A photoluminescent and electrochemiluminescent probe based on an iridium(III) complex with a boronic acid-functionalised ancillary ligand for the selective detection of mercury(II) ions.

Exposure to mercury(II) ions (Hg2+) can cause various diseases such as Minamata disease, acrodynia, Alzheimer's disease, and Hunter-Russell syndrome, and even organ damage. Therefore, real-time and accurate monitoring of Hg2+ in environmental samples is crucial. In this study, we report a photoluminescent (PL) and electrochemiluminescent (ECL) probe based on a cyclometalated Ir(III) complex for the selective detection of Hg2+. The introduction of a reaction site, o-aminomethylphenylboronic acid, on the ancillary ligands allowed a prompt transmetalation reaction to take place between Hg2+ and boronic acid. This reaction resulted in significant decreases of the PL and ECL signals due to the photo-induced electron transfer from the Ir(III) complex to the Hg2+ ions. The probe was applied to the selective detection of Hg2+, and the signal changes revealed a linear correlation with Hg2+ concentrations in the range of 0-10 µM (LOD = 0.72 µM for PL, 8.03 nM for ECL). The designed probe allowed the successful quantification of Hg2+ in tap water samples, which proves its potential for the selective detection of Hg2+ in environmental samples.

Highly Selective Electrochemiluminescence Chemosensor for Sulfide Enabled by Hierarchical Reactivity.

Hydrogen sulfide (H2S) is a well-known toxic gas with the odor of rotten eggs. Several reaction-based electrochemiluminescence (ECL) chemosensors for H2S have been developed; however, no homogeneous ECL probe with high selectivity toward H2S in aqueous media has been reported. Herein, we report an iridium(III) complex-based ECL chemodosimetric probe employing two 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) groups known as a photo-induced electron transfer quencher and a reaction site for the selective detection of H2S; the detection mechanism involves H2S being clearly distinguished from biothiols based on the different cleavage rates of the two NBD groups and extremely weak ECL interferences caused by reaction by-products. The probe was rationally designed to improve selectivity toward H2S within the ECL analysis platform by enabling the removal of nonspecific background signals observed via fluorescence analysis. This analytical system exhibited remarkable selectivity toward H2S, a rapid reaction rate, and high sensitivity (LOD = 57 nM) compared to conventional fluorescence methods. Furthermore, the probe could successfully quantify H2S in tap water samples and commercial ammonium sulfide solutions, which demonstrates the effectiveness of this probe in field monitoring.

The Stringent Response Contributes to Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection Through the Purine Biosynthetic Pathway.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a significant clinical-therapeutic challenge. Of particular concern is antibiotic treatment failure in infections caused by MRSA that are "susceptible" to antibiotic in vitro. In the current study, we investigate specific purine biosynthetic pathways and stringent response mechanism(s) related to this life-threatening syndrome using genetic matched persistent and resolving MRSA clinical bacteremia isolates (PB and RB, respectively), and isogenic MRSA strain sets. We demonstrate that PB isolates (vs RB isolates) have significantly higher (p)ppGpp production, phenol-soluble-modulin expression, polymorphonuclear leukocyte lysis and survival, fibronectin/endothelial cell (EC) adherence, and EC damage. Importantly, an isogenic strain set, including JE2 parental, relP-mutant and relP-complemented strains, translated the above findings into significant outcome differences in an experimental endocarditis model. These observations indicate a significant regulation of purine biosynthesis on stringent response, and suggest the existence of a previously unknown adaptive genetic mechanism in persistent MRSA infection.

Potential-Dependent Electrochemiluminescence for Selective Molecular Sensing of Cyanide.

