In situ analysis of osmolyte mechanisms of proteome thermal stabilization.

Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.

Atmospheric Humidity Underlies Irreproducibility of Formamidinium Lead Iodide Perovskites.

Metal halide perovskite solar cells (PSCs) are infamous for their batch-to-batch and lab-to-lab irreproducibility in terms of stability and performance. Reproducible fabrication of PSCs is a critical requirement for market viability and practical commercialization. PSC irreproducibility plagues all levels of the community; from institutional research laboratories, start-up companies, to large established corporations. In this work, the critical function of atmospheric humidity to regulate the crystallization and stabilization of formamidinium lead triiodide (FAPbI3) perovskites is unraveled. It is demonstrated that the humidity content during processing induces profound variations in perovskite stoichiometry, thermodynamic stability, and optoelectronic quality. Almost counterintuitively, it is shown that the presence of humidity is perhaps indispensable to reproduce phase-stable and efficient FAPbI3-based PSCs.

Gas Vesicle-Blood Interactions Enhance Ultrasound Imaging Contrast.

Gas vesicles (GVs) are genetically encoded, air-filled protein nanostructures of broad interest for biomedical research and clinical applications, acting as imaging and therapeutic agents for ultrasound, magnetic resonance, and optical techniques. However, the biomedical applications of GVs as systemically injectable nanomaterials have been hindered by a lack of understanding of GVs' interactions with blood components, which can significantly impact in vivo behavior. Here, we investigate the dynamics of GVs in the bloodstream using a combination of ultrasound and optical imaging, surface functionalization, flow cytometry, and mass spectrometry. We find that erythrocytes and serum proteins bind to GVs and shape their acoustic response, circulation time, and immunogenicity. We show that by modifying the GV surface we can alter these interactions and thereby modify GVs' in vivo performance. These results provide critical insights for the development of GVs as agents for nanomedicine.

Gas vesicle-blood interactions enhance ultrasound imaging contrast.

Gas vesicles (GVs) are genetically encoded, air-filled protein nanostructures of broad interest for biomedical research and clinical applications, acting as imaging and therapeutic agents for ultrasound, magnetic resonance, and optical techniques. However, the biomedical applications of GVs as a systemically injectable nanomaterial have been hindered by a lack of understanding of GVs' interactions with blood components, which can significantly impact in vivo performance. Here, we investigate the dynamics of GVs in the bloodstream using a combination of ultrasound and optical imaging, surface functionalization, flow cytometry, and mass spectrometry. We find that erythrocytes and serum proteins bind to GVs and shape their acoustic response, circulation time, and immunogenicity. We show that by modifying the GV surface, we can alter these interactions and thereby modify GVs' in vivo performance. These results provide critical insights for the development of GVs as agents for nanomedicine.

Poly(trehalose methacrylate) as an Excipient for Insulin Stabilization: Mechanism and Safety.

Insulin, the oldest U.S. Food and Drug Administration (FDA)-approved recombinant protein and a World Health Organization (WHO) essential medicine for treating diabetes globally, faces challenges due to its storage instability. One approach to stabilize insulin is the addition of poly(trehalose methacrylate) (pTrMA) as an excipient. The polymer increases the stability of the peptide to heat and mechanical agitation and has a low viscosity suitable for injection and pumps. However, the safety and stabilizing mechanism of pTrMA is not yet known and is required to understand the potential suitability of pTrMA as an insulin excipient. Herein is reported the immune response, biodistribution, and insulin plasma lifetime in mice, as well as investigation into insulin stabilization. pTrMA alone or formulated with ovalbumin did not elicit an antibody response over 3 weeks in mice, and there was no observable cytokine production in response to pTrMA. Micropositron emission tomography/microcomputer tomography of 64Cu-labeled pTrMA showed excretion of 78-79% ID/cc within 24 h and minimal liver accumulation at 6-8% ID/cc when studied out to 120 h. Further, the plasma lifetime of insulin in mice was not altered by added pTrMA. Formulating insulin with 2 mol equiv of pTrMA improved the stability of insulin to standard storage conditions: 46 weeks at 4 °C yielded 87.0% intact insulin with pTrMA present as compared to 7.8% intact insulin without the polymer. The mechanism by which pTrMA-stabilized insulin was revealed to be a combination of inhibiting deamidation of amino acid residues and preventing fibrillation, followed by aggregation of inactive and immunogenic amyloids all without complexing insulin into its hexameric state, which could delay the onset of insulin activity. Based on the data reported here, we suggest that pTrMA stabilizes insulin as an excipient without adverse effects in vivo and is promising to investigate further for the safe formulation of insulin.

