"Superchiral" Spectroscopy: Detection of Protein Higher Order Hierarchical Structure with Chiral Plasmonic Nanostructures.

Optical spectroscopic methods do not routinely provide information on higher order hierarchical structure (tertiary/quaternary) of biological macromolecules and assemblies. This necessitates the use of time-consuming and material intensive techniques, such as protein crystallography, NMR, and electron microscopy. Here we demonstrate a spectroscopic phenomenon, superchiral polarimetry, which can rapidly characterize ligand-induced changes in protein higher order (tertiary/quaternary) structure at the picogram level, which is undetectable using conventional CD spectroscopy. This is achieved by utilizing the enhanced sensitivity of superchiral evanescent fields to mesoscale chiral structure.

Synthesis of Bis(oxazoline) Ligands Possessing C-5 gem-Disubstitution and Their Application in Asymmetric Friedel-Crafts Alkylations.

A series of eight novel bis(oxazoline) ligands incorporating gem-disubstitution on one of the oxazoline rings were prepared from (S)-valine. These ligands are designed as a cost-effective alternative to similar ligands possessing an oxazolinyl C(5)-tert-butyl group derived from expensive (S)-tert-leucine. Four of the ligands possess a C(4)-gem-dimethyl group and four a C(4)-gem-diphenyl group adjacent to the C(5)-isopropyl substituent. Zinc complexes of ligands 11a-h, along with non-C(4)-gem-disubstituted analogues 1a-g, were effective in the Friedel-Crafts alkylation of both indole (up to 74% ee) and 2-methoxyfuran (up to 95% ee) with a series of nitroalkenes. Three of the ligands (11a-c), an iron dichloride complex of ligand 11d and two zinc dichloride complexes, were characterized by X-ray crystallography, one with ligand 11d and the second a bis-tert-butyl-substituted N-methylamine ligand. A direct comparison of the latter structures clearly illustrates the gem-dimethyl effect.

Evidence-Based Criteria for Differential Treatment Planning of Implant Restorations for the Maxillary Edentulous Patient.

Since the introduction of the endosseous concept to North America in 1982, there have been new permutations of the original ad modum Branemark design to meet the unique demands of treating the edentulous maxilla with an implant restoration. While there is a growing body of clinical evidence to assist the student, faculty, and private practitioner in the algorithms for design selection, confusion persists because of difficulty in assessing the external and internal validity of the relevant studies. The purpose of this article is to review clinician- and patient-mediated factors for implant restoration of the edentulous maxilla in light of the hierarchical level of available evidence, with the aim of elucidating the benefit/risk calculus of various treatment modalities.

Protein-mediated dethreading of a biotin-functionalised pseudorotaxane.

In this article, we describe the synthesis of new biotin-functionalised naphthalene derivatives 3 and 4 and their complexation behaviour with avidin and neutravidin using a range of analytical techniques. We have shown using 2-(4'-hydroxyazobenzene)benzoic acid displacement and ITC experiments, that compounds 3 and 4 have the propensity to form reasonably high-affinity bioconjugates with avidin and neutravidin. We have also demonstrated using (1)H NMR, UV-vis and fluorescence spectroscopy that the naphthalene moiety of 3 and 4 facilitates the formation of pseudorotaxane-like structures with 1 in water. We have then investigated the ability of avidin and neutravidin to modulate the complexation between 1 and 3 or 4. UV-vis and fluorescence spectroscopy has shown that in both cases the addition of the protein disrupts complexation between the naphthalene moieties of 3 and 4 with 1.

Precursory elements of safety culture: Exploratory analyses of engineering students' safety attitudes.

INTRODUCTION: Safety culture as a concept has been well-researched in the literature. There is less work, however, on how individuals entering the workforce acquire and partake in safety culture over time and how they might be primed to partake in the positive elements of safety culture. METHOD: We begin this exploration by surveying engineering students' attitudes toward safety and experiences with safety education at the Georgia Institute of Technology (n = 316). RESULTS: We find disparities among engineering disciplines, where some majors have more negative views toward safety than others. This may point to the need for more [effective] safety education to prevent negative attitudes toward safety in the workplace. In addition to describing trends among engineering students' attitudes, this study also uses factor analysis to characterize the latent constructs of precursory safety culture: the safety-related attitudes that may direct how people engage with safety culture as early-career engineers. PRACTICAL APPLICATIONS: The analysis provides a conceptual construction of precursor safety culture attitudes, which can serve as a guide to future measurement efforts.

