Comprehensive Characterization of an "Off/On" Rhodol-Based Lysosomal Tracker for Orthogonal Cellular Analysis by Confocal Imaging.

Two florescent xanthene-cyanamide lysosomal trackers emitting strongly at ∼525 nm were prepared from fluorescein and rhodol methyl esters in microwave-assisted reactions. Both forms named "off" (nonfluorescent lactam) and "on" (strongly fluorescent ring-opened amide) have been comprehensively characterized out by using a combination of NMR spectroscopy, X-ray analysis, fluorimetry and confocal microscopy. Known rhodamines bearing electron-withdrawing groups (EWGs) exhibit an equilibrium between non-fluorescent (off) and fluorescent (on) depending on the dielectric constant of the medium. Here, cyanamide was introduced as EWG amine into the fluorescein and rhodol framework. Unlike rhodamine-type dyes, the ring-opened forms of fluorescein- and rhodol-cyanamides are stable in protic solvents under circumneutral and basic pH conditions. The osteoblastic cell line MC3T3-E1 from C57BL/6 mouse calvaria was used for confocal imaging where the different organelles and nuclei were distinguished by using an orthogonal combination of fluorescent dyes.

Morphological and Mechanical Characterization of Extracellular Vesicles and Parent Human Synoviocytes under Physiological and Inflammatory Conditions.

The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This "gel-in" vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant. RA, and to a lesser extent OA, synovial vesicles had altered morphology, corresponding to a "gel-out" situation with vesicles surrounded by a viscous gel, poor mechanical resistance, and poor lubricating qualities. When subjected to inflammatory conditions, healthy cells developed phenotypes similar to that of RA samples, which reinforces the importance of inflammatory processes in the loss of lubricating properties of SF.

Synovial Extracellular Vesicles: Structure and Role in Synovial Fluid Tribological Performances.

The quality of the lubricant between cartilaginous joint surfaces impacts the joint's mechanistic properties. In this study, we define the biochemical, ultrastructural, and tribological signatures of synovial fluids (SF) from patients with degenerative (osteoarthritis-OA) or inflammatory (rheumatoid arthritis-RA) joint pathologies in comparison with SF from healthy subjects. Phospholipid (PL) concentration in SF increased in pathological contexts, but the proportion PL relative to the overall lipids decreased. Subtle changes in PL chain composition were attributed to the inflammatory state. Transmission electron microscopy showed the occurrence of large multilamellar synovial extracellular vesicles (EV) filled with glycoprotein gel in healthy subjects. Synovial extracellular vesicle structure was altered in SF from OA and RA patients. RA samples systematically showed lower viscosity than healthy samples under a hydrodynamic lubricating regimen whereas OA samples showed higher viscosity. In turn, under a boundary regimen, cartilage surfaces in both pathological situations showed high wear and friction coefficients. Thus, we found a difference in the biochemical, tribological, and ultrastructural properties of synovial fluid in healthy people and patients with osteoarthritis and arthritis of the joints, and that large, multilamellar vesicles are essential for good boundary lubrication by ensuring a ball-bearing effect and limiting the destruction of lipid layers at the cartilage surface.

Mineralization Profile of Annexin A6-Harbouring Proteoliposomes: Shedding Light on the Role of Annexin A6 on Matrix Vesicle-Mediated Mineralization.

The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles' nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS-CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS-CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles' nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS-CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.

Monomethyl Auristatin E Grafted-Liposomes to Target Prostate Tumor Cell Lines.

Novel nanomedicines have been engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or short half-life. Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. A Monomethyl Auristatin E (MMAE) warhead was grafted on a lipid derivative and integrated in fusogenic liposomes, following the model of antibody drug conjugates. By modulating the liposome composition, we designed a set of particles characterized by different membrane fluidities as a key parameter to obtain selective uptake from fibroblast or prostate tumor cells. Only the fluid liposomes made of palmitoyl-oleoyl-phosphatidylcholine and dioleoyl-phosphatidylethanolamine, integrating the MMAE-lipid derivative, showed an effect on prostate tumor PC-3 and LNCaP cell viability. On the other hand, they exhibited negligible effects on the fibroblast NIH-3T3 cells, which only interacted with rigid liposomes. Therefore, fluid liposomes grafted with MMAE represent an interesting example of drug carriers, as they can be easily engineered to promote liposome fusion with the target membrane and ensure drug selectivity.

