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Surface energy is a fundamental property of materials and is particularly important in describing nanomaterials where atoms or molecules at the surface constitute a large fraction of the material. Traditionally, surface energy is considered to be a positive quantity, where atoms or molecules at the surface are less thermodynamically stable than their counterparts in the interior of the material because they have fewer bonds or interactions at the surface. Using calorimetric methods, we show that the surface energy is negative in some prototypical colloidal semiconductor nanocrystals, or quantum dots with organic ligand coatings. This implies that the surface atoms are more thermodynamically stable than those on the interior due to the strong bonds between these atoms and surfactant molecules, or ligands, that coat their surface. In addition, we extend this work to core/shell indium phosphide/zinc sulfide nanocrystals and show that the interfacial energy between these materials is highly thermodynamically favorable in spite of their large lattice mismatch. This work challenges many of the assumptions that have guided thinking about colloidal nanomaterial thermodynamics, investigates the fundamental stability of many technologically relevant colloidal nanomaterials, and paves the way for future experimental and theoretical work on nanocrystal thermodynamics., Competing Interests: Competing interests statement:The authors declare no competing interest.

Graphene liquid cell transmission electron microscopy is a powerful technique to visualize nanoscale dynamics and transformations at atomic resolution. However, the solution in liquid cells is known to be affected by radiolysis, and the stochastic formation of graphene liquid cells raises questions about the solution chemistry in individual pockets. In this study, electron energy loss spectroscopy (EELS) was used to evaluate a model encapsulated solution, aqueous CeCl 3 . First, the ratio between the O K-edge and Ce M-edge was used to approximate the concentration of cerium salt in the graphene liquid cell. It was determined that the ratio between oxygen and cerium was orders of magnitude lower than what is expected for a dilute solution, indicating that the encapsulated solution is highly concentrated. To probe how this affects the chemistry within graphene liquid cells, the oxidation of Ce 3+ was measured using time-resolved parallel EELS. It was determined that Ce 3+ oxidizes faster under high electron fluxes, but reaches the same steady-state Ce 4+ concentration regardless of flux. The time-resolved concentration profiles enabled direct comparison to radiolysis models, which indicate rate constants and g -values of certain molecular species are substantially different in the highly concentrated environment. Finally, electron flux-dependent gold nanocrystal etching trajectories showed that gold nanocrystals etch faster at higher electron fluxes, correlating well with the Ce 3+ oxidation kinetics. Understanding the effects of the highly concentrated solution in graphene liquid cells will provide new insight on previous studies and may open up opportunities to systematically study systems in highly concentrated solutions at high resolution.

The size and shape of semiconductor nanocrystals govern their optical and electronic properties. Liquid cell transmission electron microscopy (LCTEM) is an emerging tool that can directly visualize nanoscale chemical transformations and therefore inform the precise synthesis of nanostructures with desired functions. However, it remains difficult to controllably investigate the reactions of semiconductor nanocrystals with LCTEM, because of the highly reactive environment formed by radiolysis of liquid. Here, we harness the radiolysis processes and report the single-particle etching trajectories of prototypical semiconductor nanomaterials with well-defined crystalline facets. Lead selenide nanocubes represent an isotropic structure that retains the cubic shape during etching via a layer-by-layer mechanism. The anisotropic arrow-shaped cadmium selenide nanorods have polar facets terminated by either cadmium or selenium atoms, and the transformation trajectory is driven by etching the selenium-terminated facets. LCTEM trajectories reveal how nanoscale shape transformations of semiconductors are governed by the reactivity of specific facets in liquid environments.

Liquid cell electron microscopy enables the study of nanoscale transformations in solvents with high spatial and temporal resolution, but for the technique to achieve its potential requires a new level of control over the reactivity caused by radical generation under electron beam irradiation. An understanding of how to control electron-solvent interactions is needed to further advance the study of structural dynamics for complex materials at the nanoscale. We developed an approach that scavenges radicals with redox species that form well-defined redox couples and control the electrochemical potential in situ . This approach enables the observation of electrochemical structural dynamics at near-atomic resolution with precise control of the liquid environment. Analysis of nanocrystal etching trajectories indicates that this approach can be generalized to several chemical systems. The ability to simultaneously observe heterogeneous reactions at near-atomic resolution and precisely control the electrochemical potential enables the fundamental study of complex nanoscale dynamics with unprecedented detail.

