Bioactive butenolides from Streptomyces antibioticus TÜ 99: absolute configurations and synthesis of analogs

The four furanones (butenolides) 1-4, which had been isolated from the fermentation broth of Streptomyces antibioticus TÜ 99 and in preliminary tests had been shown to be biologically active, were synthesized by reaction of the readily available furanones 9 or 16 with 2-methylpropanal or 2-methylbutanal. In addition, a series of analogs was prepared in a similar way from 16 using different aldehydes. The hitherto unknown absolute configurations of the natural products 1-4 as well as those of all the analogs prepared were determined with Mosher's NMR method and/or X-ray crystallography. Some of the compounds synthesized proved to be active in the quorum sensing system of Chromobacterium violaceum.     

High dielectric constant PrYxOy sensing films electrolyte-insulator-semiconductor pH-sensor for the detection of urea

In this paper, we describe the structural and sensing properties of high-k PrY_xO_y sensing films deposited on Si substrates through reactive co-sputtering. Secondary ion mass spectrometry and atomic force microscopy were employed to analyze the compositional and morphological features of these films after annealing at various temperatures. The electrolyte-insulator-semiconductor (EIS) device incorporating a PrY_xO_y sensing membrane that had been annealed at 800 °C exhibited good sensing characteristics, including a high sensitivity (59.07 mV pH⁻¹ in solutions from pH 2 to 12), a low hysteresis voltage (2.4 mV in the pH loop 7 → 4 → 7 → 10 → 7), and a small drift rate (0.62 mV h⁻¹ in the buffer solution at pH 7). The PrY_xO_y EIS device also showed a high selective response towards H⁺. This improvement can be attributed to the small number of crystal defects and the large surface roughness. In addition, the enzymatic EIS-based urea biosensor incorporating a high-k PrY_xO_y sensing film annealed at 800 °C allowed the potentiometric analysis of urea, at concentrations ranging from 1 to 16 mM, with a sensitivity of 9.59 mV mM⁻¹.     

Quorum sensing in <Emphasis Type="Italic">Erwinia</Emphasis> species

The term quorum sensing (QS) refers to the ability of bacteria to regulate gene expression according to the accumulation of signalling molecules that are made by every cell in the population. The erwiniae group of bacteria are often phytopathogens and the expression of a number of their important virulence determinants and secondary metabolites is under QS control. The erwiniae utilise two types of QS signalling molecules: N-acyl homoserine lactones and AI-2-type signalling molecules. Here, we review the regulatory networks involving QS in the soft rot erwiniae.     

Quorum-sensing systems in staphylococci as therapeutic targets

The staphylococci are an ever-present threat in our world, capable of causing a wide range of infections, and are a persistent presence in the clinical environment. As the number of antimicrobial compounds effective against staphylococci decreases, because of the acquisition and spread of antibiotic resistance, there is a growing need for novel therapeutic molecules. Intra and inter-species communication (quorum sensing) is a biologically significant phenomenon that has been associated with virulence, intracellular survival, and biofilm formation. Quorum sensing molecules of staphylococci and other species (e.g. Pseudomonas aeruginosa) can inhibit virulence factor production and/or growth of staphylococci, leading to the possibility that interference with staphylococcal quorum-sensing systems could be a way of controlling the diverse infections caused by the staphylococci. In this article, we discuss the potential of quorum-sensing systems of staphylococci as therapeutic targets.     

Detection of bacterial quorum sensing <Emphasis Type="Italic">N</Emphasis>-acyl homoserine lactones in clinical samples

Bacteria communicate among themselves using certain chemical signaling molecules. These signaling molecules generally are N-acyl homoserine lactones (AHLs) in Gram-negative bacteria and oligopeptides in Gram-positive bacteria. In addition, both Gram-positive and Gram-negative bacteria produce a family of signaling molecules known as autoinducer-2 that they employ for their communications. Bacteria coordinate their behavior by releasing and responding to the chemical signaling molecules present in proportion to their population density. This phenomenon is known as quorum sensing. The role of bacteria in the pathogenesis of several diseases, including gastrointestinal (GI) disorders, is well established. Moreover, rather recently bacterial quorum sensing has been implicated in the onset of bacterial pathogenicity. Thus, we hypothesized that the signaling molecules involved in bacterial communication may serve as potential biomarkers for the diagnosis and management of several bacteria-related diseases. For that, we previously developed a method based on genetically engineered whole-cell sensing systems for the rapid, sensitive, cost-effective and quantitative detection of AHLs in biological samples, such as saliva and stool, from both healthy and diseased individuals with GI disorders. Although various analytical methods, based on physical-chemical techniques and bacterial whole-cell biosensors, have been developed for the detection of AHLs in the supernatants of bacterial cultures, only a few of them have been applied to AHL monitoring in real samples. In this paper, we report work performed in our laboratory and review that from others that describes the detection of AHLs in biological, clinical samples, and report some of our recent experimental results.     