Synthesis and characterization of naphthalenediimide-functionalized flavin derivatives.

Two acceptor-acceptor dyads have been synthesized featuring a flavin moiety and a naphthalenediimide (NDI) unit. The NDI unit is linked to the flavin through a short spacer group via either the N(3) or N(10) positions of the flavin. We have investigated the UV-Vis and redox properties of these multi-electron accepting systems which indicate that these materials display the collective properties of their component systems. Fluorescence spectroscopy measurements have revealed that their emission properties are dominated by the flavin unit.

Elucidating the Biological Activity of Fish-Derived Collagen and Gelatine Hydrolysates using Animal Cell Culture - A Review.

A large percentage of a fish's weight is generally discarded during fish processing. Reducing the waste products of marine origin is a subject of great interest within the scientific community. Pelagic byproducts, such as the structural protein collagen, which can be generated during the processing of fish, have been proposed as an alternative to terrestrial, mammalian sources due to advantages including high availability and low risk of zoonotic disease transmission. Gelatine has multiple possible applications, ranging from nutraceutical applications to cosmetics and has the advantage of being generally regarded as safe. In this multidisciplinary review, the chemistry of gelatine and its parent protein collagen, the chemical reactions to generate their hydrolysates, and studies on their biological activities using animal cell culture are discussed.

Proteomic analysis of polymeric salivary mucins: no evidence for MUC19 in human saliva.

MUC5B is the predominant polymeric mucin in human saliva [Thornton, Khan, Mehrotra, Howard, Veerman, Packer and Sheehan (1999) Glycobiology 9, 293-302], where it contributes to oral cavity hydration and protection. More recently, the gene for another putative polymeric mucin, MUC19, has been shown to be expressed in human salivary glands [Chen, Zhao, Kalaslavadi, Hamati, Nehrke, Le, Ann and Wu (2004) Am. J. Respir. Cell Mol. Biol. 30, 155-165]. However, to date, the MUC19 mucin has not been isolated from human saliva. Our aim was therefore to purify and characterize the MUC19 glycoprotein from human saliva. Saliva was solubilized in 4 M guanidinium chloride and the high-density mucins were purified by density-gradient centrifugation. The presence of MUC19 was investigated using tandem MS of tryptic peptides derived from this mucin preparation. Using this approach, we found multiple MUC5B-derived tryptic peptides, but were unable to detect any putative MUC19 peptides. These results suggest that MUC19 is not a major component in human saliva. In contrast, using the same experimental approach, we identified Muc19 and Muc5b glycoproteins in horse saliva. Moreover, we also identified Muc19 from pig, cow and rat saliva; the saliva of cow and rat also contained Muc5b; however, due to the lack of pig Muc5b genomic sequence data, we were unable to identify Muc5b in pig saliva. Our results suggest that unlike human saliva, which contains MUC5B, cow, horse and rat saliva are a heterogeneous mixture of Muc5b and Muc19. The functional consequence of these species differences remains to be elucidated.

iNOS as a therapeutic target for treatment of human tumors.

Nitric oxide synthase (NOS) has been shown to be overexpressed in a number of human tumors compared to normal tissues and therefore potentially represents an exploitable target in future anticancer therapies. To achieve this, there will be a need to profile tumors to identify those expressing high levels of NOS; alternatively, endogenous (low) levels of NOS could be modulated by induction or through gene therapy approaches. NOS consists of a reductase domain which shares a high degree of sequence homology with P450 reductase and this domain supplies reducing equivalents to a haem containing oxygenase domain that is responsible for the production of nitric oxide. Thus, there are a number of routes of exploitation. Firstly, to take advantage of the reductase domain to activate bioreductive drugs as has been exemplified with tirapazamine and now extended to AQ4N (1,4-bis{2-(dimethylamino-N-oxide)ethylamino}5,8-dihydroxy-anthracene-9,10-dione). Secondly, to take advantage of nitric oxide production for its ability to increase the sensitivity of resistant hypoxic cells to radiation. Lastly, to utilize inhibition of HIF-1 to amplify NO based therapies. In this review we provide examples/evidence of how these objectives can be achieved.