Graphene liquid cell transmission electron microscopy (TEM) has enabled the observation of a variety of nanoscale transformations. Yet understanding the chemistry of the liquid cell solution and its impact on the observed transformations remains an important step toward translating insights from liquid cell TEM to benchtop chemistry. Gold nanocrystal etching can be used as a model system to probe the reactivity of the solution. FeCl 3 has been widely used to promote gold oxidation in bulk and liquid cell TEM studies, but the roles of the halide and iron species have not been fully elucidated. In this work, we observed the etching trajectories of gold nanocrystals in different iron halide solutions. We observed an increase in gold nanocrystal etch rate going from Cl - - to Br - - to I - -containing solutions. This is consistent with a mechanism in which the dominant role of halides is as complexation agents for oxidized gold species. Additionally, the mechanism through which FeCl 3 induces etching in liquid cell TEM remains unclear. Ground-state bleaching of the Fe(III) absorption band observed through pulse radiolysis indicates that iron may react with Cl 2 · - radicals to form an oxidized transient species under irradiation. Complete active space self-consistent field (CASSCF) calculations indicate that the FeCl 3 complex is oxidized to an Fe species with an OH radical ligand. Together our data indicate that an oxidized Fe species may be the active oxidant, while halides modulate the etch rate by tuning the reduction potential of gold nanocrystals.

Powder X-ray diffraction is one of the key techniques used to characterize the inorganic structure of colloidal nanocrystals. The comparatively low scattering factor of nuclei of the organic capping ligands and their propensity to be disordered has led investigators to typically consider them effectively invisible to this technique. In this report, we demonstrate that a commonly observed powder X-ray diffraction peak around [Formula: see text] observed in many small, colloidal quantum dots can be assigned to well-ordered aliphatic ligands bound to and capping the nanocrystals. This conclusion differs from a variety of explanations ascribed by previous sources, the majority of which propose an excess of organic material. Additionally, we demonstrate that the observed ligand peak is a sensitive probe of ligand shell ordering. Changes as a function of ligand length, geometry, and temperature can all be readily observed by X-ray diffraction and manipulated to achieve desired outcomes for the final colloidal system.

Surface ligands impact the properties and chemistry of nanocrystals, but observing ligand binding locations and their effect on nanocrystal shape transformations is challenging. Using graphene liquid cell electron microscopy and the controllable, oxidative etching of gold nanocrystals, the effect of different ligands on nanocrystal etching can be tracked with nanometer spatial resolution. The chemical environment of liquids irradiated with high-energy electrons is complex and potentially harsh, yet it is possible to observe clear evidence for differential binding properties of specific ligands to the nanorods' surface. Exchanging CTAB ligands for PEG-alkanethiol ligands causes the nanorods to etch at a different, constant rate while still maintaining their aspect ratio. Adding cysteine ligands that bind preferentially to nanorod tips induces etching predominantly on the sides of the rods. This etching at the sides leads to Rayleigh instabilities and eventually breaks apart the nanorod into two separate nanoparticles. The shape transformation is controlled by the interplay between atom removal and diffusion of surface atoms and ligands. These in situ observations are confirmed with ex situ colloidal etching reactions of gold nanorods in solution. The ability to monitor the effect of ligands on nanocrystal shape transformations will enable future in situ studies of nanocrystals surfaces and ligand binding positions.

Superatom electron shell and/or geometric shell filling underlies the thermodynamic stability of coinage and alkali metal clusters in both theoretical and experimental results. Factors beyond simple shell filling contribute substantially to the lifetime of ligated clusters in solution. Such factors include the nature of the solvent, the atmosphere and the steric size of the ligand shell. Here we systematically lay out a 'practical' stability model for ligated metal clusters, which includes both shell-closing aspects and colloidal stability aspects. Cluster decomposition may follow either fusion or fission pathways. Solvent polarity can be determinative of the decomposition pathway.

We investigated the effects of chronic mineralocorticoid receptor blockade with eplerenone on the development and progression of hypertension and end organ damage in Dahl salt-sensitive rats. Eplerenone significantly attenuated the progressive rise in systolic blood pressure (SBP) (204 ± 3 vs. 179±3 mmHg, p < 0.05), reduced proteinuria (605.5 ± 29.6 vs. 479.7 ± 26.1 mg/24h, p < 0.05), improved injury scores of glomeruli, tubules, renal interstitium, and vasculature in Dahl salt-sensitive rats fed a high-salt diet. These results demonstrate that mineralocorticoid receptor antagonism provides target organ protection and attenuates the development of elevated blood pressure (BP) in a model of salt-sensitive hypertension.