Toward plasmonic monitoring of surface effects on bacterial quorum-sensing

Biofilm formation is facilitated by cell–cell communication processes known as quorum sensing, which enable collective behavior and metabolic coordination. Surface topography and chemistry play a significant role in bacterial adhesion and biofilm formation, yet methods for monitoring quorum sensing in situ suffer limitations. Herein we suggest the use of surface-enhanced Raman scattering to study the effects of surface topography and chemistry on quorum sensing signals involved in biofilm growth.     

Controlling the activity of quorum sensing autoinducers with light

Bacteria use a communication system, called quorum sensing (QS), to organize into communities and synchronize gene expression to promote virulence and secure survival. Here we report on a proof-of-principle for externally interfering with this bacterial communication system, using light. By employing photoswitchable small molecules, we were able to photocontrol the QS-related bioluminescence in an Escherichia coli reporter strain, and the expression of target QS genes and pyocyanin production in Pseudomonas aeruginosa.     

Synthesis of 3-halo-analogues of HHQ, subsequent cross-coupling and first crystal structure of Pseudomonas quinolone signal (PQS)

2-Aryl- and 2-alkyl-quinolin-4-ones and their N-substituted derivatives have several important biological functions such as the Pseudomonas quinolone signal (PQS) molecule participation in quorum sensing. Herein, we report the synthesis of its biological precursor, 2-heptyl-4-hydroxy-quinoline (HHQ) and possible isosteres of PQS; the C-3 Cl, Br and I analogues. N-Methylation of the iodide was also feasible and the usefulness of this compound showcased in Pd-catalysed cross-coupling reactions, thus allowing access to a diverse set of biologically important molecules. The first crystal structure of PQS is also included.     

Experimental and theoretical study of the structure of N,N-dimethyl-4-nitroaniline derivatives as model compounds for non-linear optical organic materials

Molecular and crystal structure of a series of derivatives of N,N-dimethyl-4-nitroaniline has been studied by both X-ray diffraction method and high-level ab initio calculations. According to these data, the dimethylamino groups were found to have a trigonal-pyramidal configuration and are considerably turned with respect to the ring plane in all molecules having a substituent in the ortho-position; on the contrary, this group is planar in the meta-substituted molecules. Topological analysis of the electron density function for all molecules studied within the framework of Bader's ‘atoms in molecules’ (AIM) theory revealed that introduction of a substituent into the ortho- or meta-position of the ring results in increasing of the contribution of the resonance forms different from the quinoid one. Contribution of the latter form is predominant for the structure of N,N-dimethyl-4-nitroaniline (1). Topological analysis of the electron density distribution was used to explain a decreasing of the molecular hyperpolarisabilites of the ortho- and meta-substituted compounds as compared with those for 1.     

Kappa-casein based electrochemical and surface plasmon resonance biosensors for the assessment of the clotting activity of rennet

We report for the first time the development of kappa-casein (κ-CN)-based electrochemical and surface plasmon resonance (SPR) biosensors for the assessment of the clotting activity of rennet. Electrochemical biosensors were developed over gold electrodes modified with a self-assembled monolayer of dithiobis-N-succinimidyl propionate, while SPR measurements were performed on regenerated carboxymethylated dextran gold surfaces. In both types of biosensor, κ-CN molecules were immobilized onto modified gold surfaces through covalent bonding. In electrochemical biosensors, interactions between the immobilized κ-CN molecules and chymosin (the active component of rennet) were studied by performing cyclic voltammetry, differential pulsed voltammetry, and electrochemical impedance spectroscopy (EIS) measurements, using hexacyanoferrate(II)/(III) couple as a redox probe. κ-CN is cleaved by rennet at the Phe105–Met106 bond, producing a soluble glycomacropeptide, which is released to the electrolyte, and the positively charged insoluble para-κ-casein molecule, which remains attached to the surface of the electrode. This induced reduction of the net negative charge of the sensing surface, along with the partial degradation of the sensing layer, results in an increase of the flux of the redox probe, which exists in the solution, and consequently, to signal variations, which are associated with the increased electrocatalysis of the hexacyanoferrate(II)/(III) couple on the gold surface. SPR experiments were performed in the absence of the redox probe and the observed SPR angle alterations were solely attributed to the cleavage of the immobilized κ-CN molecules. Various experimental variables were investigated and under the selected conditions the proposed biosensors were successfully tried to real samples. The ratios of the clotting power units in various commercial solid or liquid samples, as they are calculated by the EIS-based data, were almost identical to those obtained with a reference method. In addition, EIS measurements showed an excellent reproducibility, lower than 5%.     

