Time-Resolved Mechanistic Depiction of Photoinduced CO2 Reduction Catalysis on a Urea-Modified Iron Porphyrin.

The development of functional artificial photosynthetic devices relies on the understanding of mechanistic aspects involved in specialized photocatalysts. Modified iron porphyrins have long been explored as efficient catalysts for the light-induced reduction of carbon dioxide (CO2) towards solar fuels. In spite of the advancements in homogeneous catalysis, the development of the next generation of catalysts requires a complete understanding of the fundamental photoinduced processes taking place prior to and after activation of the substrate by the catalyst. In this work, we employ a state-of-the-art nanosecond optical transient absorption spectroscopic setup with a double excitation capability to induce charge accumulation and trigger the reduction of CO2 to carbon monoxide (CO). Our biomimetic system is composed of a urea-modified iron(III) tetraphenylporphyrin (UrFeIII) catalyst, the prototypical [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) used as a photosensitizer, and sodium ascorbate as an electron donor. Under inert atmosphere, we show that two electrons can be successively accumulated on the catalyst as the fates of the photogenerated UrFeII and UrFeI reduced species are tracked. In the presence of CO2, the catalytic cycle is kick-started providing further evidence on CO2 activation by the UrFe catalyst in its formal FeI oxidation state.

Photoinduced electron transfer between a noble-metal-free [Mo6I8Cl6]2- cluster and polyoxometalates.

Evidence for photoinduced intermolecular electron transfer from the excited state of the [Mo6I8Cl6]2- electron-rich cluster to polyoxometalates (POMs) is reported. We demonstrate that the global charge density of POMs affects the efficiency of electron transfer. This work paves the way for the rational design of photocatalytic systems using cluster-based complexes as robust noble-metal-free photosensitizers.

A Novel Pump-Pump-Probe Resonance Raman Approach Featuring Light-Induced Charge Accumulation on a Model Photosystem.

Light-induced charge accumulation is at the heart of biomimetic systems aiming at solar fuel production in the realm of artificial photosynthesis. Understanding the mechanisms upon which these processes operate is a necessary condition to drive down the rational catalyst design road. We have built a nanosecond pump-pump-probe resonance Raman setup to witness the sequential charge accumulation process while probing vibrational features of different charge-separated states. By employing a reversible model system featuring methyl viologen (MV) as a dual electron acceptor, we have been able to watch the photosensitized production of its neutral form, MV0, resulting from two sequential electron transfer reactions. We have found that, upon double excitation, a fingerprint vibrational mode corresponding to the doubly reduced species appears at 992 cm-1 and peaks at 30 µs after the second excitation. This has been further confirmed by simulated resonance Raman spectra which fully support our experimental findings in this unprecedented buildup of charge seen by a resonance Raman probe.

Selective population of triplet excited states in heavy-atom-free BODIPY-C60 based molecular assemblies.

Photophysical studies on a BODIPY-fullerene-distyryl BODIPY triad (BDP-C60-DSBDP) and its reference dyads (BODIPY-fullerene; BDP-C60 and distyryl BODIPY-fullerene; DSBDP-C60) are presented herein. In the triad, the association of the two chromophore units linked by a fullerene moiety leads to strong near UV-Visible light absorption from 300 to 700 nm. The triplet-excited state was observed upon visible excitation in all these assemblies, and shown to be localized on the C60 or BODIPY moieties. Using quantitative nanosecond transient absorption, we provide a complete investigation on the lifetime and formation quantum yield of the triplet-excited state. In the BDP-C60 dyad, the triplet excited state of C60 (τ = 7 ± 1 µs) was obtained with a quantum yield of 40 ± 8%. For the DSBDP-C60 dyad and BDP-C60-DSBDP triad, a longer-lived triplet excited state with a lifetime of around 250 ± 20 µs centered on the DSBDP moiety was formed, with respective quantum yields of 37 ± 8 and 20 ± 4%. Triplet-triplet annihilation up-conversion is characterized in the BDP-C60 dyad and the bichromophoric triad in the presence of perylene and DSBDP-monomer as respective annihilators. The photo-induced formation of a long-lived 3DSBDP* in the triad coupled with panchromatic light absorption offers potential applications as a heavy-atom-free organic triplet photosensitizer.

Time-Resolved Spectroscopy and High-Efficiency Light-Driven Hydrogen Evolution of a {Mo3 S4 }-Containing Polyoxometalate-Based System.