Safety and Biodistribution Profile of Poly(styrenyl acetal trehalose) and Its Granulocyte Colony Stimulating Factor Conjugate.

Poly(styrenyl acetal trehalose) (pSAT), composed of trehalose side chains linked to a polystyrene backbone via acetals, stabilizes a variety of proteins and enzymes against fluctuations in temperature. A promising application of pSAT is conjugation of the polymer to therapeutic proteins to reduce renal clearance. To explore this possibility, the safety of the polymer was first studied. Investigation of acute toxicity of pSAT in mice showed that there were no adverse effects of the polymer at a high (10 mg/kg) concentration. The immune response (antipolymer antibody and cytokine production) in mice was also studied. No significant antipolymer IgG was detected for pSAT, and only a transient and low level of IgM was elicited. pSAT was also safe in terms of cytokine response. The polymer was then conjugated to a granulocyte colony stimulating factor (GCSF), a therapeutic protein that is approved by the Federal Drug Administration, in order to study the biodistribution of a pSAT conjugate. A site-selective, two-step synthesis approach was developed for efficient conjugate preparation for the biodistribution study resulting in 90% conjugation efficiency. The organ distribution of GCSF-pSAT was measured by positron emission tomography and compared to controls GCSF and GCSF-poly(ethylene glycol), which confirmed that the trehalose polymer conjugate improved the in vivo half-life of the protein by reducing renal clearance. These findings suggest that trehalose styrenyl polymers are promising for use in therapeutic protein-polymer conjugates for reduced renal clearance of the biomolecule.

Self-Immolative Hydroxybenzylamine Linkers for Traceless Protein Modification.

Traceless self-immolative linkers are widely used for the reversible modification of proteins and peptides. This article describes a new class of traceless linkers based on ortho- or para-hydroxybenzylamines. The introduction of electron-donating substituents on the aromatic core stabilizes the quinone methide intermediate, thus providing a platform for payload release that can be modulated. To determine the extent to which the electronics affect the rate of release, we prepared a small library of hydroxybenzylamine linkers with varied electronics in the aromatic core, resulting in half-lives ranging from 20 to 144 h. Optimization of the linker design was carried out with mechanistic insights from density functional theory (DFT) and the in silico design of an intramolecular trapping agent through the use of DFT and intramolecular distortion energy calculations. This resulted in the development of a faster self-immolative linker with a half-life of 4.6 h. To demonstrate their effectiveness as traceless linkers for bioconjugation, reversible protein-polyethylene glycol conjugates with a model protein lysozyme were prepared, which had reduced protein activity but recovered ≥94% activity upon traceless release of the polymer. This new class of linkers with tunable release rates expands the traceless linkers toolbox for a variety of bioconjugation applications.

Bulky Cyclometalated Ruthenium Nitrates for Challenging Z-Selective Metathesis: Efficient One-Step Access to α-Oxygenated Z-Olefins from Acrylates and Allyl Alcohols.

α-Oxygenated Z-olefins are ubiquitous in biologically active molecules and serve as versatile handles for organic synthesis, but their syntheses are often tedious and less selective. Here we report the efficient Z-selective metathesis of various terminal acrylates and allyl alcohols, which enables facile and selective construction of high value-added α-oxygenated Z-olefins from readily available feedstock chemicals. These challenging metathesis transformations are enabled by novel cyclometalated Ru-carbene-nitrate complexes bearing bulky-yet-flexible side arms, whose assembly was unlocked by new organometallic syntheses.

Selective CO2 Electrochemical Reduction Enabled by a Tricomponent Copolymer Modifier on a Copper Surface.