Although tremendous efforts have been devoted to providing specificity for molecular sensors, most of the methods focus on the structural variation of the binding or reaction site to improve selectivity. Herein, we report a new approach in which a chemical probe, possessing a mediocre recognition site, can successfully discriminate a target among various interferences only with electrochemical manipulation. The synthetic probe (1) was designed to react with a cyanide anion (CN-), and its dicyanovinyl group has selectivity toward CN- along with sulfides and biothiols resulting in similar adducts. However, the binding adduct between 1 and CN- (1-CN-) has significantly different energy levels that are only able to undergo electrochemical oxidation under ∼1.2 V (vs Ag/AgCl), generating strong electrochemiluminescence (ECL). The ECL emission from 1-CN- successfully discriminates CN- without any interferences from other analytes including sulfides and thiols and exhibits a linear correlation with CN- in a range of 1-400 µM (LOD = 0.04 µM, n = 5). Density functional theory (DFT) calculations and electrochemical studies supported the mechanism of CN- discrimination. The approach was finally applied to direct trace analysis of CN- in tap water (≥1 µM) and showed excellent performance suggesting a new, versatile, and rapid determination method for molecular toxins in real samples.

Electrogenerated Chemiluminescent Chemodosimeter Based on a Cyclometalated Iridium(III) Complex for Sensitive Detection of Thiophenol.

Thiophenol is the simplest aromatic thiol that is utilized for various applications in industry and agriculture. However, it should be used with care because thiophenol is readily absorbed into the human body by inhalation and ingestion, which leads to serious internal injuries. Thus, there is an urgent need for real-time and accurate monitoring of thiophenol. Despite remarkable advantages of electrogenerated chemiluminescence (ECL) analysis, ECL thiophenol probes have never been reported. Herein, a new strategy for the rapid detection of thiophenol by use of an ECL turn-on chemodosimeter based on a cyclometalated Ir(III) complex is described. This analytical system showed superior sensitivity [limit of detection (LOD) value, 3.8 nM] in comparison to the conventional fluorescence method. In addition, our system exhibited remarkable selectivity and reaction rate toward thiophenol over other analytes. Moreover, it was successfully applied to quantify thiophenol in real water samples, providing a new proof-of-concept for field monitoring based on ECL.

Molecular Recognition of Pyrophosphate with Extended Bis(Zn(II)-DPA) Derivatives.

A series of pyrophosphate (PPi) receptors were synthesized, and their binding affinities toward both PPi and adenosine triphosphate were evaluated in N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid-buffered solution. The presence of two hydrogen bond donors slightly retarded the reaction rate of anionic guest exchange, while four had a significant retardation effect. A macrocyclic receptor, 17, exhibited superior selectivity toward PPi compared with acyclic receptors, presumably due to effective steric interactions. Isothermal titration calorimetry experiments and potentiometric titration experiments revealed the effect of the substituent structure on the degree of anion guest stabilization and the deprotonation of water molecules bound to zinc(II) ions, as well as the guest binding thermodynamics.

Electrostatic Modification for Promotion of Flavin-Mediated Oxidation of a Probe for Flavin Detection.

Electrostatic effects on the redox photochemistry of synthetic probes (1, 2, and 1-Zn) are examined by adjusting the thermodynamic driving force of their oxidation reactions. The redox photochemistry was simply controlled by introducing a zinc binding site (2,2'-dipicolylamine (DPA)) on the coumarin moiety of probe 2. Zinc complexation produced a positively charged environment on the coumarin (1-Zn), which lowered the electron density of a nearby 9 H-xanthene ring, attenuating the auto-oxidation of 1-Zn by 45 % compared with that of probe 1 at 298 K. The positive net charge of 1-Zn also provided an attractive Coulombic force toward the phosphate of flavin mononucleotide and flavin adenine dinucleotide, which lowered the reduction potential of the electron acceptor (isoalloxazine) and improved intermolecular electron transfer from the 9 H-xanthene ring to isoalloxazine. The flavin-mediated oxidation rate of 1-Zn was increased to 1.5 times that of probe 2. Probe 1-Zn showed highly selective sensing behaviour toward flavins, producing an intense brightness (ϵΦF =2.80×103 m-1  cm-1 ) in the long-wavelength regions (λmax =588 nm) upon flavin-mediated oxidation. Furthermore, probes 1-Zn and 2 were successfully applied to eosinophil imaging and the differential diagnosis of eosinophilia; this demonstrates their use as diagnostic tools.

