Role of the phospholipid binding sites, PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2.

In phagocytes, superoxide anion (O2-), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40phox, p47phox, p67phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme. To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47phox. We also used a fused chimera consisting of p47phox(aa 1-286), p67phox(aa 1-212) and Rac1Q61L, as well as mutated versions in the p47phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.

Enzymatic cascade reaction in simple-coacervates.

The design of enzymatic droplet-sized reactors constitutes an important challenge with many potential applications such as medical diagnostics, water purification, bioengineering, or food industry. Coacervates, which are all-aqueous droplets, afford a simple model for the investigation of enzymatic cascade reaction since the reactions occur in all-aqueous media, which preserve the enzymes integrity. However, the question relative to how the sequestration and the proximity of enzymes within the coacervates might affect their activity remains open. Herein, we report the construction of enzymatic reactors exploiting the simple coacervation of ampholyte polymer chains, stabilized with agar. We demonstrate that these coacervates have the ability to sequester enzymes such as glucose oxidase and catalase and preserve their catalytic activity. The study is carried out by analyzing the color variation induced by the reduction of resazurin. Usually, phenoxazine molecules acting as electron acceptors are used to characterize glucose oxidase activity. Resazurin (pink) undergoes a first reduction to resorufin (salmon) and then to dihydroresorufin (transparent) in presence of glucose oxidase and glucose. We have observed that resorufin is partially regenerated in the presence of catalase, which demonstrates the enzymatic cascade reaction. Studying this enzymatic cascade reaction within coacervates as reactors provide new insights into the role of the proximity, confinement towards enzymatic activity.

Direct Sensing of Superoxide and Its Relatives Reactive Oxygen and Nitrogen Species in Phosphate Buffers during Cold Atmospheric Plasmas Exposures.

This study aims at sensing in situ reactive oxygen and nitrogen species (RONS) and specifically superoxide anion (O2•-) in aqueous buffer solutions exposed to cold atmospheric plasmas (CAPs). CAPs were generated by ionizing He gas shielded with variable N2/O2 mixtures. Thanks to ultramicroelectrodes protected against the high electric fields transported by the ionization waves of CAPs, the production of superoxide and several RONS was electrochemically directly detected in liquids during their plasma exposure. Complementarily, optical emissive spectroscopy (OES) was used to study the plasma phase composition and its correlation with the chemistry in the exposed liquid. The specific production of O2•-, a biologically reactive redox species, was analyzed by cyclic voltammetry (CV), in both alkaline (pH 11), where the species is fairly stable, and physiological (pH 7.4) conditions, where it is unstable. To understand its generation with respect to the plasma chemistry, we varied the shielding gas composition of CAPs to directly impact on the RONS composition at the plasma-liquid interface. We observed that the production and accumulation of RONS in liquids, including O2•-, depends on the plasma composition, with N2-based shieldings providing the highest superoxide concentrations (few 10s of micromolar at most) and of its derivatives (hundreds of micromolar). In situ spectroscopic and electrochemical analyses provide a high resolution kinetic and quantitative understanding of the interactions between CAPs and physiological solutions for biomedical applications.

Electroanalysis at a Single Giant Vesicle Generating Enzymatically a Reactive Oxygen Species.

In the framework of artificial or synthetic cell development, giant liposomes are common basic structures. Their enclosed membrane allows encapsulating proteins, DNA, reactants, etc., while its phospholipid nature allows some exchanges with the surrounding medium. Biochemical reactions induced inside giant liposomes or vesicles are often monitored or imaged by fluorescence microscopy techniques. Here, we show that electrochemistry performed with ultramicroelectrodes is perfectly suitable to monitor an enzymatic reaction occurring in a single giant unilamellar vesicle. Glucose oxidase (GOx) was microinjected inside individual vesicles containing 1 mM glucose. H2O2 was thus generated in the vesicle and progressively diffused across the membrane toward the surrounding environment. An ultramicroelectrode sensitive to H2O2 (black platinum-modified carbon surface) was placed next to the membrane and provided a direct detection of the hydrogen peroxide flux generated by the enzyme activity. Electrochemistry offered a highly sensitive (in situ detection), selective (potential applied at the electrode), time-resolved analysis (chronoamperometry) of the GOx activity over an hour duration, without modifying the internal giant unilamellar vesicles (GUV) medium. These results demonstrate that electroanalysis with microsensors is well adapted and complementary to fluorescence microscopy to sense enzymatic activities, for instance, generating reactive oxygen species, at single vesicles further used to develop artificial cells.

Dynamic monitoring of a bi-enzymatic reaction at a single biomimetic giant vesicle.

Giant unilamellar vesicles were used as individual biomimetic micro-reactors wherein a model bi-enzymatic reaction involving a glucose oxidase (GOx) and horseradish peroxidase (HRP) was monitored by confocal microscopy. These giant vesicles were formed from a natural mix of phospholipids in physiological conditions of pH and osmolarity (phosphate buffer, pH 7.4, 330 mOsm). The so-called Amplex Red assay, which generates the highly fluorescent resorufin species, was performed in individual vesicles and used to report on the progress of the whole reaction. We aimed at controlling kinetically and quantitatively the different steps of the bi-enzymatic reaction in vesicles. To do so, substrates (glucose and Amplex Red) were provided in individual reactors by two ways. Electro-microinjection allowed the control of volume variations owing to a reservoir of lipids connected to the vesicle membrane. Alternatively, substrates could passively diffuse from the outer solution to the vesicle compartment. The semi-permeability feature of the phospholipid membrane was characterized for all substrates and products while we demonstrated that enzymes remain sequestrated in the vesicles after their injection. The Amplex Red assay was thus achieved in individual vesicles under steady-state conditions, and could pursue over tens of minutes. Such giant vesicles are stable, fully compatible with media used for bioanalyses and allow out-of-equilibrium reactions at time scales compatible with living reaction dynamics, making them a good choice for the development of minimal cell-like systems.

