2-Alkyl-4-quinolone quorum sensing molecules are biomarkers for culture-independent Pseudomonas aeruginosa burden in adults with cystic fibrosis.

Introduction. Pseudomonas aeruginosa produces quorum sensing signalling molecules including 2-alkyl-4-quinolones (AQs), which regulate virulence factor production in the cystic fibrosis (CF) airways.Hypothesis/Gap statement. Culture can lead to condition-dependent artefacts which may limit the potential insights and applications of AQs as minimally-invasive biomarkers of bacterial load.Aim. We aimed to use culture-independent methods to explore the correlations between AQ levels and live P. aeruginosa load in adults with CF.Methodology. Seventy-five sputum samples at clinical stability and 48 paired sputum samples obtained at the beginning and end of IV antibiotics for a pulmonary exacerbation in adults with CF were processed using a viable cell separation technique followed by quantitative P. aeruginosa polymerase chain reaction (qPCR). Live P. aeruginosa qPCR load was compared with the concentrations of three AQs (HHQ, NHQ and HQNO) detected in sputum, plasma and urine.Results. At clinical stability and the beginning of IV antibiotics for pulmonary exacerbation, HHQ, NHQ and HQNO measured in sputum, plasma and urine were consistently positively correlated with live P. aeruginosa qPCR load in sputum, compared to culture. Following systemic antibiotics live P. aeruginosa qPCR load decreased significantly (P<0.001) and was correlated with a reduction in plasma NHQ (plasma: r=0.463, P=0.003).Conclusion. In adults with CF, AQ concentrations correlated more strongly with live P. aeruginosa bacterial load measured by qPCR compared to traditional culture. Prospective studies are required to assess the potential of systemic AQs as biomarkers of P. aeruginosa bacterial burden.

Chemical constituents from Waltheria indica exert in vitro activity against Trypanosoma brucei and T. cruzi.

Six extracts from the roots and the aerial parts of Waltheria indica L. (Malvaceae) were screened for their in vitro antitrypanosomal activity towards Trypanosoma brucei brucei STIB 427 strain, T. brucei rhodesiense STIB 900 and Trypanosoma cruzi Tulahuen C4. The dichloromethane extract from the roots showed the highest activity against T. cruzi (IC50=0.74 µg/mL) as well as a good selectivity index (SI value of 35). Based on these results, this extract was fractionated and led to the isolation of three alkaloids (adouetin X (1), waltheriones A (2) and C (3)) and three pentacyclic triterpene derivatives (betulinic acid (4), 3ß-acetoxy-27-trans-caffeoyloxyolean-12-en-28-oic acid methyl ester (5) and 3ß-acetoxy-27-cis-caffeoyloxyolean-12-en-28-oic acid methyl ester (6)) identified by 1D and 2D NMR, UV, IR and MS analyses. Among these, waltherione C exhibited the highest and selective antitrypanosomal activity towards T. cruzi (IC50=1.93 µM) with low cytotoxicity (IC50=101.23 µM), resulting in a selectivity index value of 52. Waltherione C conforms to hit activity criteria with respect to T. cruzi as required by the WHO/TDR.

Nematicidal activities of 4-quinolone alkaloids isolated from the aerial part of Triumfetta grandidens against Meloidogyne incognita.

The methanol extract of the aerial part of Triumfetta grandidens (Tiliaceae) was highly active against Meloidogyne incognita, with second-stage juveniles (J2s) mortality of 100% at 500 µg/mL at 48 h post-exposure. Two 4-quinolone alkaloids, waltherione E (1), a new alkaloid, and waltherione A (2), were isolated and identified as nematicidal compounds through bioassay-guided fractionation and instrumental analysis. The nematicidal activities of the isolated compounds against M. incognita were evaluated on the basis of mortality and effect on egg hatching. Compounds 1 and 2 exhibited high mortalities against J2s of M. incognita, with EC50 values of 0.09 and 0.27 µg/mL at 48 h, respectively. Compounds 1 and 2 also exhibited a considerable inhibitory effect on egg hatching, which inhibited 91.9 and 87.4% of egg hatching, respectively, after 7 days of exposure at a concentration of 1.25 µg/mL. The biological activities of the two 4-quinolone alkaloids were comparable to those of abamectin. In addition, pot experiments using the crude extract of the aerial part of T. grandidens showed that it completely suppressed the formation of gall on roots of plants at a concentration of 1000 µg/mL. These results suggest that T. grandidens and its bioactive 4-quinolone alkaloids can be used as a potent botanical nematicide in organic agriculture.

4-Quinolone alkaloids from Melochia odorata.

The methanol extract of Melochia odorata yielded three 4-quinolone alkaloids including waltherione A (1) and two new alkaloids, waltherione C (2) and waltherione D (3). Waltheriones A and C showed significant activities in an in vitro anti-HIV cytoprotection assay at concentrations of 56.2 and 0.84 µM and inhibition of HIV P24 formation of more than 50% at 1.7 and 0.95 µM, respectively. The structures of the alkaloids were established by spectroscopic data interpretation.

Unusual pyrrolyl 4-quinolinone alkaloids from the marine-derived fungus Penicillium sp. ghq208.

One new pyrrolyl 4-quinolinone alkaloid, penicinoline E (1), together with three known deriverites, methyl-penicinoline (2), penicinoline (3), and quinolactacide (4), were isolated from the marine-derived fungus Penicillium sp. ghq208. The structures of these isolated compounds were elucidated by spectroscopic methods. Compounds (2, 3) exhibited moderate cytotoxicities against the HepG2 cell line with IC(50) values of 11.3 and 13.2 µM, respectively.

Quantifying Pseudomonas aeruginosa quinolones and examining their interactions with lipids.

Pseudomonas aeruginosa produces a quorum sensing molecule termed the Pseudomonas Quinolone Signal (2-heptyl-3-hydroxy-4-quinolone; PQS) that regulates an array of genes involved in virulence. This chapter addresses four related techniques useful for detecting and quantifying PQS. First, extraction of PQS from complex mixtures (e.g. cell cultures) is described. Separation of PQS from extracts by Thin-Layer Chromatography (TLC) is used in combination with the natural fluorescence of the molecule for quantification. A second separation technique for the PQS precursor HHQ using High-Performance Liquid Chromatography (HPLC) is also described, and this assay exploits the molecule's characteristic absorbance for quantification. A third method for quantification of PQS from simple mixtures (e.g. enzyme assays) using fluorescence is outlined. Finally, a protocol for determining PQS interactions with membrane lipids through Fluorescence Resonance Energy Transfer (FRET) is presented. These techniques allow for quantification and characterization of PQS from diverse environments, a prerequisite to understanding the biological functions of QS molecules.