Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing tile PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive width MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.

Molecular mimicry: Can epitope mimicry induce autoimmune disease?

Mimicry of host antigens by infectious agents may induce cross-reactive autoimmune responses to epitopes within host proteins which, in susceptible individuals, may tip the balance of immunological response versus tolerance toward response and subsequently lead to autoimmune disease. Epitope mimicry may indeed be involved in the pathogenesis of several diseases such as post-viral myocarditis or Chagas disease, but for many other diseases in which it has been implicated, such as insulin-dependent diabetes mellitis or rheumatoid arthritis, convincing evidence is still lacking. Even if an epitope mimic can support a cross-reactive T or B cell response in vitro, its ability to induce an autoimmune disease in vivo will depend upon the appropriate presentation of the mimicked host antigen in the target tissue and, in the case of T cell mimics, the ability of the mimicking epitope to induce a proliferative rather than anergizing response upon engagement of the MHC-peptide complex with the T cell receptor. B cell presentation of mimicking foreign antigen to T cells is a possible mechanism for instigating an autoimmune response to self antigens that in turn can lead to autoimmune disease under particular conditions of antigen presentation, secondary signalling and effector cell repertoire. In this review evidence in support of epitope mimicry is examined in the light of the necessary immunological considerations of the theory.

Analysis of complex molecular systems: The impact of multivariate analysis for resolving the interactions of small molecules with biopolymers - a review

This review is focused on the impact of chemometrics for resolving data sets collected from investigations of the interactions of small molecules with biopolymers. These samples have been analyzed with various instrumental techniques, such as fluorescence, ultraviolet–visible spectroscopy, and voltammetry. The impact of two powerful and demonstrably useful multivariate methods for resolution of complex data—multivariate curve resolution–alternating least squares (MCR–ALS) and parallel factor analysis (PARAFAC)—is highlighted through analysis of applications involving the interactions of small molecules with the biopolymers, serum albumin, and deoxyribonucleic acid. The outcomes illustrated that significant information extracted by the chemometric methods was unattainable by simple, univariate data analysis. In addition, although the techniques used to collect data were confined to ultraviolet–visible spectroscopy, fluorescence spectroscopy, circular dichroism, and voltammetry, data profiles produced by other techniques may also be processed. Topics considered including binding sites and modes, cooperative and competitive small molecule binding, kinetics, and thermodynamics of ligand binding, and the folding and unfolding of biopolymers. Applications of the MCR–ALS and PARAFAC methods reviewed were primarily published between 2008 and 2013.

Pathogens penetrating the central nervous system: Infection pathways and the cellular and molecular mechanisms of invasion

The brain is well protected against microbial invasion by cellular barriers, such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). In addition, cells within the central nervous system (CNS) are capable of producing an immune response against invading pathogens. Nonetheless, a range of pathogenic microbes make their way to the CNS, and the resulting infections can cause significant morbidity and mortality. Bacteria, amoebae, fungi, and viruses are capable of CNS invasion, with the latter using axonal transport as a common route of infection. In this review, we compare the mechanisms by which bacterial pathogens reach the CNS and infect the brain. In particular, we focus on recent data regarding mechanisms of bacterial translocation from the nasal mucosa to the brain, which represents a little explored pathway of bacterial invasion but has been proposed as being particularly important in explaining how infection with Burkholderia pseudomallei can result in melioidosis encephalomyelitis.

Calibration, estimation, and sampling issues of car-following parameters

Drivers behave in different ways, and these different behaviors are a cause of traffic disturbances. A key objective for simulation tools is to correctly reproduce this variability, in particular for car-following models. From data collection to the sampling of realistic behaviors, a chain of key issues must be addressed. This paper discusses data filtering, robustness of calibration, correlation between parameters, and sampling techniques of acceleration-time continuous car-following models. The robustness of calibration is systematically investigated with an objective function that allows confidence regions around the minimum to be obtained. Then, the correlation between sets of calibrated parameters and the validity of the joint distributions sampling techniques are discussed. This paper confirms the need for adapted calibration and sampling techniques to obtain realistic sets of car-following parameters, which can be used later for simulation purposes.