Membrane Fluidity as a New Means to Selectively Target Cancer Cells with Fusogenic Lipid Carriers.

Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. Their clinical success relies on their composition, similar to that of the cell membrane. Their cellular specificity often relies on a ligand-receptor interaction. Although differences in the physicochemical properties of the cell membrane between tumor and nontumor cells have been reported, they are not systematically used for drug delivery purposes. In this report, a new approach was developed to ensure selective targeting based on physical compatibility between the target and the carrier membranes. By modulating the liposome composition and thus its membrane fluidity, we achieved selective targeting on four cancer cell lines of varying aggressiveness. Furthermore, using membrane-embedded and inner core-encapsulated fluorophores, we assessed the mechanism of this interaction to be based on the fusion of the liposome with the cell membranes. Membrane fluidity is therefore a major parameter to be considered when designing lipid drug carriers as a promising, lower cost alternative to current targeting strategies based on covalent grafting.

Localization of Annexin A6 in Matrix Vesicles During Physiological Mineralization.

Annexin A6 (AnxA6) is the largest member of the annexin family of proteins present in matrix vesicles (MVs). MVs are a special class of extracellular vesicles that serve as a nucleation site during cartilage, bone, and mantle dentin mineralization. In this study, we assessed the localization of AnxA6 in the MV membrane bilayer using native MVs and MV biomimetics. Biochemical analyses revealed that AnxA6 in MVs can be divided into three distinct groups. The first group corresponds to Ca2+-bound AnxA6 interacting with the inner leaflet of the MV membrane. The second group corresponds to AnxA6 localized on the surface of the outer leaflet. The third group corresponds to AnxA6 inserted in the membrane's hydrophobic bilayer and co-localized with cholesterol (Chol). Using monolayers and proteoliposomes composed of either dipalmitoylphosphatidylcholine (DPPC) to mimic the outer leaflet of the MV membrane bilayer or a 9:1 DPPC:dipalmitoylphosphatidylserine (DPPS) mixture to mimic the inner leaflet, with and without Ca2+, we confirmed that, in agreement with the biochemical data, AnxA6 interacted differently with the MV membrane. Thermodynamic analyses based on the measurement of surface pressure exclusion (πexc), enthalpy (ΔH), and phase transition cooperativity (Δt1/2) showed that AnxA6 interacted with DPPC and 9:1 DPPC:DPPS systems and that this interaction increased in the presence of Chol. The selective recruitment of AnxA6 by Chol was observed in MVs as probed by the addition of methyl-ß-cyclodextrin (MßCD). AnxA6-lipid interaction was also Ca2+-dependent, as evidenced by the increase in πexc in negatively charged 9:1 DPPC:DPPS monolayers and the decrease in ΔH in 9:1 DPPC:DPPS proteoliposomes caused by the addition of AnxA6 in the presence of Ca2+ compared to DPPC zwitterionic bilayers. The interaction of AnxA6 with DPPC and 9:1 DPPC:DPPS systems was distinct even in the absence of Ca2+ as observed by the larger change in Δt1/2 in 9:1 DPPC:DPPS vesicles as compared to DPPC vesicles. Protrusions on the surface of DPPC proteoliposomes observed by atomic force microscopy suggested that oligomeric AnxA6 interacted with the vesicle membrane. Further work is needed to delineate possible functions of AnxA6 at its different localizations and ways of interaction with lipids.

Evidence of proteolipid domain formation in an inner mitochondrial membrane mimicking model.