Sulfatase activity assay using an activity-based probe by generation of N-methyl isoindole under reducing conditions.

Sulfatases catalyze the hydrolysis of sulfate esters that are present in a range of biomolecules. This is an important step in several biological processes such as cellular degradation, hormone regulation, and cell signaling. We have developed a new activity-based sulfatase probe (probe 1) that generates a fluorescent N-methylisoindole upon hydrolysis by sulfatase. Because of the autoxidation of N-methylisoindole, the sulfatase activity was also tested under reducing conditions, containing either glutathione (GSH) or tris(2-carboxyethyl)phosphine (TCEP), exhibiting little change in kinetic parameters compared to non-reducing conditions. Probe 1 displayed reasonable kinetic parameters under both non-reducing and reducing conditions, among which the use of Tris buffer and Tris buffer containing GSH appeared to be appropriate conditions for inhibitor screening. Probe 1 showed stronger intensity upon treatment with sulfatase under neutral conditions than under acidic conditions, but it still has limitations in the selectivity for a specific sulfatase. Nevertheless, the fluorescent signal generated as a result of the release of N-methylisoindole after treatment of probe 1 with sulfatase provides a new assay for measuring sulfatase activity that could be adapted for high throughput screening.

The bacterial alarmone (p)ppGpp activates the type III secretion system in Erwinia amylovora.

UNLABELLED: The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp(0)) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp(0) and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp(0) and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCE: The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of an spoT null mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional.

A regulatory feedback loop between RpoS and SpoT supports the survival of Legionella pneumophila in water.

Legionella pneumophila is a waterborne pathogen, and survival in the aquatic environment is central to its transmission to humans. Therefore, identifying genes required for its survival in water could help prevent Legionnaires' disease outbreaks. In the present study, we investigate the role of the sigma factor RpoS in promoting survival in water, where L. pneumophila experiences severe nutrient deprivation. The rpoS mutant showed a strong survival defect compared to the wild-type strain in defined water medium. The transcriptome of the rpoS mutant during exposure to water revealed that RpoS represses genes associated with replication, translation, and transcription, suggesting that the mutant fails to shut down major metabolic programs. In addition, the rpoS mutant is transcriptionally more active than the wild-type strain after water exposure. This could be explained by a misregulation of the stringent response in the rpoS mutant. Indeed, the rpoS mutant shows an increased expression of spoT and a corresponding decrease in the level of (p)ppGpp, which is due to the presence of a negative feedback loop between RpoS and SpoT. Therefore, the lack of RpoS causes an aberrant regulation of the stringent response, which prevents the induction of a successful response to starvation.

Homogeneous electrochemical assay for protein kinase activity.

Herein, we report a homogeneous assay for protein kinase activity using an electrochemistry-based probe. The approach involves a peptide substrate conjugated with a redox tag and the phosphate-specific receptor immobilized on an electrode surface. The peptide substrate phosphorylated by a protein kinase binds to the receptor site of the probe, which results in a redox current under voltammetric measurement. Our method was successfully applied even in the presence of citrated human blood and modified to enable a single-use, chip-based electrochemical assay for kinase activity.

Phosphorescent sensor for phosphorylated peptides based on an iridium complex.