Design, synthesis and biological evaluation of non-natural modulators of quorum sensing in Pseudomonas aeruginosa

Many species of bacteria employ a mechanism of intercellular communication known as quorum sensing which is mediated by small diffusible signalling molecules termed autoinducers. The most common class of autoinducer used by Gram-negative bacteria are N-acylated-L-homoserine lactones (AHLs). Pseudomonas aeruginosa is a clinically important bacterium which is known to use AHL-mediated quorum sensing systems to regulate a variety of processes associated with virulence. Thus the selective disruption of AHL-based quorum sensing represents a strategy to attenuate the pathogenicity of this bacterium. Herein we describe the design, synthesis and biological evaluation of a collection of structurally novel AHL mimics. A number of new compounds capable of modulating the LasR-dependent quorum sensing system of P. aeruginosa were identified, which could have value as molecular tools to study and manipulate this signalling pathway. Worthy of particular note, this research has delivered novel potent quorum sensing antagonists, which strongly inhibit the production of virulence factors in a wild type strain of this pathogenic bacterium.     

(Z)-N-(4-Decenoyl)homoserine lactone, a new quorum-sensing molecule, produced by endobacteria associated with Mortierella alpina A-178

N-Acylhomoserine lactones (AHLs) are the conserved quorum-sensing signal molecules in Gram-negative bacteria. (Z)-N-(4-Decenoyl)homoserine lactone (1), a new AHL, was isolated from the culture broth of the fungus Mortierella alpina A-178 harboring bacterial endosymbionts, called endobacteria. The structure and absolute configuration of 1 were elucidated by EI-MS, chemical synthesis, and chiral GC analysis. The compound induced the expression of a QS-regulated reporter gene in Agrobacterium tumefaciens NTL4, although its activity was lower than that of N-decanoylhomoserine lactone (6).     

Rapid differentiation of Panax ginseng and Panax quinquefolius by matrix-assisted laser desorption/ionization mass spectrometry

A matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based method has been developed for rapid differentiation between Panax ginseng and Panax quinquefolius, two herbal medicines with similar chemical and physical properties but different therapeutic effects. This method required only a small quantity of samples, and the herbal medicines were analyzed by MALDI-MS either after a brief extraction step, or directly on the powder form or small pieces of raw samples. The acquired MALDI-MS spectra showed different patterns of ginsenosides and small chemical molecules between P. ginseng and P. quinquefolius, thus allowing unambiguous differentiation between the two Panax species based on the specific ions, intensity ratios of characteristic ions or principal component analysis. The approach could also be used to differentiate red ginseng or P. quinquefolius adulterated with P. ginseng from pure P. ginseng and pure Panax quinquefolium. The intensity ratios of characteristic ions in the MALDI-MS spectra showed high reproducibility and enabled quantitative determination of ginsenosides in the herbal samples and percentage of P. quinquefolius in the adulterated binary mixture. The method is simple, rapid, robust, and can be extended for analysis of other herbal medicines.     

Novel microporous zirconium silicate (K2ZrSi3O9·2H2O) from high temperature phase transformation

A new microporous zirconosilicate K₂ZrSi₃O₉·2H₂O (AV-15) has been prepared by high-temperature phase transformation at 910 °C. Its structure has been determined ab initio from powder X-ray diffraction data. The unit cell is orthorhombic, space group C222₁ (no. 20), Z=4 with cell dimensions: a=8.105(3), b=10.684(5), c=12.030(5) Å, V=1041.76(7) Å³. The framework connection of AV-15 is essentially the same as the previously reported sodium stannosilicate AV-10 while the locations of potassium and water molecules in the former are quite different from those of the sodium and water molecules in AV-10. In AV-10 sodium and water molecules form a sinucoidal chain, while potassium and water molecules build up a linear chain in AV-15. The water molecules in AV-15 are lost on heating with a typical zeolitic behaviour. SEM shows that the particle sizes and habits of AV-15 and parent umbite material are the same. The ²⁹Si MAS NMR spectrum of AV-15 displays two resonances at ca. −89.4 and −90.1 ppm in a 1:2 intensity ratio. Thermogravimetry analysis confirms the existence of water in this material.     