A flavin-based [2]catenane.

We report the synthesis, solid-state and preliminary solution properties of a flavin-based [2]catenane.

Manufacturing history of etanercept (Enbrel®): Consistency of product quality through major process revisions.

Etanercept (ETN) (Enbrel®) is a soluble protein that binds to, and specifically inhibits, tumor necrosis factor (TNF), a proinflammatory cytokine. ETN is synthesized in Chinese hamster ovary cells by recombinant DNA technology as a fusion protein, with a fully human TNFRII ectodomain linked to the Fc portion of human IgG1. Successful manufacture of biologics, such as ETN, requires sophisticated process and product understanding, as well as meticulous control of operations to maintain product consistency. The objective of this evaluation was to show that the product profile of ETN drug substance (DS) has been consistent over the course of production. Multiple orthogonal biochemical analyses, which included evaluation of attributes indicative of product purity, potency, and quality, were assessed on >2,000 batches of ETN from three sites of DS manufacture, during the period 1998-2015. Based on the key quality attributes of product purity (assessed by hydrophobic interaction chromatography HPLC), binding activity (to TNF by ELISA), potency (inhibition of TNF-induced apoptosis by cell-based bioassay) and quality (N-linked oligosaccharide map), we show that the integrity of ETN DS has remained consistent over time. This consistency was maintained through three major enhancements to the initial process of manufacturing that were supported by detailed comparability assessments, and approved by the European Medicines Agency. Examination of results for all major quality attributes for ETN DS indicates a highly consistent process for over 18 years and throughout changes to the manufacturing process, without affecting safety and efficacy, as demonstrated across a wide range of clinical trials of ETN in multiple inflammatory diseases.

Variability of intended copies for etanercept (Enbrel®): Data on multiple batches of seven products.

Fusion protein and monoclonal antibody-based tumor necrosis factor (TNF) inhibitors represent established treatment options for a range of inflammatory diseases. Regulatory authorities have outlined the structural characterization and clinical assessments necessary to establish biosimilarity of a new biotherapeutic product with the innovator biologic drug. Biologic products that would not meet the minimum World Health Organization's standard for evaluation of similar biotherapeutic products are available in some countries; in some cases relevant data to assess biosimilarity and appropriate regulatory approval pathways are lacking. Batches of seven intended copy (IC) products for etanercept (Enbrel®) were subjected to a subset of test methods used in the routine release and heightened characterization of Enbrel®, to determine key attributes of identity, quality, purity, strength, and activity. While a number of quality attributes of the IC lots tested met the release specifications for Enbrel®, none fell within these limits across all methods performed, and there were no IC lots that satisfied the criteria typically applied by the innovator to support comparability with Enbrel®. Although the consequences of these differences are largely unknown, the potential for unanticipated clinical outcomes should not be overlooked.

Sub-wavelength focusing of acoustic waves in bubbly media.

The purpose of this paper is to investigate acoustic wave scattering by a large number of bubbles in a liquid at frequencies near the Minnaert resonance frequency. This bubbly media has been exploited in practice to obtain super-focusing of acoustic waves. Using layer potential techniques, we derive the scattering function for a single spherical bubble excited by an incident wave in the low frequency regime. We then propose a point scatterer approximation for N bubbles, and describe several numerical simulations based on this approximation, that demonstrate the possibility of achieving super-focusing using bubbly media.

Rapid analysis of coumarins using surface plasmon resonance.

Coumarin molecules are ubiquitous in nature. Several have come to prominence as potential clinical therapeutic candidates. The principal example is warfarin, which is a very widely prescribed anticoagulant. Other coumarin derivatives, such as aflatoxin B1, are insidious contaminants in crop-derived foodstuffs. Extreme potency is a common feature of all biochemically active coumarins and, thus reliable methods for their rapid and sensitive detection are of paramount importance. Accordingly, this review examines the current methods used in the analysis of these molecules and compares them with immunoassay-based strategies. As a case study, we report on our experiences with using coumarin-specific polyclonal, monoclonal, and recombinant antibodies in conjunction with a surface plasmon resonance-based biosensor for analysis of coumarins. We chart the assay development process and demonstrate high sensitivity and reproducibility that compares favorably with established methodologies.