Impacts of vesicular environment on Nox2 activity measurements in vitro.

BACKGROUND: The production of superoxide anions (O2•-) by the phagocyte NADPH oxidase complex has a crucial role in the destruction of pathogens in innate immunity. Majority of in vitro studies on the functioning of NADPH oxidase indirectly follows the enzymatic reaction by the superoxide reduction of cytochrome c (cyt c). Only few reports mention the alternative approach consisting in measuring the NADPH consumption rate. When using membrane vesicles of human neutrophils, the enzyme specific activity is generally found twice higher by monitoring the NADPH oxidation than by measuring the cyt c reduction. Up to now, the literature provides only little explanations about such discrepancy despite the critical importance to quantify the exact enzyme activity. METHODS: We deciphered the reasons of this disparity in studying the role of key parameters, including. cyt c and arachidonic acid concentrations, in conjunction with an ionophore, a detergent and using Clark electrode to measure the O2 consumption rates. RESULTS: Our results show that the O2•- low permeability of the vesicle membrane as well as secondary reactions (O2•- and H2O2 disproportionations) are strong clues to shed light on this inconsistency. CONCLUSION AND GENERAL SIGNIFICANCE: These results altogether indicate that the cyt c reduction method underestimates the accurate Nox2 activity.

Reactive Oxygen Species Generated by Cold Atmospheric Plasmas in Aqueous Solution: Successful Electrochemical Monitoring in Situ under a High Voltage System.

Many investigations are dedicated to the detection and quantification of reactive oxygen and nitrogen species (RONS), particularly when generated in liquids exposed to cold atmospheric plasmas (CAPs). CAPs are partially ionized gases that can be obtained by applying a high electric field to a gas. A challenge is to get better insights on the plasma-liquid interactions in order to understand the induced effects on different targets (liquid, cells, tissues, etc.). As RONS are biochemically reactive, the difficulty lies in finding efficient methods to get both dynamic and quantitative data. Herein, we developed an innovative setup aimed at performing an in situ electrochemical monitoring of redox species generated by CAPs in a physiological buffer (PBS, pH 7.4). The challenge was to apply millivolt-potential variations and measure nanoampere Faradaic currents in the presence of ionization waves generated by micropulsed electric fields of some 10 kV·cm-1 amplitude and ampere-transient currents. This was fulfilled by using dedicated working ultramicroelectrodes (Pt-black UMEs) and protecting them, as well as the reference and counter electrodes, within insulated-earthed containers. In this condition, we succeeded in performing both cyclic voltammetry and chronoamperometry in situ, with a resolution equivalent to working in a static solution (subnanoampere currents). Thus, we monitored the accumulation over time of species (H2O2, NO2-) generated by CAPs in PBS and observed the mean dynamic of RONS chemistry during and after plasma exposition, particularly through the detection of a short-living species.

Physicochemical considerations for bottom-up synthetic biology.

The bottom-up construction of synthetic cells from molecular components is arguably one of the most challenging areas of research in the life sciences. We review the impact of confining biological systems in synthetic vesicles. Complex cell-like systems require control of the internal pH, ionic strength, (macro)molecular crowding, redox state and metabolic energy conservation. These physicochemical parameters influence protein activity and need to be maintained within limits to ensure the system remains in steady-state. We present the physicochemical considerations for building synthetic cells with dimensions ranging from the smallest prokaryotes to eukaryotic cells.

Electroformation of phospholipid giant unilamellar vesicles in physiological phosphate buffer.

Phospholipid Giant Unilamellar Vesicles (GUVs) are usually prepared by electroformation in water, that is in a low-conductivity solution. We developed a protocol allowing their electroformation in the most common physiological buffer, phosphate-buffered saline (PBS). This was achieved based on a specific sequence of increasing electrical fields and for the two usual electrode types for electroformation, namely Indium Tin oxide-coated glass slides and Pt electrodes. These GUVs are stable over time (hour time-scale) and they can be isolated or micro-injected. The membrane composition was modified by adding cholesterol in order to adjust its mechanical properties. The optimal proportion of cholesterol vs. total phospholipid concentration was a ratio of 20 mol%, which increases membrane rigidity and facilitates vesicle microinjection.

Direct oxidative pathway from amplex red to resorufin revealed by in situ confocal imaging.

Amplex Red (AR) is a very useful chemical probe that is employed in biochemical assays. In these assays, the non-fluorescent AR is converted to resorufin (RS), which strongly absorbs in the visible region (λabs = 572 nm) and yields strong fluorescence (λfluo = 583 nm). Even if AR is commonly used to report on enzymatic oxidase activities, an increasing number of possible interferences have been reported, thus lowering the accuracy of the so-called AR assay. As a redox-based reaction, we propose here to directly promote the conversion of AR to RS by means of electrochemistry. The process was first assessed by classic electrochemical and spectroelectrochemical investigations. In addition, we imaged the electrochemical conversion of AR to RS at the electrode surface by in situ confocal microscopy. The coupling of methodologies allowed to demonstrate that RS is directly formed from AR by an oxidation step, unlike what was previously reported. This gives a new insight in the deciphering of AR assays' mechanism and about their observed discrepancy.