernatants of these mutant strains. These findings offer novel insights into the mechanisms underlying how the lasI/rhlI mutation attenuates cytotoxicity, oxidative stress, inflammation, and apoptosis in macrophages induced by P. aeruginosa.IMPORTANCEP. aeruginosa is classified as one of the ESKAPE pathogens and poses a global public health concern. The QS system of this versatile pathogen contributes to a broad spectrum of virulence, thereby constraining therapeutic options for serious infections. This study illustrated that the lasI/rhlI mutation of the QS system plays a prominent role in attenuating the virulence of P. aeruginosa by affecting bacterial adhesion, biofilm formation, extracellular polymeric substances synthesis, bacterial motility, and virulence factors' production. Notably, THP-1 macrophages infected with mutant strains exhibited increased phagocytic activity in eliminating intracellular bacteria and enhanced resistance to cytotoxicity, oxidative stress, inflammation, and apoptosis. These findings suggest that targeted intervention toward the QS system is anticipated to diminish the pathogenicity of P. aeruginosa to THP-1 macrophages.

Quercetin: a promising virulence inhibitor of Pseudomonas aeruginosa LasB in vitro.

With the inappropriate use of antibiotics, antibiotic resistance has emerged as a major dilemma for patients infected with Pseudomonas aeruginosa. Elastase B (LasB), a crucial extracellular virulence factor secreted by P. aeruginosa, has been identified as a key target for antivirulence therapy. Quercetin, a natural flavonoid, exhibits promising potential as an antivirulence agent. We aim to evaluate the impact of quercetin on P. aeruginosa LasB and elucidate the underlying mechanism. Molecular docking and molecular dynamics simulation revealed a rather favorable intermolecular interaction between quercetin and LasB. At the sub-MICs of ≤256 µg/ml, quercetin was found to effectively inhibit the production and activity of LasB elastase, as well as downregulate the transcription level of the lasB gene in both PAO1 and clinical strains of P. aeruginosa. Through correlation analysis, significant positive correlations were shown between the virulence gene lasB and the QS system regulatory genes lasI, lasR, rhlI, and rhlR in clinical strains of P. aeruginosa. Then, we found the lasB gene expression and LasB activity were significantly deficient in PAO1 ΔlasI and ΔlasIΔrhlI mutants. In addition, quercetin significantly downregulated the expression levels of regulated genes lasI, lasR, rhlI, rhlR, pqsA, and pqsR as well as effectively attenuated the synthesis of signaling molecules 3-oxo-C12-HSL and C4-HSL in the QS system of PAO1. Quercetin was also able to compete with the natural ligands OdDHL, BHL, and PQS for binding to the receptor proteins LasR, RhlR, and PqsR, respectively, resulting in the formation of more stabilized complexes. Taken together, quercetin exhibits enormous potential in combating LasB production and activity by disrupting the QS system of P. aeruginosa in vitro, thereby offering an alternative approach for the antivirulence therapy of P. aeruginosa infections. KEY POINTS: • Quercetin diminished the content and activity of LasB elastase of P. aeruginosa. • Quercetin inhibited the QS system activity of P. aeruginosa. • Quercetin acted on LasB based on the QS system.

Effect of piperine on the inhibitory potential of MexAB-OprM efflux pump and imipenem resistance in carbapenem-resistant Pseudomonas aeruginosa.

