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Intravaginal rings (IVRs) represent a sustained-release approach to drug delivery and have long been used and investigated for hormones and microbicides delivery. For decades, IVRs have been manufactured by injection molding and hot-melt extrusion with very limited design and material capabilities. Additive manufacturing (AM), specifically digital light synthesis (DLS), represents an opportunity to harness the freedom of design to expand control and tunability of drug release properties from IVRs. We report a novel approach to IVR design and manufacturing that results in geometrically complex internal architectures through the incorporation of distinct unit cells using computationally-aided design (CAD) software. We developed a systematic approach to design through the generation of an IVR library and investigated the effects of these parameters on ring properties. We demonstrate the ability to precisely and predictably control the compressive properties of the IVR independent of the internal architecture with which control of drug release kinetics can be achieved, thus opening the door for a 'plug-and-play' platform approach to IVR fabrication.
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The performance of binary electrolytes is governed by three transport properties: conductivity, salt diffusion coefficient, and transference number. Rigorous methods for measuring conductivity and the salt diffusion coefficient are well established and used routinely in the literature. The commonly used methods for measuring transference number are the steady-state current method, t+,id, and pulsed field gradient NMR, t+,NMR. These methods yield the transference number only if the electrolyte is ideal, i.e., the salt dissociates completely into non-interacting anions and cations. In this work, we present a complete set of ion transport properties for mixtures of a functionalized perfluoroether, dimethyl carbonate terminated perfluorinated tetraethylene ether, and lithium bis(fluorosulfonyl)imide (LiFSI). The equations used to determine these properties from experimental data are based on Newman's concentrated solution theory. The concentrated-solution-theory-based transference number, t, is negative across all salt concentrations, and it increases with increasing salt concentration. In contrast, the ideal transference number, t+,id, is positive across all salt concentrations and it decreases with salt concentration. The NMR-based transference number, t+,NMR, is approximately 0.5, independent of salt concentration. The disparity between the three transference numbers, which indicates the dominance of ion clustering, is resolved by the use of Newman's concentrated solution theory.
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OBJECTIVES: To discuss the current state of measuring value-added services in emerging value-based payment practice models and their impact on the pharmacy profession. SUMMARY: Value-based care models require a focus on population health. In value-based care settings, providers are held accountable for risk that goes beyond the clinic encounter, and payment is tied to quality measures that reflect clinical and humanistic outcomes across the health care spectrum. This creates opportunities for pharmacist integration into value-based care settings through addressing gaps in care, optimizing medication use, and developing physician-pharmacist team-based care practice models. CONCLUSION: Scalable and sustainable pharmacist integration into value-based care settings should involve expansion of preventive care into the community. The need for measurement of the value added by pharmacy services is a priority.
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Serviços Comunitários de Farmácia/organização & administração , Equipe de Assistência ao Paciente/organização & administração , Farmacêuticos/organização & administração , Atenção Primária à Saúde/organização & administração , Humanos , Papel ProfissionalRESUMO
Perfluoropolyethers (PFPEs) are polymer electrolytes with fluorinated carbon backbones that have high flash points and have been shown to exhibit moderate conductivities and high cation transference numbers when mixed with lithium salts. Ion transport in four PFPE electrolytes with different endgroups was characterized by differential scanning calorimetry (DSC), ac impedance, and pulsed-field gradient NMR (PFG-NMR) as a function of salt concentration and temperature. In spite of the chemical similarity of the electrolytes, salt diffusion coefficients measured by PFG-NMR and the glass transition temperature measured by DSC appear to be uncorrelated to ionic conductivity measured by ac impedance. We calculate a non-dimensional parameter, ß, that depends on the salt diffusion coefficients and ionic conductivity. We also use the Vogel-Tammann-Fulcher relationship to fit the temperature dependence of conductivity. We present a linear relationship between the prefactor in the VTF fit and ß; both parameters vary by four orders of magnitude in our experimental window. Our analysis suggests that transport in electrolytes with low dielectric constants (low ß) is dictated by ion hopping between clusters.
