Dense liquid phases, metastable with respect to a solid phase, form in solutions of proteins and small-molecule materials. They have been shown to serve as a prerequisite for the nucleation of crystals and other ordered solid phases. Here, using crystals of the protein lumazine synthase from Bacillus subtilis, which grow by the generation and spreading of layers, we demonstrate that within a range of supersaturations the only mechanism of generation of growth layers involves the association of submicrometer-size droplets of the dense liquid to the crystal surface. The dense liquid is metastable not only with respect to the crystals, but also with respect to the low-concentration solution: dynamic light scattering reveals that the droplets' lifetime is limited to several seconds, after which they decay into the low-concentration solution. The short lifetime does not allow growth to detectable dimensions so that liquid-liquid phase separation is not observed within a range of conditions broader than the one used for crystallization. If during their lifetime the droplets encounter a crystal surface, they lower their free energy not by decay, but by transformation into crystalline matter, ensuring perfect registry with the substrate. These observations illustrate two novel features of phase transformations in solutions: the existence of doubly metastable, short-lifetime dense phases and their crucial role for the growth of an ordered solid phase.

Dense liquid phases, metastable with respect to a solid phase, but stable with respect to the solution, have been known to form in solutions of proteins and small-molecule substances. Here, with the protein lumazine synthase as a test system, using dynamic and static light scattering and atomic force microscopy, we demonstrate submicron size clusters of dense liquid. In contrast to the macroscopic dense liquid, these clusters are metastable not only with respect to the crystals, but also with respect to the low-concentration solution: the characteristic cluster lifetime is limited to approximately 10 s, after which they decay. The cluster population is detectable only if they occupy >10(-6) of the solution volume and have a number density >105 cm-3 for 3 to 11% of the monitored time. The cluster volume fraction varies within wide limits and reaches up to 10(-3). Increasing protein concentration increases the frequency of cluster detection but does not affect the ranges of the cluster sizes, suggesting that a preferred cluster size exists. A simple Monte Carlo model with protein-like potentials reproduces the metastable clusters of dense liquid with limited lifetimes and variable sizes and suggests that the mean cluster size is determined by the kinetics of growth and decay and not by thermodynamics.

Seeking new insights into the homeostasis, modulation and plasticity of cortical synaptic networks, we have analyzed results from a single-cell RNA-seq study of 22,439 mouse neocortical neurons. Our analysis exposes transcriptomic evidence for dozens of molecularly distinct neuropeptidergic modulatory networks that directly interconnect all cortical neurons. This evidence begins with a discovery that transcripts of one or more neuropeptide precursor (NPP) and one or more neuropeptide-selective G-protein-coupled receptor (NP-GPCR) genes are highly abundant in all, or very nearly all, cortical neurons. Individual neurons express diverse subsets of NP signaling genes from palettes encoding 18 NPPs and 29 NP-GPCRs. These 47 genes comprise 37 cognate NPP/NP-GPCR pairs, implying the likelihood of local neuropeptide signaling. Here, we use neuron-type-specific patterns of NP gene expression to offer specific, testable predictions regarding 37 peptidergic neuromodulatory networks that may play prominent roles in cortical homeostasis and plasticity.