BACKGROUND: Mitochondrial creatine kinase (mtCK) is highly abundant in mitochondria; its quantity is equimolecular to the Adenylic Nucleotide Translocator and represents 1% of the mitochondrial proteins. It is a multitask protein localized in the mitochondria intermembrane space where it binds to the specific cardiolipin (CL) phospholipid. If mtCK was initially thought to be exclusively implicated in energy transfer between mitochondria and cytosol through a mechanism referred to as the phosphocreatine shuttle, several recent studies suggested an additional role in maintaining mitochondria membrane structure. METHODS: To further characterized mtCK binding process we used multiphoton excitation fluorescence microscopy coupled with Giant Unilamellar Vesicles (GUV) and laurdan as fluorescence probe. RESULTS: We gathered structural and dynamical information on the molecular events occurring during the binding of mtCK to the mitochondria inner membrane. We present the first visualization of mtCK-induced CL segregation on a bilayer model forming micrometer-size proteolipid domains at the surface of the GUV. Those microdomains, which only occurred when CL is included in the lipid mixture, were accompanied by the formation of protein multimolecular assembly, vesicle clamping, and changes in both vesicle curvature and membrane fluidity CONCLUSION: Those results highlighted the importance of the highly abundant mtCK in the lateral organization of the mitochondrial inner membrane. GENERAL SIGNIFICANCE: Microdomains were induced in mitochondria-mimicking membranes composed of natural phospholipids without cholesterol and/or sphingolipids differing from the proposed cytoplasmic membrane rafts. Those findings as well as membrane curvature modification were discussed in relation with protein-membrane interaction and protein cluster involvement in membrane morphology.

Two-Step Membrane Binding of NDPK-B Induces Membrane Fluidity Decrease and Changes in Lipid Lateral Organization and Protein Cluster Formation.

Nucleoside diphosphate kinases (NDPKs) are crucial elements in a wide array of cellular physiological or pathophysiological processes such as apoptosis, proliferation, or metastasis formation. Among the NDPK isoenzymes, NDPK-B, a cytoplasmic protein, was reported to be associated with several biological membranes such as plasma or endoplasmic reticulum membranes. Using several membrane models (liposomes, lipid monolayers, and supported lipid bilayers) associated with biophysical approaches, we show that lipid membrane binding occurs in a two-step process: first, initiation by a strong electrostatic adsorption process and followed by shallow penetration of the protein within the membrane. The NDPK-B binding leads to a decrease in membrane fluidity and formation of protein patches. The ability of NDPK-B to form microdomains at the membrane level may be related to protein-protein interactions triggered by its association with anionic phospholipids. Such accumulation of NDPK-B would amplify its effects in functional platform formation and protein recruitment at the membrane.

Protein "amyloid-like" networks at the phospholipid membrane formed by 4-hydroxy-2-nonenal-modified mitochondrial creatine kinase.

4-Hydroxy-2-nonenal (4-HNE) is a reactive aldehyde and a lipid peroxidation product formed in biological tissues under physiological and pathological conditions. Its concentration increases with oxidative stress and induces deleterious modifications of proteins and membranes. Mitochondrial and cytosolic isoforms of creatine kinase were previously shown to be affected by 4-HNE. In the present study, we analyzed the effect of 4-HNE on mitochondrial creatine kinase, an abundant protein from the mitochondrial intermembrane space with a key role in mitochondrial physiology. We show that this effect is double: 4-HNE induces a step-wise loss of creatine kinase activity together with a fast protein aggregation. Protein-membrane interaction is affected and amyloid-like networks formed on the biomimetic membrane. These fibrils may disturb mitochondrial organisation both at the membrane and in the inter membrane space.

Carrier-inside-carrier: polyelectrolyte microcapsules as reservoir for drug-loaded liposomes.

Conventional liposomes have a short life-time in blood, unless they are protected by a polymer envelope, most often polyethylene glycol. However, these stabilizing polymers frequently interfere with cellular uptake, impede liposome-membrane fusion and inhibit escape of liposome content from endosomes. To overcome such drawbacks, polymer-based systems as carriers for liposomes are currently developed. Conforming to this approach, we propose a new and convenient method for embedding small size liposomes, 30-100 nm, inside porous calcium carbonate microparticles. These microparticles served as templates for deposition of various polyelectrolytes to form a protective shell. The carbonate particles were then dissolved to yield hollow polyelectrolyte microcapsules. The main advantage of using this method for liposome encapsulation is that carbonate particles can serve as a sacrificial template for deposition of virtually any polyelectrolyte. By carefully choosing the shell composition, bioavailability of the liposomes and of the encapsulated drug can be modulated to respond to biological requirements and to improve drug delivery to the cytoplasm and avoid endosomal escape.