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Incipient microphase separation is observed by wide angle X-ray scattering (WAXS) in short chain multiblock copolymers consisting of perfluoropolyether (PFPE) and poly(ethylene oxide) (PEO) segments. Two PFPE-PEO block copolymers were studied; one with dihydroxyl end groups and one with dimethyl carbonate end groups. Despite having a low degree of polymerization (N â¼ 10), these materials exhibited significant scattering intensity, due to disordered concentration fluctuations between their PFPE-rich and PEO-rich domains. The disordered scattering intensity was fit to a model based on a multicomponent random phase approximation to determine the value of the interaction parameter, χ, and the radius of gyration, Rg. Over the temperature range 30-90 °C, the values of χ were determined to be very large (â¼2-2.5), indicating a high degree of immiscibility between the PFPE and PEO blocks. In PFPE-PEO, due to the large electron density contrast between the fluorinated and non-fluorinated block and the high value of χ, disordered scattering was detected at intermediate scattering angles, (q â¼ 2 nm-1) for relatively small polymer chains. Our ability to detect concentration fluctuations was enabled by both a relatively large value of χ and significant scattering contrast.
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BACKGROUND: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. METHODS: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. RESULTS: Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2µM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. CONCLUSIONS: Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. GENERAL SIGNIFICANCE: Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.
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Antígenos de Neoplasias/biossíntese , Fator de Ligação a CCAAT/metabolismo , Dano ao DNA , DNA Topoisomerases Tipo II/biossíntese , Proteínas de Ligação a DNA/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Nylons/farmacologia , Regiões Promotoras Genéticas , Células A549 , Animais , Antígenos de Neoplasias/genética , Fator de Ligação a CCAAT/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Etoposídeo/efeitos adversos , Etoposídeo/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Camundongos , Células NIH 3T3 , Nylons/químicaRESUMO
We use a large laboratory, modeling, and field dataset to investigate the isoprene + O3 reaction, with the goal of better understanding the fates of the C1 and C4 Criegee intermediates in the atmosphere. Although ozonolysis can produce several distinct Criegee intermediates, the C1 stabilized Criegee (CH2OO, 61 ± 9%) is the only one observed to react bimolecularly. We suggest that the C4 Criegees have a low stabilization fraction and propose pathways for their decomposition. Both prompt and non-prompt reactions are important in the production of OH (28% ± 5%) and formaldehyde (81% ± 16%). The yields of unimolecular products (OH, formaldehyde, methacrolein (42 ± 6%) and methyl vinyl ketone (18 ± 6%)) are fairly insensitive to water, i.e., changes in yields in response to water vapor (≤4% absolute) are within the error of the analysis. We propose a comprehensive reaction mechanism that can be incorporated into atmospheric models, which reproduces laboratory data over a wide range of relative humidities. The mechanism proposes that CH2OO + H2O (k(H2O)â¼ 1 × 10(-15) cm(3) molec(-1) s(-1)) yields 73% hydroxymethyl hydroperoxide (HMHP), 6% formaldehyde + H2O2, and 21% formic acid + H2O; and CH2OO + (H2O)2 (k(H2O)2â¼ 1 × 10(-12) cm(3) molec(-1) s(-1)) yields 40% HMHP, 6% formaldehyde + H2O2, and 54% formic acid + H2O. Competitive rate determinations (kSO2/k(H2O)n=1,2â¼ 2.2 (±0.3) × 10(4)) and field observations suggest that water vapor is a sink for greater than 98% of CH2OO in a Southeastern US forest, even during pollution episodes ([SO2] â¼ 10 ppb). The importance of the CH2OO + (H2O)n reaction is demonstrated by high HMHP mixing ratios observed over the forest canopy. We find that CH2OO does not substantially affect the lifetime of SO2 or HCOOH in the Southeast US, e.g., CH2OO + SO2 reaction is a minor contribution (<6%) to sulfate formation. Extrapolating, these results imply that sulfate production by stabilized Criegees is likely unimportant in regions dominated by the reactivity of ozone with isoprene. In contrast, hydroperoxide, organic acid, and formaldehyde formation from isoprene ozonolysis in those areas may be significant.
