Glioma stem cell proliferation and tumor growth are promoted by nitric oxide synthase-2.

Malignant gliomas are aggressive brain tumors with limited therapeutic options, and improvements in treatment require a deeper molecular understanding of this disease. As in other cancers, recent studies have identified highly tumorigenic subpopulations within malignant gliomas, known generally as cancer stem cells. Here, we demonstrate that glioma stem cells (GSCs) produce nitric oxide via elevated nitric oxide synthase-2 (NOS2) expression. GSCs depend on NOS2 activity for growth and tumorigenicity, distinguishing them from non-GSCs and normal neural progenitors. Gene expression profiling identified many NOS2-regulated genes, including the cell-cycle inhibitor cell division autoantigen-1 (CDA1). Further, high NOS2 expression correlates with decreased survival in human glioma patients, and NOS2 inhibition slows glioma growth in a murine intracranial model. These data provide insight into how GSCs are mechanistically distinct from their less tumorigenic counterparts and suggest that NOS2 inhibition may be an efficacious approach to treating this devastating disease.

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 µg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 µg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

Bioenabled Platform to Access Polyamides with Built-In Target Properties.

The diversification of platform chemicals is key to today's petroleum industry. Likewise, the flourishing of tomorrow's biorefineries will rely on molecules with next-generation properties from biomass. Herein, we explore this opportunity with a novel approach to monomers with custom property enhancements. Cyclic diacids with alkyl and aromatic decorations were synthesized from muconic acid by Diels-Alder cycloaddition, and copolymerized with hexamethylenediamine and adipic acid to yield polyamides with built-in hydrophobicity and flame retardancy. Testing shows a 70% reduction in water uptake and doubling of char production while largely retaining other key properties of the parent Nylon-6,6. The present approach can be generalized to access a wide range of performance-advantaged polyamides.

Protein denitrosylation: enzymatic mechanisms and cellular functions.

S-Nitrosylation, the redox-based modification of Cys thiol side chains by nitric oxide, is a common mechanism in signal transduction. Dysregulated S-nitrosylation contributes to a range of human pathologies. New roles for protein denitrosylation in regulating S-nitrosylation are being revealed. Recently, several denitrosylases - the enzymes that mediate Cys denitrosylation - have been discovered, of which two enzyme systems in particular, the S-nitrosoglutathione reductase and thioredoxin systems, have been shown to be physiologically relevant. These highly conserved enzymes regulate signalling through multiple classes of receptors and influence diverse cellular responses. In addition, they protect from nitrosative stress in microorganisms, mammals and plants, thereby exerting profound effects on host-microbe interactions and innate immunity.

ERK-dependent proteasome degradation of Txnip regulates thioredoxin oxidoreductase activity.

Dynamic control of thioredoxin (Trx) oxidoreductase activity is essential for balancing the need of cells to rapidly respond to oxidative/nitrosative stress and to temporally regulate thiol-based redox signaling. We have previously shown that cytokine stimulation of the respiratory epithelium induces a precipitous decline in cell S-nitrosothiol, which depends upon enhanced Trx activity and proteasome-mediated degradation of Txnip (thioredoxin-interacting protein). We now show that tumor necrosis factor-α-induced Txnip degradation in A549 respiratory epithelial cells is regulated by the extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinase pathway and that ERK inhibition augments both intracellular reactive oxygen species and S-nitrosothiol. ERK-dependent Txnip ubiquitination and proteasome degradation depended upon phosphorylation of a PXTP motif threonine (Thr349) located within the C-terminal α-arrestin domain and proximal to a previously characterized E3 ubiquitin ligase-binding site. Collectively, these findings demonstrate the ERK mitogen-activated protein kinase pathway to be integrally involved in regulating Trx oxidoreductase activity and that the regulation of Txnip lifetime via ERK-dependent phosphorylation is an important mediator of this effect.

Next-generation neural mass and field modeling.

The Wilson-Cowan population model of neural activity has greatly influenced our understanding of the mechanisms for the generation of brain rhythms and the emergence of structured brain activity. As well as the many insights that have been obtained from its mathematical analysis, it is now widely used in the computational neuroscience community for building large-scale in silico brain networks that can incorporate the increasing amount of knowledge from the Human Connectome Project. Here, we consider a neural population model in the spirit of that originally developed by Wilson and Cowan, albeit with the added advantage that it can account for the phenomena of event-related synchronization and desynchronization. This derived mean-field model provides a dynamic description for the evolution of synchrony, as measured by the Kuramoto order parameter, in a large population of quadratic integrate-and-fire model neurons. As in the original Wilson-Cowan framework, the population firing rate is at the heart of our new model; however, in a significant departure from the sigmoidal firing rate function approach, the population firing rate is now obtained as a real-valued function of the complex-valued population synchrony measure. To highlight the usefulness of this next-generation Wilson-Cowan style model, we deploy it in a number of neurobiological contexts, providing understanding of the changes in power spectra observed in electro- and magnetoencephalography neuroimaging studies of motor cortex during movement, insights into patterns of functional connectivity observed during rest and their disruption by transcranial magnetic stimulation, and to describe wave propagation across cortex.

