Working Memory Content Is Distorted by Its Use in Perceptual Comparisons.

Visual information around us is rarely static. To perform a task in such a dynamic environment, we often have to compare current visual input with our working memory (WM) representation of the immediate past. However, little is known about what happens to a WM representation when it is compared with perceptual input. To test this, we asked young adults (N = 170 total in three experiments) to compare a new visual input with a WM representation prior to reporting the WM representation. We found that the perceptual comparison biased the WM report, especially when the input was subjectively similar to the WM representation. Furthermore, using computational modeling and individual-differences analyses, we found that this similarity-induced memory bias was driven by representational integration, rather than incidental confusion, between the WM representation and subjectively similar input. Together, our findings highlight a novel source of WM distortion and suggest a general mechanism that determines how WM interacts with new visual input.

Enhanced Ribozyme-Catalyzed Recombination and Oligonucleotide Assembly in Peptide-RNA Condensates.

The ability of RNA to catalyze RNA ligation is critical to its central role in many prebiotic model scenarios, in particular the copying of information during self-replication. Prebiotically plausible ribozymes formed from short oligonucleotides can catalyze reversible RNA cleavage and ligation reactions, but harsh conditions or unusual scenarios are often required to promote folding and drive the reaction equilibrium towards ligation. Here, we demonstrate that ribozyme activity is greatly enhanced by charge-mediated phase separation with poly-L-lysine, which shifts the reaction equilibrium from cleavage in solution to ligation in peptide-RNA coaggregates and coacervates. This compartmentalization enables robust isothermal RNA assembly over a broad range of conditions, which can be leveraged to assemble long and complex RNAs from short fragments under mild conditions in the absence of exogenous activation chemistry, bridging the gap between pools of short oligomers and functional RNAs.

Controlling Protein Nanocage Assembly with Hydrostatic Pressure.

Controlling the assembly and disassembly of nanoscale protein cages for the capture and internalization of protein or non-proteinaceous components is fundamentally important to a diverse range of bionanotechnological applications. Here, we study the reversible, pressure-induced dissociation of a natural protein nanocage, E. coli bacterioferritin (Bfr), using synchrotron radiation small-angle X-ray scattering (SAXS) and circular dichroism (CD). We demonstrate that hydrostatic pressures of 450 MPa are sufficient to completely dissociate the Bfr 24-mer into protein dimers, and the reversibility and kinetics of the reassembly process can be controlled by selecting appropriate buffer conditions. We also demonstrate that the heme B prosthetic group present at the subunit dimer interface influences the stability and pressure lability of the cage, despite its location being discrete from the interdimer interface that is key to cage assembly. This indicates a major cage-stabilizing role for heme within this family of ferritins.

Role of imaging biomarkers for prognostic prediction in patients with pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumours. PDAC has a poor prognosis; therefore, it is necessary to perform further risk stratification. Identifying prognostic factors before treatment might help to implement suitable and personalised treatment for individuals and avoid side effects. Conventional staging systems and tumour biomarkers are fundamental to establish prognosis; however, they have obvious limitations. Novel imaging biomarkers extracted from advanced imaging techniques offer opportunities to evaluate underlying tumour physiological characteristics, such as mutational status, cellular composition, local microenvironment, tumour metabolism, and biological behaviour. Thus, imaging biomarkers might help the decision making of oncologists and surgeons. The present review discusses the functions of imaging biomarkers for prognostic prediction in patients with PDAC and their potential value for further translation in clinical practice.

Reversible pH-Responsive Coacervate Formation in Lipid Vesicles Activates Dormant Enzymatic Reactions.

In situ, reversible coacervate formation within lipid vesicles represents a key step in the development of responsive synthetic cellular models. Herein, we exploit the pH responsiveness of a polycation above and below its pKa , to drive liquid-liquid phase separation, to form single coacervate droplets within lipid vesicles. The process is completely reversible as coacervate droplets can be disassembled by increasing the pH above the pKa . We further show that pH-triggered coacervation in the presence of low concentrations of enzymes activates dormant enzyme reactions by increasing the local concentration within the coacervate droplets and changing the local environment around the enzyme. In conclusion, this work establishes a tunable, pH responsive, enzymatically active multi-compartment synthetic cell. The system is readily transferred into microfluidics, making it a robust model for addressing general questions in biology, such as the role of phase separation and its effect on enzymatic reactions using a bottom-up synthetic biology approach.