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Speciated particle-phase organic nitrates (pONs) were quantified using online chemical ionization MS during June and July of 2013 in rural Alabama as part of the Southern Oxidant and Aerosol Study. A large fraction of pONs is highly functionalized, possessing between six and eight oxygen atoms within each carbon number group, and is not the common first generation alkyl nitrates previously reported. Using calibrations for isoprene hydroxynitrates and the measured molecular compositions, we estimate that pONs account for 3% and 8% of total submicrometer organic aerosol mass, on average, during the day and night, respectively. Each of the isoprene- and monoterpenes-derived groups exhibited a strong diel trend consistent with the emission patterns of likely biogenic hydrocarbon precursors. An observationally constrained diel box model can replicate the observed pON assuming that pONs (i) are produced in the gas phase and rapidly establish gas-particle equilibrium and (ii) have a short particle-phase lifetime (â¼2-4 h). Such dynamic behavior has significant implications for the production and phase partitioning of pONs, organic aerosol mass, and reactive nitrogen speciation in a forested environment.
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The design, synthesis, and DNA binding properties of azaHx-PI or p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (5) are described. AzaHx, 2-(p-anisyl)-4-aza-benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, ΔT(M), and SPR studies provided evidence that an azaHx/IP pairing, formed from antiparallel stacking of two azaHx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C-G doublet. AzaHx-PI was found to target 5'-ACGCGT-3', the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity (K(eq) 4.0±0.2×10(7) M(-1)).
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Benzimidazóis/química , DNA/metabolismo , Corantes Fluorescentes/química , Nylons/química , Pirróis/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Pareamento de Bases , Benzimidazóis/síntese química , Benzimidazóis/metabolismo , Sítios de Ligação , Técnicas de Química Sintética , Dicroísmo Circular , DNA/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/química , Desenho de Fármacos , Corantes Fluorescentes/metabolismo , Nylons/síntese química , Regiões Promotoras Genéticas , Pirróis/síntese química , Pirróis/metabolismoRESUMO
Secondary organic aerosol (SOA) constitutes a substantial fraction of fine particulate matter and has important impacts on climate and human health. The extent to which human activities alter SOA formation from biogenic emissions in the atmosphere is largely undetermined. Here, we present direct observational evidence on the magnitude of anthropogenic influence on biogenic SOA formation based on comprehensive ambient measurements in the southeastern United States (US). Multiple high-time-resolution mass spectrometry organic aerosol measurements were made during different seasons at various locations, including urban and rural sites in the greater Atlanta area and Centreville in rural Alabama. Our results provide a quantitative understanding of the roles of anthropogenic SO2 and NOx in ambient SOA formation. We show that isoprene-derived SOA is directly mediated by the abundance of sulfate, instead of the particle water content and/or particle acidity as suggested by prior laboratory studies. Anthropogenic NOx is shown to enhance nighttime SOA formation via nitrate radical oxidation of monoterpenes, resulting in the formation of condensable organic nitrates. Together, anthropogenic sulfate and NOx can mediate 43-70% of total measured organic aerosol (29-49% of submicron particulate matter, PM1) in the southeastern US during summer. These measurements imply that future reduction in SO2 and NOx emissions can considerably reduce the SOA burden in the southeastern US. Updating current modeling frameworks with these observational constraints will also lead to more accurate treatment of aerosol formation for regions with substantial anthropogenic-biogenic interactions and consequently improve air quality and climate simulations.
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Hx-amides are fluorescent hybrids of imidazole (I)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx-I/P-I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and ΔTM studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T-A/T; Hx/IP prefers C-A/T; Hx/PI prefers A/T-C; and Hx/II prefers C-C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts.
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Amidas/química , Anisóis/química , Benzimidazóis/química , DNA/química , Imidazóis/química , Pirróis/química , Pareamento de Bases , Dicroísmo Circular , DNA/metabolismo , Corantes Fluorescentes/química , Conformação de Ácido NucleicoRESUMO
Peptide libraries have proven to be useful in applications such as substrate profiling, drug candidate screening and identifying protein-protein interaction partners. However, issues of fidelity, peptide length, and purity have been encountered when peptide libraries are chemically synthesized. Biochemically produced libraries, on the other hand, circumvent many of these issues due to the fidelity of the protein synthesis machinery. Using thioredoxin as an expression partner, a stably folded peptide scaffold (avian pancreatic polypeptide) and a compatible cleavage site for human rhinovirus 3C protease, we report a method that allows robust expression of a genetically encoded peptide library, which yields peptides of high purity. In addition, we report the use of methodological synchronization, an experimental design created for the production of a library, from initial cloning to peptide characterization, within a 5-week period of time. Total peptide yields ranged from 0.8% to 16%, which corresponds to 2-70 mg of pure peptide. Additionally, no correlation was observed between the ability to be expressed or overall yield of peptide-fusions and the intrinsic chemical characteristics of the peptides, indicating that this system can be used for a wide variety of peptide sequences with a range of chemical characteristics.
