Molecule In Question Research Articles

670. Probing the Stability of rAAV Capsids and Genomic Constructs Using Differential Scanning Calorimetry in Concert With Mobile Phase Modifiers

The recent resurgence of using rAAV vectors as commercially viable therapeutic agents has highlighted the need to identify stable capsid/genomic construct combinations which can both endure a typical cGMP manufacturing process and possess a long product shelf life. In this work we investigate the inherent stability of various capsids and genomic constructs using differential scanning calorimetry (DSC). DSC measures the amount of energy required to increase the temperature of a sample and reference and can be used to determine the Tm of the molecule in question. Numerous commercially relevant rAAV serotypes containing several genomic constructs (self-complimentary and oversized genomes) were subjected to melting curve analysis using DSC to compare stability. Additionally, various mobile phase modifiers were added to solution in order to probe the stability of rAAV vector, using organics and kosmotropic/chaotropic salts. These results demonstrate DSC as an effective tool to examine inherent rAAV vector capsid stability and support a role for the serotype and vector genome structure to affect the observed stability.

Energy maps for glycosidic linkage conformations.

Glycosidic linkage conformations are the main factors in determining the shapes of disaccharide, oligosaccharide, and polysaccharide molecules. The conformations are expressed in terms of the torsion angles about the bonds from each ring of the disaccharide moiety to its glycosidic oxygen atom, and the probability of a given conformation is often expressed in terms of its free or potential energy. The energy surface or map for a disaccharide is a display of the energy plotted against the two torsion angles. Successful mapping allows a particular kind of energy calculation to provide the energy values for each conformation and avoids possible pitfalls. Although different methods are discussed, the main emphasis of this chapter is on the technical production of the maps and their exploitation in further understanding the shape of the molecule in question.

Cell types important for fostering normal thymic medullary epithelial cell development (HEM4P.242)

Abstract Tolerance in developing T cells is dependent on medullary thymic epithelial cells (mTEC) and mTEC development in turns requires signals from mature SP thymocytes. Several TNF family receptor-ligand pairs have been shown to be important players in the thymic crosstalk required to foster mTEC development including RANK-RANKL, LTαβ-LTβR and CD40L-CD40. In addition to confirming the important roles of TNF family members in thymic crosstalk, we have recently identified CD28-CD80/86 interactions as an additional pathway important for normal thymic medullary compartment development. CD80 and CD86, similar to LTβR, CD40 and RANK, are expressed by mTEC as well as by bone marrow (BM) derived cells in the thymus. To address the question of which cell type(s) need to express CD80/86, LTβR, CD40 and RANK in order to support mTEC development, we have generated and analyzed BM chimeras or conditional knockout mice in which either non-BM-derived cells (including thymic epithelium) or BM-derived cells express the molecule in question. For LTβR and RANK, we find that expression on radiation-resistant non-BM-derived cells, is required. Interestingly, however, we find that for CD80/86 and CD40, expression on either non-BM-derived or BM-derived cells is sufficient to promote mTEC development. Our findings suggest that models of thymic crosstalk must consider not only communication between TEC and thymocytes but with other bone marrow derived cell types as well.

The intrinsic view of ionization equilibria of polyprotic molecules

The described intrinsic approach massively reduces the parameter number to describe microequilibria. The resulting intrinsic parameters yield insight into the protonation of polyprotic molecules.

Polyethyleneimine-functionalized large pore ordered silica materials for poorly water-soluble drug delivery