Rationale: Arginine methylation by protein N-arginine methyltransferases (PRMTs) is an important posttranslational modification in the regulation of protein signaling. PRMT2 contains a highly conserved catalytic Ado-Met binding domain, but the enzymatic function of PRMT2 with respect to methylation is unknown. The JAK-STAT pathway is proposed to be regulated through direct arginine methylation of STAT transcription factors, and STAT3 signaling is known to be required for leptin regulation of energy balance., Objective: To identify the potential role of STAT3 arginine methylation by PRMT2 in the regulation of leptin signaling and energy homeostasis., Methods and Results: We identified that PRMT2(-/-) mice are hypophagic, lean, and have significantly reduced serum leptin levels. This lean phenotype is accompanied by resistance to food-dependent obesity and an increased sensitivity to exogenous leptin administration. PRMT2 colocalizes with STAT3 in hypothalamic nuclei, where it binds and methylates STAT3 through its Ado-Met binding domain. In vitro studies further clarified that the Ado-Met binding domain of PRMT2 induces STAT3 methylation at the Arg31 residue. Absence of PRMT2 results in decreased methylation and prolonged tyrosine phosphorylation of hypothalamic STAT3, which was associated with increased expression of hypothalamic proopiomelanocortin following leptin stimulation., Conclusions: These data elucidate a molecular pathway that directly links arginine methylation of STAT3 by PRMT2 to the regulation of leptin signaling, suggesting a potential role for PRMT2 antagonism in the treatment of obesity and obesity-related syndromes.

Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS-/- mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS-/- VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.

The cyclin-dependent kinase inhibitor p27(Kip1) arrests cell cycle progression through G1/S phases and is regulated by phosphorylation of serine/threonine residues. Recently, we identified the serine/threonine kinase, KIS, which phosphorylates p27(Kip1) on serine 10 leading to nuclear export of p27(Kip1) and protein degradation. However, the molecular mechanisms of transcriptional activation of the human KIS gene and its biological activity are not known. We mapped the transcription initiation site approximately 116 bp 5' to the translation start site, and sequences extending to -141 were sufficient for maximal promoter activity. Mutation in either of two Ets-binding sites in this region resulted in an approximately 75-80% decrease in promoter activity. These sites form at least 3 specific complexes, which contained GA-binding protein (GABP). Knocking down GABPalpha by siRNA in vascular smooth muscle cells (VSMCs) diminished KIS gene expression and reduced cell migration. Correspondingly, in serum stimulated GABPalpha-deficient mouse embryonic fibroblasts (MEFs), KIS gene expression was also significantly reduced, which was associated with an increase in p27(Kip1) protein levels and a decreased percentage of cells in S-phase. Consistent with these findings, following vascular injury in vivo, GABPalpha-heterozygous mice demonstrated reduced KIS gene expression within arterial lesions and these lesions were significantly smaller compared to GABP+/+ mice. In summary, serum-responsive GABP binding to Ets-binding sites activates the KIS promoter, leading to KIS gene expression, cell migration, and cell cycle progression.

The retinoblastoma gene product (RB) is an important regulator of E2F activity. RB recruits a number of proteins, including HDACs, SWI/SNF complex, lysine methyl transferase (SUV39H1) and DNA methyltransferase (DNMT1), all of which negatively regulate E2F activity with RB. Here, we show that RB interacts with PRMT2, a member of the protein arginine methyltransferase family, to regulate E2F activity. PRMT2 directly bound and interacted with RB through its AdoMet binding domain, in contrast to other PRMT proteins, including PRMT1, PRMT3 and PRMT4. In reporter assays, PRMT2 repressed E2F1 transcriptional activity in an RB-dependent manner. PRMT2 formed a ternary complex with E2F1 in the presence of RB. To further explore the role of endogenous PRMT2 in the regulation of E2F activity, the PRMT2 gene was ablated in mice by gene targeting. Compared with PRMT2(+/+) mouse embryonic fibroblasts (MEFs), PRMT2(-/-) MEFs demonstrated increased E2F activity and early S phase entry following release of serum starvation. Vascular injury to PRMT2(-/-) arteries results in a hyperplastic response, consistent with increased G1-S phase progression. Taken together, these findings demonstrate a novel mechanism for the regulation of E2F activity by a member of the protein arginine methyltransferase family.