Polyoxothiometalate ions (ThioPOM) are active hydrogen-evolution reaction (HER) catalysts based on modular assembly built from electrophilic clusters {MoSx } and vacant polyoxotungstates. Herein, the dumbbell-like anion [{(PW11 O39 )Mo3 S4 (H2 O)3 (OH)}2 ]8- exhibits very high light-driven HER activity, while the active cores {Mo3 S4 } do not contain any exposed disulfido ligands, which were suspected to be the origin of the HER activity. Moreover, in the catalyst architecture, the two central {Mo3 S4 } cores are sandwiched by two {PW11 O39 }7- subunits that act as oxidant-resistant protecting groups and behave as electron-collecting units. A detailed photophysical study was carried out confirming the reductive quenching mechanism of the photosensitizer [Ir(ppy)2 (dtbbpy)]+ by the sacrificial donor triethanolamine (TEOA) and highlighting the very high rate constant of the electron transfer from the reduced photosensitizer to the ThioPOM catalyst. Such results provide new insights into the field of molecular catalytic systems able to promote high HER activity.

Fluorescence Emitted by Papanicolaou-Stained Urothelial Cells Improves Sensitivity of Urinary Conventional Cytology for Detection of Urothelial Tumors.

BACKGROUND: Urinary conventional cytology (UCCy) is easy to perform, but its low sensitivity, especially for low-grade urothelial neoplasms (LGUNs), limits its indications in the management of patients at risk of bladder cancer. The authors aim at obtaining a complementary test that would effectively increase the sensitivity of UCCy on voided urines by analyzing fluorescence of Papanicolaou-stained urothelial cells with no change of method in slide preparation. METHODS: In this retrospective study of 155 patients, 91 Papanicolaou-stained voided urines were considered satisfactory under fluorescence microscopy (FMi). The results of FMi were compared with UCCy (using transmission microscopy) and correlated to cystoscopy, histology and follow-up data. RESULTS: The results are given for all patients and for two groups of them according to the patients' main complaints (group 1: 33 patients followed up for a previously treated bladder tumor; group 2: 58 patients with persistent urinary symptoms). Overall negative predictive value (NPV) and sensitivity of FMi were 100% vs. 73.7% and 64.3% respectively for UCCy (P = 0.0001). Sensitivity of FMi for LGUN was unexpectedly high with a value of 100% vs. 46.2% for UCCy (P = 0.0002). FMi was significantly superior to UCCy for detecting urothelial tumors in every group of patients and would allow a better characterization of atypical urothelial cells (AUCs) defined by the Paris System for Reporting Urine Cytology (TPS). CONCLUSIONS: Because of its sensitivity and NPV of 100%, FMi could complement UCCy to screen voided urines allowing a better detection of primary urothelial tumors or early recurrences of previously treated urothelial carcinoma. Moreover, this "dual screening" would allow completing efficiently cystoscopy to detect flat dysplasia, carcinoma in situ (CIS) and extra bladder carcinoma.

Self-assembled multifunctional core-shell highly porous metal-organic framework nanoparticles.

Core-shell nanoparticles (NPs) are attracting increasing interest in nanomedicine as they exhibit unique properties arising from the combined assets of core and shell materials. Porous nanoscale metal-organic frameworks (nanoMOFs) are able to incorporate with high payloads a large variety of drugs. Like other types of NPs, nanoMOFs need to be functionalized with engineered coatings to ensure colloidal stability, control in vivo fate and drug release. To do so, a novel biodegradable cyclodextrin (CD)-based shell was designed in this study. Water soluble γ-CD-citrate oligomers grafted or not with fluorophores were successfully synthesized using citric acid as crosslinker and efficiently anchored onto the surface of porous nanoMOFs. As compared to monomeric CDs, the oligomeric CD coatings could offer higher interaction possibilities with the cores and better possibilities to graft functional moieties such as fluorescent molecules. The amounts of γ-CD-citrate oligomers onto the nanoMOFs were as high as 53 ± 8 wt%. The yield reached up to 86% in the optimized system. These core-shell nanocomposites were stable upon storage, in contrast to the naked nanoMOFs. In addition, the presence of the coating prevented the doxorubicin (DOX)-loaded nanoMOFs from aggregation. Moreover, due to the presence of fluorophores conjugated to the shell, fluorescence-lifetime microscopy enabled deciphering the coating mechanism. DOX loadings reached 48 ± 10 wt% after 24 h incubation with the drug solution. After coating for additional 24 h, DOX loadings reached 65 ± 8 wt%.

Direct observation of the cell-wall remodeling in adhering Staphylococcus aureus 27217: An AFM study supported by SEM and TEM.