Self-assembly of Janus Dumbbell Nanocrystals and Their Enhanced Surface Plasmon Resonance.

Self-assembly is a critical process that can greatly expand the existing structures and lead to new functionality of nanoparticle systems. Multicomponent superstructures self-assembled from nanocrystals have shown promise as multifunctional materials for various applications. Despite recent progress in assembly of homogeneous nanocrystals, synthesis and self-assembly of Janus nanocrystals with contrasting surface chemistry remains a significant challenge. Herein, we designed a novel Janus nanocrystal platform to control the self-assembly of nanoparticles in aqueous solutions by balancing the hydrophobic and hydrophilic moieties. A series of superstructures have been assembled by systematically varying the Janus balance and assembly conditions. Janus Au-Fe3O4 dumbbell nanocrystals (<20 nm) were synthesized with the hydrophobic ligands coated on the Au lobe and negatively charged hydrophilic ligands coated on the Fe3O4 lobe. We systematically fine-tune the lobe size ratio, surface coating, external conditions, and even additional growth of Au nanocrystal domains on the Au lobe of dumbbell nanoparticles (Au-Au-Fe3O4) to harvest self-assembly structures including clusters, chains, vesicles, and capsules. It was discovered that in all these assemblies the hydrophobic Au lobes preferred to stay together. In addition, these superstructures clearly demonstrated different levels of enhanced surface plasmon resonance that is directly correlated with the Au coupling in the assembly structure. The strong interparticle plasmonic coupling displayed a red-shift in surface plasmon resonance, with larger structures formed by Au-Au-Fe3O4 assembly extending into the near-infrared region. Self-assembly of Janus dumbbell nanocrystals can also be reversible under different pH values. The biphasic Janus dumbbell nanocrystals offer a platform for studying the novel interparticle coupling and open up opportunities in applications including sensing, disease diagnoses, and therapy.

How children aged 2;6 tailor verbal expressions to interlocutor informational needs.

Although preschoolers are pervasively underinformative in their actual usage of verbal reference, a number of studies have shown that they nonetheless demonstrate sensitivity to listener informational needs, at least when environmental cues to this are obvious. We investigated two issues. The first concerned the types of visual cues to interlocutor informational needs which children aged 2;6 can process whilst producing complex referring expressions. The second was whether performance in experimental tasks related to naturalistic conversational proficiency. We found that 2;6-year-olds used fewer complex expressions when the objects were dissimilar compared to highly similar objects, indicating that they tailor their verbal expressions to the informational needs of another person, even when the cue to the informational need is relatively opaque. We also found a correlation between conversational skills as rated by the parents and the degree to which 2;6-year-olds could learn from feedback to produce complex referring expressions.

Thioredoxin-mediated denitrosylation regulates cytokine-induced nuclear factor κB (NF-κB) activation.

S-nitrosylation of nuclear factor κB (NF-κB) on the p65 subunit of the p50/p65 heterodimer inhibits NF-κB DNA binding activity. We have recently shown that p65 is constitutively S-nitrosylated in the lung and that LPS-induced injury elicits a decrease in SNO-p65 levels concomitant with NF-κB activation in the respiratory epithelium and initiation of the inflammatory response. Here, we demonstrate that TNFα-mediated activation of NF-κB in the respiratory epithelium similarly induces p65 denitrosylation. This process is mediated by the denitrosylase thioredoxin (Trx), which becomes activated upon cytokine-induced degradation of thioredoxin-interacting protein (Txnip). Similarly, inhibition of Trx activity in the lung attenuates LPS-induced SNO-p65 denitrosylation, NF-κB activation, and airway inflammation, supporting a pathophysiological role for this mechanism in lung injury. These data thus link stimulus-coupled activation of NF-κB to a specific, protein-targeted denitrosylation mechanism and further highlight the importance of S-nitrosylation in the regulation of the immune response.

Solid-phase capture for the detection and relative quantification of S-nitrosoproteins by mass spectrometry.