A bis[(4,6-difluorophenyl)pyridinato-N,C(2')]iridium(III) picolinate (FIrpic) derivative coupled with bis(Zn(2+)-dipicolylamine) (ZnDPA) was developed as a sensor (1) for phosphorylated peptides, which are related to many cellular mechanisms. As a control, a fluorescent sensor (2) based on anthracene coupled to ZnDPA was also prepared. When the total negative charge on the phosphorylated peptides was changed to -2, -4, and -6, the emission intensity of sensor 1 gradually increased by factors of up to 7, 11, and 16, respectively. In contrast, there was little change in the emission intensity of sensor 1 upon the addition of a neutral phosphorylated peptide, non-phosphorylated peptides, or various anions such as CO3(2-), NO3(-), SO4(2-), phosphate, azide, and pyrophosphate. Furthermore, sensor 1 could be used to visually discriminate between phosphorylated peptides and adenosine triphosphate in aqueous solution under a UV-vis lamp, unlike fluorescent sensor 2. This enhanced luminance of phosphorescent sensor 1 upon binding to a phosphorylated peptide is attributed to a reduction in the repulsion between the Zn(2+) ions due to the phenoxy anion, its strong metal-to-ligand charge transfer character, and a reduction in self-quenching.

Fluorescent probes designed for detecting human serum albumin on the basis of its pseudo-esterase activity.

We developed activity-based fluorescent probes for detecting human serum albumin (HSA) on the basis of its pseudo-esterase activity. These probes could also detect HSA in blood-contaminated tissue samples.

Reaction-based energy level modulation of a cyclometalated iridium complex for electrochemiluminescent detection of formaldehyde.

Formaldehyde is a toxic compound present in both the environment and living systems, and its detection is important due to its association with various pathological process. In this study, we report a new electrochemiluminescence (ECL) probe based on a cyclometalated iridium complex (IrHAA) for the selective detection of formaldehyde. The homoallylamine moiety in IrHAA reacts with formaldehyde, undergoing a 2-aza-Cope-rearrangement reaction to form a formyl group. Significant changes in the electronic properties and molecular orbital energies of the iridium complex through the functional group transformation result in enhanced ECL and radiometric phosphorescence changes, enabling the quantitative and selective detection of formaldehyde. The energetic requirements for ECL sensing were investigated, highlighting the importance of the excited state energy for achieving efficient ECL. The sensing mechanism was elucidated using NMR spectroscopy and MALDI-TOF analysis.

Ambient-Stable n-Type Carbon Nanotube/Organic Small-Molecule Thermoelectrics Enabled by Energy Level Control.

The stability of n-type organic and hybrid thermoelectric materials is limited in terms of their practical application to p-n parallel thermoelectric devices. We demonstrate the ambient stability of an n-type single-walled carbon nanotube/organic small-molecule (SWNT/OSM) hybrid by deepening the lowest occupied molecular orbital energy level. This hybrid exhibited the best figure of merit (0.032) among n-type SWNT/OSM hybrid thermoelectrics and an enhanced power factor of 291.0 µW m-1 K-2. Furthermore, we observed that the n-type thermoelectric stability of a hybrid of SWNT and pip containing two N-ethylpiperidinyl groups on both sides of a naphthalenediimide core was retained at 87% over 7 months (220 days) under ambient conditions without encapsulation.

Molecular Engineering for Enhanced Thermoelectric Performance of Single-Walled Carbon Nanotubes/π-Conjugated Organic Small Molecule Hybrids.

Hybridizing single-walled carbon nanotubes (SWCNTs) with π-conjugated organic small molecules (π-OSMs) offers a promising approach for producing high-performance thermoelectric (TE) materials through the facile optimization of the molecular geometry and energy levels of π-OSMs. Designing a twisted molecular structure for the π-OSM with the highest occupied molecular orbital energy level comparable to the valence band of SWCNTs enables effective energy filtering between the two materials. The SWCNTs/twisted π-OSM hybrid exhibits a high Seebeck coefficient of 110.4 ± 2.6 µV K-1 , leading to a significantly improved power factor of 2,136 µW m-1 K-2 , which is 2.6 times higher than that of SWCNTs. Moreover, a maximum figure of merit over 0.13 at room temperature is achieved via the efficient TE transport of the SWCNTs/twisted π-OSM hybrid. The study highlights the promising potential of optimizing molecular engineering of π-OSMs for hybridization with SWCNTs to create next-generation, efficient TE materials.

