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.     

Complexes of aminosquarate ligands with first-row transition metals and lanthanides: new insights into their hydrolysis

Attempts at synthesizing first-row transition-metal complexes of the 3-hydroxy-4-[(1'S,2'R)-(2-hydroxy-1',2'-diphenylethyl)amino]-3-cyclobutene-1,2-dione ligand in alcoholic solutions resulted in the formation of the monomers [M(NH(2)C(4)O(3))(2)(H(2)O)(4)] [M = Mn (1), Co (2), Ni (3), Cu (4), Zn (5)] instead, as a result of the hydrolysis of the ligand. 1, 2, and 3 are isomorphous (C2/c), with the metal atoms octahedrally coordinated to four aqua and two cis aminosquarate ligands. The copper and zinc complexes (4 and 5) have the same molecular formula as 1-3 but belong to the C2/m and P2(1)/c space groups respectively. 4 has square-pyramidal geometry with trans-oriented aminosquarate ligands in the basal plane; aqua ligands complete the coordination sphere. 5 has octahedral geometry, with four aqua and two trans-oriented aminosquarate ligands. Reaction of aqueous solutions of the anilinosquarate ligand with Ln(NO(3))(3) x xH(2)O produced the eight-coordinate complexes {Sm(mu-C(6)H(5)NHC(4)O(3))(3)(H(2)O)(4) x 3H(2)O}n (6), {[M(mu(2)-C(4)O(4))(H(2)O)(6)][C(6)H(5)NHC(4)O(3)] x 4H(2)O}n [M = Er (7), Yb (8)], {Sm(C(6)H(5)NHC(4)O(3)) (mu(3)-C(4)O(4))(H(2)O)(4) x H(2)O}(n) (9), and {[{(C(6)H(5)NHC(4)O(3))(2)(H(2)O)(5)Yb}(2)(mu-C(4)O(4))] x 4H(2)O}n (10). 7 and 8 are isomorphous with the previously reported analogues Eu, Gd, and Tb ionic polymers. The presence of the squarate ligand in 7-10 is indicative of some form of hydrolysis of the anilinosquarate ligand during their syntheses. However, hydrolysis was not evident in the synthesis of 6. The mechanism for the hydrolysis in the syntheses of 1-5 is apparently different from that for 7-10.     

Single drop microextraction of homoserine lactones based quorum sensing signal molecules, and the separation of their enantiomers using gas chromatography mass spectrometry in the presence of biological matrices

N-acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the “quorum sensing” response of bacterial communities. AHLs are optically active components and the L-form is known to have activity in the quorum sensing. However, the knowledge regarding the stereospecific production of the AHLs in bacterial cultures is limited; therefore, there is a need for a fast and easy method for their chiral analysis. A method was developed for the preconcentration of the AHLs using single drop microextraction or liquid-liquid microphase extraction in toluene for their analysis using GC-MS. The performance of the method were determined and discussed for the chiral separation of these autoinducers using a capillary column coated with heptakis-(2,3-di-O-acetyl-6-O-t-butyldimethyl-silyl)-β-cyclodextrin. The salient feature of this study is the demonstration, that Burkholderia cepacia LA3 produced D-decanoyl-homoserine lactone beside L-decanoyl- and L-octaonyl- enantiomers.     

Submicrometer intermediates in the citrate synthesis of gold nanoparticles: new insights into the nucleation and crystal growth mechanisms

The reduction of tetrachloroaurate by citrate ions in aqueous solutions yielding gold nanoparticles (GNPs) has been studied using in situ tapping mode atomic force microscopy (AFM), UV-vis absorption and dynamic light scattering (DLS) spectroscopies, small-angle X-ray scattering (SAXS) along with ex situ TEM, EDX and XPS. Special attention is given to mesoscale intermediates responsible for the intense coloring of the transient solutions and their role in nucleation and crystal growth. AFM detects liquid droplet-like domains, globules 30-50 nm in diameter arranged in submicrometer aggregates in the gray and blue solutions, and well separated individual particles in the final red sols. DLS shows abrupt appearance of species about 30 nm and larger but not growing Au nanoparticles, while SAXS reveals gradually increasing nanoparticles and no aggregates. The mesoscale structures observed in TEM become looser as the reaction proceeds; they contain signatures of oxidized Au and other solutes. The results are interpreted in terms of decomposition of supersaturated solutions to afford domains ("dense droplets") enriched by gold, and then, after nucleation and coalescence of Au nuclei inside them, rather slow growth of gold nanoparticles within the associated globules; the color changes of the transient solutions are due to increasing interparticle distances.     