Carrier-Tumor Cell Membrane Interactions for Optimized Delivery of a Promising Drug, 4(RS)-4-F4t-Neuroprostane.

Nanomedicines engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or a short half-life, are targeted towards their cellular destination either passively or through various elements of cell membranes. The differences in the physicochemical properties of the cell membrane between tumor and nontumor cells have been reported, but they are not systematically used for drug delivery purposes. Thus, in this study, a new approach based on a match between the liposome compositions, i.e., membrane fluidity, to selectively interact with the targeted cell membrane was used. Lipid-based carriers of two different fluidities were designed and used to deliver 4(RS)-4-F4t-Neuroprostane (F4t-NeuroP), a potential antitumor molecule derived from docosahexaenoic acid (DHA). Based on its hydrophobic character, F4t-NeuroP was added to the lipid mixture prior to liposome formation, a protocol that yielded over 80% encapsulation efficiency in both rigid and fluid liposomes. The presence of the active molecule did not modify the liposome size but increased the liposome negative charge and the liposome membrane fluidity, which suggested that the active molecule was accommodated in the lipid membrane. F4t-NeuroP integration in liposomes with a fluid character allowed for the selective targeting of the metastatic prostate cell line PC-3 vs. fibroblast controls. A significant decrease in viability (40%) was observed for the PC-3 cancer line in the presence of F4t-NeuroP fluid liposomes, whereas rigid F4t-NeuroP liposomes did not alter the PC-3 cell viability. These findings demonstrate that liposomes encapsulating F4t-NeuroP or other related molecules may be an interesting model of drug carriers based on membrane fluidity.

Hybrid core-shell particles for mRNA systemic delivery.

mRNA based infectious disease vaccines have opened the venue for development of novel nucleic acids-based therapeutics. For all mRNA therapeutics dedicated delivery systems are required, where different functionalities and targeting abilities need to be optimized for the respective applications. One option for advanced formulations with tailored properties are lipid-polymer hybrid nanoparticles with complex nanostructure, which allow to combine features of several already well described nucleic acid delivery systems. Here, we explored hyaluronic acid (HA) as coating of liposome-mRNA complexes (LRCs) to investigate effects of the coating on surface charge, physicochemical characteristics and biological activity. HA was electrostatically attached to positively charged complexes, forming hybrid LRCs (HLRCs). At different N/P ratios, physico-chemical characterization of the two sets of particles showed similarity in size (around 200 nm) and mRNA binding abilities, while the presence of the HA shell conferred a negative surface charge to otherwise positive complexes. High transfection efficiency of LRCs and HLRCs in vitro has been obtained in THP-1 and human monocytes derived from PBMC, an interesting target cell population for cancer and immune related pathologies. In mice, quantitative biodistribution of radiolabeled LRC and HLRC particles, coupled with bioluminescence studies to detect the protein translation sites, hinted towards both particles' accumulation in the hepatic reticuloendothelial system (RES). mRNA translated proteins though was found mainly in the spleen, a major source for immune cells, with preference for expression in macrophages. The results showed that surface modifications of liposome-mRNA complexes can be used to fine-tune nanoparticle physico-chemical characteristics. This provides a tool for assembly of stable and optimized nanoparticles, which are prerequisite for future therapeutic interventions using mRNA-based nanomedicines.

Acyl chain composition determines cardiolipin clustering induced by mitochondrial creatine kinase binding to monolayers.

It has been recently shown that mitochondrial creatine kinase (mtCK) organizes mitochondrial model membrane by modulating the state and fluidity of lipids and by promoting the formation of protein-cardiolipin clusters. This report shows, using Brewster angle microscopy, that such clustering is largely dependent on the acyl chain composition of phospholipids. Indeed, mtCK-cardiolipin domains were observed not only with unsaturated cardiolipins, but also with the cardiolipin precursor phosphatidylglycerol. On the other hand, in the case of saturated dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin, mtCK was homogeneously distributed underneath the monolayer. However, an overall decrease in membrane fluidity was indicated by infrared spectroscopy as well as by extrinsic fluorescence spectroscopy using Laurdan as a fluorescent probe, both for tetramyristoylcardiolipin and bovine heart cardiolipin containing liposomes. The binding mechanism implicated the insertion of protein segments into monolayers, as evidenced from alternative current polarography, regardless of the chain unsaturation for the phosphatidylglycerols and cardiolipins tested.