Special Issue on Bottom-Up Synthetic Biology.

Bottom-up synthetic biology uses both biological and artificial chemical building blocks to create biomimetic systems, including artificial cells. Existing and new technologies such as microfluidics are being developed and applied to the assembly processes. In this special issue, experts present and review the latest progress in this rapidly expanding and diverse field.

The geometry of consciousness.

Conscious experience implies a reference-frame or vantage, which is often important in scientific models. Control models of ball-interception are used as an example. Models that use viewer-dependent egocentric reference-frames are contrasted with viewer-independent allocentric ones. Allocentric reference-frames serve well for models like Newtonian physics, which utilize static coordinate-systems that allow forces and object-movements to be compartmentalized. In contrast, egocentric reference-frames are natural for modeling mobile organisms or robots when controlling perception-action behavior. Lower-level perception-action behavior is often characterized using egocentric coordinate-systems that optimize processing-speed, while higher-level cognitive-processes use allocentric frames that provide a stationary spatial reference. Brain-behavior models like the Ventral-Stream What System, and Dorsal-Stream Where-How System, also respectively utilize allocentric and egocentric reference-frames. Reference-frame clarification can resolve disputes about models of control-tasks like running to catch baseballs, and can provide insights for biomimetic-robots. Confusion regarding geometry and reference-frames contributes to a lack of clarity between how and when egocentric versus allocentric geometries are imposed, with perception-actions generally being more egocentric and conscious experience more allocentric.

MaxSynBio: Avenues Towards Creating Cells from the Bottom Up.

A large German research consortium mainly within the Max Planck Society ("MaxSynBio") was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to synthetic biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnology.

Structural studies of the lamellar to bicontinuous gyroid cubic (Q(G)(II)) phase transitions under limited hydration conditions.

Non-equilibrium pathways of lyotropic phase transitions such as the lamellar to inverse bicontinuous cubic phase transition are important dynamical processes resembling cellular fusion and fission processes which can be exploited in biotechnological processes such as drug delivery. However, utilising and optimising these structural transformations for applications require a detailed understanding of the energetic pathways which drive the phase transition. We have used the high pressure X-ray diffraction technique to probe the lamellar to Q(G)(II) phase transition in limited hydration monolinolein on the millisecond time scale. Our results show that the phase transition goes via a structural intermediate and once the Q(G)(II) phase initially forms the elastic energy in the bilayer drives this structure to its equilibrium lattice parameter.

Hydrophobic nanoparticles promote lamellar to inverted hexagonal transition in phospholipid mesophases.

This study focuses on how the mesophase transition behaviour of the phospholipid dioleoyl phosphatidylethanolamine (DOPE) is altered by the presence of 10 nm hydrophobic and 14 nm hydrophilic silica nanoparticles (NPs) at different concentrations. The lamellar to inverted hexagonal phase transition (Lα-HII) of phospholipids is energetically analogous to the membrane fusion process, therefore understanding the Lα-HII transition with nanoparticulate additives is relevant to how membrane fusion may be affected by these additives, in this case the silica NPs. The overriding observation is that the HII/Lα boundaries in the DOPE p-T phase diagram were shifted by the presence of NPs: the hydrophobic NPs enlarged the HII phase region and thus encouraged the inverted hexagonal (HII) phase to occur at lower temperatures, whilst hydrophilic NPs appeared to stabilise the Lα phase region. This effect was also NP-concentration dependent, with a more pronounced effect for higher concentration of the hydrophobic NPs, but the trend was less clear cut for the hydrophilic NPs. There was no evidence that the NPs were intercalated into the mesophases, and as such it was likely that they might have undergone microphase separation and resided at the mesophase domain boundaries. Whilst the loci and exact roles of the NPs invite further investigation, we tentatively discuss these results in terms of both the surface chemistry of the NPs and the effect of their curvature on the elastic bending energy considerations during the mesophase transition.

Microfluidic Formation of Membrane-Free Aqueous Coacervate Droplets in Water.