Quorum sensing in Candida albicans: probing farnesol's mode of action with 40 natural and synthetic farnesol analogs

The dimorphic fungus Candida albicans produces extracellular farnesol (3,7,11-trimethyl-2,6,10-dodecatriene-1-ol) which acts as a quorum-sensing molecule (QSM) to suppress filamentation. Of four possible geometric isomers of farnesol, only the E,E isomer possesses QSM activity. We tested 40 natural and synthetic analogs of farnesol for their activity in an N-acetylglucosamine-induced differentiation assay for germ tube formation (GTF). Modified structural features include the head group, chain length, presence or absence of the three double bonds, substitution of a backbone carbon by S, O, N, and Se heteroatoms, presence or absence of a 3-methyl branch, and the bulkiness of the hydrophobic tail. Of the 40 compounds, 22 showed QSM activity by their ability to reduce GTF by 50%. However, even the most active of the analogs tested had only 7.3% of the activity of E,E-farnesol. Structure-activity relationships were examined in terms of the likely presence in C. albicans of a farnesol binding receptor protein.     

Design of dinuclear copper species with carboranylcarboxylate ligands: study of their steric and electronic effects

A series of new mononuclear and carboranylcarboxylate‐bridged dinuclear copper(II) compounds containing the 1‐CH₃‐2‐CO₂H‐1,2‐closo‐C₂B₁₀H₁₀ carborane ligand ( L H) has been synthesized. Reaction of different copper salts with L H at room temperature leads to dinuclear compounds of the general formula [Cu₂(μ‐ L )₄( L_t )₂] ( L_t =thf ( 1 ), L_t =H₂O ( 1′ )). The reaction of 1 and 1′ with different terminal pyridyl (py) ligands leads to the formation of a series of structurally analogous complexes by substitution of the terminal ligand thf or H₂O ( L_t =py ( 2 ), p‐CF₃‐py ( 3 ), p‐CH₃‐py ( 4 ), pz ( 6 ), and 4,4′‐bpy ( 7 )), which maintain the structural Cu₂(μ‐O₂CR)₄ core in the majority of the cases except for o‐(CH₃)₂‐py, where a mononuclear compound ( 5 ) is exclusively obtained. These compounds have been characterized through analytical, spectroscopic (NMR, IR, UV‐visible, ESI‐MS) and magnetic techniques. X‐ray structural analysis revealed a paddle‐wheel structure for the dinuclear compounds, with a square‐pyramidal geometry around each copper ion and the carboranylcarboxylate ions bridging two copper atoms in syn–syn mode. The mononuclear complex obtained with the o‐(CH₃)₂‐py ligand presents a square‐planar structure, in which the carboranylcarboxylate ligand adopts a monodentate coordination mode. The magnetic properties of the dinuclear compounds 1 , 3 , 4 , and 6 show a strong antiferromagnetic coupling in all cases (J=?261 ( 1 ), ?255 ( 3 ), ?241 ( 4 ), ?249 cm^?1 ( 6 )). Computational studies based on hybrid density functional methods have been used to study the magnetic properties of the complexes and also to evaluate their relative stability on the basis of the strength of the bond between each Cu^II and the terminal ligand.     

Deciphering the DNAzyme activity of multimeric quadruplexes: insights into their actual role in the telomerase activity evaluation assay

The end of human telomeres is comprised of a long G-rich single-stranded DNA (known as 3'-overhang) able to adopt an unusual three-dimensional "beads-on-the-string" organization made of consecutively stacked G-quadruplex units (so-called quadruplex multimers). It has been widely demonstrated that, upon interaction with hemin, discrete quadruplexes acquire peroxidase-mimicking properties, oxidizing several organic probes in H(2)O(2)-rich conditions; this property, known as DNAzyme, has found tens of applications in the last two decades. However, little is known about the DNAzyme activity of multimeric quadruplexes; this is an important question to address, especially in light of recent reports that exploit the DNAzyme process to optically assess the activity of an enzyme that elongates the telomeric overhang, the telomerase. Herein, we thoroughly investigate the DNAzyme activity of long telomeric fragments, with a particular focus on both the nature of the hemin/multimeric quadruplex interactions and the putative higher-order fold of the studied fragments; in light of our results, we also propose possible ways that may be followed to improve the use of DNAzyme to evaluate the telomerase activity.     

