pH-dependent structural transitions in cationic ionizable lipid mesophases are critical for lipid nanoparticle function.

Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), cationic ionizable lipid (CIL), cholesterol (chol), and mRNA. Yet the mechanism of pH-dependent response that is believed to cause endosomal release of LNPs is not well understood. Here, we show that eGFP (enhanced green fluorescent protein) protein expression in the mouse liver mediated by the ionizable lipids DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD) ranks MC3 ≥ KC2 > DD despite similar delivery of mRNA per cell in all cell fractions isolated. We hypothesize that the three CIL-LNPs react differently to pH changes and hence study the structure of CIL/chol bulk phases in water. Using synchrotron X-ray scattering a sequence of ordered CIL/chol mesophases with lowering pH values are observed. These phases show isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal [Formula: see text] and bicontinuous cubic Pn3m symmetry. If polyadenylic acid, as mRNA surrogate, is added to CIL/chol, excess lipid coexists with a condensed nucleic acid lipid [Formula: see text] phase. The next-neighbor distance in the excess phase shows a discontinuity at the Fd3m inverse micellar to inverse hexagonal [Formula: see text] transition occurring at pH 6 with distinctly larger spacing and hydration for DD vs. MC3 and KC2. In mRNA LNPs, DD showed larger internal spacing, as well as retarded onset and reduced level of DD-LNP-mediated eGFP expression in vitro compared to MC3 and KC2. Our data suggest that the pH-driven Fd3m-[Formula: see text] transition in bulk phases is a hallmark of CIL-specific differences in mRNA LNP efficacy.

Geometry-Sensitive Protrusion Growth Directs Confined Cell Migration.

The migratory dynamics of cells can be influenced by the complex microenvironment through which they move. It remains unclear how the motility machinery of confined cells responds and adapts to their microenvironment. Here, we propose a biophysical mechanism for a geometry-dependent coupling between cellular protrusions and the nucleus that leads to directed migration. We apply our model to geometry-guided cell migration to obtain insights into the origin of directed migration on asymmetric adhesive micropatterns and the polarization enhancement of cells observed under strong confinement. Remarkably, for cells that can choose between channels of different size, our model predicts an intricate dependence for cellular decision making as a function of the two channel widths, which we confirm experimentally.

On the adhesion-velocity relation and length adaptation of motile cells on stepped fibronectin lanes.

The biphasic adhesion-velocity relation is a universal observation in mesenchymal cell motility. It has been explained by adhesion-promoted forces pushing the front and resisting motion at the rear. Yet, there is little quantitative understanding of how these forces control cell velocity. We study motion of MDA-MB-231 cells on microlanes with fields of alternating Fibronectin densities to address this topic and derive a mathematical model from the leading-edge force balance and the force-dependent polymerization rate. It reproduces quantitatively our measured adhesion-velocity relation and results with keratocytes, PtK1 cells, and CHO cells. Our results confirm that the force pushing the leading-edge membrane drives lamellipodial retrograde flow. Forces resisting motion originate along the whole cell length. All motion-related forces are controlled by adhesion and velocity, which allows motion, even with higher Fibronectin density at the rear than at the front. We find the pathway from Fibronectin density to adhesion structures to involve strong positive feedbacks. Suppressing myosin activity reduces the positive feedback. At transitions between different Fibronectin densities, steady motion is perturbed and leads to changes of cell length and front and rear velocity. Cells exhibit an intrinsic length set by adhesion strength, which, together with the length dynamics, suggests a spring-like front-rear interaction force. We provide a quantitative mechanistic picture of the adhesion-velocity relation and cell response to adhesion changes integrating force-dependent polymerization, retrograde flow, positive feedback from integrin to adhesion structures, and spring-like front-rear interaction.

Learning the dynamics of cell-cell interactions in confined cell migration.