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Biblioteca de Peptídeos , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Proteases Virais 3C , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Evolução Molecular Direcionada/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Marcação por Isótopo , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Polipeptídeo Pancreático/genética , Polipeptídeo Pancreático/metabolismo , Peptídeos/química , Peptídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Accumulating evidence suggests that specific metabolites of estrogens, namely, catechol estrogen quinones, react with DNA to form adducts and generate apurinic sites, which can lead to the mutations that induce breast cancer. Oxidation of estradiol (E(2)) produces 2 catechol estrogens, 4-hydroxyestradiol (4-OHE(2)) and 2-OHE(2) among the major metabolites. These, in turn, are oxidized to the quinones, E(2)-3,4-quinone (E(2)-3,4-Q) and E(2)-2,3-Q, which can react with DNA. Oxidation of E(2) to 2-OHE(2) is mainly catalyzed by cytochrome P450 (CYP) 1A1, and CYP3A4, whereas oxidation of E(2) to 4-OHE(2) in extrahepatic tissues is mainly catalyzed by CYP1B1 as well as some CYP3As. The potential involvement of CYP isoforms in the further oxidation of catechols to semiquinones and quinones has, however, not been investigated in detail. In this project, to identify the potential function of various CYPs in oxidizing catechol estrogens to quinones, we used different recombinant human CYP isoforms, namely, CYP1A1, CYP1B1, and CYP3A4, with the scope of oxidizing the catechol estrogens 2-OHE(2) and 4-OHE(2) to their respective estrogen quinones, which then reacted with DNA. The depurinating adducts 2-OHE(2)-6-N3Ade, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua were observed in the respective reaction systems by ultraperformance liquid chromatography/tandem mass spectrometry. Furthermore, more than 100-fold higher levels of estrogen-glutathione (GSH) conjugates were detected in the reactions. Glutathione conjugates were observed, in much smaller amounts, when control microsomes were used. Depurinating adducts, as well as GSH conjugates, were obtained when E(2)-3,4-Q was incubated with CYP1B1 or control microsomes in a 30-minute reaction, further demonstrating that GSH is present in these recombinant enzyme preparations. These experiments demonstrated that CYP1A1, CYP1B1, and CYP3A4 are able to oxidize catechol estrogens to their respective quinones, which can further react with GSH, protein, and DNA, the last resulting in depurinating adducts that can lead to mutagenesis.
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Hidrocarboneto de Aril Hidroxilases/fisiologia , Citocromo P-450 CYP1A1/fisiologia , Sistema Enzimático do Citocromo P-450/fisiologia , DNA/metabolismo , Estrogênios de Catecol/metabolismo , Quinonas/metabolismo , Catálise , Citocromo P-450 CYP1B1 , Citocromo P-450 CYP3A , Glutationa/metabolismoRESUMO
Increased awareness about the importance of protein folding and trafficking to the etiology of gain-of-function diseases has driven extensive efforts to understand the cell and molecular biology underlying the life cycle of normal secretory pathway proteins and the detrimental effects of abnormal proteins. In this regard, the quality-control machinery in the endoplasmic reticulum (ER) has emerged as a major mechanism by which cells ensure that secreted and transmembrane proteins either adopt stable secondary, tertiary, and quaternary structures or are retained in the ER and degraded. Here we examine cellular and molecular aspects of ER retention in transfected fibroblasts expressing missense mutations in the Proteolipid Protein-1 (PLP1) gene that cause mild or severe forms of neurodegenerative disease in humans. Mild mutations cause protein retention in the ER that is partially dependent on the presence of a cytoplasmically exposed heptapeptide, KGRGSRG. In contrast, retention associated with severe mutations occurs independently of this peptide. Accordingly, the function of this novel heptapeptide has a significant impact on pathogenesis and provides new insight into the functions of the two splice isoforms encoded by the PLP1 gene, PLP1 and DM-20.