Influential parameters on particle concentration and size distribution in the mainstream of e-cigarettes

Electronic cigarette-generated mainstream aerosols were characterized in terms of particle number concentrations and size distributions through a Condensation Particle Counter and a Fast Mobility Particle Sizer spectrometer, respectively. A thermodilution system was also used to properly sample and dilute the mainstream aerosol. Different types of electronic cigarettes, liquid flavors, liquid nicotine contents, as well as different puffing times were tested. Conventional tobacco cigarettes were also investigated. The total particle number concentration peak (for 2-s puff), averaged across the different electronic cigarette types and liquids, was measured equal to 4.39 ± 0.42 × 109 part. cm−3, then comparable to the conventional cigarette one (3.14 ± 0.61 × 109 part. cm−3). Puffing times and nicotine contents were found to influence the particle concentration, whereas no significant differences were recognized in terms of flavors and types of cigarettes used. Particle number distribution modes of the electronic cigarette-generated aerosol were in the 120–165 nm range, then similar to the conventional cigarette one.

Molecular and pathological insights into Chlamydia pecorum-associated sporadic bovine encephalomyelitis (SBE) in Western Australia

Background Despite its global recognition as a ruminant pathogen, cases of Chlamydia pecorum infection in Australian livestock are poorly documented. In this report, a C. pecorum specific Multi Locus Sequence Analysis scheme was used to characterise the C. pecorum strains implicated in two cases of sporadic bovine encephalomyelitis confirmed by necropsy, histopathology and immunohistochemistry. This report provides the first molecular evidence for the presence of mixed infections of C. pecorum strains in Australian cattle. Case presentation Affected animals were two markedly depressed, dehydrated and blind calves, 12 and 16 weeks old. The calves were euthanized and necropsied. In one calf, a severe fibrinous polyserositis was noted with excess joint fluid in all joints whereas in the other, no significant lesions were seen. No gross abnormalities were noted in the brain of either calf. Histopathological lesions seen in both calves included: multifocal, severe, subacute meningoencephalitis with vasculitis, fibrinocellular thrombosis and malacia; diffuse, mild, acute interstitial pneumonia; and diffuse, subacute epicarditis, severe in the calf with gross serositis. Immunohistochemical labelling of chlamydial antigen in brain, spleen and lung from the two affected calves and brain from two archived cases, localised the antigen to the cytoplasm of endothelium, mesothelium and macrophages. C. pecorum specific qPCR, showed dissemination of the pathogen to multiple organs. Phylogenetic comparisons with other C. pecorum bovine strains from Australia, Europe and the USA revealed the presence of two genetically distinct sequence types (ST). The predominant ST detected in the brain, heart, lung and liver of both calves was identical to the C. pecorum ST previously described in cases of SBE. A second ST detected in an ileal tissue sample from one of the calves, clustered with previously typed faecal bovine isolates. Conclusion This report provides the first data to suggest that identical C. pecorum STs may be associated with SBE in geographically separated countries and that these may be distinct from those found in the gastrointestinal tract. This report provides a platform for further investigations into SBE and for understanding the genetic relationships that exist between C. pecorum strains detected in association with other infectious diseases in livestock.

Synthesis of a multimodal molecular imaging probe based on a hyperbranched polymer architecture

Molecular imaging is utilised in modern medicine to aid in the diagnosis and treatment of disease by allowing its spatiotemporal state to be examined in vivo. This study focuses on the development of novel multimodal molecular imaging agents based on hyperbranched polymers that combine the complementary capabilities of optical fluorescence imaging and positron emission tomography-computed tomography (PET/CT) into one construct. RAFT-mediated polymerisation was used to prepare two hydrophilic hyperbranched polymers that were differentiated by their size and level of branching. The multiple functional end-groups facilitated covalent attachment of both near infrared fluorescent dyes for optical imaging, as well as a copper chelator allowing binding of 64Cu as a PET radio nuclei. In vivo multimodal imaging of mice using PET/CT and planar optical imaging was first used to assess the biodistribution of the polymeric materials and it was shown that the larger and more branched polymer had a significantly longer circulation time. The larger constructs were also shown to exhibit enhanced accumulation in solid tumours in a murine B16 melanoma model. Importantly, it was demonstrated that the PET modality gave rise to high sensitivity immediately after injection of the agent, while the optical modality facilitated extended longitudinal studies, thus highlighting how the complementary capabilities of the molecular imaging agents can be useful for studying various diseases, including cancer.