Electrochemical CO2 reduction over Cu could provide value-added multicarbon hydrocarbons and alcohols. Despite recent breakthroughs, it remains a significant challenge to design a catalytic system with high product selectivity. Here we demonstrate that a high selectivity of ethylene (55%) and C2+ products (77%) could be achieved by a highly modular tricomponent copolymer modified Cu electrode, rivaling the best performance using other modified polycrystalline Cu foil catalysts. Such a copolymer can be conveniently prepared by a ring-opening metathesis polymerization, thereby offering a new degree of freedom for tuning the selectivity. Control experiments indicate all three components are essential for the selectivity enhancement. A surface characterization showed that the incorporation of a phenylpyridinium component increased the film robustness against delamination. It was also shown that its superior performance is not due to a morphology change of the Cu underneath. Molecular dynamics (MD) simulations indicate that a combination of increased local CO2 concentration, increased porosity for gas diffusion, and the local electric field effect together contribute to the increased ethylene and C2+ product selectivity.

Efficient Z-Selective Olefin-Acrylamide Cross-Metathesis Enabled by Sterically Demanding Cyclometalated Ruthenium Catalysts.

The efficient Z-selective cross-metathesis between acrylamides and common terminal olefins has been developed by the use of novel cyclometalated ruthenium catalysts with bulky N-heterocyclic carbene (NHC) ligands. Superior reactivity and stereoselectivity are realized for the first time in this challenging transformation, allowing streamlined access to an important class of cis-Michael acceptors from readily available feedstocks. The kinetic preference for cross-metathesis is enabled by a pivalate anionic ligand, and the origin of this effect is elucidated by density functional theory calculations.

Synthesis of Zwitterionic and Trehalose Polymers with Variable Degradation Rates and Stabilization of Insulin.

Polymers that stabilize biomolecules are important as excipients in protein formulation. Herein, we describe a class of degradable polymers that have tunable degradation rates depending on the polymer backbone and can stabilize proteins to aggregation. Specifically, zwitterion- and trehalose-substituted polycaprolactone, polyvalerolactone, polycarbonate, and polylactide were prepared and characterized with regards to their hydrolytic degradation and ability to stabilize insulin to mechanical agitation during heat. Ring-opening polymerization (ROP) of allyl-substituted monomers was performed by using organocatalysis, resulting in well-defined alkene-substituted polymers with good control over molecular weight and dispersity. The polymers were then modified by using photocatalyzed thiol-ene reactions to install protein-stabilizing carboxybetaine and trehalose side chains. The resulting polymers were water-soluble and exhibited a wide range of half-lives, from 12 h to more than 3 months. The polymers maintained the ability to stabilize the therapeutic protein insulin from activity loss due to aggregation, demonstrating their potential as degradable excipients for protein formulation.

A guide to maximizing the therapeutic potential of protein-polymer conjugates by rational design.

Proteins are an important class of therapeutics that have advantages including high target specificity, but challenges to their use include rapid clearance and low physical stability. Conjugation of synthetic polymers is an effective approach to address the drawbacks and enhance other properties such as solubility. In this review, we present various considerations in synthesizing protein-polymer conjugates for therapeutic applications with a focus on the choice of polymer, protein, and conjugation method, as well as characterization and evaluation of the resulting conjugate in order to maximize the therapeutic potential of the protein drug.

Tuning Molecular Interactions for Highly Reproducible and Efficient Formamidinium Perovskite Solar Cells via Adduct Approach.

The Lewis acid-base adduct approach has been widely used to form uniform perovskite films, which has provided a methodological base for the development of high-performance perovskite solar cells. However, its incompatibility with formamidinium (FA)-based perovskites has impeded further enhancement of photovoltaic performance and stability. Here, we report an efficient and reproducible method to fabricate highly uniform FAPbI3 films via the adduct approach. Replacement of the typical Lewis base dimethyl sulfoxide (DMSO) with N-methyl-2-pyrrolidone (NMP) enabled the formation of a stable intermediate adduct phase, which can be converted into a uniform and pinhole-free FAPbI3 film. Infrared and computational analyses revealed a stronger interaction between NMP with the FA cation than DMSO, which facilitates the formation of a stable FAI·PbI2·NMP adduct. On the basis of the molecular interactions with different Lewis bases, we proposed criteria for selecting the Lewis bases. Owed to the high film quality, perovskite solar cells with the highest PCE over 20% (stabilized PCE of 19.34%) and average PCE of 18.83 ± 0.73% were demonstrated.

Glucose-Responsive Trehalose Hydrogel for Insulin Stabilization and Delivery.