We report on the formation of coacervate droplets from poly(diallyldimethylammonium chloride) with either adenosine triphosphate or carboxymethyl-dextran using a microfluidic flow-focusing system. The formed droplets exhibit improved stability and narrower size distributions for both coacervate compositions when compared to the conventional vortex dispersion techniques. We also demonstrate the use of two parallel flow-focusing channels for the simultaneous formation and co-location of two distinct populations of coacervate droplets containing different DNA oligonucleotides, and that the populations can coexist in close proximity up to 48 h without detectable exchange of genetic information. Our results show that the observed improvements in droplet stability and size distribution may be scaled with ease. In addition, the ability to encapsulate different materials into coacervate droplets using a microfluidic channel structure allows for their use as cell-mimicking compartments.

The effects of pressure and temperature on the energetics and pivotal surface in a monoacylglycerol/water gyroid inverse bicontinuous cubic phase.

We have studied the effect of pressure and temperature on the location of the pivotal surface in a lipid inverse bicontinuous gyroid cubic phase (Q(G)(II)), described by the area at the pivotal surface (An), the volume between the pivotal surface and the bilayer midplane (Vn), and the molecular volume of the lipid (V). Small angle X-ray scattering (SAXS) was used to measure the swelling behaviour of the lipid, monolinolein, as a function of pressure and temperature, and the data were fitted to two different geometric models: the parallel interface model (PIM), and the constant mean curvature model (CMCM). The results show that an increase in temperature leads to a shift in the location of the pivotal surface towards the bilayer midplane, whilst an increase in pressure causes the pivotal surface to move towards the interfacial region. In addition, we describe the relevance of An, Vn and V for modeling the energetics of curved mesophases with specific reference to the mean curvature at the pivotal surface and discuss the significance of this parameter for modelling the energetics of curved mesophases.

Cell Free Expression in Proteinosomes Prepared from Native Protein-PNIPAAm Conjugates.

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalization with an open, semi-permeable membrane. Critically, to date, there has been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, a novel approach to generate proteinosomes that can support cell free transcription and translation is presented. This approach generates proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. These native proteinosomes offer an excellent alternative as a synthetic cell chassis to other membrane bound compartments. Significantly, the native proteinosomes are stable under high salt conditions that enables the ability to support cell free transcription and translation and offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, the integration of native proteinosomes into higher order synthetic cellular architectures with membrane free compartments such as liposomes is demonstrated. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

Statistical mechanics of biomolecular condensates via cavity methods.

Physical mechanisms of phase separation in living systems play key physiological roles and have recently been the focus of intensive studies. The strongly heterogeneous nature of such phenomena poses difficult modeling challenges that require going beyond mean-field approaches based on postulating a free energy landscape. The pathway we take here is to calculate the partition function starting from microscopic interactions by means of cavity methods, based on a tree approximation for the interaction graph. We illustrate them on the binary case and then apply them successfully to ternary systems, in which simpler one-factor approximations are proved inadequate. We demonstrate the agreement with lattice simulations and contrast our theory with coacervation experiments of associative de-mixing of nucleotides and poly-lysine. Different types of evidence are provided to support cavity methods as ideal tools for modeling biomolecular condensation, giving an optimal balance between the consideration of spatial aspects and fast computational results.

Hydrostatic pressure effects on the lamellar to gyroid cubic phase transition of monolinolein at limited hydration.

Monoacylglycerol based lipids are highly important model membrane components and attractive candidates for drug encapsulation and as delivery agents. However, optimizing the properties of these lipids for applications requires a detailed understanding of the thermodynamic factors governing the self-assembled structures that they form. Here, we report on the effects of hydrostatic pressure, temperature, and water composition on the structural behavior and stability of inverse lyotropic liquid crystalline phases adopted by monolinolein (an unsaturated monoacylglycerol having cis-double bonds at carbon positions 9 and 12) under limited hydration conditions. Six pressure-temperature phase diagrams have been determined using small-angle X-ray diffraction at water contents between 15 wt % and 27 wt % water, in the range 10-40 °C and 1-3000 bar. The gyroid bicontinuous cubic (Q(II)(G)) phase is formed at low pressure and high temperatures, transforming to a fluid lamellar (L(α)) phase at high pressures and low temperature via a region of Q(II)(G)/L(α) coexistence. Pressure stabilizes the lamellar phase over the Q(II)(G) phase; at fixed pressure, increasing the water content causes the coexistence region to move to lower temperature. These trends are consistent throughout the hydration range studied. Moreover, at fixed temperature, increasing the water composition increases the pressure at which the Q(II)(G) to L(α) transition takes place. We discuss the qualitative effect of pressure, temperature, and water content on the stability of the Q(II)(G) phase.