We took benefit from Atomic Force Microscopy (AFM) in the force spectroscopy mode to describe the time evolution - over 24 h - of the surface nanotopography and mechanical properties of the strain Staphylococcus aureus 27217 from bacterial adhesion to the first stage of biofilm genesis. In addition, Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) experiments allowed identifying two types of self-adhering subpopulations (the so-called "bald" and "hairy" cells) and revealed changes in their relative populations with the bacterial culture age and the protocol of preparation. We indeed observed a dramatic evanescing of the "hairy" subpopulation for samples that underwent centrifugation and resuspension processes. When examined by AFM, the "hairy" cell surface resembled to a herringbone structure characterized by upper structural units with lateral dimensions of ∼70 nm and a high Young modulus value (∼2.3 MPa), a mean depth of the trough between them of ∼15 nm and a resulting roughness of ∼5 nm. By contrast, the "bald" cells appeared much softer (∼0.35 MPa) with a roughness one order of magnitude lower. We observed too the gradual detachment of the herringbone patterns from the "hairy" bacterial envelope of cell harvested from a 16 h old culture and their progressive accumulation between the bacteria in the form of globular clusters. The secretion of a soft extracellular polymeric substance was also identified that, in addition to the globular clusters, may contribute to the initiation of the biofilm spatial organization.

Food-grade TiO2 is trapped by intestinal mucus in vitro but does not impair mucin O-glycosylation and short-chain fatty acid synthesis in vivo: implications for gut barrier protection.

BACKGROUND: Titanium dioxide (TiO2) particles are commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, the risk of gut barrier disruption is an increasing concern because of the presence of a nano-sized fraction. Food-grade E171 may interact with mucus, a gut barrier protagonist still poorly explored in food nanotoxicology. To test this hypothesis, a comprehensive approach was performed to evaluate in vitro and in vivo interactions between TiO2 and intestinal mucus, by comparing food-grade E171 with NM-105 (Aeroxyde P25) OECD reference nanomaterial. RESULTS: We tested E171-trapping properties of mucus in vitro using HT29-MTX intestinal epithelial cells. Time-lapse confocal laser scanning microscopy was performed without labeling to avoid modification of the particle surface. Near-UV irradiation of E171 TiO2 particles at 364 nm resulted in fluorescence emission in the visible range, with a maximum at 510 nm. The penetration of E171 TiO2 into the mucoid area of HT29-MTX cells was visualized in situ. One hour after exposure, TiO2 particles accumulated inside "patchy" regions 20 µm above the substratum. The structure of mucus produced by HT29-MTX cells was characterized by MUC5AC immunofluorescence staining. The mucus layer was thin and organized into regular "islands" located approximately 20 µm above the substratum. The region-specific trapping of food-grade TiO2 particles was attributed to this mucus patchy structure. We compared TiO2-mediated effects in vivo in rats after acute or sub-chronic oral daily administration of food-grade E171 and NM-105 at relevant exposure levels for humans. Cecal short-chain fatty acid profiles and gut mucin O-glycosylation patterns remained unchanged, irrespective of treatment. CONCLUSIONS: Food-grade TiO2 is trapped by intestinal mucus in vitro but does not affect mucin O-glycosylation and short-chain fatty acid synthesis in vivo, suggesting the absence of a mucus barrier impairment under "healthy gut" conditions.

Live intramacrophagic Staphylococcus aureus as a potential cause of antibiotic therapy failure: observations in an in vivo mouse model of prosthetic vascular material infections.

Objectives: To evaluate the significant role played by biofilms during prosthetic vascular material infections (PVMIs). Methods: We developed an in vivo mouse model of Staphylococcus aureus PVMI allowing its direct observation by confocal microscopy to describe: (i) the structure of biofilms developed on Dacron® vascular material; (ii) the localization and effect of antibiotics on these biostructures; and (iii) the interaction between bacteria and host tissues and cells during PVMI. Results: In this model we demonstrated that the biofilm structures are correlated to the activity of antibiotics. Furthermore, live S. aureus bacteria were visualized inside the macrophages present at the biofilm sites, which is significant as antibiotics do not penetrate these immune cells. Conclusions: This intracellular situation may explain the limited effect of antibiotics and also why PVMIs can relapse after antibiotic therapy.

Impact of Bacterial Membrane Fatty Acid Composition on the Failure of Daptomycin To Kill Staphylococcus aureus.