Comparative proteomic analysis of B. cenocepacia using two-dimensional liquid separations coupled with mass spectrometry

Burkholderia cenocepacia is an important respiratory pathogen in persons with cystic fibrosis. We compared the proteomes of clinical and environmental isolates of B. cenocepacia by using a 2D liquid separation method coupled with mass spectrometry. Proteome maps of four B. cenocepacia isolates were generated. In the first dimension, 5 mg of protein from each isolate was fractionated by chromatofocusing (CF) in the range of pH 4.0-7.0. In the second dimension, each CF fraction was separated by NPS-RP-HPLC. Results of the 2D liquid separation were visualized as 2D UV maps, which allowed direct comparison of proteomes with high resolution and reproducibility. From the proteomic comparison of the four isolates, 38 of 96 differentially abundant proteins were identified by peptide mass fingerprinting and MS/MS sequence analysis using a partially annotated B. cenocepacia protein database. Many of the identified proteins in the clinical isolates are involved in gene translation and bacterial virulence such as transmissibility, resistance, and quorum sensing.     

Flexible NH3 sensors fabricated by in situ self-assembly of polypyrrole

Novel flexible NH₃ gas sensors were formed by the in situ self-assembly of polypyrrole (PPy) on plastic substrates. A negatively charged substrate was prepared by the formation of an organic monolayer (3-mercapto-1-propanesulfonic acid sodium salt—MPS) on a polyester (PET) substrate using a pair of comb-like Au electrodes. Two-cycle poly(4-styrenesulfonic acid) sodium salt/poly(allylamine hydrochloride) (PSS/PAH) bilayers (precursor layer) were then layer-by-layer (LBL) deposited on an MPS-modified substrate. Finally, a monolayer of PPy self-assembled in situ and PPy multilayer thin films self-assembled LBL in situ on a (PSS/PAH)₂/MPS/Au/Cr/PET substrate. The thin films were analyzed by atomic force microscopy (AFM). The effects of the precursor layer (PSS), the deposition time of the monolayer of PPy and the number of PPy multilayers on the gas sensing properties (response) and the flexibility of the sensors were investigated to optimize the fabrication of the film. Additionally, other sensing properties such as sensing linearity, reproducibility, response and recovery times, as well as cross-sensitivity effects were studied. The flexible NH₃ gas sensor exhibited a strong response that was comparable to or even greater than that of sensors that were fabricated on rigid substrate at room temperature.     

Small molecule inhibitors of bacterial quorum sensing and biofilm formation

Bacteria monitor their local population densities using small molecules (or autoinducers) in a process known as quorum sensing. Here, we report a new and efficient synthetic route to naturally occurring bacterial autoinducers [N-acyl l-homoserine lactones (AHLs)] that is readily amenable to the synthesis of analogues. This route has been applied in the first synthesis of a library of non-native AHLs. Evaluation of these compounds in bacterial reporter gene and biofilm assays has revealed a potent set of quorum sensing antagonists. These ligands will serve as valuable new tools to explore the role of quorum sensing in bacterial pathogenesis.     

Localized quorum sensing in Vibrio fischeri

Quorum sensing is almost always regarded as a population density effect in three-dimensional bulk samples of bacteria. Here we create two-dimensional samples of Vibrio fischeri cells adhered onto glass surfaces to examine the effect of local population densities on quorum sensing. This is done by measuring the luminescent response. The 2-D bacterial populations enable us to simultaneously account for time and distance effects on quorum sensing, which were previously very challenging to access in typical three-dimensional bulk samples. Thus, we are able to consider quorum sensing in terms of signal diffusion. A diffusion model of quorum sensing signals guides the experiments and shows that for a given cell spacing (density) and diffusion time there exists a “true quorum”— a number of cells necessary for quorum sensing. We find that quorum sensing can occur locally in 2-D surface samples and is a function of cell population density as well as signal diffusion time.     

Toward a fragment-based approach to MMPs inhibitors: an expedite and efficient synthesis of N-hydroxylactams

Matrix metalloproteinases (MMPs), a class of zinc-enzymes over-activated in many pathologies, such as arthritis and cancer, can be efficiently inhibited by a variety of molecules bearing zinc-binding groups (ZBGs). The hydroxamic acid moiety represents one of the most potent and widely exploited ZBG but the poor target selectivity and in vivo toxicity have tempered the initial enthusiasm for this class of potential therapeutics. These drawbacks might be circumvented, at least in part, by increasing the structural constraints around the hydroxamic moiety. Following this strategy we designed and prepared N-hydroxylactam molecules of different size through a synthetic protocol based on a ring closing metathesis amenable to a fragment-based approach potentially leading to a large molecular diversity.