Cell-Free Gene Expression Dynamics in Synthetic Cell Populations.

The ability to build synthetic cellular populations from the bottom-up provides the groundwork to realize minimal living tissues comprising single cells which can communicate and bridge scales into multicellular systems. Engineered systems made of synthetic micron-sized compartments and integrated reaction networks coupled with mathematical modeling can facilitate the design and construction of complex and multiscale chemical systems from the bottom-up. Toward this goal, we generated populations of monodisperse liposomes encapsulating cell-free expression systems (CFESs) using double-emulsion microfluidics and quantified transcription and translation dynamics within individual synthetic cells of the population using a fluorescent Broccoli RNA aptamer and mCherry protein reporter. CFE dynamics in bulk reactions were used to test different coarse-grained resource-limited gene expression models using model selection to obtain transcription and translation rate parameters by likelihood-based parameter estimation. The selected model was then applied to quantify cell-free gene expression dynamics in populations of synthetic cells. In combination, our experimental and theoretical approaches provide a statistically robust analysis of CFE dynamics in bulk and monodisperse synthetic cell populations. We demonstrate that compartmentalization of CFESs leads to different transcription and translation rates compared to bulk CFE and show that this is due to the semipermeable lipid membrane that allows the exchange of materials between the synthetic cells and the external environment.

Programmable synthetic cell networks regulated by tuneable reaction rates.

Coupled compartmentalised information processing and communication via molecular diffusion underpin network based population dynamics as observed in biological systems. Understanding how both compartmentalisation and communication can regulate information processes is key to rational design and control of compartmentalised reaction networks. Here, we integrate PEN DNA reactions into semi-permeable proteinosomes and characterise the effect of compartmentalisation on autocatalytic PEN DNA reactions. We observe unique behaviours in the compartmentalised systems which are not accessible under bulk conditions; for example, rates of reaction increase by an order of magnitude and reaction kinetics are more readily tuneable by enzyme concentrations in proteinosomes compared to buffer solution. We exploit these properties to regulate the reaction kinetics in two node compartmentalised reaction networks comprised of linear and autocatalytic reactions which we establish by bottom-up synthetic biology approaches.

Charge-density reduction promotes ribozyme activity in RNA-peptide coacervates via RNA fluidization and magnesium partitioning.

It has long been proposed that phase-separated compartments can provide a basis for the formation of cellular precursors in prebiotic environments. However, we know very little about the properties of coacervates formed from simple peptides, their compatibility with ribozymes or their functional significance. Here we assess the conditions under which functional ribozymes form coacervates with simple peptides. We find coacervation to be most robust when transitioning from long homopeptides to shorter, more pre-biologically plausible heteropeptides. We mechanistically show that these RNA-peptide coacervates display peptide-dependent material properties and cofactor concentrations. We find that the interspacing of cationic and neutral amino acids increases RNA mobility, and we use isothermal calorimetry to reveal sequence-dependent Mg2+ partitioning, two critical factors that together enable ribozyme activity. Our results establish how peptides of limited length, homogeneity and charge density facilitate the compartmentalization of active ribozymes into non-gelating, magnesium-rich coacervates, a scenario that could be applicable to cellular precursors with peptide-dependent functional phenotypes.

Non-equilibrium conditions inside rock pores drive fission, maintenance and selection of coacervate protocells.

Key requirements for the first cells on Earth include the ability to compartmentalize and evolve. Compartmentalization spatially localizes biomolecules from a dilute pool and an evolving cell, which, as it grows and divides, permits mixing and propagation of information to daughter cells. Complex coacervate microdroplets are excellent candidates as primordial cells with the ability to partition and concentrate molecules into their core and support primitive and complex biochemical reactions. However, the evolution of coacervate protocells by fusion, growth and fission has not yet been demonstrated. In this work, a primordial environment initiated the evolution of coacervate-based protocells. Gas bubbles inside heated rock pores perturb the coacervate protocell distribution and drive the growth, fusion, division and selection of coacervate microdroplets. Our findings provide a compelling scenario for the evolution of membrane-free coacervate microdroplets on the early Earth, induced by common gas bubbles within heated rock pores.