TAR RNA Mediated Folding of a Single-Arginine-Mutant HIV-1 Tat Protein within HeLa Cells Experiencing Intracellular Crowding.

The various effects of native protein folding on the stability and folding rate of intrinsically disordered proteins (IDPs) in crowded intracellular environments are important in biomedicine. Although most studies on protein folding have been conducted in vitro, providing valuable insights, studies on protein folding in crowded intracellular environments are scarce. This study aimed to explore the effects of intracellular molecular crowding on the folding of mutant transactivator HIV-1 Tat based on intracellular interactions, including TAR RNA, as proof of the previously reported chaperna-RNA concept. Considering that the Tat-TAR RNA motif binds RNA, we assessed the po tential function of TAR RNA as a chaperna for the refolding of R52Tat, a mutant in which the argi nine (R) residues at R52 have been replaced with alanine (A) by site-directed mutagenesis. We mon itored Tat-EGFP and Tat folding in HeLa cells via time-lapse fluorescence microscopy and biolayer interferometry using EGFP fusion as an indicator for folding status. These results show that the refolding of R52A Tat was stimulated well at a 0.3 µM TAR RNA concentration; wild-type Tat refolding was essentially abolished because of a reduction in the affinity for TAR RNA at that con centration. The folding and refolding of R52Tat were mainly promoted upon stimulation with TAR RNA. Our findings provide novel insights into the therapeutic potential of chaperna-mediated fold ing through the examination of as-yet-unexplored RNA-mediated protein folding as well as viral genetic variants that modulate viral evolutionary linkages for viral diseases inside a crowded intra cellular environment.

Self-diffusion of nonspherical particles fundamentally conflicts with effective sphere models.

Modeling diffusion of nonspherical particles presents an unsolved and considerable challenge, despite its importance for the understanding of crowding effects in biology, food technology and formulation science. A common approach in experiment and simulation is to map nonspherical objects on effective spheres to subsequently use the established predictions for spheres to approximate phenomena for nonspherical particles. Using numerical evaluation of the hydrodynamic mobility tensor, we show that this so-called effective sphere model fundamentally fails to represent the self-diffusion in solutions of ellipsoids as well as rod-like assemblies of spherical beads. The effective sphere model drastically overestimates the slowing down of self-diffusion down to volume fractions below 0.01. Furthermore, even the linear term relevant at lower volume fraction is inaccurate, linked to a fundamental misconception of effective sphere models. To overcome the severe problems related with the use of effective sphere models, we suggest a protocol to predict the short-time self-diffusion of rod-like systems, based on simulations with hydrodynamic interactions that become feasible even for more complex molecules as the essential observable shows a negligible system-size effect.

Unpacking the Allee effect: determining individual-level mechanisms that drive global population dynamics.

We present a solid theoretical foundation for interpreting the origin of Allee effects by providing the missing link in understanding how local individual-based mechanisms translate to global population dynamics. Allee effects were originally proposed to describe population dynamics that cannot be explained by exponential and logistic growth models. However, standard methods often calibrate Allee effect models to match observed global population dynamics without providing any mechanistic insight. By introducing a stochastic individual-based model, with proliferation, death and motility rates that depend on local density, we present a modelling framework that translates particular global Allee effects to specific individual-based mechanisms. Using data from ecology and cell biology, we unpack individual-level mechanisms implicit in an Allee effect model and provide simulation tools for others to repeat this analysis.

Accurate and efficient discretizations for stochastic models providing near agent-based spatial resolution at low computational cost.

Understanding how cells proliferate, migrate and die in various environments is essential in determining how organisms develop and repair themselves. Continuum mathematical models, such as the logistic equation and the Fisher-Kolmogorov equation, can describe the global characteristics observed in commonly used cell biology assays, such as proliferation and scratch assays. However, these continuum models do not account for single-cell-level mechanics observed in high-throughput experiments. Mathematical modelling frameworks that represent individual cells, often called agent-based models, can successfully describe key single-cell-level features of these assays but are computationally infeasible when dealing with large populations. In this work, we propose an agent-based model with crowding effects that is computationally efficient and matches the logistic and Fisher-Kolmogorov equations in parameter regimes relevant to proliferation and scratch assays, respectively. This stochastic agent-based model allows multiple agents to be contained within compartments on an underlying lattice, thereby reducing the computational storage compared to existing agent-based models that allow one agent per site only. We propose a systematic method to determine a suitable compartment size. Implementing this compartment-based model with this compartment size provides a balance between computational storage, local resolution of agent behaviour and agreement with classical continuum descriptions.