The migratory dynamics of cells in physiological processes, ranging from wound healing to cancer metastasis, rely on contact-mediated cell-cell interactions. These interactions play a key role in shaping the stochastic trajectories of migrating cells. While data-driven physical formalisms for the stochastic migration dynamics of single cells have been developed, such a framework for the behavioral dynamics of interacting cells still remains elusive. Here, we monitor stochastic cell trajectories in a minimal experimental cell collider: a dumbbell-shaped micropattern on which pairs of cells perform repeated cellular collisions. We observe different characteristic behaviors, including cells reversing, following, and sliding past each other upon collision. Capitalizing on this large experimental dataset of coupled cell trajectories, we infer an interacting stochastic equation of motion that accurately predicts the observed interaction behaviors. Our approach reveals that interacting noncancerous MCF10A cells can be described by repulsion and friction interactions. In contrast, cancerous MDA-MB-231 cells exhibit attraction and antifriction interactions, promoting the predominant relative sliding behavior observed for these cells. Based on these experimentally inferred interactions, we show how this framework may generalize to provide a unifying theoretical description of the diverse cellular interaction behaviors of distinct cell types.

On multistability and constitutive relations of cell motion on fibronectin lanes.

Cell motility on flat substrates exhibits coexisting steady and oscillatory morphodynamics, the biphasic adhesion-velocity relation, and the universal correlation between speed and persistence (UCSP) as simultaneous observations common to many cell types. Their universality and concurrency suggest a unifying mechanism causing all three of them. Stick-slip models for cells on one-dimensional lanes suggest multistability to arise from the nonlinear friction of retrograde flow. This study suggests a mechanical mechanism controlled by integrin signaling on the basis of a biophysical model and analysis of trajectories of MDA-MB-231 cells on fibronectin lanes, which additionally explains the constitutive relations. The experiments exhibit cells with steady or oscillatory morphodynamics and either spread or moving with spontaneous transitions between the dynamic regimes, spread and moving, and spontaneous direction reversals. Our biophysical model is based on the force balance at the protrusion edge, the noisy clutch of retrograde flow, and a response function of friction and membrane drag to integrin signaling. The theory reproduces the experimentally observed cell states, characteristics of oscillations, and state probabilities. Analysis of experiments with the biophysical model establishes a stick-slip oscillation mechanism, and explains multistability of cell states and the statistics of state transitions. It suggests protrusion competition to cause direction reversal events, the statistics of which explain the UCSP. The effect of integrin signaling on drag and friction explains the adhesion-velocity relation and cell behavior at fibronectin density steps. The dynamics of our mechanism are nonlinear flow mechanics driven by F-actin polymerization and shaped by the noisy clutch of retrograde flow friction, protrusion competition via membrane tension, and drag forces. Integrin signaling controls the parameters of the mechanical system.

Self-Assembly of Tunable Intrinsically Disordered Peptide Amphiphiles.

Intrinsically disordered peptide amphiphiles (IDPAs) present a novel class of synthetic conjugates that consist of short hydrophilic polypeptides anchored to hydrocarbon chains. These hybrid polymer-lipid block constructs spontaneously self-assemble into dispersed nanoscopic aggregates or ordered mesophases in aqueous solution due to hydrophobic interactions. Yet, the possible sequence variations and their influence on the self-assembly structures are vast and have hardly been explored. Here, we measure the nanoscopic self-assembled structures of four IDPA systems that differ by their amino acid sequence. We show that permutations in the charge pattern along the sequence remarkably alter the headgroup conformation and consequently alter the pH-triggered phase transitions between spherical, cylindrical micelles and hexagonal condensed phases. We demonstrate that even a single amino acid mutation is sufficient to tune structural transitions in the condensed IDPA mesophases, while peptide conformations remain unfolded and disordered. Furthermore, alteration of the peptide sequence can render IDPAs to become susceptible to enzymatic cleavage and induce enzymatically activated phase transitions. These results hold great potential for embedding multiple functionalities into lipid nanoparticle delivery systems by incorporating IDPAs with the desired properties.

Combining molecular dynamics simulations and x-ray scattering techniques for the accurate treatment of protonation degree and packing of ionizable lipids in monolayers.

The pH-dependent change in protonation of ionizable lipids is crucial for the success of lipid-based nanoparticles as mRNA delivery systems. Despite their widespread application in vaccines, the structural changes upon acidification are not well understood. Molecular dynamics simulations support structure prediction but require an a priori knowledge of the lipid packing and protonation degree. The presetting of the protonation degree is a challenging task in the case of ionizable lipids since it depends on pH and on the local lipid environment and often lacks experimental validation. Here, we introduce a methodology of combining all-atom molecular dynamics simulations with experimental total-reflection x-ray fluorescence and scattering measurements for the ionizable lipid Dlin-MC3-DMA (MC3) in POPC monolayers. This joint approach allows us to simultaneously determine the lipid packing and the protonation degree of MC3. The consistent parameterization is expected to be useful for further predictive modeling of the action of MC3-based lipid nanoparticles.