The escalating prevalence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant threat to global public health through the spread of its 'high-risk' clones. Immediate and decisive research into antimicrobial agents against CRPA is crucial for the development of effective measures and interventions. Overexpression of the MexAB-OprM efflux pump is one of the major mechanisms of CRPA. Since the active efflux of antibacterial agents plays a significant role in mediating drug resistance in CRPA, the inhibition of efflux pumps has become a promising strategy to restore antibacterial potency. Piperine (PIP) has been proven to be a promising efflux pump inhibitor in some bacteria. However, there are no studies on whether PIP can act as a potential efflux pump inhibitor in CRPA. The present study aimed to identify the antibacterial activity of PIP against CRPA and to evaluate the effect on the MexAB-OprM efflux pump. Molecular docking was used to analyze the possible interaction of PIP with the proteins of the MexAB-OprM efflux pump in CRPA. The effect of PIP on the expression of the MexAB-OprM efflux pump was investigated by real-time quantitative PCR (qPCR) and ethidium bromide accumulation efflux assay. The effect of PIP on CRPA imipenem (IPM) resistance was investigated by the checkerboard dilution method. The results demonstrated that PIP exhibited the lowest binding affinity of -9.1 kcal towards efflux pump proteins. A synergistic effect between PIP and IPM on CRPA was observed. More importantly, PIP effectively hindered the efflux of ethidium bromide and IPM by up-regulating MexR gene expression while down-regulating MexA, MexB, and OprM gene expressions. In conclusion, PIP could enhance the antibacterial activity of IPM by inhibiting the MexAB-OprM efflux pump. Our work proved that PIP had the potential to be an efflux pump inhibitor of CRPA.

Characterization and genomic analysis of a broad-spectrum lytic phage HZ2201 and its antibiofilm efficacy against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a clinically common conditionally pathogenic bacterium, and the abuse of antibiotics has exacerbated its drug resistance in recent years. This has resulted in extensive reports about the usage of Pseudomonas aeruginosa phage as a novel antibacterial drug. In this study, we isolated a novel phage HZ2201 with a broad lytic spectrum. The lytic rate of this phage against Pseudomonas aeruginosa reached 78.38% (29/37), including 25 multi-drug- and carbapenem-resistant Pseudomonas aeruginosa strains. Transmission electron microscopy revealed that phage HZ2201 belongs to the class Caudoviricetes. Biological characterization showed that phage HZ2201 had an latent period of 40 min, a lytic period of 20 min, and a burst size of 440 PFU/cell, with improved tolerance to temperature and pH. Considering genomic analysis, the HZ2201 genome was a circular double-stranded DNA with a size of 45,431 bp and a guanine-cytosine (G + C) content of 52.16%, and contained 3 tRNAs. 27 of the 74 open reading frames (ORFs) annotated by the Rapid Annotation using Subsystem Technology (RAST) tool could be matched to the genomes of known functions, and no genes related to virulence and antibiotic resistance were found. The phylogenetic tree suggests that phage HZ2201 is highly related to the phage ZCPS1 and PaP3, and ORF57 and ORF17 are predicted to encode a holin and an endolysin, respectively. Cell lysis by HZ2201 proceeds through the holin-endolysin system, suggesting that it is a novel phage. Additionally, we demonstrated that phage HZ2201 has a high inhibitory capacity against Pseudomonas aeruginosa biofilms. The results of our study suggest that phage HZ2201 is a novel potential antimicrobial agent for treating drug-resistant Pseudomonas aeruginosa infection.

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL-1 and the linear dynamic detection range was 0.015 ~ 158.5 pg mL-1. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

Electrochemical immunosensor for ultrasensitive detection of human papillomaviruse type 16 L1 protein based on Ag@AuNPs-GO/SPA.

Human papillomaviruse type 16 (HPV16) is a high-risk serotype. As the main protective antigen protein, L1 protein is also the target protein for diagnosis. A simple label free electrochemical immunosensor (ECIS) was fabricated for ultrasensitive detection of HPV16 L1 protein in this work. Quasi-spherical Ag@Au core-shell nanoparticles on graphene oxide (Ag@AuNPs-GO) was developed as current response amplifier and characterized by UV-Vis Spectroscopy, Transmission Electron Microscopy and energy dispersive X-ray spectroscopy. Staphylococcal protein A was decorated on the modified electrode and utilized to immobilized the Fc portion of the monoclonal antibody specific for HPV16 L1 protein. Cyclic Voltammetry, Differential Pulse Voltammetry and Electrochemical Impedance Spectroscopy were used to verify the electrochemical performance and interfacial kinetic property. The increased concentration of HPV16 L1 protein led to slow electron transport and linearly decreased differential pulse voltammetry peak current with a detection limit of 0.002 ng mL-1 and a wide linear relationship in the range of 0.005-400 ng mL-1at a regression coefficient (R2) of 0.9948. Furthermore, this ECIS demonstrated acceptable accuracy with good reproducibility, stability and selectivity, suggesting a promising immunological strategy for HPV typing and early screening.