Selective recognition of a saccharide-type tumor marker with natural and synthetic ligands: a new trend in cancer diagnosis

It is well known that saccharides and their glycoconjugates can have an important influence on various serious pathologic stages such as cancer. They can regulate tumor proliferation, invasion, hematogenous metastasis, and angiogenesis. These facts clearly show the importance of cancer saccharide recognition. In medicine, sensor analysis is one of the best methods for recognition and determination of biologically important analytes. The development and study of sensors for saccharide tumor markers can open a new way for their detection. Therefore, this review is focused on recognition of saccharide-based cancer markers by natural or synthetic selective ligands working as bio- and chemosensors. The design and application of these ligands for cancer diagnosis is a useful direction of research. Moreover, it also opens the possibility of using these agents for the targeted drug transport required for advanced anticancer therapy.     

Structure and dynamics of a complex of cellulose with EDA: insights into the action of amines on cellulose

The neutron structure of a complex of EDA with cellulose has been determined to reveal the location of hydrogen atoms involved in hydrogen-bonding. EDA disrupts the hydrogen-bonding pattern of naturally occurring cellulose by accepting a strong hydrogen-bond from the O6 hydroxymethyl group as the conformation of this group is rotated from tg to gt. The O3-H·O5 intrachain hydrogen-bond commonly found in cellulose allomorphs is observed to be disordered in the neutron structure, and quantum chemistry and molecular dynamics calculations show that O3 prefers to donate to EDA. The hydrogen-bonding arrangement is highly dynamic with bonds continually being formed and broken thus explaining the difficulty in locating all of the hydrogen atoms in the neutron scattering density maps. Comparison with other polysaccharide-amino complexes supports a common underlying mechanism for amine disruption of cellulose.     

Electrochemical sensing of nitric oxide for biological systems: methodological approach and new insights in examining interfering compounds

We report here on the appropriate analysis of some examples of interfering compounds that should be done to assess the specificity of the electrochemical sensing of nitric oxide in solution. To do so, we describe the design of a nickel porphyrin and Nafion®-coated carbon microfibre and discuss the methodological approach in examining interfering compounds.     

Decomposition of dinitrosyl iron complex with thioformaldehyde ligands in water: reaction mechanisms and the role of chemical hardness of ligands

The mechanisms of hydrolysis of a model cationic dinitrosyl iron complex with a prototypic thioformaldehyde ligand have been studied using the density functional theory and polarizable continuum water model. The free-energy calculations have predicted that the associative mechanism of the thioformaldehyde ligand removal has a ～34 kJ mol^-1 lower activation barrier in water than the dissociative mechanism. The additional estimates of chemical hardness have provided useful qualitative characterization of the thio ligands binding.     

Synthesis of iridium complexes with new planar chiral chelating phosphinyl-imidazolylidene ligands and their application in asymmetric hydrogenation

The synthesis of planar chiral phosphinoimidazolium salts such as (R_p)-3-(4-diphenyl-phosphino[2.2]paracyclophan-12-ylmethyl)-1-(2,6-diisopropylphenyl)imidazolium bromide (R_p)-11c starting from enantiopure 4,12-dibromo[2.2]paracyclophane (R_p)-6 is reported. After deprotonation of these salts and a subsequent reaction with [Ir(COD)Cl]₂, chelating iridium imidazolylidene complexes (R_p)-5a–c are obtained. These complexes catalyze the asymmetric hydrogenation of functionalized and simple alkenes with up to 89% ee.     

Probing the motional behavior of eumelanin and pheomelanin with solid-state NMR spectroscopy: new insights into the pigment properties