Four ordered mesoporous silica supports with different pore structure characteristics were investigated for their drug loading and release abilities with regard to their structural variabilities as well as implications of surface modification. The (model) drug molecule in question was the poorly water-soluble glucocorticoid Prednisolone, composed of a steroid skeleton with functional groups in the form of carbonyls and hydroxyls. Under non-aqueous conditions, such as those applied for drug loading, these functional groups are expected to interact with the surface silanols of the silica supports, but this interaction could possibly also be enhanced by introducing amino groups to the silica surfaces. Thus, all four supports were further functionalized by surface hyperbranching of polyethyleneimine), PEI, which was successfully incorporated to all supports in high amounts (>30 wt%). However, the accessibility of the pore system after organic modification was dependent on the pore sizes and structures, highlighting the importance of using large-pore mesophases with adequate structures when aiming for applications involving (bulky) guest molecules. Additionally, after incorporation of large amounts of guest molecules (40 wt%), full water accessibility was retained in that the loaded cargo could be rapidly released from the carrier matrixes, which is a crucial requirement when formulating poorly soluble substances. Results displayed that the release of Prednisolone from the silica supports occurred faster than the dissolution of the pure drug. All silica materials released more than 85 % of the adsorbed drug in 5 h, independently of the support material. Thus, the confinement of Prednisolone inside the mesopores seems to be the main reason for the faster kinetic release rate. These constraints imply that Prednisolone becomes more mobile inside the pores, and therefore more soluble in release medium. These results confirm the potential of silica supports as drug delivery carriers for drugs with limited water solubility such as steroids.

Coarse-Grained Atomistic Models from Affine Transformations of Geometric Shapes Applied to Small-Angle Scattering

Simplified geometric modeling can serve as a valuable source of information about the structure and function of biological macromolecules, particularly when an atomic resolution model of the molecule in question is not available. Indeed, using simple geometric forms to approximate solute particles has long been a hallmark of x-ray and neutron scattering studies, and continues to figure in a variety of other disciplines. Here we demonstrate that the molecular envelope of a biological molecule of interest may be represented by the affine transformation of a single geometric shape, and show that models thus obtained show excellent agreement to experimental small-angle scattering data. In one example, each of the theoretical small-angle scattering curves of the Fab and Fc units of immunoglobulins, structures which contain central structural cavities, was convincingly modeled as the single linear affine transformation of a torus, optimized against the experimental scattering from that molecule; a more complex toroidal model, comprising three tori representing the intact immunoglobulin, also well fit the corresponding experimental data. The same method of affine transformation of a geometric shape to determine an overall shape model was then shown to generalize to other proteins and macromolecular assemblies, suggesting that the method of may prove widely applicable.

Don't ditch the NMR just yet: Using atomic force microscopy as an aid to solving problematic structures

Images of chemical structures abound in the literature, and we use different formats to explain different features of the molecules in question. Recent advances in atomic force microscopy (AFM), using a carbon monoxide terminated tips have brought about a spectacular enhancement in the resolution attainable.1 It is now feasible to distinguish chemical features such as atoms, bond lengths and bond angles at the Ångstrom scale. The images observed are very close to textbook stick structures and we postulated that this might be useful to assist organic structure determination. To test the possibility of using this technique for this purpose, we used a planar molecule to obtain NMR and AFM data and assembled the substructures guided by the AFM image (Figure 1).2 Two solutions were deemed possible, and one was excluded based on density functional theory calculations leaving only one structure remaining. More recent studies have dealt with non-planar structures as well as the combined use of NMR, AFM and computer assisted structure elucidation to solve structures. The presentation will include the amount of material needed to perform such studies as well as the current limitations of the method. Finally, prospects for the future will be discussed for the integration of AFM into the structure determination armoury.

Unusual co-crystal of isonicotinamide: the structural landscape in crystal engineering

The idea of a structural landscape is based on the fact that a large number of crystal structures can be associated with a particular organic molecule. Taken together, all these structures constitute the landscape. The landscape includes polymorphs, pseudopolymorphs and solvates. Under certain circumstances, it may also include multi-component crystals (or co-crystals) that contain the reference molecule as one of the components. Under still other circumstances, the landscape may include the crystal structures of molecules that are closely related to the reference molecule. The idea of a landscape is to facilitate the understanding of the process of crystallization. It includes all minima that can, in principle, be accessed by the molecule in question as it traverses the path from solution to the crystal. Isonicotinamide is a molecule that is known to form many co-crystals. We report here a 2:1 co-crystal of this amide with 3,5-dinitrobenzoic acid, wherein an unusual N-H···N hydrogen-bonded pattern is observed. This crystal structure offers some hints about the recognition processes between molecules that might be implicated during crystallization. Also included is a review of other recent results that illustrate the concept of the structural landscape.