Development of a label free electrochemical sensor based on a sensitive monoclonal antibody for the detection of tiamulin.

Based on a murine monoclonal antibody (mAb) against tiamulin (TML), an electrochemical immunosensor was proposed using silver-graphene oxide (Ag-GO) nanocomposites and gold nanocomposites (AuNPs) to detect tiamulin (TML). Due to the synergetic properties of Ag-GO nanocomposites and AuNPs, the conductivity of the immunosensor was significantly enhanced. On account of the specific mAb and conductive nanocomposites, the proposed electrochemical immunosensor exhibited a low LOD of 0.003 ng mL-1 for the detection of TML in a wide linear range of 0.01 to 1000 ng mL-1. In addition, the immunosensor did not involve additional redox species. Furthermore, the efficient and simple electrochemical immunosensor was employed to detect TML in real samples with high accuracy, suggesting a potential detection platform for other veterinary antibiotics in animal derived foods.

A novel electrochemical immunosensor for the sensitive detection of tiamulin based on staphylococcal protein A and silver nanoparticle-graphene oxide nanocomposites.

Tiamulin (TML) is a pleuromutilin antibiotic and mainly used to treat pulmonary and gastrointestinal infections. However, excessive use of TML can bring health threats to consumers. In this work, a label-free electrochemical immunosensor was proposed for sensitive detection of TML in pork and pork liver. Silver nanoparticles (AgNPs) were synthesized in situ on graphene oxide (GO), in which GO acted as a carrier for loading more AgNPs and AgNPs exhibited both strong conductivity and good redox property. In addition, staphylococcal protein A (SPA) was applied to oriented immobilization of fragment crystallizable (Fc) region of the TML monoclonal antibody. Under the optimal condition, the developed electrochemical immunosensor exhibited a good linear response with a concentration of TML ranging from 0.05 ng mL-1 to 100 ng mL-1 and the limit of detection (LOD) was 0.04 ng mL-1. Furthermore, the designed immunosensor was applied to detect TML in real samples with a good accuracy. Therefore, the label-free electrochemical immunosensor could be used as a potential method to detect TML and other antibiotic residues in animal derived foods.

An Electrochemical Immunosensor Based on SPA and rGO-PEI-Ag-Nf for the Detection of Arsanilic Acid.

A sensitive electrochemical immunosensor was prepared for rapid detection of ASA based on arsanilic acid (ASA) monoclonal antibody with high affinity. In the preparation of nanomaterials, polyethyleneimine (PEI) improved the stability of the solution and acted as a reducing agent to generate reduced graphene oxide (rGO) with relatively strong conductivity, thereby promoting the transfer of electrons. The dual conductivity of rGO and silver nanoparticles (AgNPs) improved the sensitivity of the sensor. The synthesis of nanomaterials were confirmed by UV-Vis spectroscopy, X-ray diffraction, transmission electron microscopy and scanning electron microscopy. In the optimal experiment conditions, the sensor could achieve the detection range of 0.50-500 ng mL-1 and the limit of detection (LOD) of 0.38 ng mL-1 (S/N = 3). Moreover, the sensor exhibited excellent specificity and acceptable stability, suggesting that the proposed sensor possessed a good potential in ASA detection. Thus, the as-prepared biosensor may be a potential way for detecting other antibiotics in meat and animal-derived foods.

Using Natural Biomacromolecules for Adsorptive and Enzymatic Removal of Aniline Blue from Water.