The proteomic analysis of S-nitrosylated protein (SNO-proteins) has long depended on the biotin switch technique (BST), which requires blocking of free thiols, ascorbate-based denitrosylation of SNO-Cys, biotinylation of nascent thiol and avidin-based affinity isolation. A more recent development is resin assisted-capture of SNO-proteins (SNO-RAC), which substitutes thiopropyl Sepharose (TPS) for biotin-avidin, thus reducing the number of steps required for enrichment of S-nitrosylated proteins. In addition, SNO-RAC facilitates on-resin proteolytic digestion following SNO-protein capture, greatly simplifying the purification of peptides containing sites of S-nitrosylation ("SNO-sites"). This resin-based approach has also now been applied to detection of alternative Cys-based modifications, including S-palmitoylation (Acyl-RAC) and S-oxidation (Ox-RAC). Here, we review the important steps to minimize false-positive identification of SNO-proteins, give detailed methods for processing of protein-bound TPS for mass spectrometry (MS) based analysis, and discuss the various quantitative MS methods that are compatible with SNO-RAC. We also discuss strategies to overcome the current limitations surrounding MS-based SNO-site localization in peptides containing more than one potential target Cys residue. This article therefore serves as a starting point and guide for the MS-focused exploration of SNO-proteomes by SNO-RAC.

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 micrograms of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g. H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 micrograms of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamlines the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

Accelerated discovery of perovskite solid solutions through automated materials synthesis and characterization.

Accelerating perovskite solid solution discovery and sustainable synthesis is crucial for addressing challenges in wireless communication and biosensors. However, the vast array of chemical compositions and their dependence on factors such as crystal structure, and sintering temperature require time-consuming manual processes. To overcome these constraints, we introduce an automated materials discovery approach encompassing machine learning (ML) assisted material screening, robotic synthesis, and high-throughput characterization. Our proposed platform for rapid sintering and dielectric analysis streamlines the characterization of perovskites and the discovery of disordered materials. The setup has been successfully validated, demonstrating processing materials within minutes, in stark contrast to conventional procedures that can take hours or days. Following setup validation with established samples, we showcase synthesizing single-phase solid solutions within the barium family, such as (BaxSr1-x)CeO3, identified through ML-guided chemistry.

An integrated experimental and theoretical approach to probe Cr(vi) uptake using decorated halloysite nanotubes for efficient water treatment.

Halloysite nanotubes (HNTs) were surface functionalized using four distinct chemical moieties (amidoxime, hydrazone, ethylenediamine (EDA), and diethylenetriamine (DETA)), producing modified HNTs (H1-H4) capable of binding with Cr(vi) ions. Advanced techniques like FTIR, XRD, SEM, and EDX provided evidence of the successful functionalization of these HNTs. Notably, the functionalization occurred on the surface of HNTs, rather than within the interlayer or lumen. These decorated HNTs were effective in capturing Cr(vi) ions at optimized sorption parameters, with adsorption rates ranging between 58-94%, as confirmed by atomic absorption spectroscopy (AAS). The mechanism of adsorption was further scrutinized through the Freundlich and Langmuir isotherms. Langmuir isotherms revealed the nearest fit to the data suggesting the monolayer adsorption of Cr(vi) ions onto the nanotubes, indicating a favorable adsorption process. It was hypothesized that Cr(vi) ions are primarily attracted to the amine groups on the modified nanotubes. Quantum chemical calculations further revealed that HNTs functionalized with hydrazone structures (H2) demonstrated a higher affinity (interaction energy -26.33 kcal mol-1) for the Cr(vi) ions. This can be explained by the formation of stronger hydrogen bonds with the NH moieties of the hydrazone moiety, than those established by the OH of oxime (H1) and longer amine chains (H3 and H4), respectively. Overall, the findings suggest that these decorated HNTs could serve as an effective and cost-efficient solution for treating water pollution.

Proteomic analysis of the NOS2 interactome in human airway epithelial cells.