Cells responding to chemoattractant on a structured substrate.

Cell migration on an adhesive substrate surface comprises actin-based protrusion at the front and retraction of the tail in combination with coordinated adhesion to, and detachment from, the substrate. To study the effect of cell-to-substrate adhesion on the chemotactic response of Dictyostelium discoideum cells, we exposed the cells to patterned substrate surfaces consisting of adhesive and inert areas, and forced them by a gradient of chemoattractant to enter the border between the two areas. Wild-type as well as myosin II-deficient cells stop at the border of an adhesive area. They do not detach with their rear part, while on the nonadhesive area they protrude pseudopods at their front toward the source of chemoattractant. Avoidance of the nonadhesive area may cause a cell to move in tangential direction relative to the attractant gradient, keeping its tail at the border of the adhesive surface.

3D-printed SAXS chamber for controlled in situ dialysis and optical characterization.

3D printing changes the scope of how samples can be mounted for small-angle X-ray scattering (SAXS). In this paper a 3D-printed X-ray chamber, which allows for in situ exchange of buffer and in situ optical transmission spectroscopy, is presented. The chamber is made of cyclic olefin copolymers (COC), including COC X-ray windows providing ultra-low SAXS background. The design integrates a membrane insert for in situ dialysis of the 100 µl sample volume against a reservoir, which enables measurements of the same sample under multiple conditions using an in-house X-ray setup equipped with a 17.4 keV molybdenum source. The design's capabilities are demonstrated by measuring reversible structural changes in lipid and polymer systems as a function of salt concentration and pH. In the same chambers optical light transmission spectroscopy was carried out measuring the optical turbidity of the mesophases and local pH values using pH-responsive dyes. Microfluidic exchange and optical spectroscopy combined with in situ X-ray scattering enables vast applications for the study of responsive materials.

DNA binds to a specific site of the adhesive blood-protein von Willebrand factor guided by electrostatic interactions.

Neutrophils release their intracellular content, DNA included, into the bloodstream to form neutrophil extracellular traps (NETs) that confine and kill circulating pathogens. The mechanosensitive adhesive blood protein, von Willebrand Factor (vWF), interacts with the extracellular DNA of NETs to potentially immobilize them during inflammatory and coagulatory conditions. Here, we elucidate the previously unknown molecular mechanism governing the DNA-vWF interaction by integrating atomistic, coarse-grained, and Brownian dynamics simulations, with thermophoresis, gel electrophoresis, fluorescence correlation spectroscopy (FCS), and microfluidic experiments. We demonstrate that, independently of its nucleotide sequence, double-stranded DNA binds to a specific helix of the vWF A1 domain, via three arginines. This interaction is attenuated by increasing the ionic strength. Our FCS and microfluidic measurements also highlight the key role shear-stress has in enabling this interaction. Our simulations attribute the previously-observed platelet-recruitment reduction and heparin-size modulation, upon establishment of DNA-vWF interactions, to indirect steric hindrance and partial overlap of the binding sites, respectively. Overall, we suggest electrostatics-guiding DNA to a specific protein binding site-as the main driving force defining DNA-vWF recognition. The molecular picture of a key shear-mediated DNA-protein interaction is provided here and it constitutes the basis for understanding NETs-mediated immune and hemostatic responses.

Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells.

Cell signaling is initiated by characteristic protein patterns in the plasma membrane, but tools to decipher their molecular organization and activation are hitherto lacking. Among the well-known signaling pattern is the death inducing signaling complex with a predicted hexagonal receptor architecture. To probe this architecture, DNA origami-based nanoagents with nanometer precise arrangements of the death receptor ligand FasL are introduced and presented to cells. Mimicking different receptor geometries, these nanoagents act as signaling platforms inducing fastest time-to-death kinetics for hexagonal FasL arrangements with 10 nm inter-molecular spacing. Compared to naturally occurring soluble FasL, this trigger is faster and 100× more efficient. Nanoagents with different spacing, lower FasL number or higher coupling flexibility impede signaling. The results present DNA origami as versatile signaling scaffolds exhibiting unprecedented control over molecular number and geometry. They define molecular benchmarks in apoptosis signal initiation and constitute a new strategy to drive particular cell responses.