Daptomycin is a last-resort membrane-targeting lipopeptide approved for the treatment of drug-resistant staphylococcal infections, such as bacteremia and implant-related infections. Although cases of resistance to this antibiotic are rare, increasing numbers of clinical, in vitro, and animal studies report treatment failure, notably against Staphylococcus aureus The aim of this study was to identify the features of daptomycin and its target bacteria that lead to daptomycin treatment failure. We show that daptomycin bactericidal activity against S. aureus varies significantly with the growth state and strain, according to the membrane fatty acid composition. Daptomycin efficacy as an antibiotic relies on its ability to oligomerize within membranes and form pores that subsequently lead to cell death. Our findings ascertain that daptomycin interacts with tolerant bacteria and reaches its membrane target, regardless of its bactericidal activity. However, the final step of pore formation does not occur in cells that are daptomycin tolerant, strongly suggesting that it is incapable of oligomerization. Importantly, membrane fatty acid contents correlated with poor daptomycin bactericidal activity, which could be manipulated by fatty acid addition. In conclusion, daptomycin failure to treat S. aureus is not due to a lack of antibiotic-target interaction, but is driven by its capacity to form pores, which depends on membrane composition. Manipulation of membrane fluidity to restore S. aureus daptomycin bactericidal activity in vivo could open the way to novel antibiotic treatment strategies.

How do fluorescence spectroscopy and multimodal fluorescence imaging help to dissect the enhanced efficiency of the vancomycin-rifampin combination against Staphylococcus aureus infections?

Staphylococcus aureus is one of the most frequent pathogens responsible for biofilm-associated infections. Among current clinical antibiotics, very few enable long-term successful treatment. Thus, it becomes necessary to better understand antibiotic failures and successes in treating infections in order to master the use of proper antibiotic therapies. In this context, we took benefit from a set of fluorescence spectroscopy and imaging methods, with the support of conventional microbiological tools to better understand the vancomycin-rifampin combination (in)efficiency against S. aureus biofilms. It was shown that both antibiotics interacted by forming a complex. This latter allowed a faster penetration of the drugs before dissociating from each other to interact with their respective biological targets. However, sufficiently high concentrations of free vancomycin should be maintained, either by increasing the vancomycin concentration or by applying repetitive doses of the two drugs, in order to eradicate rifampin-resistant mutants.

New Insight into Daptomycin Bioavailability and Localization in Staphylococcus aureus Biofilms by Dynamic Fluorescence Imaging.

Staphylococcus aureus is one of the most frequent pathogens responsible for biofilm-associated infections (BAI), and the choice of antibiotics to treat these infections remains a challenge for the medical community. In particular, daptomycin has been reported to fail against implant-associated S. aureus infections in clinical practice, while its association with rifampin remains a good candidate for BAI treatment. To improve our understanding of such resistance/tolerance toward daptomycin, we took advantage of the dynamic fluorescence imaging tools (time-lapse imaging and fluorescence recovery after photobleaching [FRAP]) to locally and accurately assess the antibiotic diffusion reaction in methicillin-susceptible and methicillin-resistant S. aureus biofilms. To provide a realistic representation of daptomycin action, we optimized an in vitro model built on the basis of our recently published in vivo mouse model of prosthetic vascular graft infections. We demonstrated that at therapeutic concentrations, daptomycin was inefficient in eradicating biofilms, while the matrix was not a shield to antibiotic diffusion and to its interaction with its bacterial target. In the presence of rifampin, daptomycin was still present in the vicinity of the bacterial cells, allowing prevention of the emergence of rifampin-resistant mutants. Conclusions derived from this study strongly suggest that S. aureus biofilm resistance/tolerance toward daptomycin may be more likely to be related to a physiological change involving structural modifications of the membrane, which is a strain-dependent process.

Natural Rubber-Filler Interactions: What Are the Parameters?

Reinforcement of a polymer matrix through the incorporation of nanoparticles (fillers) is a common industrial practice that greatly enhances the mechanical properties of the composite material. The origin of such mechanical reinforcement has been linked to the interaction between the polymer and filler as well as the homogeneous dispersion of the filler within the polymer matrix. In natural rubber (NR) technology, knowledge of the conditions necessary to achieve more efficient NR-filler interactions is improving continuously. This study explores the important physicochemical parameters required to achieve NR-filler interactions under dilute aqueous conditions by varying both the properties of the filler (size, composition, surface activity, concentration) and the aqueous solution (ionic strength, ion valency). By combining fluorescence and electron microscopy methods, we show that NR and silica interact only in the presence of ions and that heteroaggregation is favored more than homoaggregation of silica-silica or NR-NR. The interaction kinetics increases with the ion valence, whereas the morphology of the heteroaggregates depends on the size of silica and the volume percent ratio (dry silica/dry NR). We observe dendritic structures using silica with a diameter (d) of 100 nm at a ∼20-50 vol % ratio, whereas we obtain raspberry-like structures using silica with d = 30 nm particles. We observe that in liquid the interaction is controlled by the hydrophilic bioshell, in contrast to dried conditions, where hydrophobic polymer dominates the interaction of NR with the fillers. A good correlation between the nanoscopic aggregation behavior and the macroscopic aggregation dynamics of the particles was observed. These results provide insight into improving the reinforcement of a polymer matrix using NR-filler films.