A fluid-physics approach for determining the strength of the coupling between a multiwalled carbon nanotube and a liquid metal contact

We propose a novel fluid-physics formulation to calculate the strength of the coupling between a multiwalled carbon nanotube and a reservoir represented by a liquid-metal contact. Typically, this liquid metal can be mercury or gallium so that the molecules of these two metals are diatomic in liquid state allowing to treat the molecules in question as Morse oscillators whose quantum energy levels are the starting point for evaluating the tube–metal contact coupling.

PHP12 Then and Now: The Evolution of International Reference Pricing Globally

This study assesses the evolution of international reference pricing (IRP) across 34 countries, from 2006 to 2011. Its current influence on innovative drug pricing in the leading five European Union (EU) markets was also considered. An international reference pricing matrix was created and reviewed to see if the basket of countries referred by nations to price their pharmaceuticals had changed. Pharmaceutical prices were also used to review 2011 prices of five randomly selected innovative blockbuster molecules across EU-5 countries; the molecules in question were bevacizumab, adalimumab, etanercept, rosuvastatin and infliximab. The EU-5 markets lead the reference basket used by countries in their price setting process both in 2006 and 2011. Countries that reference these markets are varied and not limited to economically similar markets both within and outside the EU. While there have been additions and deletions, many countries have largely maintained their reference basket of countries. Since 2006, more emerging markets have become IRP prescribers. Unlike Brazil, and Turkey, which followed IRP prior to 2006 and exclusively use developed country prices to price their own products, the newer emerging market followers have also chosen to include neighbouring countries and/or economically similar country prices in their mechanism. A comparison of 2011 prices across the EU-5 markets showed less price variation between countries that followed IRP compared to those that followed free pricing, but prices were not necessarily lower. Countries using IRP still rely on EU-5 drug prices to price their medicines. However, new adopters of the mechanism are including similar and neighbouring countries to arrive at affordable rates and prevent parallel export. With more emerging markets rolling out IRP, it is notable that in the absence of a set formula that identifies the lowest prices, this technique is one of cost harmonization rather than cost containment.

Computation of magnetic shielding to simultaneously validate a crystal structure and assign a solid-state NMR spectrum

Abstract The validity of the structure of diterbutaline sulphate diacetic acid solvate in the Cambridge Crystallographic Database (code ZIYXAG) was checked using 13C solid-state NMR together with shielding computations and was found to be in error as far as the geometry of one acetic acid molecule is concerned. A new X-ray diffraction study confirmed the existence of an error and showed there was disorder in the molecule in question. Techniques of NMR crystallography were used to further understand the situation. Two-dimensional (13C, 1H) heteronuclear correlation experiments enabled the 1H chemical shifts to be determined, including those of the hydrogen-bonded protons. NMR was also used to monitor partial desolvation and the 13C spectrum of a new solvate, diterbutaline sulphate monoacetic acid, is reported. Heteronuclear correlation experiments on this new compound allowed most of its proton chemical shifts to be determined and showed that it had one hydrogen bond stronger than any of those in the diacetic acid solvate.

English

This study, based on mere considerations induced by the Special Theory of Relativity, has previously established the following relationship between the “minimum electronic energy” , and the related “classical vibration frequency” , in regards to electronic states of a given diatomic molecule: . Where is the reduced mass of the molecule, the “internuclear distance” associated with , and a Lorentz invariant dimensionless coefficient, insuring the equality; it depends only on the electronic structure of the molecule; therefore for electronic states configured similarly, we expect the coefficient , to remain practically the same; it takes values, roughly around unity. The framework in question is interesting, given that, for alike electronic states of a given molecule, versus , should behave linearly. This further, should allow the determination of, for the states in consideration. The expression is anyway valid for any diatomic molecule, along with a given . On the other hand, the “ground states” of bonds delineating chemical similarities, display “alike electronic configurations”. This means that, for such bonds, should remain practically the same. Thus, regarding the ground states of such molecules, versus should further be expected to behave linearly (the quantities of concern, now being exclusively assigned to the ground states of the molecules in question). We check this prediction successfully for the entire body of diatomic molecules and calculate , for different “chemical families”. The relationship we discover has got as much predictive power as that provided by the classical quantum mechanical tools; it is though incomparably simpler and faster. Key words: Special theory of relativity, quantum mechanics.