Area and Geometry Dependence of Cell Migration in Asymmetric Two-State Micropatterns.

Microstructured surfaces provide a unique framework to probe cell migration and cytoskeletal dynamics in a standardized manner. Here, we report on the steady-state occupancy probability of cells in asymmetric two-state microstructures that consist of two fibronectin-coated adhesion sites connected by a thin guidance cue. In these dumbbell-like structures, cells transition between the two sites in a repeated and stochastic manner, and average dwell times in the respective microenvironments are determined from the cell trajectories. We study the dynamics of human breast carcinoma cells (MDA-MB-231) in these microstructures as a function of area, shape, and orientation of the adhesion sites. On square adhesive sites with different areas, we find that the occupancy probability ratio is directly proportional to the ratio of corresponding adhesion site areas. These asymmetries are well captured by a simple model for the stochastic nonlinear dynamics of the cells, which reveals generic features of the motion. Sites of equal area but different shape lead to equal occupancy if shapes are isotropic (e.g., squared or circular). In contrast, an asymmetry in the occupancy is induced by anisotropic shapes like rhombi, triangles, or rectangles that enable motion in the direction perpendicular to the transition axis. Analysis of the two-dimensional motion of cells between two rectangles with orthogonal orientation suggests that cellular transition rates depend on the cell polarization induced by anisotropic micropatterns. Taken together, our results illustrate how two-state micropatterns provide a dynamic migration assay with distinct dwell times and relative cell occupancy as readouts, which may be useful to probe cell-microenvironment interactions.

Micropatterning as a tool to identify regulatory triggers and kinetics of actin-mediated endothelial mechanosensing.

Developmental processes, such as angiogenesis, are associated with a constant remodeling of the actin cytoskeleton in response to different mechanical stimuli. The mechanosensitive transcription factors MRTF-A (MKL1) and YAP (also known as YAP1) are important mediators of this challenging adaptation process. However, it is as yet unknown whether both pathways respond in an identical or in a divergent manner to a given microenvironmental guidance cue. Here, we use a micropatterning approach to dissect single aspects of cellular behavior in a spatiotemporally controllable setting. Using the exemplary process of angiogenesis, we show that cell-cell contacts and adhesive surface area are shared regulatory parameters of MRTF and YAP on rigid 2D surfaces. By analyzing MRTF and YAP under laminar flow conditions and during cell migration on dumbbell-shaped microstructures, we demonstrate that they exhibit different translocation kinetics. In conclusion, our work promotes the application of micropatterning techniques as a cell biological tool to study mechanosensitive signaling in the context of angiogenesis.

Interference Disturbance Analysis Enables Single-Cell Level Growth and Mobility Characterization for Rapid Antimicrobial Susceptibility Testing.

To support the emerging battle against antimicrobial resistance (AMR), detection methods that allow fast and accurate antimicrobial susceptibility testing (AST) are urgently needed. The early identification and application of an appropriate antibiotic treatment leads to lower mortality rates and substantial cost savings and prevents the development of resistant pathogens. In this work, we present a diffraction-based method, which is capable of quantitative bacterial growth, mobility, and susceptibility measurements. The method is based on the temporal analysis of the intensity of a light diffraction peak, which arises due to interference at a periodic pattern of gold nanostructures. The presence of bacteria disturbs the constructive interference, leading to an intensity decrease and thus allows the monitoring of bacterial growth in very low volumes. We demonstrate the direct correlation of the decrease in diffraction peak intensity with bacterial cell number starting from single cells and show the capability for rapid high-throughput AST measurements by determining the minimum inhibitory concentration for three different antimicrobials in less than 2-3 h as well as the susceptibility in less than 30-40 min. Furthermore, bacterial mobility is obtained from short-term fluctuations of the diffraction peak intensity and is shown to decrease by a factor of 3 during bacterial attachment to a surface. This multiparameter detection method allows for rapid AST of planktonic and of biofilm-forming bacterial strains in low volumes and in real-time without the need of high initial cell numbers.

Age related changes in cell stiffness of tendon stem/progenitor cells and a rejuvenating effect of ROCK-inhibition.

