Distribution of gold nanoparticles into the brain: a systematic review and meta-analysis.

Despite the widespread use of gold nanoparticles (GNPs), there is no consensus on their distribution to different tissues and organs. The present systematic review and meta-analysis addresses the accumulation of GNPs in brain tissue. Extensive searches were conducted in electronic databases, Medline, Web of Science, EMBASE, and Scopus. Based on inclusion and exclusion criteria, primary and secondary screening was performed. The value of brain accumulation of gold nanoparticle (the percentage of the injection dose of GNPs/gram of brain tissue that applied as effect size (ES) in analysis) and the standard error of the mean were extracted from articles and analyzed by calculating the pooled ES and the pooled confidence interval (CI) using STATA software. p ≤ 0.05 was considered significant. Thirty-eight studies were included in the meta-analysis. The results showed that the amount of GNPs was 0.06% of the injection dose/gram of brain tissue (ES = 0.06, %95 CI: 0.06-0.06, p < 0.0001). Considering the time between injection and tissue harvest (follow-up time), after 1 h the GNPs in brain tissue was 0.288% of the injection dose/gram of tissue (ES = 0.29, 95% CI: 0.25-0.33, p < 0.0001), while after four weeks it was only 0.02% (ES = 0.02, 95% CI: 0.01-0.03, p < 0.0001) of the injection dose/gram of tissue. The amount of GNPs in brain tissue was higher for PEG-coated GNPs compared to uncoated GNPs, and it was 5.6 times higher for rod-shaped GNPs compared to spherical GNPs. The mean amount of GNPs in the brain tissues of animals bearing a tumor was 5.8 times higher than in normal animals.

Frustrated peptide chains at the fibril tip control the kinetics of growth of amyloid-ß fibrils.

Amyloid fibrillization is an exceedingly complex process in which incoming peptide chains bind to the fibril while concertedly folding. The coupling between folding and binding is not fully understood. We explore the molecular pathways of association of Aß40 monomers to fibril tips by combining time-resolved in situ scanning probe microscopy with molecular modeling. The comparison between experimental and simulation results shows that a complex supported by nonnative contacts is present in the equilibrium structure of the fibril tip and impedes fibril growth in a supersaturated solution. The unraveling of this frustrated state determines the rate of fibril growth. The kinetics of growth of freshly cut fibrils, in which the bulk fibril structure persists at the tip, complemented by molecular simulations, indicate that this frustrated complex comprises three or four monomers in nonnative conformations and likely is contained on the top of a single stack of peptide chains in the fibril structure. This pathway of fibril growth strongly deviates from the common view that the conformational transformation of each captured peptide chain is templated by the previously arrived peptide. The insights into the ensemble structure of the frustrated complex may guide the search for suppressors of Aß fibrillization. The uncovered dynamics of coupled structuring and assembly during fibril growth are more complex than during the folding of most globular proteins, as they involve the collective motions of several peptide chains that are not guided by a funneled energy landscape.

Interaction of spindle assembly factor TPX2 with importins-α/ß inhibits protein phase separation.

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/ß until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/ß with nanomolar affinity in a 1:1:1 monodispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-ß via dispersed, weak interactions. We show that interactions of both importin-α and -ß with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.

Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils.

The protein p53 is a crucial tumor suppressor, often called "the guardian of the genome"; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.

Steady, Symmetric, and Reversible Growth and Dissolution of Individual Amyloid-ß Fibrils.

Oligomers and fibrils of the amyloid-ß (Aß) peptide are implicated in the pathology of Alzheimer's disease. Here, we monitor the growth of individual Aß40 fibrils by time-resolved in situ atomic force microscopy and thereby directly measure fibril growth rates. The measured growth rates in a population of fibrils that includes both single protofilaments and bundles of filaments are independent of the fibril thickness, indicating that cooperation between adjacent protofilaments does not affect incorporation of monomers. The opposite ends of individual fibrils grow at similar rates. In contrast to the "stop-and-go" kinetics that has previously been observed for amyloid-forming peptides, growth and dissolution of the Aß40 fibrils are relatively steady for peptide concentration of 0-10 µM. The fibrils readily dissolve in quiescent peptide-free solutions at a rate that is consistent with the microscopic reversibility of growth and dissolution. Importantly, the bimolecular rate coefficient for the association of a monomer to the fibril end is significantly smaller than the diffusion limit, implying that the transition state for incorporation of a monomer into a fibril is associated with a relatively high free energy.

Anomalous Dense Liquid Condensates Host the Nucleation of Tumor Suppressor p53 Fibrils.