Gyration- and Inertia-Tensor-Based Collective Coordinates for Metadynamics. Application on the Conformational Behavior of Polyalanine Peptides and Trp-Cage Folding

Effective simulations of proteins, their complexes, and other amino-acid polymers such as peptides or peptoids are critically dependent on the performance of the simulation methods and their ability to map the conformational space of the molecule in question. The most important step in this process is the choice of the coordinates in which the conformational sampling will be executed and their uniqueness regarding the capability to unambiguously determine the energy minimum on the free-energy hypersurface. In the presented study, we show that metadynamics and chosen collective coordinates-the principal moments of the tensors of gyration and inertia, the principal radii of gyration around the principal axes, asphericity, acylindricity, and anisotropy-can be used as a powerful combination to map the conformational space of peptides and proteins. We show that the combination of these coordinates with metadynamics produces a powerful tool for the study of biologically relevant molecules.

Spectral investigation of the intramolecular charge-transfer in some aminotriazole Schiff bases

3-Amino-1,2,4-triazole Schiff bases were reported to contain intramolecular charge-transfer. The enhancing and depressing effects were remarkable as the substituent was changed from electron-donating to electron-withdrawing groups. The path of the resonating delocalization was reversed in the case of the p-NO 2 group. To validate these results we effectively used Weinhold et al's natural bond orbital analysis to assess the UV and FT-IR spectrophotometric monitoring of the change reflected in this phenomenon when the substituent in the benzene ring is altered. The NBO analysis was simulated by ab inito computations at the HF/6-31G(d) level of theory, in order to properly detect any possible presence of a hydrogen bond association. The changes occurring in electron occupancies of double-centered bonds, antibonding orbitals and in lone-pair orbitals appraised the results, as did the s and p character listings of the two-centered bonds and the simultaneous changes occurring in the geometric parameters of the molecules in question. Contrary to its normal preference, in these molecules the nitrogen used sp 2 hybrid orbitals for its interaction, housing its electron lone-pair in the third p hybrid orbital. Furthermore, NBO analysis reflected the presence of a very soft intramolecular hydrogen association (C–H⋯π), labelled by UV and FT-IR assignments, between the benzene and triazole rings in all Schiff bases but p-N(Me) 2. The n–π* stabilization energy decreased in the order: p-OH > p-OCH 3 > p-Cl > p-CH 3 > H > p-NO 2 > o-OH. The relation between the band position and Hammett substitution constant is interpreted in relation to the molecular structure.

HSQC–ADEQUATE correlation: a new paradigm for establishing a molecular skeleton

Various experimental methods have been developed to unequivocally identify vicinal neighbor carbon atoms. Variants of the HMBC experiment intended for this purpose have included 2J3J-HMBC and H2BC. The 1,1-ADEQUATE experiment, in contrast, was developed to accomplish the same goal but relies on the (1) J(CC) coupling between a proton-carbon resonant pair and the adjacent neighbor carbon. Hence, 1,1-ADEQUATE can identify non-protonated adjacent neighbor carbons, whereas the 2J3J-HMBC and H2BC experiments require both neighbor carbons to be protonated to operate. Since 1,1-ADEQUATE data are normally interpreted with close reference to an HSQC spectrum of the molecule in question, we were interested in exploring the unsymmetrical indirect covariance processing of multiplicity-edited GHSQC and 1,1-ADEQUATE spectra to afford an HSQC-ADEQUATE correlation spectrum that facilitates the extraction of carbon-carbon connectivity information. The HSQC-ADEQUATE spectrum of strychnine is shown and the means by which the carbon skeleton can be conveniently traced is discussed.

The centrosome and spindle as a ribonucleoprotein complex

The notion of nucleic acids in the spindle, and particularly, the centrosome has a long history of inquiry, doubt, and debate. However, the association of specific RNAs with these structures is now confirmed by several investigators. What their presence means for the assembly, function, and evolution of the cell division apparatus is not known; but with newly available information and probes, these are questions that can finally be addressed. The present article summarizes the history of this field, what we know about the molecules in question, and in light of these findings, emphasizes the need to view the cell division apparatus for what it is by definition, a ribonucleoprotein complex.