The cyclin-dependent kinase inhibitors are key regulators of cell cycle progression. Although implicated in carcinogenesis, they inhibit the proliferation of a variety of normal cell types, and their role in diverse human diseases is not fully understood. Here, we report that p27(Kip1) plays a major role in cardiovascular disease through its effects on the proliferation of bone marrow-derived (BM-derived) immune cells that migrate into vascular lesions. Lesion formation after mechanical arterial injury was markedly increased in mice with homozygous deletion of p27(Kip1), characterized by prominent vascular infiltration by immune and inflammatory cells. Vascular occlusion was substantially increased when BM-derived cells from p27(-/-) mice repopulated vascular lesions induced by mechanical injury in p27(+/+) recipients, in contrast to p27(+/+) BM donors. To determine the contribution of immune cells to vascular injury, transplantation was performed with BM derived from RAG(-/-) and RAG(+/+) mice. RAG(+/+) BM markedly exacerbated vascular proliferative lesions compared with what was found in RAG(-/-) donors. Taken together, these findings suggest that vascular repair and regeneration is regulated by the proliferation of BM-derived hematopoietic and nonhematopoietic cells through a p27(Kip1)-dependent mechanism and that immune cells largely mediate these effects.

Vascular smooth muscle cell (VSMC) proliferation after arterial injury results in neointima formation and plays an important role in the pathogenesis of restenosis after angioplasty, in-stent restenosis, vascular bypass graft occlusion, and allograft vasculopathy. Major progress has been made recently in elucidating the cellular and molecular mechanisms underlying neointima formation. However, no known curative treatment currently exists. In cases in which pharmacologic and surgical interventions have had limited success, gene therapy remains a potential strategy for the treatment of such vascular proliferative diseases. To date, recombinant adenoviral vectors continue to be the most efficient methods of gene transfer into the arterial wall. However, concerns over the safety of using viral vectors in a clinical situation have inspired the considerable progress that has been made in improving both viral and nonviral modes of gene transfer. This review discusses some of the recent insights and outstanding progress in vascular gene therapeutic approaches to inhibit neointima both from a biologic and therapeutic perspective.

The cyclin-dependent kinase inhibitor, p27(Kip1), which regulates cell cycle progression, is controlled by its subcellular localization and subsequent degradation. p27(Kip1) is phosphorylated on serine 10 (S10) and threonine 187 (T187). Although the role of T187 and its phosphorylation by Cdks is well-known, the kinase that phosphorylates S10 and its effect on cell proliferation has not been defined. Here, we identify the kinase responsible for S10 phosphorylation as human kinase interacting stathmin (hKIS) and show that it regulates cell cycle progression. hKIS is a nuclear protein that binds the C-terminal domain of p27(Kip1) and phosphorylates it on S10 in vitro and in vivo, promoting its nuclear export to the cytoplasm. hKIS is activated by mitogens during G(0)/G(1), and expression of hKIS overcomes growth arrest induced by p27(Kip1). Depletion of KIS using small interfering RNA (siRNA) inhibits S10 phosphorylation and enhances growth arrest. p27(-/-) cells treated with KIS siRNA grow and progress to S/G(2 )similar to control treated cells, implicating p27(Kip1) as the critical target for KIS. Through phosphorylation of p27(Kip1) on S10, hKIS regulates cell cycle progression in response to mitogens.

Metalloproteinase secretion by macrophages is believed to play a key role in the matrix degradation that underlies atherosclerotic plaque instability and aneurysm formation. We studied the hypothesis that nuclear factor-kappaB (NF-kappaB), a transcription factor, is necessary for metalloproteinase secretion and, hence, is a target for pharmacological intervention. Adenovirus-mediated gene transfer of the inhibitory NF-kappaB subunit, I-kappa Balpha, was achieved into human monocyte-derived macrophages in vitro and into foam cells produced in vivo in cholesterol-fed rabbits. Human macrophages and rabbit foam cells secreted matrix-degrading metalloproteinase (MMP)-9 without further stimulation, and this was not inhibited by I-kappaBalpha (11+/-16% and 8+/-10%, respectively; P> 0.05). MMP-1 secretion from human macrophages increased in response to recombinant human CD40 ligand and was inhibited 92+/-5% by I-kappaBalpha (n=3, P<0.05). Rabbit foam cells secreted MMP-1 and -3 without further stimulation, and this was inhibited 83+/-12% and 69+/-11%, respectively, by I-kappaBalpha (n=6 or 7, P<0.001). I-kappaBalpha did not significantly affect the expression or activity of tissue inhibitor of metalloproteinases-1 or -2. Overexpression of I-kappaBalpha inhibited collagenolytic and beta-caseinolytic activity by 42+/-2% and 41+/-7%, respectively (n=3, P<0.05). Secretion of MMP-1 and MMP-3 from macrophages stimulated in vitro or in vivo depends on the activation of NF-kappaB. Because the inhibition of NF-kappaB reduces proteolytic activity, it appears to be an attractive pharmacological target in unstable atheromas.