Fluorescence evidence of annexin A6 translocation across membrane in model matrix vesicles during apatite formation.

Matrix vesicles (MVs) are 100-300 nm spherical structures released by mineralization competent cells to initiate formation of apatite, the mineral component in bones. Among proteins present in MVs, annexin A6 (AnxA6) is thought to be ubiquitously distributed in the MVs' lumen, on the surface of the internal and external leaflets of the membrane and also inserted in the lipid bilayer. To determine the molecular mechanism(s) that lead to the different locations of AnxA6, we hypothesized the occurrence of a pH drop during the mineralization. Such a change would induce the AnxA6 protonation, which in turn, and because of its isoelectric point of 5.41, would change the protein hydrophobicity facilitating its insertion into the MVs' bilayer. The various distributions of AnxA6 are likely to disturb membrane phospholipid organization. To examine this possibility, we used fluorescein as pH reporter, and established that pH decreased inside MVs during apatite formation. Then, 4-(14-phenyldibenzo[a,c]phenazin-9(14H)-yl)-phenol, a vibration-induced emission fluorescent probe, was used as a reporter of changes in membrane organization occurring with the varying mode of AnxA6 binding. Proteoliposomes containing AnxA6 and 1,2-Dimyristoyl-sn-glycero-3phosphocholine (DMPC) or 1,2-Dimyristoyl-sn-glycero-3phosphocholine: 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (DMPC:DPPS 9:1), to mimic the external and internal MV membrane leaflet, respectively, served as biomimetic models to investigate the nature of AnxA6 binding. Addition of Anx6 to DMPC at pH 7.4 and 5.4, or DMPC:DPPS (9:1) at pH 7.4 induced a decrease in membrane fluidity, consistent with AnxA6 interactions with the bilayer surface. In contrast, AnxA6 addition to DMPC:DPPS (9:1) at pH 5.4 increased the fluidity of the membrane. This latest result was interpreted as reflecting the insertion of AnxA6 into the bilayer. Taken together, these findings point to a possible mechanism of AnxA6 translocation in MVs from the surface of the internal leaflet into the phospholipid bilayer stimulated upon acidification of the MVs' lumen during formation of apatite.

Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of approximately 25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains approximately 15 mobile TatA complexes and a pool of approximately 100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.

Mitochondrial creatine kinase interaction with cardiolipin-containing biomimetic membranes is a two-step process involving adsorption and insertion.

Mitochondrial creatine kinase (mtCK) binding to the mitochondrial inner membrane largely determines its biological functions in cellular energy homeostasis, mitochondrial physiology, and dynamics. The membrane binding mechanism is, however, not completely understood. Recent data suggest that a hydrophobic component is involved in mtCK binding to cardiolipin at the outer face of the inner mitochondrial membrane, in addition to the well known electrostatically driven process. In this manuscript, using an electrochemical method derived from alternating current polarography for differential capacity measurements, we distinctly reveal that protein-cardiolipin interaction has a two-step mechanism. For short incubation time, protein adsorption to the phospholipid charged headgroup was the only process detected, whereas on a longer time scale evidence of protein insertion was observed.

Morphology modifications in negatively charged lipid monolayers upon mitochondrial creatine kinase binding.

