Glycine N-Thiocarboxyanhydride: A Key to Glycine-Rich Protein Mimics.

Glycine-rich proteins (GRPs) containing a high content of glycine residues (>30%) possess unique structural stability. However, the controllable synthesis of glycine-rich poly(amino acid)s (PAAs) to mimic GRPs has not been realized yet due to the poor solubility of polyglycine segments. We developed a novel method to synthesize glycine-rich PAAs via the controlled ring-opening copolymerization of glycine-N-thiocarboxyanhydrides (Gly-NTA) with sarcosine-N-carboxyanhydride and ε-Cbz-l-lysine-N-carboxyanhydride. The random copolymerization is evidenced by a kinetic study that shows that the propagation rate constant of Gly-NTA is close to those of comonomers. The copolymers exhibit predictable molecular weights between 4.5 and 24.6 kg/mol and tunable glycine incorporation, varying from 10.3 to 59.2%. Poly(Gly-r-Sar) samples with various glycine contents form nanoparticles or a hydrogel in water. Remarkably, the ß-sheet folding of poly(Gly-r-Lys) remains intact in a neutral environment where the amine groups are protonated. Overall, the strategy paves the way to engineer glycine-rich PAAs and thereby expands their applications.

Fe3+@PDOPA­b­PSar Nanoparticles for Magnetic Resonance Imaging and Cancer Chemotherapy.

Purpose: Chemotherapy treatments for cancer are always accompanied by a low concentration of drug delivered in the tumor area and severe side effects including systemic toxicity. Improving the concentration, biocompatibility, and biodegradability of regional chemotherapy drugs is a pressing challenge in the field of materials. Methods: N-Phenyloxycarbonyl-amino acids (NPCs) which exhibit significant tolerance to nucleophiles, such as water and hydroxyl-containing compounds, are promising monomers for the synthesis of polypeptides and polypeptoids. Cell line and mouse models were used to comprehensively explore how to enhance the tumor MRI signal and evaluate the therapeutic effect of Fe@POS-DOX nanoparticles. Results: In this study, poly(3,4-dihydroxy-L-phenylalanine)-b-polysarcosine (PDOPA-b-PSar, simplified as POS) was synthesized by the block copolymerization of DOPA-NPC with Sar-NPC. Fe@POS-DOX nanoparticles were prepared in order to utilize the strong chelation of catechol ligands to iron (III) cations and the hydrophobic interaction between DOX and DOPA block to deliver chemotherapeutics to tumor tissue. The Fe@POS-DOX nanoparticles exhibit high longitudinal relaxivity (r 1 = 7.06 mM-1·s-1) and act as T 1-weighted magnetic resonance (MR) imaging contrast agents. Further, the main focus was improving tumor site-specific bioavailability and achieving therapeutic effects through the biocompatibility and biodegradability of Fe@POS-DOX NPs. The Fe@POS-DOX treatment exhibited excellent antitumor effects. Conclusion: Upon intravenous injection, Fe@POS-DOX delivers DOX specifically to the tumor tissues, as revealed by MR, and leads to the inhibition of tumor growth without overt toxicity to normal tissues, thus displaying considerable potential for use in clinical applications.

Chemical Vapor Deposition of Uniform and Large-Domain Molybdenum Disulfide Crystals on Glass/Al2O3 Substrates.

Two-dimensional molybdenum disulfide (MoS2) has attracted significant attention for next-generation electronics, flexible devices, and optical applications. Chemical vapor deposition is the most promising route for the production of large-scale, high-quality MoS2 films. Recently, the chemical vapor deposition of MoS2 films on soda-lime glass has attracted great attention due to its low cost, fast growth, and large domain size. Typically, a piece of Mo foil or graphite needs to be used as a buffer layer between the glass substrates and the CVD system to prevent the glass substrates from being fragmented. In this study, a novel method was developed for synthesizing MoS2 on glass substrates. Inert Al2O3 was used as the buffer layer and high-quality, uniform, triangular monolayer MoS2 crystals with domain sizes larger than 400 µm were obtained. To demonstrate the advantages of glass/Al2O3 substrates, a direct comparison of CVD MoS2 on glass/Mo and glass/Al2O3 substrates was performed. When Mo foil was used as the buffer layer, serried small bilayer islands and bright core centers could be observed on the MoS2 domains at the center and edges of glass substrates. As a control, uniform MoS2 crystals were obtained when Al2O3 was used as the buffer layer, both at the center and the edge of glass substrates. Raman and PL spectra were further characterized to show the merit of glass/Al2O3 substrates. In addition, the thickness of MoS2 domains was confirmed by an atomic force microscope and the uniformity of MoS2 domains was verified by Raman mapping. This work provides a novel method for CVD MoS2 growth on soda-lime glass and is helpful in realizing commercial applications of MoS2.

Understanding Acid-Promoted Polymerization of the N-Substituted Glycine N-Thiocarboxyanhydride in Polar Solvents.

Polymerization of N-substituted glycine N-thiocarboxyanhydrides (NNTAs) is a promising pathway to prepare functional polypeptoids benefiting from their tolerance to nucleophilic impurities. However, controlled NNTA polymerization is hard to achieve in amide polar solvents, including N,N-dimethylacetamide (DMAc), N,N-dimethylformamide (DMF), and N-methyl pyrrolidone (NMP), the only aprotic solvents for many biomacromolecules and polypeptoids. In the present work, we successfully achieve controlled NNTA polymerization in amide polar solvents by adding acetic acid as a promoter. The promotion is applied to the polymerization of sarcosine NTA, N-ethyl glycine NTA, and N-butyl glycine NTA. DMAc, DMF, and NMP are suitable solvents to prepare polypeptoids with designable molecular weights and low dispersities (1.06-1.21). The polysarcosines with high molecular weights are prepared up to 35.2 kg/mol. A kinetic investigation quantitatively reveals that the presence of acetic acid not only accelerates the polymerization, but also suppresses H2S-catalyzed decomposition of NNTAs by decreasing the concentration of H2S dissolved in polar solvents. Benzoic acid is also able to promote the polymerization, while trifluoroacetic acid, phosphoric acid, and phenol are not appropriate promoters. The moderate acidity of acids is essential. l-Methionine, l-tryptophan, and l-phenylalanine, which are dissolved in DMF, initiate the controlled polymerization of sarcosine-NTA in the presence of acetic acid and introduce functional end groups to polysarcosines quantitatively. In DMAc, hydrophilic vancomycin is grafted by poly(N-butyl glycine). The amphiphilic product dissolves in dichloromethane and stabilizes water-in-oil emulsion.

Ring-Opening Polymerization of CO2-Based Disubstituted δ-Valerolactone toward Sustainable Functional Polyesters.

3-Ethylidene-6-vinyltetrahydro-2H-pyran-2-one (EVL) is a disubstituent δ-lactone derived from CO2 and 1,3-butadiene. In this contribution, we report the ring-opening polymerization (ROP) of EVL with ß-butyrolactone (BBL) as the comonomer catalyzed by scandium triflate [Sc(OTf)3]. The obtained polyester bearing active unsaturated bonds has the weight-average molecular weight (Mw) of 4.1 kg/mol, in which the EVL content is 38 mol % in accordance with the initial ratio of 40 mol %. The copolymers are characterized in detail and the cationic ROP mechanism has been confirmed by kinetic study, chain end analysis and density functional theory (DFT) calculation. The modification of the unsaturated bonds in EVL repeating units via the thio-ene click reaction with mercapto-ended polysarcosine polysarcosine yields the amphiphilic grafting polymers. It is a CO2 fixation approach toward the functional poly(EVL-r-BBL) that is promising as a degradable polyester precursor for adhesive or surface-coating materials.

Dynamic Porous Pattern through Controlling Noncovalent Interactions in Polyelectrolyte Film for Sequential and Regional Encapsulation.

Inspired by nature, many functional surfaces have been developed with special structures in biology, chemistry, and materials. Many research studies have been focused on the preparation of surfaces with static structure. Achieving dynamical manipulation of surface structure is desired but still a great challenge. Herein, a polyelectrolyte film capable of regional and reversible changes in the microporous structure is presented. Our proposal is based on the combination of azobenzene (Azo) π-π stacking and electrostatic interaction, which could be affected respectively by ultraviolet (UV) irradiation and water plasticization, to tune the mobility of polyelectrolyte chains. The porous patterns can be obtained after regional ultraviolet irradiation and acid treatment. Owing to the reversibility of Azo π-π stacking and electrostatic interaction, the patterns can be repeatedly created and erased in the polyelectrolyte film made by layer-by-layer (LbL) self-assembly of poly(ethyleneimine)-azo and poly(acrylic acid). Furthermore, through two rounds of porous pattern formation and erasure, different functional species can be loaded separately and confined regionally within the film, showing potential applications in the functional surface. This work highlights the coordination of two noncovalent interactions in thin films for regional and reversible controlling its structure, opening a window for more in-depth development of functional surfaces.

Discoidin domain receptor 1 deficiency in vascular smooth muscle cells leads to mislocalisation of N-cadherin contacts.

N-cadherin mediates cell-cell contacts in vascular smooth muscle cells (VSMCs), and regulates VSMC behaviours including migration and proliferation. Discoidin domain receptor 1 (DDR1) is a collagen binding receptor also implicated in these processes. Previous studies have shown that both N-cadherin and DDR1 are upregulated after vascular injury, but it is not known whether there is a relationship between the two molecules. In the current study we found that N-cadherin was mislocalised from cell-cell junctions in the absence of DDR1. This occurred in spite of the fact that there was no significant difference in total cell lysate levels of N-cadherin between DDR1+/+ and DDR1-/- VSMCs. Analysis of lipid raft fractions revealed decreased N-cadherin and associated junctional complex catenins in DDR1-/- compared to DDR1+/+ VSMCs. Treatment with cholesterol oxidase or methyl-ß-cyclodextrin to disrupt lipid rafts removed N-cadherin and DDR1 from the raft fractions. Reciprocal co-immunoprecipitations suggested the association of DDR1 and N-cadherin. Importantly, transfection of DDR1-/- cells with full-length DDR1b rescued the formation of N-cadherin junctions. Together, these data reveal that N-cadherin cell-cell contacts in VSMCs are regulated through interactions with DDR1 and both molecules are located in lipid rafts.

The progression of calcific aortic valve disease through injury, cell dysfunction, and disruptive biologic and physical force feedback loops.

Calcific aortic valve disease (CAVD) is the most common form of heart valve disease in Western society and results in the second most common cardiovascular surgery performed. Despite its prevalence, high morbidity, and high mortality, the pathogenesis of CAVD still eludes our understanding. This review article brings together experimental in vivo and in vitro as well as human in vivo research in cell and molecular pathobiology to construct an overarching hypothesis regarding the development and progression of CAVD. We focus on injury, cell dysfunction, and disruptive biologic and physical forces, and how they function in positive feedback loops that result in the eventual calcification of the valve. We propose that injury, inflammation, matrix remodeling, and physical forces are all processes that influence each other and alter the normal physiologic functions of a key player in the pathogenesis of CAVD: the valve interstitial cell. We propose that the different phenotypes of the valve interstitial cell play essential roles in the pathogenesis of CAVD. We describe important physiologic processes which become dysfunctional including proliferation, migration, secretion of growth factors, chemokines and cytokines, and matrix remodeling. We also describe the emergence of chondrogenesis and osteogenesis in the fibrotic valve that lead to the severe clinical conditions of CAVD. CAVD appears to have a complex pathogenesis which fortunately can be studied in vitro and in vivo to identify ways to detect, treat, and prevent CAVD.

Wnt3a/ß-catenin increases proliferation in heart valve interstitial cells.

BACKGROUND: Valve interstitial cells (VICs), the most prevalent cells in the heart valve, mediate normal valve function and repair in valve injury and disease. The Wnt3a/ß-catenin pathway, important for proliferation and endothelial-to-mesenchymal transition in endocardial cushion formation in valve development, is up-regulated in adult valves with calcific aortic stenosis. Therefore, we tested the hypothesis that Wnt3a/ß-catenin signaling regulates proliferation in adult VICs. METHODS: Porcine VICs were treated with 150 ng/ml of exogenous Wnt3a. To measure proliferation, cells were counted on day 4 posttreatment and stained for bromodeoxyuridine (BrdU) at 24 h posttreatment. ß-Catenin small interfering RNA (siRNA) was used to knock down ß-catenin expression. Apoptosis was measured with terminal deoxynucleotidyl transferase dUTP nick end labeling assay. To assess changes in ß-catenin, cells were stained for ß-catenin at days 1, 3, 6, and 9 posttreatment. Western blot for ß-catenin was performed on whole cell, cytoplasmic, and nuclear extracts at day 4 posttreatment. To measure ß-catenin-mediated transcription, TOPFLASH/FOPFLASH reporter assay was performed at 24 h posttreatment. RESULTS: Wnt3a produced a significant increase in cell number at day 4 posttreatment and in the percentage of BrdU-positive nuclei at 24 h posttreatment. The increase in proliferation was abolished by ß-catenin siRNA. Apoptosis was minimal in all conditions. Wnt3a produced progressively greater ß-catenin staining as treatment length increased from 1 to 9 days. Wnt3a produced a significant increase in ß-catenin protein in both whole cell and nuclear lysates after 4 days of treatment. Wnt3a significantly increased TOPFLASH/FOPFLASH reporter activity after 24 h of treatment. CONCLUSION: Wnt3a/ß-catenin signaling pathway is an important regulator of proliferation in adult VICs.

Cell density regulates in vitro activation of heart valve interstitial cells.

BACKGROUND: Valve interstitial cells, the most prominent cell type in the heart valve, are activated and express α-smooth muscle actin in valve repair and in diseased valves. We hypothesize that cell density, time in culture, and the establishment of cell-cell contacts may be involved in regulating valve interstitial cell activation in vitro. METHODS: To study cell density, valve interstitial cells were plated at passages 3 to 5, at a density of 17,000 cells/22 × 22 mm(2) coverslip, and grown for 1, 2, 4, 7, and 10 days. Valve interstitial cells were stained for α-smooth muscle actin and viewed under confocal microscopy to characterize the intensity of staining. To study time in culture, valve interstitial cells were plated at a 10-fold higher density to achieve similar growth densities over a shorter time period compared with valve interstitial cells plated at low density. α-Smooth muscle actin staining was compared at the same time points between those plated at high and low densities. To confirm valve interstitial cell activation as indicated by α-smooth muscle actin staining, valve interstitial cells were stained for cofilin at days 2, 5, 8, and 14 days postplating. To study the association of transforming growth factor ß with valve interstitial cell activation with respect to cell density, valve interstitial cells were stained for α-smooth muscle actin and transforming growth factor ß at 2, 4, 6, and 8 days postplating. To study the activation of the transforming growth factor ß signaling pathway, valve interstitial cells were stained for pSmad2/3 at days 2, 4, 6, 8, 10, and 12 days postplating. To study cell contacts and activation, subconfluent and confluent cultures of valve interstitial cells were stained for ß-catenin, N-cadherin, and α-smooth muscle actin. Also, whole-cell lysates of subconfluent and confluent valve interstitial cell cultures were probed by Western blot analysis for phospho-ß-catenin at Ser33/37/Thr41, which is the form of ß-catenin targeted for proteosomal degradation. RESULTS: The percentage valve interstitial cells with high-intensity α-smooth muscle actin staining decreases significantly between days 1 and 4, and at confluency, most cells show absent or low-intensity staining, regardless of time in culture. Similar results are obtained with cofilin staining. Transforming growth factor ß and nuclear pSmad2/3 staining in valve interstitial cells decreases concurrently with valve interstitial cell activation as cell density increases. Examining ß-catenin and N-cadherin staining, single valve interstitial cells show no cell-cell contact with strong cytoplasmic staining, with some showing nuclear staining of ß-catenin, while confluent monolayers show strong staining of fully established cell-cell contacts, weak cytoplasmic staining, and absent nuclear staining. The presence of cell-cell contacts is associated with a decreased α-smooth muscle actin. The level of phospho-ß-catenin at Ser33/37/Thr41 is lower in confluent cultures compared with low-density subconfluent valve interstitial cell cultures. CONCLUSION: Cell-cell contacts may inhibit valve interstitial cell activation, while absence of cell-cell contacts may contribute to activation.

The response to valve injury. A paradigm to understand the pathogenesis of heart valve disease.

Human heart valve diseases have become an important topic in cardiovascular pathology and medicine. These diseases have different etiologies and manifestations. However, the most common ones including calcific aortic stenosis have histopathological features that are best characterized as a "response to tissue injury" similar to ones seen in numerous tissues and organs. The valve interstitial cell is the prevalent cell type in the valve and is likely the master cell which ultimately regulates cell and molecular repair processes within the valve that involve autocrine and paracrine processes as well as interactions with the matrix components of the valve. This presentation explores the concept of "response to tissue injury" in understanding the pathogenesis of calcific aortic stenosis.

Common pathogenic features of atherosclerosis and calcific aortic stenosis: role of transforming growth factor-beta.

Calcific aortic stenosis and atherosclerosis have been investigated separately in experimental in vitro and in vivo studies and in clinical studies. The similarities identified in both diseases suggest that similar pathogenic pathways are involved in both conditions. Most current therapeutic studies are focused on statins. The evidence suggests that statin effects on valves may, in large part, be independent of the lipid lowering effects of the drug. There are several molecules that play significant regulatory roles on the development and progression of valve sclerosis and calcification and on growth and complications of atherosclerotic plaques. The purpose of this review is to discuss the pathogenic features of the two conditions, highlight the important similarities, and then review the data that suggest that transforming growth factor-beta may play a key regulatory role in both diseases and that this is worthy of study as a potential therapeutic target for both conditions.

Quantitation of CD36 (platelet glycoprotein IV) expression on platelets and monocytes by flow cytometry: application to the study of Plasmodium falciparum malaria.

BACKGROUND: The expression of CD36 (platelet glycoprotein IV) is variable among different individuals and cannot be determined by gene analysis. Previous studies suggest that CD36 expression plays a central role in the pathophysiology of Plasmodium falciparum malaria, a disease of global significance. METHODS: We developed a flow cytometric method to quantitatively measure CD36 on monocytes and platelets from whole blood using antibodies to CD36, CD14, and CD61 directly conjugated to different fluorochromes. Commercially available fluorescent beads were used to quantify CD36 expression. RESULTS: The assay was successfully run at three different centers. African-Americans (n = 57), nonAfrican-Americans (n = 33), individuals with and without hemoglobin S (n = 15 and n = 12), and children with P falciparum malaria (n = 97) were tested. Platelet-monocyte aggregates, present to varying degrees in different anticoagulants, were eliminated from final analysis. The median fluorescence intensity (MFI) of CD36 among different subjects followed a log-normal distribution. Among African-Americans, 5% were CD36-deficient (logMFI < 1.5; MFI < 32). Expression of platelet CD36 paralleled monocyte CD36. CONCLUSIONS: Flow cytometry can be used to quantify the expression of CD36 of platelets and monocytes in EDTA whole blood. The assay will allow investigation of the relationship between CD36 and clinical outcome in malaria and other disease states.