Precise electrochemical fabrication of sub-20 nm solid-state nanopores for single-moleculebiosensing

It has recently been shown that solid-state nanometer-scale pores (‘nanopores’) can be usedas highly sensitive single-molecule sensors. For example, electrophoretic translocation ofDNA, RNA and proteins through such nanopores has enabled both detection andstructural analysis of these complex biomolecules. Control over the nanopore size is criticalas the pore must be comparable in size to the analyte molecule in question. The mostwidely used fabrication methods are based on focused electron or ion beams and thusrequire (scanning) transmission electron microscopy and focused ion beam (FIB)instrumentation. Even though very small pores have been made using these approaches,several issues remain. These include the requirement of being restricted to ratherthin, mechanically less stable membranes, particularly for pore diameters in thesingle-digit nanometer range, lack of control of the surface properties at and insidethe nanopore, and finally, the fabrication cost. In the proof-of-concept study, wereport on a novel and simple route for fabricating metal nanopores with apparentdiameters below 20 nm using electrodeposition and real-time ionic current feedback insolution. This fabrication approach inserts considerable flexibility into the kindsof platforms that can be used and the nanopore membrane material. Startingfrom much larger pores, which are straightforward to make using FIB or othersemiconductor fabrication methods, we electrodeposit Pt at the nanopore interfacewhile monitoring its ionic conductance at the same time in a bi-potentiostaticsetup. Due to the deposition of Pt, the nanopore decreases in size, resulting in adecrease of the pore conductance. Once a desired pore conductance has beenreached, the electrodeposition process is stopped by switching the potential of themembrane electrode and the fabrication process is complete. Furthermore, wedemonstrate that these pores can be used for single-biomolecule analysis, such as that ofλ-DNA, which we use in a proof-of-concept study. Importantly, our approach is applicable tosingle nanopores as well as nanopore arrays, and can easily be extended to deposits ofmetal other than Pt.

Quantification of the Antioxidant Capacity of Different Molecules and Their Kinetic Antioxidant Efficiencies

A kinetic method has been developed to determine the antioxidant capacity of a variety of molecules. In this method, named the enzymatic kinetic method, the free radical of ABTS is generated continuously in the reaction medium by a peroxidase/ABTS/H(2)O(2) system. The presence of an antioxidant in the solution provokes a lag period in the accumulation of the free radical in the medium, and by studying this lag period it is possible to calculate the antioxidant capacity of the molecule in question. This antioxidant capacity, named the primary antioxidant capacity, will be quantified by n, the number of electrons donated per molecule of antioxidant, the effective concentration, EC50, and the antioxidant or antiradical power (ARP) (ARP = 1/EC50 = 2n). If the products arising from the reaction between the antioxidant and the free radical evolve by consuming more radical, a secondary antioxidant capacity is generated. To calculate this, a nonenzymatic test is proposed.

Raman Spectroscopic Detection of an Optically Trapped Single DNA Molecule

Optical trapping has opened up a number of biophysical fields because of its ability to hold and manipulate single cells and molecules. In addition, the force sensitivity of an optical trap has allowed for a number of studies in to the mechanics of the most basic biological systems such as DNA. However, the majority of these experiments are based on a measured force correlated to a detected displacement or extension of the molecule in question. Due to the low optical cross section of a single DNA molecule, for example, interacting light directly with the structure, in order to obtain a detailed spectrum, has not been possible.

The first-row hydrides and influences of orbital scaling on formally charged valence bond structures

A new technique has been used to make valence-bond calculations of the ground states of the hydrides of the first-row elements in their equilibrium geometries. A minimal atomic orbital basis set was used and the calculation was done with the orbitals scaled to their best atom value and also optimally scaled in the molecule. It was found that the amount of polar character contained in the calculated wave function varies strongly with the scaling. The direction of variance depends on the molecule in question. A set of restricted calculations with the configurations selected by an automatic method have also been done. The polarity of these wave functions is also calculated for comparison with the full valence-shell VB calculations.