Electrochemiluminescence of dimethylaminonaphthalene-oxazaborine donor-acceptor luminophores.

Donor-acceptor (D-A) type molecules with a skeleton consisting of a dimethylaminonaphthalene donor and an oxazaborine acceptor were designed as efficient electrochemiluminescence (ECL) luminophores with tunable intramolecular charge transfer (ICT). The D-A ECL luminophores demonstrated that the ICT characteristics play a critical role in the electrochemistry and ECL of luminophores in the presence of tri-n-propylamine, which was rationalised experimentally and computationally. Furthemore, dual-peaked ECL-potential behaviours of the luminophores were rationalised using two competitive pathway ECL mechanisms, elucidated through the use of spooling ECL spectroscopy.

Gp05, a Prophage-Encoded Virulence Factor, Contributes to Persistent Methicillin-Resistant Staphylococcus aureus Endovascular Infection.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) endovascular infections represent a serious public health threat. We recently demonstrated that the presence of a novel prophage ϕSA169 was associated with vancomycin (VAN) treatment failure in experimental MRSA endocarditis. In this study, we assessed the role of a ϕSA169 gene, ϕ80α_gp05 (gp05), in VAN-persistent outcome using gp05 isogenic MRSA strain sets. Of note, Gp05 significantly influences the intersection of MRSA virulence factors, host immune responses, and antibiotic treatment efficacy, including the following: (i) activity of the significant energy-yielding metabolic pathway (e.g., tricarboxylic acid cycle); (ii) carotenoid pigment production; (iii) (p)ppGpp (guanosine tetra- and pentaphosphate) production, which activates the stringent response and subsequent downstream functional factors (e.g., phenol-soluble modulins and polymorphonuclear neutrophil bactericidal activity); and (iv) persistence to VAN treatment in an experimental infective endocarditis model. These data suggest that Gp05 is a significant virulence factor which contributes to the persistent outcomes in MRSA endovascular infection by multiple pathways. IMPORTANCE Persistent endovascular infections are often caused by MRSA strains that are susceptible to anti-MRSA antibiotics in vitro by CLSI breakpoints. Thus, the persistent outcome represents a unique variant of traditional antibiotic resistance mechanisms and a significant therapeutic challenge. Prophage, a critical mobile genetic element carried by most MRSA isolates, provides their bacterial host with metabolic advantages and resistance mechanisms. However, how prophage-encoded virulence factors interact with the host defense system and antibiotics, driving the persistent outcome, is not well known. In the current study, we demonstrated that a novel prophage gene, gp05, significantly impacts tricarboxylic acid cycle activity, stringent response, and pigmentation, as well as vancomycin treatment outcome in an experimental endocarditis model using isogenic gp05 overexpression and chromosomal deletion mutant MRSA strain sets. The findings significantly advance our understanding of the role of Gp05 in persistent MRSA endovascular infection and provide a potential target for development of novel drugs against these life-threatening infections.

Efficient fluorescence "turn-on" sensing of dissolved oxygen by electrochemical switching.

We report on a novel method for sensing oxygen that is based on the use of a perylene diimide dye (1) which is electrochemically reduced to its nonfluorescent dianion form (1(2-)). In the presence of oxygen, the dianion is oxidized to its initial form via an electron-transfer reaction with oxygen upon which fluorescence is recovered. As a result, the fluorescence intensity of the dianion solution increases upon the addition of oxygen gas. Results demonstrate that high sensitivity is obtained, and the emission intensity shows a linear correlation with oxygen content (0.0-4.0% v/v) at ambient barometric pressure. In addition, using electrochemical reduction, oxygen determination becomes regenerative, and no significant degradation is observed over several turnovers. The limit of detection is 0.4% oxygen in argon gas.

Two novel point mutations in clinical Staphylococcus aureus reduce linezolid susceptibility and switch on the stringent response to promote persistent infection.

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3', 5'-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens.