The preparation and identification of an unusually stable viologen radical has been demonstrated. The intramolecular interactions of bis-viologen species such as 1,1 ''-[1,2-phenylenebis(methylene)]bis[1'-methyl-4,4'-bipyridinium] (1) and 1,1 ''-(1,3-propanediyl)bis[1'-methyl-4,4'-bipyridinium] (2) upon reduction and complexation by cucurbit[8]uril (CB[8]) result in the formation of a unique monoradical species. Cyclic voltammetric responses for 1 and 2 in the presence of CB[8] showed a splitting of the degenerate 2 e(-) redox wave (corresponding to the 4+/2+ transition of the bis-viologen) into two separate 1 e- redox waves indicating the stabilization of the 3+ form of the bis-viologens. Visible light absorbance showed that this species had a distinct electronic structure, imparting a blue color unlike the 2+ state of the bis-viologen or the 1+ state of partially reduced methylviologen. The persistence of the blue color over time, even in the presence of dissolved O-2, illustrated a significant stabilization of these monoradical species in comparison to any other partially reduced viologen radical. Molecular orbital calculations indicated that the origin of this stability arises from the SOMO of the monoradical that features the electron density as buried between the two cofacial viologen units, hindering electronic coupling with oxidants such as O-2 and thereby providing enhanced stability for energy storage and electrochromic applications. (c) 2019 The Electrochemical Society.

Background This study aimed to quantitatively evaluate the heavy metal content of several types of mosquito nets used in hernia repair surgery and to determine whether autoclave sterilization weakens the mosquito net material. Methods We extracted the metallic contents of a commercial polypropylene surgical mesh as well as polyester, low density polypropylene, and polyethylene mosquito nets via a wet digestion procedure. The extractions were analysed by microwave plasma atomic emis­sion spectroscopy. The breaking strength of the mosquito nets was measured using a tensile tester. Results The polypropylene mosquito net metal content was comparable to or less than the metal contents found in commercial surgical meshes and textile fibers.1 The polyester and polyethylene mosquito net metal contents were higher yet below toxic thresholds. The structural integrity of the nets was not compromised by autoclave sterilization. Conclusions The metal content of mosquito nets is below toxic thresholds and is comparable to a commercially available surgical mesh. The variability in metal contents found among the mosquito nets is likely due to the manufacturing process. However, there is no evidence that their metal contents are grounds for disqualification as an alternative to commercial surgical mesh.

Multivalent ligand-receptor interaction provides the fundamental basis for the hypothetical notion that high binding avidity relates to the strong force of adhesion. Despite its increasing importance in the design of targeted nanoconjugates, an understanding of the physical forces underlying the multivalent interaction remains a subject of urgent investigation. In this study, we designed three vancomycin (Van)-conjugated dendrimers G5(Van) n ( n = mean valency = 0, 1, 4) for bacterial targeting with generation 5 (G5) poly(amidoamine) dendrimer as a multivalent scaffold and evaluated both their binding avidity and physical force of adhesion to a bacterial model surface by employing surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. The SPR experiment for these conjugates was performed in a biosensor chip surface immobilized with a bacterial cell-wall peptide Lys-d-Ala-d-Ala. Of these, G5(Van)4 bound most tightly with a KD of 0.34 nM, which represents an increase in avidity by 2 or 3 orders of magnitude relative to a monovalent conjugate G5(Van)1 or free vancomycin, respectively. By single-molecule force spectroscopy, we measured the adhesion force between G5(Van) n and the same cell-wall peptide immobilized on the surface. The distribution of adhesion forces increased in proportion to vancomycin valency with the mean force of 134 pN at n = 4 greater than 96 pN at n = 1 at a loading rate of 5200 pN/s. In summary, our results are strongly supportive of the positive correlation between the avidity and adhesion force in the multivalent interaction of vancomycin nanoconjugates.

Riboflavin receptors (RFRs) are overexpressed in several malignant cells, and have been characterized as an emerging tumor surface biomarker. In this article, we discuss the design principles of a RFR-targeted nanoparticle system and illustrate its applications with studies performed in our laboratories. This system is based on a poly(amidoamine) (PAMAM) dendritic polymer which is modified on the surface by conjugation with riboflavin (RF) as the targeting ligand. First, we discuss the application of this system for targeted drug delivery by its conjugation with methotrexate as an antitumor payload. In cell-based experiments performed in vitro, this drug conjugate displayed RF-dependent, potent inhibition of cell growth in RFR(+) KB carcinoma cells. Second, the use of the RF-conjugated dendrimer for gene delivery applications through the formation of polyplexes with plasmid DNA is described. The ability of this targeted system to significantly enhance gene transfection in epithelial cells points to its potential as a promising new class of nonviral vectors. Third, the tunability of the functional properties of the dendrimer through modular integration is illustrated with an optically active gold nanoparticle (AuNP). The resultant dendrimer-coated AuNPs have a unique capability for tumor cell imaging via surface plasmon resonance scattering. Finally, we discuss the biophysical basis of the multivalent mechanism involved in the tight and specific binding of a RF-conjugated multivalent dendrimer to RFRs on the cell surface. The design principles and proof of concept studies presented here are strongly supportive of the promising potential of RF-conjugated nanoparticles for delivery and imaging applications in tumors.

Prions, characterized by self-propagating protease-resistant prion protein (PrP) conformations, are agents causing prion disease. Recent studies generated several such self-propagating protease-resistant recombinant PrP (rPrP-res) conformers. While some cause prion disease, others fail to induce any pathology. Here we showed that although distinctly different, the pathogenic and non-pathogenic rPrP-res conformers were similarly recognized by a group of conformational antibodies against prions and shared a similar guanidine hydrochloride denaturation profile, suggesting a similar overall architecture. Interestingly, two independently generated non-pathogenic rPrP-res were almost identical, indicating that the particular rPrP-res resulted from cofactor-guided PrP misfolding, rather than stochastic PrP aggregation. Consistent with the notion that cofactors influence rPrP-res conformation, the propagation of all rPrP-res formed with phosphatidylglycerol/RNA was cofactor-dependent, which is different from rPrP-res generated with a single cofactor, phosphatidylethanolamine. Unexpectedly, despite the dramatic difference in disease-causing capability, RT-QuIC assays detected large increases in seeding activity in both pathogenic and non-pathogenic rPrP-res inoculated mice, indicating that the non-pathogenic rPrP-res is not completely inert in vivo. Together, our study supported a role of cofactors in guiding PrP misfolding, indicated that relatively small structural features determine rPrP-res’ pathogenicity, and revealed that the in vivo seeding ability of rPrP-res does not necessarily result in pathogenicity.

The conversion of normal prion protein (PrP) into pathogenic PrP conformers is central to prion disease, but the mechanism remains unclear. The α-helix 2 of PrP contains a string of four threonines, which is unusual due to the high propensity of threonine to form β-sheets. This structural feature was proposed as the basis for initiating PrP conversion, but experimental results have been conflicting. We studied the role of the threonine string on PrP conversion by analyzing mouse Prnpa and Prnpb polymorphism that contains a polymorphic residue at the beginning of the threonine string, and PrP mutants in which threonine 191 was replaced by valine, alanine, or proline. The PMCA (protein misfolding cyclic amplification) assay was able to recapitulate the in vivo transmission barrier between PrPa and PrPb. Relative to PMCA, the amyloid fibril growth assay is less restrictive, but it did reflect certain properties of in vivo prion transmission. Our results suggest a plausible theory explaining the apparently contradictory results in the role of the threonine string in PrP conversion and provide novel insights into the complicated relationship among PrP stability, seeded conformational change, and prion structure, which is critical for understanding the molecular basis of prion infectivity.

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5(RF)n nanoparticle. By varying the average RF ligand valency (n = 0, 3, 5), the rupture force was measured between G5(RF)n and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (n = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell-Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates.

Atomic force microscopy force-pulling experiments have been used to measure the binding forces between folic acid (FA) conjugated poly(amidoamine) (PAMAM) dendrimers and folate binding protein (FBP). The generation 5 (G5) PAMAM conjugates contained an average of 2.7, 4.7, and 7.2 FA per dendrimer. The most probable rupture force was measured to be 83, 201, and 189 pN for G5-FA2.7, G5-FA4.7, and G5-FA7.2, respectively. Folic acid blocking experiments for G5-FA7.2 reduced the frequency of successful binding events and increased the magnitude of the average rupture force to 274 pN. The force data are interpreted as arising from a network of van der Waals and electrostatic interactions that form between FBP and G5 PAMAM dendrimer, resulting in a binding strength far greater than that expected for an interaction between FA and FBP alone.

Riboflavin receptors are overexpressed in malignant cells from certain human breast and prostate cancers, and they constitute a group of potential surface markers important for cancer targeted delivery of therapeutic agents and imaging molecules. Here we report on the fabrication and atomic force microscopy (AFM) characterization of a core–shell nanocomposite consisting of a gold nanoparticle (AuNP) coated with riboflavin receptor-targeting poly(amido amine) dendrimer. We designed this nanocomposite for potential applications such as a cancer targeted imaging material based on its surface plasmon resonance properties conferred by AuNP. We employed AFM as a technique for probing the binding interaction between the nanocomposite and riboflavin binding protein (RfBP) in solution. AFM enabled precise measurement of the AuNP height distribution before (13.5 nm) and after chemisorption of riboflavin-conjugated dendrimer (AuNP–dendrimer; 20.5 nm). Binding of RfBP to the AuNP–dendrimer caused a height increase to 26.7 nm, which decreased to 22.8 nm when coincubated with riboflavin as a competitive ligand, supporting interaction of AuNP–dendrimer and its target protein. In summary, physical determination of size distribution by AFM imaging can serve as a quantitative approach to monitor and characterize the nanoscale interaction between a dendrimer-covered AuNP and target protein molecules in vitro.

The formation of guanine (G)-quadruplex structures in the guanine-rich tandem repeats of the insulin-linked polymorphic region (ILPR) have potential effects on transcription of the insulin gene. Recent studies demonstrate that the ILPR G-quadruplexes can bind to insulin. The energetics of the binding between insulin and the G-quadruplexes formed by the most common ILPR repeat sequence have also been characterized in prior work. We have studied the proton transfer involved in the interaction between insulin and this DNA sequence by conducting isothermal titration calorimetry (ITC) experiments in various buffers and analyzing the observed enthalpy change. The transcriptional activity of a number of ILPR repeat sequences, including the consensus sequence, have been measured previously. Transcriptional activity for less common repeats is significantly lower than that of the consensus sequence but can be increased substantially by varying only one or two nucleotides. To determine the potential role of G-quadruplex formation and stability in regulating transcription, we have studied the second and third most common ILPR repeats as well as their variant sequences that exhibit increased transcriptional activity. ITC was used to characterize the energetics of the binding interaction between insulin and each of the four ILPR repeat sequences. The bulk thermodynamic measurements performed at various temperatures from 20 - 37 degrees Celsius provide insight into these biomolecular interactions.

Riboflavin ligands present an alternative pathway for targeted drug delivery as riboflavin receptors are over-expressed in breast and prostate cancer cells. We have examined a riboflavin-conjugated PAMAM dendrimer (generation 5) for targeting riboflavin binding protein (RFBP), which acts as a model protein for the riboflavin receptor. A single molecule force spectroscopy study using Atomic Force Microscopy (AFM) was performed to examine the binding interactions between riboflavin-conjugated dendrimers and RFBP. Using multiple theoretical models, the apparent kinetic and thermodynamic parameters for the riboflavin-conjugated dendrimer system were estimated.

Generation 5 poly(amidoamine) (G5 PAMAM) methotrexate (MTX) conjugates employing two small molecular linkers, G5-(COG-MTX)n, G5-(MFCO-MTX)n were prepared along with the conjugates of the G5-G5 (D) dimer, D-(COG-MTX)n, D-(MFCO-MTX)n. The monomer G5-(COG-MTX)n conjugates exhibited only a weak, rapidly reversible binding to folate binding protein (FBP) consistent with monovalent MTX binding. The D-(COG-MTX)n conjugates exhibited a slow onset, tight-binding mechanism in which the MTX first binds to the FBP, inducing protein structural rearrangement, followed by polymer-protein van der Waals interactions leading to tight-binding. The extent of irreversible binding is dependent on total MTX concentration and no evidence of multivalent MTX binding was observed.

The binding of insulin to the G-quadruplexes formed by the consensus sequence of the insulin-linked polymorphic region (ILPR) was investigated with differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The thermal denaturation temperature of insulin was increased by almost 4 °C upon binding to ILPR G-quadruplex DNA as determined by DSC. The thermodynamic parameters (K(D), ΔH, ΔG, and ΔS) of the insulin-G-quadruplex complex were further investigated by temperature-dependent ITC measurement over the range of 10-37 °C. The binding of insulin to the ILPR consensus sequence displays micromolar affinity in phosphate buffer at pH 7.4, which is mainly driven by entropic factors below 25 °C but by enthalpic terms above 30 °C. The interaction was also examined in several different buffers, and results showed that the observed ΔH is dependent on the ionization enthalpy of the buffer used. This indicates proton release upon the binding of G-quadruplex DNA to insulin. Additionally, the large negative change in heat capacity for this interaction may be associated with the dominant hydrophobicity of the amino acid sequence of insulin's β subunit, which is known to bind to the ILPR G-quadruplex DNA.

Background: Cosmetic skin lightening is practiced worldwide. Mercury is a well-documented melanotoxin added to some lightening products. However, mercury can cause many dermatologic, renal, and neurologic problems. The Food and Drug Administration limits the amount of mercury in cosmetic products to trace amounts, 1 ppm.Objective: The objective of this study was to quantitatively evaluate a large international sample of lightening products for mercury content, focusing on products available to US consumers either online or in stores.Methods: A total of 549 skin-lightening products, manufactured in 32 countries, were purchased online in the United States, Taiwan, and Japan and in stores in the United States, China, Taiwan, Thailand, Japan, and Sri Lanka. Cosmetics were screened for mercury content above 200 ppm using a low-cost portable x-ray fluorescence spectrometer.Results: Of the 549 tested products, 6.0% (n = 33) contained mercury above 1000 ppm. In all, 45% of mercury-containing samples contained mercury in excess of 10,000 ppm. Of lightening products purchased in the United States, 3.3% were found to contain mercury in excess of 1000 ppm.Limitations: Our study did not evaluate creams for other melanosuppressive ingredients. Only 1 sample of each product was tested.Conclusion: Our study confirms the national and global presence of mercury in skin-lightening products.

The formation of guanine (G)-quadruplex structures in the guanine-rich tandem repeats of the insulin-linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that the ILPR G-quadruplexes can bind to insulin while the energetics of this interaction with the most common ILPR repeat sequences have been characterized. Studies have also measured the transcriptional activity of less common ILPR repeats. Transcriptional activity for these repeats is significantly lower than that of the consensus sequence but can be increased substantially by varying only one or two nucleotides. To determine the potential role of G-quadruplex formation and stability in regulating transcription, we have studied the second and third most common ILPR repeats as well as their variant sequences that exhibit increased transcriptional activity. Circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC) have been used to characterize the binding interaction between insulin and each of the four ILPR repeat sequences. The bulk thermodynamic measurements performed at various temperatures from 20 - 37 degrees Celsius provide insight into these biomolecular interactions. Correction et al.Biophysical JournalApril 16, 2013In Brief2013. Characterizing the Biomolecular Interactions between Insulin and G-Quadruplex DNA. Nicole L. Michmerhuizen, Christine M. Timmer, Kumar Sinniah. Biophys. J. 104(2)s: 419a. Full-Text PDF Open Archive

Riboflavin ligands present an alternative pathway for targeted drug delivery as riboflavin receptors are over-expressed in breast and prostate cancer cells. We have examined a riboflavin conjugated PAMAM dendrimer (generation 5) for targeting riboflavin binding protein (RFBP), which acts as a model protein for the riboflavin receptor. By characterizing the binding interactions between riboflavin dendrimer gold nanoparticle conjugates and RFBP, the efficacy of this platform for a targeted approach of drug delivery can be predicted more accurately. Atomic Force Microscopy (AFM) was used for biological imaging studies of these riboflavin-dendrimer complexes conjugated with gold-nanoparticles. using a systematic "building block" approach, the size distribution of riboflavin dendrimer gold nanoparticle conjugates was mapped. Changes in height upon binding to RFBP and subsequent removal by competitive binding ligands demonstrate that this method could present a novel approach to screening the binding of drugs to drug targets.

2846-Pos Board B1 Volumetric Characterization of Tri-N-Acetylglucosamine Binding to Lysozyme Ikbae Son, Yuen Lai Shek, David N. Dubins, Tigran V. Chalikian. University of Toronto, Toronto, ON, Canada. Volumetric characteristics of protein recognition events determine the direction of pressure-induced shifts in the recognition reaction, while also providing insights into the structural, dynamic, and hydration changes. We report changes in volume, aV, and adiabatic compressibility, aKS, accompanying the binding of tri-N-acetylglucosamine [(GlcNAc)3] to lysozyme at 25 C in a pH 5.5 sodium acetate buffer. We interpret our measured changes in volume and compressibility in terms of changes in hydration and dynamic properties of the protein. Based on our aV data, we find that 79544 water molecules are released to the bulk from the hydration shells of the protein and the ligand. Our aKS data suggest a 452 % decrease in the mean-square fluctuations of the intrinsic volume of the protein, (or a 2 % decrease in dVM). Thus, the trisaccharide-bound state of the enzyme is less hydrated, more rigid, and less dynamic compared to the unbound state. In general, we discuss the importance of volumetric insights into the molecular origins of protein recognition events.

The formation of guanine (G)‐quadruplex structures in the guanine‐rich tandem repeats of the insulin‐linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that the ILPR G‐quadruplexes can bind to insulin, and while this may impact the transcription of insulin, little is known about the binding mechanism. We have performed single molecule atomic force microscopy (AFM), differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and fluorescence quenching spectroscopy to characterize the binding interaction between G‐quadruplex DNA and insulin. In this presentation, I will describe the bulk thermodynamic measurements performed at various temperatures from 10 to 37 °C. The experimental results based on KD demonstrate a moderate affinity by the ILPR consensus sequence to insulin. In addition to thermodynamic measurements, kinetic off‐rates were measured by single molecule force‐pulling measurements and these results will be discussed in relation to kinetic off‐rates measured from the unfolding of G‐quadruplex DNA.

The formation of guanine (G)-quadruplex structures in the guanine-rich tandem repeats of the insulin-linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that the ILPR G-quadruplexes can bind to insulin, and while this may impact the transcription of insulin, little is known about the binding mechanism. We have performed single molecule atomic force microscopy (AFM), differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and fluorescence quenching spectroscopy measurements to characterize the binding interaction between G-quadruplex DNA and insulin. The bulk thermodynamic measurements performed at various temperatures from 10 - 37 degree celcius highlight a moderate affinity by the ILPR consensus sequence to insulin. In addition to thermodynamic measurements, kinetic off-rates were measured by single molecule force-pulling measurements.

Artemisinins are proposed to act in the malaria parasite cytosol by oxidizing dihydroflavin cofactors of redox-active flavoenzymes, and under aerobic conditions by inducing their autoxidation. Perturbation of redox homeostasis coupled with the generation of reactive oxygen species (ROS) ensues. Ascorbic acid-methylene blue (MB), N-benzyl-1,4-dihydronicotinamide (BNAH)-MB, BNAH-lumiflavine, BNAH-riboflavin (RF), and NADPH-FAD-E. coli flavin reductase (Fre) systems at pH 7.4 generate leucomethylene blue (LMB) and reduced flavins that are rapidly oxidized in situ by artemisinins. These oxidations are inhibited by the 4-aminoquinolines piperaquine (PPQ), chloroquine (CQ), and others. In contrast, the arylmethanols lumefantrine, mefloquine (MFQ), and quinine (QN) have little or no effect. Inhibition correlates with the antagonism exerted by 4-aminoquinolines on the antimalarial activities of MB, RF, and artemisinins. Lack of inhibition correlates with the additivity/synergism between the arylmethanols and artemisinins. We propose association via pi complex formation between the 4-aminoquinolines and LMB or the dihydroflavins; this hinders hydride transfer from the reduced conjugates to the artemisinins. The arylmethanols have a decreased tendency to form pi complexes, and so exert no effect. The parallel between chemical reactivity and antagonism or additivity/synergism draws attention to the mechanism of action of all drugs described herein. CQ and QN inhibit the formation of hemozoin in the parasite digestive vacuole (DV). The buildup of heme-Fe-III results in an enhanced efflux from the DV into the cytosol. In addition, the lipophilic heme-Fe-III complexes of CQ and QN that form in the DV are proposed to diffuse across the DV membrane. At the higher pH of the cytosol, the complexes decompose to liberate heme-Fe-III. The quinoline or arylmethanol reenters the DV, and so transfers more heme-Fe-III out of the DV. In this way, the 4-aminoquinolines and arylmethanols exert antimalarial activities by enhancing heme-Fe-III and thence free Fe-III concentrations in the cytosol. The iron species enter into redox cycles through reduction of Fe-III to Fe-II largely mediated by reduced flavin cofactors and likely also by NAD(P)H-Fre. Generation of ROS through oxidation of Fe-II by oxygen will also result. The cytotoxicities of artemisinins are thereby reinforced by the iron. Other aspects of drug action are emphasized. In the cytosol or DV, association by pi complex formation between pairs of lipophilic drugs must adversely influence the pharmacokinetics of each drug. This explains the antagonism between PPQ and MFQ, for example. The basis for the antimalarial activity of RF mirrors that of MB, wherein it participates in redox cycling that involves flavoenzymes or Fre, resulting in attrition of NAD(P)H. The generation of ROS by artemisinins and ensuing Fenton chemistry accommodate the ability of artemisinins to induce membrane damage and to affect the parasite SERCA PfATP6 Ca2+ transporter. Thus, the effect exerted by artemisinins is more likely a downstream event involving ROS that will also be modulated by mutations in PfATP6. Such mutations attenuate, but cannot abrogate, antimalarial activities of artemisinins. Overall, parasite resistance to artemisinins arises through enhancement of antioxidant defense mechanisms.