Use of molecular crowding for the detection of protein self-association by size-exclusion chromatography.

The feasibility of employing molecular crowding cosolutes to facilitate the detection of protein self-association by zonal size exclusion chromatography is investigated. Theoretical considerations have established that although the cosolute-induced displacement of a self-association equilibrium towards the oligomeric state invariably occurs in the mobile phase of the column, that displacement is only manifested as a decreased protein elution volume for cosolutes sufficiently small to partition between the mobile and stationary phases. Indeed, the use of a crowding agent sufficiently large to be confined to the mobile phase gives rise to an increased elution volume that could be misconstrued as evidence of cosolute-induced protein dissociation. Those theoretical considerations are reinforced by experimental studies of α-chymotrypsin (a reversibly dimerizing enzyme) on Superdex 200. The use of cosolutes such as sucrose and small polyethylene glycol fractions such as PEG-2000 is therefore recommended for the detection of protein self-association by molecular crowding effects in size exclusion chromatography.

Molecular crowding effects on the distribution of amphiphiles in biological media.

Biological systems are the result of the interactions established among their many distinct molecules and molecular assemblies. The high concentration of small molecules dissolved in the aqueous media alter the water properties with important consequences in the interactions established. In this work, the effects of high concentrations of the disaccharide trehalose on the solubility of a homologous series of fluorescent amphiphiles (NBD-Cn, n=4-16) and on their interaction with a lipid bilayer and a serum protein are quantitatively characterized. Both kinetic and equilibrium aspects are reported for a better understanding of the effects observed. The aqueous solubility of the most hydrophobic amphiphiles (n ≥ 8) is strongly increased by 1 M trehalose, while no signifcant effect is observed for the most polar amphiphile (n = 4). This results from a decrease in the magnitude of the hydrophobic effect at molecular crowding conditions. A small decrease is observed on the equilibrium association with serum albumin. This is most significant for amphiphiles with longer alkyl chains, in agreement with their increased solubility in the aqueous media containing trehalose. The effects on the association of the amphiphiles with lipid bilayers are influenced by both equilibrium and kinetic aspects. On the one hand, the decreased magnitude of the hydrophobic effect leads to a decrease in the affinity of the amphiphiles towards the membrane. However, this tendency may be overbalanced by the effects on the kinetics of the interaction (insertion/desorption) due to the increase in the viscosity of the aqueous media. It is shown that the distribution of amphiphilic drugs in the crowded biological media is significantly different from that predicted from studies in dilute solutions and that the effects are dependent on the solute's hydrophobicity.

Computational Study of the Forces Driving Aggregation of Ultrasmall Nanoparticles in Biological Fluids.

Nanoparticle (NP) aggregation can lead to prolonged retention in tissues or embolism, among other adverse effects. Successful use in biomedicine thus requires the capability to make NPs with limited aggregative potential. Rational design is presently a challenge due to incomplete knowledge of their interactions in biofluids. Recently, ultrasmall gold NPs passivated with endogenous antioxidant glutathione have shown promise for use in vivo. Computer simulations are here conducted to identify the forces underlying aggregation (or lack thereof) of these NPs in a cell culture. Electrostatic interactions are insufficient to induce association, but the van der Waals forces exerted by cations, anions, and net-neutral polar species can promote the formation of stable dimers. The entropic effects of depletion are negligible, but the combined effect of depletion and macromolecular crowding at physiological concentrations can stabilize aggregates containing just a few NPs. Interparticle interactions are controlled by modest changes in both the structure and dynamic of the interfacial liquid. The molecular origin of these effects and their dependence on NP size are described. The liquid is shown to be highly structured, with large and long-lived hydrogen-bonded water clusters developing often in the interparticle space; their potential role as transient, long-range proton wires connecting and enveloping neighboring NPs is discussed. The basis for a parsimonious theory of ultrasmall NPs in complex fluids is established.

Collective Cell Behaviour with Neighbour-Dependent Proliferation, Death and Directional Bias.