Deciphering biofilm structure and reactivity by multiscale time-resolved fluorescence analysis.

In natural, industrial and medical environments, microorganisms mainly live as structured and organised matrix-encased communities known as biofilms. In these communities, microorganisms demonstrate coordinated behaviour and are able to perform specific functions such as dramatic resistance to antimicrobials, which potentially lead to major public health and industrial problems. It is now recognised that the appearance of such specific biofilm functions is intimately related to the three-dimensional organisation of the biological edifice, and results from multifactorial processes. During the last decade, the emergence of innovative optical microscopy techniques such as confocal laser scanning microscopy in combination with fluorescent labelling has radically transformed imaging in biofilm research, giving the possibility to investigate non-invasively the dynamic mechanisms of formation and reactivity of these biostructures. In this chapter, we discuss the contribution of fluorescence analysis and imaging to the study at different timescales of various processes: biofilm development (hours to days), antimicrobial reactivity within the three-dimensional structure (minutes to hours) or molecular diffusion/reaction phenomena (pico- to milliseconds).

Diffusion of nanoparticles in biofilms is altered by bacterial cell wall hydrophobicity.

Diffusion of entities inside biofilm triggers most mechanisms involved in biofilm-specific phenotypes. Using genetically engineered hydrophilic and hydrophobic cells of Lactococcus lactis yielding similar biofilm architectures, we demonstrated by fluorescence correlation spectroscopy that bacterial surface properties affect diffusion of nanoparticles through the biofilm matrix.

Diffusion measurements inside biofilms by image-based fluorescence recovery after photobleaching (FRAP) analysis with a commercial confocal laser scanning microscope.

Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside "live" biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactococcus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.

Tracking the photosensitizing antibacterial activity of mono(acridyl)bis(arginyl)porphyrin (MABAP) by time-resolved spectroscopy.

Photodynamic inactivation (PDI) is currently receiving interest for its potential as an antimicrobial treatment. Although photosensitizing agents and light have been used for medical purposes for a very long time, only a little information is available about the mechanism of PDI for bacteria. Pseudomonas aeruginosa is a gram negative bacteria involved in chronic infections in cystic fibrosis patients and also one of the commonest agents of hospital acquired infections. In the present study the sensitivity of Pseudomonas aeruginosa to the phototoxic effects of the mono(acridyl)bis(arginyl)porphyrin (MABAP) has been investigated as well as the photophysical and photochemical properties of this cationic porphyrin complexed to [poly(dG-dC)](2) to investigate the mechanisms that lead to bacteria inactivation. Both picosecond time-resolved fluorescence and femtosecond to nanosecond transient absorption measurements give evidence that while MABAP can react through its triplet state and/or an ultrafast electron transfer with guanine, its intercalation between GC base pairs is not the main target of MABAP photoactivity. The analysis of both fluorescence emission and excitation spectra reveals the occurrence of an energy transfer through the DNA double helix between the acridine and porphyrin chromophores of MABAP, as previously observed for the stacked free molecule in solution. This efficient process may lead to the excitation of twice more porphyrin chromophores in MABAP by comparison to other cationic porphyrins.

Ex vivo fluorescence imaging of normal and malignant urothelial cells to enhance early diagnosis.

Urinary cytology is a noninvasive and unconstraining technique for urothelial cancer diagnosis but lacks sensitivity for detecting low-grade lesions. In this study, the fluorescence properties of classical Papanicolaou-stained urothelial cytological slides from patients or from cell lines were monitored to investigate metabolic changes in normal and tumoral cells. Time- and spectrally-resolved fluorescence imaging was performed at the single cell level to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by urothelial cells. The results reveal quite different fluorescence distributions between tumoral urothelial cells, characterized by a perimembrane fluorescence localization, and the normal cells which exhibit an intracellular fluorescence. This is not caused by differences in the fluorescence emission of the endogenous fluorophores NAD(P)H, flavoproteins or porphyrins but by various localization of the EA 50 Papanicolaou stain as revealed by both the spectral and time-resolved parameters. The present results demonstrate that the use of single-cell endofluorescence emission of Papanicolaou-stained urothelial cytological slides can allow an early ex vivo diagnosis of low-grade bladder cancers.