Effective delivery of therapeutic proteins is important for many biomedical applications. Yet, the stabilization of proteins during delivery and long-term storage remains a significant challenge. Herein, a trehalose-based hydrogel is reported that stabilizes insulin to elevated temperatures prior to glucose-triggered release. The hydrogel is synthesized using a polymer with trehalose side chains and a phenylboronic acid end-functionalized 8-arm poly(ethylene glycol) (PEG). The hydroxyls of the trehalose side chains form boronate ester linkages with the PEG boronic acid cross-linker to yield hydrogels without any further modification of the original trehalose polymer. Dissolution of the hydrogel is triggered upon addition of glucose as a stronger binder to boronic acid (Kb = 2.57 vs 0.48 m-1 for trehalose), allowing the insulin that is entrapped during gelation to be released in a glucose-responsive manner. Moreover, the trehalose hydrogel stabilizes the insulin as determined by immunobinding after heating up to 90 °C. After 30 min heating, 74% of insulin is detected by enzyme-linked immunosorbent assay in the presence of the trehalose hydrogel, whereas only 2% is detected without any additives.

Expanding the ROMP Toolbox: Synthesis of Air-Stable Benzonorbornadiene Polymers by Aryne Chemistry.

Benzonorbornadiene polymers synthesized by ring-opening metathesis polymerization (ROMP) are typically prone to oxidation at the benzylic/allylic position under ambient conditions. Accordingly, the use of benzonorbornadiene polymers in practical applications has remained limited. In this manuscript, we report the synthesis of poly(benzonorbornadiene) polymers using a strategic blend of benzyne chemistry and ROMP. Through a comparative study, we show that substitution at the benzylic/allylic position prevents oxidative deformation, yet does not inhibit polymerization by common ruthenium catalysts with good control over molecular weight dispersity. We expect the benzyne/ROMP reaction sequence will allow easy access to air-stable benzonorbornadiene polymers for various applications.

Trehalose Glycopolymer Enhances Both Solution Stability and Pharmacokinetics of a Therapeutic Protein.

Biocompatible polymers such as poly(ethylene glycol) (PEG) have been successfully conjugated to therapeutic proteins to enhance their pharmacokinetics. However, many of these polymers, including PEG, only improve the in vivo lifetimes and do not protect proteins against inactivation during storage and transportation. Herein, we report a polymer with trehalose side chains (PolyProtek) that is capable of improving both the external stability and the in vivo plasma half-life of a therapeutic protein. Insulin was employed as a model biologic, and high performance liquid chromatography and dynamic light scattering confirmed that addition of trehalose glycopolymer as an excipient or covalent conjugation prevented thermal or agitation-induced aggregation of insulin. The insulin-trehalose glycopolymer conjugate also showed significantly prolonged plasma circulation time in mice, similar to the analogous insulin-PEG conjugate. The insulin-trehalose glycopolymer conjugate was active as tested by insulin tolerance tests in mice and retained bioactivity even after exposure to high temperatures. The trehalose glycopolymer was shown to be nontoxic to mice up to at least 1.6 mg/kg dosage. These results together suggest that the trehalose glycopolymer should be further explored as an alternative to PEG for long circulating protein therapeutics.

Trehalose hydrogels for stabilization of enzymes to heat.

Enzymes can catalyze various reactions with high selectivity and are involved in many important biological processes. However, the general instability of enzymes against high temperature often limits their application. To address this, we synthesized a trehalose-based hydrogel in two steps from commercial starting materials with minimal purification procedures. Mono- and multi-functional trehalose monomers were cross-linked by redox-initiated radical polymerization to form a hydrogel. Phytase, an important enzyme utilized in animal feedstock, was employed to study the effectiveness of the trehalose hydrogel to stabilize proteins against heat. Addition of the phytase solution to the hydrogel resulted in enzyme internalization as confirmed by confocal microscopy. The phytase in the hydrogel retained 100% activity upon heating at 90 °C compared to 39% when the hydrogel was absent. The enzyme could also be recovered from the hydrogel. The trehalose hydrogel synthesis reported herein should be readily scalable for thermal stabilization of a wide variety of enzymes.

Evaluation of node-inhomogeneity effects on the functional brain network properties using an anatomy-constrained hierarchical brain parcellation.