Collective cell migration and proliferation are integral to tissue repair, embryonic development, the immune response and cancer. Central to collective cell migration and proliferation are interactions among neighbouring cells, such as volume exclusion, contact inhibition and adhesion. These individual-level processes can have important effects on population-level outcomes, such as growth rate and equilibrium density. We develop an individual-based model of cell migration and proliferation that includes these interactions. This is an extension of a previous model with neighbour-dependent directional bias to incorporate neighbour-dependent proliferation and death. A deterministic approximation to this individual-based model is derived using a spatial moment dynamics approach, which retains information about the spatial structure of the cell population. We show that the individual-based model and spatial moment model match well across a range of parameter values. The spatial moment model allows insight into the two-way interaction between spatial structure and population dynamics that cannot be captured by traditional mean-field models.

Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm.

Biological macromolecules function in highly crowded cellular environments. The structure and dynamics of proteins and nucleic acids are well characterized in vitro, but in vivo crowding effects remain unclear. Using molecular dynamics simulations of a comprehensive atomistic model cytoplasm we found that protein-protein interactions may destabilize native protein structures, whereas metabolite interactions may induce more compact states due to electrostatic screening. Protein-protein interactions also resulted in significant variations in reduced macromolecular diffusion under crowded conditions, while metabolites exhibited significant two-dimensional surface diffusion and altered protein-ligand binding that may reduce the effective concentration of metabolites and ligands in vivo. Metabolic enzymes showed weak non-specific association in cellular environments attributed to solvation and entropic effects. These effects are expected to have broad implications for the in vivo functioning of biomolecules. This work is a first step towards physically realistic in silico whole-cell models that connect molecular with cellular biology.

A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP.

Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J(15N,13Cα) couplings. This concept was realized using a unidirectional (H)NCO 13C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J(15N,13Cα) couplings were acquired for WIP residues 2-65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16-23 and 45-60, and a helical tendency at residues 28-42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP2-65 conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein-protein interactions that stabilize the unfolded state. We conclude that J-couplings are a useful measureable in characterizing structural ensembles in IDPs, and that the proposed experiment provides a practical method for accurately performing such measurements, once again emphasizing the power of NMR in studying IDP behavior.

Incentives and intrinsic motivation in healthcare.

OBJECTIVE: It has been established in the literature that workers within public organisations are intrinsically motivated. This paper is an empirical study of the healthcare sector using methods of qualitative analysis research, which aims to answer the following hypotheses: 1) doctors are intrinsically motivated; 2) economic incentives and control policies may undermine doctors' intrinsic motivation; and 3) well-designed incentives may encourage doctors' intrinsic motivation. METHOD: We conducted semi-structured interviews à-la-Bewley with 16 doctors from Navarre's Healthcare Service (Servicio Navarro de Salud-Osasunbidea), Spain. The questions were based on current theories of intrinsic motivation and incentives to test the hypotheses. Interviewees were allowed to respond openly without time constraints. Relevant information was selected, quantified and analysed by using the qualitative concepts of saturation and codification. RESULTS: The results seem to confirm the hypotheses. Evidence supporting hypotheses 1 and 2 was gathered from all interviewees, as well as indications of the validity of hypothesis 3 based on interviewees' proposals of incentives. CONCLUSIONS: The conclusions could act as a guide to support the optimal design of incentive policies and schemes within health organisations when healthcare professionals are intrinsically motivated.

Crowding of receptors induces ring-like adhesions in model membranes.

The dynamics of formation of macromolecular structures in adherent membranes is a key to a number of cellular processes. However, the interplay between protein reaction kinetics, diffusion and the morphology of the growing domains, governed by membrane mediated interactions, is still poorly understood. Here we show, experimentally and in simulations, that a rich phase diagram emerges from the competition between binding, cooperativity, molecular crowding and membrane spreading. In the cellular context, the spontaneously-occurring organization of adhesion domains in ring-like morphologies is particularly interesting. These are stabilized by the crowding of bulky proteins, and the membrane-transmitted correlations between bonds. Depending on the density of the receptors, this phase may be circumvented, and instead, the adhesions may grow homogeneously in the contact zone between two membranes. If the development of adhesion occurs simultaneously with membrane spreading, much higher accumulation of binders can be achieved depending on the velocity of spreading. The mechanisms identified here, in the context of our mimetic model, may shed light on the structuring of adhesions in the contact zones between two living cells. This article is part of a Special Issue entitled: Mechanobiology.