Mitochondrial creatine kinase (mtCK) may participate to membrane organization at the mitochondrial level by modulating lipid state and fluidity. The effect of the protein on lipid phase behaviour of different acyl chain length phosphatidylglycerol monolayers was analyzed from pressure-area isotherms and from the compressional modulus variation with respect to the surface pressure. Monolayer morphology was visualized by Brewster angle microscopy. No condensation effect was visible on dimyristoylphosphatidylglycerol (DMPG). For the other PG monolayers tested, dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylglycerol (DSPG), mtCK facilitated the formation of a liquid condensed phase. The effect depended on the surface pressure at which transition phase occurred. The effect of mtCK was more pronounced for tetramyristoylcardiolipin (TMCL) monolayers, as liquid condensed regions appeared 10 mN/m below the transition phase of the pure TMCL monolayer. The observed domains were circular and rather uniform, indicating a stabilization of the condensed phase. The same effect, namely an overall condensation of the monolayer with formation of circular domains, was observed upon protein injection beneath TMCL monolayers in different condensation states at constant area. MtCK ability to induce and stabilize a LC phase on monolayers could have important consequences in membrane organization and emphasize its structural role at mitochondrial level.

In Vivo Characterization of the Toxicological Properties of DPhP, One of the Main Degradation Products of Aryl Phosphate Esters.

BACKGROUND: Aryl phosphate esters (APEs) are widely used and commonly present in the environment. Health hazards associated with these compounds remain largely unknown and the effects of diphenyl phosphate (DPhP), one of their most frequent derivatives, are poorly characterized. OBJECTIVE: Our aim was to investigate whether DPhP per se may represent a more relevant marker of exposure to APEs than direct assessment of their concentration and determine its potential deleterious biological effects in chronically exposed mice. METHODS: Conventional animals (FVB mice) were acutely or chronically exposed to relevant doses of DPhP or to triphenyl phosphate (TPhP), one of its main precursors. Both molecules were measured in blood and other tissues by liquid chromatography-mass spectrometry (LC-MS). Effects of chronic DPhP exposure were addressed through liver multi-omics analysis to determine the corresponding metabolic profile. Deep statistical exploration was performed to extract correlated information, guiding further physiological analyses. RESULTS: Multi-omics analysis confirmed the existence of biological effects of DPhP, even at a very low dose of 0.1mg/mL in drinking water. Chemical structural homology and pathway mapping demonstrated a clear reduction of the fatty acid catabolic processes centered on acylcarnitine and mitochondrial ß-oxidation in mice exposed to DPhP in comparison with those treated with vehicle. An interesting finding was that in mice exposed to DPhP, mRNA, expression of genes involved in lipid catabolic processes and regulated by peroxisome proliferator-activated receptor alpha (PPARα) was lower than that in vehicle-treated mice. Immunohistochemistry analysis showed a specific down-regulation of HMGCS2, a kernel target gene of PPARα. Overall, DPhP absorption disrupted body weight-gain processes. CONCLUSIONS: Our results suggest that in mice, the effects of chronic exposure to DPhP, even at a low dose, are not negligible. Fatty acid metabolism in the liver is essential for controlling fast and feast periods, with adverse consequences on the overall physiology. Therefore, the impact of DPhP on circulating fat, cardiovascular pathologies and metabolic disease incidence deserves, in light of our results, further investigations. https://doi.org/10.1289/EHP6826.

Mitochondrial creatine kinase binding to phospholipid monolayers induces cardiolipin segregation.

It is well established that the octameric mitochondrial form of creatine kinase (mtCK) binds to the outer face of the inner mitochondrial membrane mainly via electrostatic interactions with cardiolipin (CL). However, little is known about the consequences of these interactions on membrane and protein levels. Brewster angle microscopy investigations provide, for the first time to our knowledge, images indicating that mtCK binding induced cluster formation on CL monolayers. The thickness of the clusters (10-12 nm) corresponds to the theoretical height of the mtCK-CL complex. Protein insertion into a condensed CL film, together with monolayer stabilization after protein addition, was observed by means of differential capacity measurements. Polarization modulation infrared reflection-absorption spectroscopy showed that the mean orientation of alpha-helices within the protein shifted upon CL binding from 30 degrees to 45 degrees with respect to the interface plane, demonstrating protein domain movements. A comparison of data obtained with CL and phosphatidylcholine/phosphatidylethanolamine/CL (2:1:1) monolayers indicates that mtCK is able to selectively recruit CL molecules within the mixed monolayer, consolidating and changing the morphology of the interfacial film. Therefore, CL-rich domains induced by mtCK binding could modulate mitochondrial inner membrane morphology into a raft-like organization and influence essential steps of mitochondria-mediated apoptosis.