To investigate functional brain networks, many graph-theoretical studies have defined nodes in a graph using an anatomical atlas with about a hundred partitions. Although use of anatomical node definition is popular due to its convenience, functional inhomogeneity within each node may lead to bias or systematic errors in the graph analysis. The current study was aimed to show functional inhomogeneity of a node defined by an anatomical atlas and to show its effects on the graph topology. For this purpose, we compared functional connectivity defined using 138 resting state fMRI data among 90 cerebral nodes from the automated anatomical labeling (AAL), which is an anatomical atlas, and among 372 cerebral nodes defined using a functional connectivity-based atlas as a ground truth, which was obtained using anatomy-constrained hierarchical modularity optimization algorithm (AHMO) that we proposed to evaluate the graph properties for anatomically defined nodes. We found that functional inhomogeneity in the anatomical parcellation induced significant biases in estimating both functional connectivity and graph-theoretical network properties. We also found very high linearity in major global network properties and nodal strength at all brain regions between anatomical atlas and functional atlas with reasonable network-forming thresholds for graph construction. However, some nodal properties such as betweenness centrality did not show significant linearity in some regions. The current study suggests that the use of anatomical atlas may be biased due to its inhomogeneity, but may generally be used in most neuroimaging studies when a single atlas is used for analysis.

Everyday conversation requires cognitive inference: neural bases of comprehending implicated meanings in conversations.

In ordinary conversations, literal meanings of an utterance are often quite different from implicated meanings and the inference about implicated meanings is essentially required for successful comprehension of the speaker's utterances. Inference of finding implicated meanings is based on the listener's assumption that the conversational partner says only relevant matters according to the maxim of relevance in Grice's theory of conversational implicature. To investigate the neural correlates of comprehending implicated meanings under the maxim of relevance, a total of 23 participants underwent an fMRI task with a series of conversational pairs, each consisting of a question and an answer. The experimental paradigm was composed of three conditions: explicit answers, moderately implicit answers, and highly implicit answers. Participants were asked to decide whether the answer to the Yes/No question meant 'Yes' or 'No'. Longer reaction time was required for the highly implicit answers than for the moderately implicit answers without affecting the accuracy. The fMRI results show that the left anterior temporal lobe, left angular gyrus, and left posterior middle temporal gyrus had stronger activation in both moderately and highly implicit conditions than in the explicit condition. Comprehension of highly implicit answers had increased activations in additional regions including the left inferior frontal gyrus, left medial prefrontal cortex, left posterior cingulate cortex and right anterior temporal lobe. The activation results indicate involvement of these regions in the inference process to build coherence between literally irrelevant but pragmatically associated utterances under the maxim of relevance. Especially, the left anterior temporal lobe showed high sensitivity to the level of implicitness and showed increased activation for highly versus moderately implicit conditions, which imply its central role in inference such as semantic integration. The right hemisphere activation, uniquely found in the anterior temporal lobe for highly implicit utterances, suggests its competence for integrating distant concepts in implied utterances under the relevance principle.

Development of MRI/NIRF 'activatable' multimodal imaging probe based on iron oxide nanoparticles.

A fabrication method of Cy5.5-MMP substrate and PEG conjugated iron oxide nanoparticles with thin silica coating (PCM-CS) and its potential as an 'activatable' dual imaging probe for tumor imaging is described in this report. PCM-CS showed an intensity-averaged diameter of 43.1 ± 6.3 nm by dynamic light scattering without any noticeable aggregation over 7 days. Fluorescence of Cy5.5 on the surface of nanoparticles was fully quenched and the quenching efficiency was 97.2%. PCM-CS showed protease specific fluorescence recovery in vitro caused from the specific peptide cleavage by MMP-2 and the probe displayed the sensitivity on 0.5 nM or less enzyme concentration. Tumor was successfully visualized by NIRF and MRI in vivo by intravenously injected PCM-CS. NIRF signal of tumor was gradually increased up to 12h post injection and the intensity of tumor was about 3-4 times higher than normal tissue. NIRF signal at MMP-2 inhibitor treated tumor was clearly lower than tumor without inhibitor due to the insufficient peptide cleavage. NIRF signal at excised tumor was 5-10 times stronger than other organs. Noticeable darkening in magnetic resonance image was observed at the tumor region and the image was gradually darkened at 12h post injection of PCM-CS. The maximum signal difference between tumor region and healthy muscle was 34%.