Redox-mediated reactions of vinylferrocene: toward redox auxiliaries.

Chemical redox reactions have been exploited to transform unreactive vinylferrocene into a powerful dienophile for the Diels-Alder reaction and reactive substrate for thiol addition reactions upon conversion to its ferrocenium state. We have further investigated the ability of these reactions to facilitate redox-auxiliary-like reactivity by further hydrogenolyisis of the Diels-Alder adduct to the corresponding cyclopentane derivative.

Differentiating biosimilarity and comparability in biotherapeutics.

The manufacture of biologics is a complex process involving numerous steps. Over time, differences may arise as a result of planned changes to the manufacturing processes of a biologic from the same manufacturer. Comparability is the regulatory process that outlines the scope of an assessment required of an already licensed biologic after a manufacturing process change made by the same manufacturer. The aim of a comparability assessment is to demonstrate that any pre-manufacturing and post-manufacturing changes have no adverse impact on quality, safety, and efficacy of the biologic. A comparability assessment is distinct from a biosimilarity assessment, which involves extensive assessment of a biologic that is highly similar to the originator (reference product) in terms of quality, safety, and efficacy. The US Food and Drug Administration, European Medicines Agency, and World Health Organization have applied the fundamental comparability concepts into their respective biosimilarity guidance documents. In this review, we examine the rationale behind the distinct, highly regulated approval processes governing changes that may occur over time to an originator biologic due to planned manufacturing changes (as described by a comparability exercise) and those that outline the approval of a proposed biosimilar drug, based on its relationship with the reference product (biosimilarity evaluations).

Spatial control of chemical processes on nanostructures through nano-localized water heating.

Optimal performance of nanophotonic devices, including sensors and solar cells, requires maximizing the interaction between light and matter. This efficiency is optimized when active moieties are localized in areas where electromagnetic (EM) fields are confined. Confinement of matter in these 'hotspots' has previously been accomplished through inefficient 'top-down' methods. Here we report a rapid 'bottom-up' approach to functionalize selective regions of plasmonic nanostructures that uses nano-localized heating of the surrounding water induced by pulsed laser irradiation. This localized heating is exploited in a chemical protection/deprotection strategy to allow selective regions of a nanostructure to be chemically modified. As an exemplar, we use the strategy to enhance the biosensing capabilities of a chiral plasmonic substrate. This novel spatially selective functionalization strategy provides new opportunities for efficient high-throughput control of chemistry on the nanoscale over macroscopic areas for device fabrication.

The impact of driving force on electron transfer rates in photovoltaic donor-acceptor blends.

The effect of acceptor energy level on electron transfer rate in blends of the polymer solar-cell material poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl]] (PTB7) is studied using time-resolved fluorescence. Fast electron transfer in less than 2 ps is observed for a driving force between 0.2 and 0.6 eV and the electron transfer is slower outside this range. This dependence is described by Marcus theory with a reorganization energy of ≈0.4 eV.

The development and application of a surface plasmon resonance-based inhibition immunoassay for the determination of warfarin in plasma ultrafiltrate.

Warfarin is the most widely prescribed oral anticoagulant for the management of a wide variety of thromboembolic disorders such as atrial fibrillation and deep vein thrombosis. A panel of warfarin-protein conjugates were produced and characterised and subsequently used for the production of monoclonal antibodies to warfarin. Following characterisation, the monoclonal antibodies were used in the development of a surface plasmon resonance-based inhibition immunoassay for the determination of the physiologically active 'nonprotein'-bound fraction of the drug in plasma ultrafiltrate. The inhibition immunoassay was compared with an existing high-performance liquid chromatography (HPLC) chromatographic technique for the determination of warfarin in plasma ultrafiltrate, and an excellent correlation was achieved between the two independent analytical techniques. The ligand-binding capacity and stability of various immobilised ligands were also compared. The BIACore-based inhibition immunoassay demonstrated an assay precision range of approximately 4-250 ng/ml, which is within the clinical range and demonstrated good reproducibility and robustness.