Tendon stem/progenitor cells (TSPC) are potential targets for regenerative medicine and the treatment of tendon injuries. The frequency of such injuries increases in elderly patients while the proportion of functional TSPCs in tendon tissue decreases, protracting tendon repair. Using atomic force microscopy (AFM), we show that cell stiffness and size increase in TSPCs isolated from elderly patients (A-TSPC) compared to TSPCs from younger patients (Y-TSPC). Additionally, two-photon excited fluorescence (TPEF) microscopy revealed a denser, well-structured actin cytoskeleton in A-TSPC, which correlates with the augmented cell stiffness. Treating A-TSPC with ROCK-inhibitor, reverses these age-related changes, and has rejuvenating effect on cell morphology and stiffness. We assume that cellular stiffness is a suitable marker for cell aging and ROCK a potential target for therapeutic applications of cell rejuvenation.

Probing the Interface Structure of Adhering Cells by Contrast Variation Neutron Reflectometry.

Cellular adhesion is a central element in tissue mechanics, biological cell-cell signaling, and cell motility. In this context, the cell-substrate distance has been investigated in the past by studying natural cells and biomimetic cell models adhering on solid substrates. The amount of water in the membrane substrate gap, however, is difficult to determine. Here, we present a neutron reflectivity (NR) structural study of confluent epithelial cell monolayers on silicon substrates. In order to ensure valid in vitro conditions, we developed a cell culture sample chamber allowing us to grow and cultivate cells under proper cell culture conditions while performing in vitro neutron reflectivity measurements. The cell chamber also enabled perfusion with cell medium and hence allowed for contrast variation in situ by sterile exchange of buffer with different H2O-to-D2O ratio. Contrast variation reduces the ambiguity of data modeling for determining the thickness and degree of hydration of the interfacial cleft between the adherent cells and the substrate. Our data suggest a three-layer interfacial organization. The first layer bound to the silicon surface interface is in agreement with a very dense protein film with a thickness of 9 ± 2 nm, followed by a highly hydrated 24 ± 4 nm thick layer, and a several tens of nanometers thick layer attributed to the composite membrane. Hence, the results provide clear evidence of a highly hydrated intermediate region between the composite cell membrane and the substrate, reminiscent of the basal lamina.

Single-cell kinetics of siRNA-mediated mRNA degradation.

RNA interference (RNAi) enables the therapeutic use of small interfering RNAs (siRNAs) to silence disease-related genes. The efficiency of silencing is commonly assessed by measuring expression levels of the target protein at a given time point post-transfection. Here, we determine the siRNA-induced fold change in mRNA degradation kinetics from single-cell fluorescence time-courses obtained using live-cell imaging on single-cell arrays (LISCA). After simultaneous transfection of mRNAs encoding eGFP (target) and CayRFP (reference), the eGFP expression is silenced by siRNA. The single-cell time-courses are fitted using a mathematical model of gene expression. Analysis yields best estimates of related kinetic rate constants, including mRNA degradation constants. We determine the siRNA-induced changes in kinetic rates and their correlations between target and reference protein expression. Assessment of mRNA degradation constants using single-cell time-lapse imaging is fast (<30 h) and returns an accurate, time-independent measure of siRNA-induced silencing, thus allowing the exact evaluation of siRNA therapeutics.

Parameter estimation for dynamical systems with discrete events and logical operations.

Motivation: Ordinary differential equation (ODE) models are frequently used to describe the dynamic behaviour of biochemical processes. Such ODE models are often extended by events to describe the effect of fast latent processes on the process dynamics. To exploit the predictive power of ODE models, their parameters have to be inferred from experimental data. For models without events, gradient based optimization schemes perform well for parameter estimation, when sensitivity equations are used for gradient computation. Yet, sensitivity equations for models with parameter- and state-dependent events and event-triggered observations are not supported by existing toolboxes. Results: In this manuscript, we describe the sensitivity equations for differential equation models with events and demonstrate how to estimate parameters from event-resolved data using event-triggered observations in parameter estimation. We consider a model for GFP expression after transfection and a model for spiking neurons and demonstrate that we can improve computational efficiency and robustness of parameter estimation by using sensitivity equations for systems with events. Moreover, we demonstrate that, by using event-outputs, it is possible to consider event-resolved data, such as time-to-event data, for parameter estimation with ODE models. By providing a user-friendly, modular implementation in the toolbox AMICI, the developed methods are made publicly available and can be integrated in other systems biology toolboxes. Availability and Implementation: We implement the methods in the open-source toolbox Advanced MATLAB Interface for CVODES and IDAS (AMICI, https://github.com/ICB-DCM/AMICI ). Contact: jan.hasenauer@helmholtz-muenchen.de. Supplementary information: Supplementary data are available at Bioinformatics online.

von Willebrand factor is dimerized by protein disulfide isomerase.