The present study investigated the adsorptive and enzymatic removal of aniline blue dye (AB) from aqueous solution using waxy riceprocessing waste (RW), peanut shell (PS), microbial waste of Aspergillus niger (MW) as low cost adsorbents, and laccase (Lac) as a biocatalyst. Commercial activated carbon (AC) was also employed to compare the adsorption performance with the three adsorbents. Dye removal was examined under various parameters in batch experiments. It was found that dye removal by RW and Lac was 89⁻94% noticeably better than that by MW and PS (20⁻70%). In any cases, AC produced the highest dye removal among the tested materials. The kinetics, isotherms, and thermodynamics were then analyzed to elucidate the adsorption process by the four adsorbents. The pseudo-second order kinetic was superior to the pseudo first order kinetic model in describing adsorption for all adsorbents. The Langmuir model fitted the adsorption process very well, indicating monolayer coverage of dyes on a solid surface. A thermodynamic analysis of enthalpy (ΔH°), entropy (ΔS°), and Gibbs free energy (ΔG°) classified the adsorption as a nonspontaneous and endothermic process. The results reveal diverse natural materials (e.g., processing waste RW) as novel substitutes for traditional activated carbon, as well as laccase as a green catalyst for the treatment of dye wastewater.

Adsorption of methylene blue on an agro-waste oiltea shell with and without fungal treatment.

A lignocellulosic waste oiltea shell (OTS) was evaluated as an inexpensive sorbent to remove methylene blue (MB) from aqueous solution. Fungal treatment of OTS increased the MB adsorption by modifying the physicochemical properties of OTS and simultaneously produced laccase as a beneficial co-product. Without fungal treatment, the maximum amount of adsorption (qm) of MB by OTS was 64.4 mg/g, whereas the treatment with fungus Pycnoporus sp. and Trametes versicolor increased qm up to 72.5 mg/g and 85.7 mg/g, respectively. This is because of the improved surface area and pore sizes as well as altered chemical compositions. The equilibrium sorption data for OTS both with and without treatment fitted to the Langmuir model, and the sorption rate data well fitted to the pseudo second-order kinetic model. The changes in free energy (ΔG°) and separation factor (RL) indicated that the sorption was spontaneous and favorable. Scanning electron microscopy and Fourier transform infrared spectroscopy showed the changes in the surface morphology and functional groups of OTS after fungal treatment. The agro-waste OTS could be utilized as a low-cost adsorbent for efficient dye removal, and fungal treatment can serve as a mild and clean technique to increase the adsorptive capacity of OTS.

The antitumor effect and mechanism of taipeinine A, a new C19-diterpenoid alkaloid from Aconitum taipeicum, on the HepG2 human hepatocellular carcinoma cell line.

PURPOSE: To investigate whether taipeinine A (JNQ2), a C19-diterpenoid alkaloid prepared from the roots of Aconitum taipeicum, has anticancer effects on hepatocellular carcinoma (HCC) and to study its probable anticancer mechanisms. METHODS: JNQ2 activities were assessed on human HCC cell line (HepG2) by proliferative assay, cell cycle arrest assay, apoptosis analysis, cell invasion assay and Western blot analysis. RESULTS: The antitumor activity tests showed that JNQ2 inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner and blocked the cell cycle at the G1/S phase. High dosage of JNQ2 induced significant apoptosis of tumor cells. The invasiveness of HepG2 cells was also inhibited by JNQ2. The mechanism of JNQ2 antitumor effect at the molecular level was presumed to be the upregulation of the protein expression of Bax and Caspase-3 and the downregulation of the protein expression of Bcl-2 and CCND1. CONCLUSION: Our study suggests that JNQ2 has anticancer effects on HepG2 cells and it is a potential reagent for the treatment of HCC that merits further investigation.

Antitumor effect and mechanism of an ellagic acid derivative on the HepG2 human hepatocellular carcinoma cell line.

In the present study, to identify the effective components of Chinese traditional herbs, Euphorbia hylonoma Hand.-Mazz. (Euphorbiaceae), a folk herb that has been used among the Qinling mountain area for hundreds of years, was investigated. 3,3'-Di-O-methyl ellagic acid-4'-O-ß-d-xylopyranoside (JNE2), an ellagic acid derivative, was isolated from the acetone extract of the herb and its antitumor activity against human hepatoma HepG2 cells was detected in vitro. The results showed that JNE2 inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner and blocked the cell cycle at the G1/S phase. A high dosage of JNE2 induced apoptosis of the tumor cells, but no significant differences were identified between the treatment groups. The invasiveness of HepG2 cells was also inhibited by JNE2. The mechanism of the antitumor effect of JNE2 at the molecular level was presumed to be due to the upregulation of the protein expression of Bax and caspase-3, and the downregulation of the protein expression of Bcl-2 and CCND1. The results suggested that JNE2 is a potential antitumor agent that merits further investigation.