Melanin is the most widespread pigment in the animal kingdom. Despite its importance, its detailed structure and overall molecular architecture remain elusive. Both eumelanin (black) and pheomelanin (red) occur in the human body. These two melanin compounds show very different responses to UV-radiation exposure, which could relate to their microscopic features. Herein, the structural properties and motional behavior of natural eu- and pheomelanin extracted from black and red human hair are investigated by means of solid-state NMR spectroscopy. Several 1D and 2D NMR spectroscopic techniques were combined to highlight the differences between the two forms of the pigment. The quantitative analysis of the (1) H?NMR wide-line spectra extracted from 2D (1) H-(13) C LG-WISE experiments revealed the presence of two dynamically distinguishable components in both forms. Remarkably, the more mobile fraction of the pigment showed a higher mobility with respect to the proteinaceous components that coexist in the melanosome, which is particularly evident for the red pigment. An explanation of the observed effects takes into account the different architecture of the proteinaceous matrix that constitutes the physical substrate onto which melanin polymerizes within the eu- and pheomelanosomes. Further insight into the molecular structure of the more mobile fraction of pheomelanin was also obtained by means of the analysis of 2D (1) H-(13) C INEPT experiments. Our view is that not only structural features inherent in the pure pigment, but also the role of the matrix structure in defining the overall melanin supramolecular arrangement and the resulting dynamic behavior of the two melanin compounds should be taken into account to explain their functions. The reported results could pave a new way toward the explanation of the molecular origin of the differences in the photoprotection activity displayed by black and red melanin pigments.     

Involvement of some large immunophilins and their ligands in the protection and regeneration of neurons: a hypothetical mode of action

The powerful immunosuppressive drugs such as FK506 and its derivatives induce some regeneration and protection of neurons from ischaemic brain injury and some other neurological disorders. The drugs form complexes with diverse FKBPs but apparently the FKBP52/FK506 complex was shown to be involved in the protection and regeneration of neurons. We used several different sequence attributes in searching diverse genomic databases for similar motifs as those present in the FKBPs. A Fortran library of algorithms (Par_Seq) has been designed and used in searching for the similarity of sequence motifs extracted from the multiple sequence alignments of diverse groups of proteins (query motifs) and the target motifs which are encoded in various genomes. The following sequence attributes were used in the establishment of the degree of convergence between: (A) amino acid (AA) sequence similarity (ID) of the query/target motifs and (B) their: (1) AA composition (AAC); (2) hydrophobicity (HI); (3) Jensen-Shannon entropy; and (4) AA propensity to form a particular secondary structure. The sequence hallmark of two different groups of peptidylprolyl cis/trans isomerases (PPIases), namely tetratricopetide repeat (TPR) motifs, which are present in the heat-shock cyclophilins and in the large FK506-binding proteins (FKBPs) were used to search various genomic databases. The Par_Seq algorithm has revealed that the TPR motifs have similar sequence attributes as a number of hydrophobic sequence segments of functionally unrelated membrane proteins, including some of the TMs from diverse G protein-coupled receptors (GPCRs). It is proposed that binding of the FKBP52/FK506 complex to the membranes via the TPR motifs and its interaction with some membrane proteins could be in part responsible for some neuro-regeneration and neuro-protection of the brain during some ischaemia-induced stresses.     

Thermal and sonochemical studies on the Diels-Alder cycloadditions of masked o-benzoquinones with furans: new insights into the reaction mechanism

What does a Diels-Alder cycloaddition look like? This question is here addressed in the case of the increasingly significant cycloadditions of masked o-benzoquinones (MOBs), which serve as versatile dienes for the construction of complex and functionalized structures. So first, what the mechanism is not: It is not (by and large) a classical, concerted [4 + 2] cycloaddition. Experimental evidence is now supported by sonochemical studies, which were instrumental in elucidating the pathway. Reactions with furans are accelerated and improved under sonication, even when conducted at -10 degrees C. Variation of the acoustic energy, temperature, and solvent composition allows us to optimize the yields and provide insights into the mechanism. Ultrasound does not cause sonochemical switching, as an alternative radical pathway should be ruled out. Results are consistent with a polar mechanism as claimed recently in a theoretical study. Moreover, this also does justice to a series of seminal papers, largely ignored, that gave a clue to the crucial issue of furan regioselectivity based on a nucleophilic addition. Most effects caused by ultrasonic agitation are of mechanochemical nature and suggest the existence of a perfectly stirred reactor with enhanced mass transfer.     

Synthesis of novel norephedrine-based chiral ligands with multiple stereogenic centers and their application in enantioselective addition of diethylzinc to aldehyde and chalcone

Novel norephedrine-based chiral ligands with multiple stereogenic centers were conveniently prepared from norephedrine and N-substituted pyrrole. These novel chiral ligands were used to catalyze the enantioselective addition of diethylzinc to aldehydes and to chalcone in high yields and with good to high enantioselectivities. The absolute configuration of products was found to be affected by the stereogenic centers on the norephedrine part of the novel chiral ligands.