Multimeric von Willebrand factor (VWF) is essential for primary hemostasis. The biosynthesis of VWF high-molecular-weight multimers requires spatial separation of each step because of varying pH value requirements. VWF is dimerized in the endoplasmic reticulum by formation of disulfide bonds between the C-terminal cysteine knot (CK) domains of 2 monomers. Here, we investigated the basic question of which protein catalyzes the dimerization. We examined the putative interaction of VWF and the protein disulfide isomerase PDIA1, which has previously been used to visualize endoplasmic reticulum localization of VWF. Excitingly, we were able to visualize the PDI-VWF dimer complex by high-resolution stochastic optical reconstruction microscopy and atomic force microscopy. We proved and quantified direct binding of PDIA1 to VWF, using microscale thermophoresis and fluorescence correlation spectroscopy (dissociation constants KD = 236 ± 66 nM and KD = 282 ± 123 nM by microscale thermophoresis and fluorescence correlation spectroscopy, respectively). The similar KD (258 ± 104 nM) measured for PDI interaction with the isolated CK domain and the atomic force microscopy images strongly indicate that PDIA1 binds exclusively to the CK domain, suggesting a key role of PDIA1 in VWF dimerization. On the basis of protein-protein docking and molecular dynamics simulations, combined with fluorescence microscopy studies of VWF CK-domain mutants, we suggest the following mechanism of VWF dimerization: PDI initiates VWF dimerization by forming the first 2 disulfide bonds Cys2771-2773' and Cys2771'-2773. Subsequently, the third bond, Cys2811-2811', is formed, presumably to protect the first 2 bonds from reduction, thereby rendering dimerization irreversible. This study deepens our understanding of the mechanism of VWF dimerization and the pathophysiological consequences of its inhibition.

Nanostructures in n-Octanol Equilibrated with Additives and/or Water.

Fluid fatty alcohols are believed to be nanostructured but broadly amorphous (i.e., noncrystalline) fluids and solvents, including the most popular fatty tissue mimetic, hydrated n-octanol (i.e., hydro-octanol). To check this premise, we studied dry octanol and hydro-octanol as a model of relatively short fluid n-alkanols with small-angle X-ray scattering (SAXS). We also combined this alkanol with the matching alkane (i.e., octane) and with a common anti-inflammatory pain killer (ketoprofen). This revealed that (hydro-)octanol and arguably any other short fatty alcohol form a mesophase. Its basic structural motif are regularly packed polar nanoclusters, reflected in the inner peak in the SAXS diffractogram of (hydro-)octanol and other fluid n-alkanols. The nanoclusters arguably resemble tiny, (inverse) hydrated bilayer fragments, located on a thermally smeared para-crystalline lattice. Additives to hydro-octanol can change the nanoclusters only moderately, if at all. For example, octane and the drug ketoprofen added to hydro-octanol enlarge the nanoclusters only little because of the mixture's packing frustration. To associate with and to bring more water into hydro-octanol, an additive must hence transform the nanoclusters: it expands them into irregularly distributed aqueous lacunae that form a proto-microemulsion, reflected in the previously unknown Guinier's SAXS signal. A "weak" (i.e., a weakly polar or nonpolar) additive can moreover create only size-limited lacunae. Coexistence of nanoclusters and lacunae as well as size variability of the latter in hydro-octanol subvert the concept of octanol-water partition coefficient, which relies on the studied compartment homogeneity. In turn, it opens new possibilities for interfacial catalysis. Reinterpreting "octanol-water partition coefficient" data in terms of octanol-water association or binding constant(s) could furthermore diminish the variability of molecular lipophilicity description and pave the ground toward a more precise theoretical quantification and prediction of molecular properties.