The cytokine-inducible isoform of nitric oxide synthase (NOS2) is constitutively expressed in human respiratory epithelia and is upregulated in inflammatory lung disease. Here, we sought to better define the protein interactions that may be important for NOS2 activity and stability, as well as to identify potential targets of NOS2-derived NO, in the respiratory epithelium. We overexpressed Flag-tagged, catalytically-inactive NOS2 in A549 cells and used mass spectrometry to qualitatively identify NOS2 co-immunoprecipitating proteins. Stable isotope labeling of amino acids in cell culture (SILAC) was used to quantify the coordinate effects of cytokine stimulation on NOS2-protein interactions. Multi-protein networks dominated the NOS2 interactome, and cytokine-inducible interactions with allosteric activators and with the ubiquitin-proteasome system were correlated with cytokine-dependent increases in NO metabolites and in NOS2 ubiquitination. The ubiquitin ligase scaffolding protein, FBXO45, was identified as a novel, direct NOS2 interactor. Similar to the SPRY domain-containing SOCS box (SPSB) proteins, FBXO45 requires Asn27 in the (23)DINNN(27) motif of NOS2 for its interaction. However, FBXO45 is unique from the SPSBs in that it recruits a distinct E3 ligase complex containing MYCBP2 and SKP1. Collectively, these findings demonstrate the general utility of interaction proteomics for defining new aspects of NOS2 physiology.

Dihydroxyterephthalate-A Trojan Horse PET Counit for Facile Chemical Recycling.

Here, low-energy poly(ethylene terephthalate) (PET) chemical recycling in water: PET copolymers with diethyl 2,5-dihydroxyterephthalate (DHTE) undergo selective hydrolysis at DHTE sites, autocatalyzed by neighboring group participation, is demonstrated. Liberated oligomeric subchains further hydrolyze until only small molecules remain. Poly(ethylene terephthalate-stat-2,5-dihydroxyterephthalate) copolymers were synthesized via melt polycondensation and then hydrolyzed in 150-200 °C water with 0-1 wt% ZnCl2 , or alternatively in simulated sea water. Degradation progress follows pseudo-first order kinetics. With increasing DHTE loading, the rate constant increases monotonically while the thermal activation barrier decreases. The depolymerization products are ethylene glycol, terephthalic acid, 2,5-dihydroxyterephthalic acid, and bis(2-hydroxyethyl) terephthalate dimer, which could be used to regenerate virgin polymer. Composition-optimized copolymers show a decrease of nearly 50% in the Arrhenius activation energy, suggesting a 6-order reduction in depolymerization time under ambient conditions compared to that of PET homopolymer. This study provides new insight to the design of polymers for end-of-life while maintaining key properties like service temperature and mechanical properties. Moreover, this chemical recycling procedure is more environmentally friendly compared to traditional approaches since water is the only needed material, which is green, sustainable, and cheap.

S-nitrosylation in cardiovascular signaling.

Well over 2 decades have passed since the endothelium-derived relaxation factor was reported to be the gaseous molecule nitric oxide (NO). Although soluble guanylyl cyclase (which generates cyclic guanosine monophosphate, cGMP) was the first identified receptor for NO, it has become increasingly clear that NO exerts a ubiquitous influence in a cGMP-independent manner. In particular, many, if not most, effects of NO are mediated by S-nitrosylation, the covalent modification of a protein cysteine thiol by an NO group to generate an S-nitrosothiol (SNO). Moreover, within the current framework of NO biology, endothelium-derived relaxation factor activity (ie, G protein-coupled receptor-mediated, or shear-induced endothelium-derived NO bioactivity) is understood to involve a central role for SNOs, acting both as second messengers and signal effectors. Furthermore, essential roles for S-nitrosylation have been implicated in virtually all major functions of NO in the cardiovascular system. Here, we review the basic biochemistry of S-nitrosylation (and denitrosylation), discuss the role of S-nitrosylation in the vascular and cardiac functions of NO, and identify current and potential clinical applications.

A protein microarray-based analysis of S-nitrosylation.

The ubiquitous cellular influence of nitric oxide (NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of alpha-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols.

Structure-function clustering in weighted brain networks.

Functional networks, which typically describe patterns of activity taking place across the cerebral cortex, are widely studied in neuroscience. The dynamical features of these networks, and in particular their deviation from the relatively static structural network, are thought to be key to higher brain function. The interactions between such structural networks and emergent function, and the multimodal neuroimaging approaches and common analysis according to frequency band motivate a multilayer network approach. However, many such investigations rely on arbitrary threshold choices that convert dense, weighted networks to sparse, binary structures. Here, we generalise a measure of multiplex clustering to describe weighted multiplexes with arbitrarily-many layers. Moreover, we extend a recently-developed measure of structure-function clustering (that describes the disparity between anatomical connectivity and functional networks) to the weighted case. To demonstrate its utility we combine human connectome data with simulated neural activity and bifurcation analysis. Our results indicate that this new measure can extract neurologically relevant features not readily apparent in analogous single-layer analyses. In particular, we are able to deduce dynamical regimes under which multistable patterns of neural activity emerge. Importantly, these findings suggest a role for brain operation just beyond criticality to promote cognitive flexibility.