Atherosclerosis underlies late occlusion of human saphenous vein (HSV) coronary artery bypass grafts. Monocyte infiltration is implicated, but its mechanisms are unclear given that HSV normally expresses the ICAM-1 but not the VCAM-1 adhesion molecule. To define the mechanisms underlying monocyte adhesion, samples of HSV taken from coronary artery bypass graft patients were co-cultured with human monocytes and adherent monocytes were quantified by immunocytochemistry for CD68 on transverse sections. Pre-treatment of veins with anti-ICAM-1 antibodies reduced monocyte adhesion (monocytes/mm of section) from 7.2 +/- 1.5 to 3.1 +/- 0.7 (p < 0.05, n = 6), but the effect of anti-VCAM-1 was not significant (4.1 +/- 0.7). Paradoxically, pre-treatment of monocytes with either anti-beta(2)-integrins, the counter-receptor of ICAM-1, or anti-alpha(4) integrins, the counter-receptor of VCAM-1, significantly reduced adhesion (1.8 +/- 0.6 and 2.4 +/- 0.7, respectively, p < 0.05). These results were clarified by immunocytochemistry, which confirmed that VCAM-1 expression was absent in harvested vein but was induced in the endothelium during co-culture. Consistent with this, when anti-ICAM-1 or anti-VCAM-1 was present throughout co-culture, either of them reduced adhesion (from 4.2 +/- 0.9 to 2.3 +/- 0.5 and 2.2 +/- 0.4, respectively, p < 0.02, n = 8) and there was no further effect of adding both (2.0 +/- 0.5). These results demonstrate that both ICAM-1/beta(2) and VCAM-1/alpha(4) integrin interactions mediate monocyte adhesion to HSV, possibly as part of a common pathway. These experiments imply that either integrin might be targeted to reduce monocyte infiltration into HSV grafts., (Copyright 2002 S. Karger AG, Basel)

Atherosclerosis causes occlusions in as many as 50% of human saphenous vein coronary artery bypass grafts. Monocyte infiltration is an early step in saphenous vein-graft atherosclerosis, however, comparatively little is known of its underlying mechanisms. As a first approach, we sought to define the occurrence, location and regulation of leukocyte adhesion molecules in human saphenous vein before and after surgical preparation for grafting, during neointima formation in culture and on stimulation with inflammatory cytokines. We compared the distribution of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and platelet endothelial cell adhesion molecule (PECAM-1 or CD-31) in endothelial cells and smooth muscle cells (SMCs), using immunocytochemistry. ICAM-1 was expressed on endothelial cells before culture and on both endothelial cells and medial or neointimal SMCs after culturing vein for 14 days in 30% foetal bovine serum or after culturing for 24 h with TNF-alpha. Relative tissue levels of ICAM-1 measured by Western blotting were significantly elevated by culturing freshly-isolated (0.02+/-0.01 to 0.18+/-0.03) and surgically-prepared (0.02+/-0.01 to 0.14+/-0.03; n=6) veins or following TNF-alpha treatment of surgically-prepared veins (0.04+/-0.01 to 0.32+/-0.11, n=7). VCAM-1 was undetectable before or after culturing but was strongly upregulated on endothelial cells by incubation with the cytokines TNF-alpha, IL-1alpha or interferon-gamma. PECAM-1 was expressed constitutively on endothelial cells. We conclude that human saphenous vein expresses several adhesion molecules capable of mediating monocyte migration. The increased expression of ICAM-1 in SMC after culturing or cytokine treatment and of VCAM-1 in endothelial cells suggests that interactions with beta1 and beta2 integrins are important pathways for stimulated monocyte ingress into human saphenous vein grafts.