Ginsenoside Re reduces insulin resistance through activation of PPAR-γ pathway and inhibition of TNF-α production.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng is a well-known traditional Chinese medicine and has been used for treatment of various diseases for more than four thousand years in Asia. Ginseng saponins or ginsenosides, the active constituents are reported to possess antidiabetic activity, but their antihyperglycemic mechanisms are not fully elucidated. In the present study, the mechanisms of action of ginsenoside Re were investigated in vitro models. MATERIALS AND METHODS: 3T3-L1 cells were chosen as the model to investigate the molecular mechanisms of action of ginsenoside Re. Influence of ginsenoside Re on the adipogenesis was examined by determining TG levels in 3T3-L1 adipocytes by the method of TG oxidation enzyme. Glucose uptake in 3T3-L1 cells stimulated by insulin in the absence or presence of ginsenoside Re were quantified by measuring (3)H-2-deoxy-d-glucose levels. Cytokine proteins released into the medium including adiponectin and TNF-α were tested using respective ELISA kits. In addition, real time RT-PCR was conducted to investigate the expression changes of PPAR-γ and its responsive genes, ap2, adiponectin, IRS-1, GLUT4 and TNF-α. And western blot analysis was performed to determine the translocation of GLUT4. Finally, effects of ginsenoside Re on NO production in 3T3-L1 adipocytes and in macrophages were investigated through measurement of nitrite concentration by Griess reagent. RESULTS: Ginsenoside Re induced adipogenesis of 3T3-L1 adipocytes by accumulating TG, increased glucose uptake and up-regulated PPAR-γ2, IRS-1, ap2 and adiponectin genes expressions. Meanwhile, Re also increased production and release of adiponectin. Although having no effects on GLUT4 gene expression, Re facilitated GLUT4 protein translocation to the membranes. In addition, Re inhibited the expression and release of TNF-α. Finally, Re did not show inhibitory effects on NO production both in 3T3-L1 cells stimulated by LPS, TNF-α and IFN-γ and in LPS-stimulated mouse peritoneal macrophages. CONCLUSIONS: Ginsenoside Re exhibited the action of reducing insulin resistance through activation of PPAR-γ pathway by directly increasing the expressions of PPAR-γ2 and its responsive genes, adiponectin, IRS-1, ap2, inhibiting TNF-α production and facilitating the translocation of GLUT4 to promote glucose uptake and disposal in 3T3-L1 adipocytes.

Structure of sorting nexin 11 (SNX11) reveals a novel extended phox homology (PX) domain critical for inhibition of SNX10-induced vacuolation.

Sorting nexins are phox homology (PX) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in these lipids. The structure of the PX domain is highly conserved, containing a three-stranded ß-sheet, followed by three α-helices. Here, we report the crystal structures of truncated human SNX11 (sorting nexin 11). The structures reveal that SNX11 contains a novel PX domain, hereby named the extended PX (PXe) domain, with two additional α-helices at the C terminus. We demonstrate that these α-helices are indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in SNX10 and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important PX domain subfamily.

[Chemical constituents of flavonoids from flowers of Koelreuteria paniculata].

OBJECTIVE: To study the chemical constituents from flowers of Koelreuteria paniculata. METHODS: Column chromatography and spectral analysis were used to isolate and identify the constituents. RESULTS: The EtOAc fraction from flowers of Koelreuteria paniculata was separated and purified. Nine compounds were obtained and identified as:sitosterol glucoside (I), gallic acid (II), kaempferol (III), luteolin (IV), kaempferol-3-O-(6"-acetyl)-beta-D-glucopyranoside (V), hyperoside-2"-O-acetyl (VI), hyperoside-2"-O-galloyl (VII), hyperoside (VIII), kaempferol-3-O-D-glucopyranoside (IX). CONCLUSION: Nine compounds are isolated for the first time from flowers of Koelreuteria paniculata. Compounds IV, V, VI and IX are isolated from this genus for the first time.