About half of human cancers are associated with mutations of the tumor suppressor p53. Gained oncogenic functions of the mutants have been related to aggregation behaviors of wild-type and mutant p53. The thermodynamic and kinetic mechanisms of p53 aggregation are poorly understood. Here we find that wild-type p53 forms an anomalous liquid phase. The liquid condensates exhibit several behaviors beyond the scope of classical phase transition theories: their size, ca. 100 nm, is independent of the p53 concentration and decoupled from the protein mass held in the liquid phase. Furthermore, the liquid phase lacks constant solubility. The nucleation of p53 fibrils deviates from the accepted mechanism of sequential association of single solute molecules. We find that the liquid condensates serve as pre-assembled precursors of high p53 concentration that facilitate fibril assembly. Fibril nucleation hosted by precursors represents a novel biological pathway, which opens avenues to suppress protein fibrillation in aggregation diseases.

Differential dynamic microscopy of bidisperse colloidal suspensions.

Research tasks in microgravity include monitoring the dynamics of constituents of varying size and mobility in processes such as aggregation, phase separation, or self-assembly. We use differential dynamic microscopy, a method readily implemented with equipment available on the International Space Station, to simultaneously resolve the dynamics of particles of radius 50 nm and 1 µm in bidisperse aqueous suspensions. Whereas traditional dynamic light scattering fails to detect a signal from the larger particles at low concentrations, differential dynamic microscopy exhibits enhanced sensitivity in these conditions by accessing smaller wavevectors where scattering from the large particles is stronger. Interference patterns due to scattering from the large particles induce non-monotonic decay of the amplitude of the dynamic correlation function with the wavevector. We show that the position of the resulting minimum contains information on the vertical position of the particles. Together with the simple instrumental requirements, the enhanced sensitivity of differential dynamic microscopy makes it an appealing alternative to dynamic light scattering to characterize samples with complex dynamics.

Polymorphism of Lysozyme Condensates.

Protein condensates play essential roles in physiological processes and pathological conditions. Recently discovered mesoscopic protein-rich clusters may act as crucial precursors for the nucleation of ordered protein solids, such as crystals, sickle hemoglobin polymers, and amyloid fibrils. These clusters challenge settled paradigms of protein condensation as the constituent protein molecules present features characteristic of both partially misfolded and native proteins. Here we employ the antimicrobial enzyme lysozyme and examine the similarities between mesoscopic clusters, amyloid structures, and disordered aggregates consisting of chemically modified protein. We show that the mesoscopic clusters are distinct from the other two classes of aggregates. Whereas cluster formation and amyloid oligomerization are both reversible, aggregation triggered by reduction of the intramolecular S-S bonds is permanent. In contrast to the amyloid structures, protein molecules in the clusters retain their enzymatic activity. Furthermore, an essential feature of the mesoscopic clusters is their constant radius of less than 50 nm. The amyloid and disordered aggregates are significantly larger and rapidly grow. These findings demonstrate that the clusters are a product of limited protein structural flexibility. In view of the role of the clusters in the nucleation of ordered protein solids, our results suggest that fine-tuning the degree of protein conformational stability is a powerful tool to control and direct the pathways of protein condensation.

Protein Conformational Flexibility Enables the Formation of Dense Liquid Clusters: Tests Using Solution Shear.

According to recently proposed two-step nucleation mechanisms, crystal nuclei form within preexisting dense liquid clusters. Clusters with radii about 100 nm, which capture from 10(-7) to 10(-3) of the total protein, have been observed with numerous proteins and shown to host crystal nucleation. Theories aiming to understand the mesoscopic size and small protein fraction held in the clusters have proposed that in solutions of single-chain proteins, the clusters consist of partially misfolded protein molecules. To test this conjecture, we perturb the protein conformation by shearing solutions of the protein lysozyme. We demonstrate that shear rates greater than a threshold applied for longer than 1 h reduce the volume of the cluster population. The likely mechanism of the observed response involves enhanced partial unfolding of lysozyme molecules, which exposes hydrophobic surfaces between the constituent domains to the aqueous solution.

Differential dynamic microscopy of weakly scattering and polydisperse protein-rich clusters.

Nanoparticle dynamics impact a wide range of biological transport processes and applications in nanomedicine and natural resource engineering. Differential dynamic microscopy (DDM) was recently developed to quantify the dynamics of submicron particles in solutions from fluctuations of intensity in optical micrographs. Differential dynamic microscopy is well established for monodisperse particle populations, but has not been applied to solutions containing weakly scattering polydisperse biological nanoparticles. Here we use bright-field DDM (BDDM) to measure the dynamics of protein-rich liquid clusters, whose size ranges from tens to hundreds of nanometers and whose total volume fraction is less than 10(-5). With solutions of two proteins, hemoglobin A and lysozyme, we evaluate the cluster diffusion coefficients from the dependence of the diffusive relaxation time on the scattering wave vector. We establish that for weakly scattering populations, an optimal thickness of the sample chamber exists at which the BDDM signal is maximized at the smallest sample volume. The average cluster diffusion coefficient measured using BDDM is consistently lower than that obtained from dynamic light scattering at a scattering angle of 90°. This apparent discrepancy is due to Mie scattering from the polydisperse cluster population, in which larger clusters preferentially scatter more light in the forward direction.