Molecular imaging with polymers

Polymers open up new possibilities in the field of molecular imaging, allowing sensitive and robust agents that can be imaged over long periods of time. This review highlights some recent advances in polymeric molecular imaging agents in both (pre)clinical and emerging applications.

Nitrogen-doped graphene films from chemical vapor deposition of pyridine: Influence of process parameters on the electrical and optical properties

Graphene films were produced by chemical vapor deposition (CVD) of pyridine on copper substrates. Pyridine-CVD is expected to lead to doped graphene by the insertion of nitrogen atoms in the growing sp2 carbon lattice, possibly improving the properties of graphene as a transparent conductive film. We here report on the influence that the CVD parameters (i.e., temperature and gas flow) have on the morphology, transmittance, and electrical conductivity of the graphene films grown with pyridine. A temperature range between 930 and 1070 °C was explored and the results were compared to those of pristine graphene grown by ethanol-CVD under the same process conditions. The films were characterized by atomic force microscopy, Raman and X-ray photoemission spectroscopy. The optical transmittance and electrical conductivity of the films were measured to evaluate their performance as transparent conductive electrodes. Graphene films grown by pyridine reached an electrical conductivity of 14.3 × 105 S/m. Such a high conductivity seems to be associated with the electronic doping induced by substitutional nitrogen atoms. In particular, at 930 °C the nitrogen/carbon ratio of pyridine-grown graphene reaches 3%, and its electrical conductivity is 40% higher than that of pristine graphene grown from ethanol-CVD.

Significant deterioration in nanomechanical quality occurs through incomplete extrafibrillar mineralization in rachitic bone: Evidence from in-situ synchrotron X-ray scattering and backscattered electron imaging

Bone diseases such as rickets and osteoporosis cause significant reduction in bone quantity and quality, which leads to mechanical abnormalities. However, the precise ultrastructural mechanism by which altered bone quality affects mechanical properties is not clearly understood. Here we demonstrate the functional link between altered bone quality (reduced mineralization) and abnormal fibrillar-level mechanics using a novel, real-time synchrotron X-ray nanomechanical imaging method to study a mouse model with rickets due to reduced extrafibrillar mineralization. A previously unreported N-ethyl-N-nitrosourea (ENU) mouse model for hypophosphatemic rickets (Hpr), as a result of missense Trp314Arg mutation of the phosphate regulating gene with homologies to endopeptidase on the X chromosome (Phex) and with features consistent with X-linked hypophosphatemic rickets (XLHR) in man, was investigated using in situ synchrotron small angle X-ray scattering to measure real-time changes in axial periodicity of the nanoscale mineralized fibrils in bone during tensile loading. These determine nanomechanical parameters including fibril elastic modulus and maximum fibril strain. Mineral content was estimated using backscattered electron imaging. A significant reduction of effective fibril modulus and enhancement of maximum fibril strain was found in Hpr mice. Effective fibril modulus and maximum fibril strain in the elastic region increased consistently with age in Hpr and wild-type mice. However, the mean mineral content was ∼21% lower in Hpr mice and was more heterogeneous in its distribution. Our results are consistent with a nanostructural mechanism in which incompletely mineralized fibrils show greater extensibility and lower stiffness, leading to macroscopic outcomes such as greater bone flexibility. Our study demonstrates the value of in situ X-ray nanomechanical imaging in linking the alterations in bone nanostructure to nanoscale mechanical deterioration in a metabolic bone disease. Copyright

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)18-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

Estimating the parameters of stochastic volatility models using option price data

This article describes a maximum likelihood method for estimating the parameters of the standard square-root stochastic volatility model and a variant of the model that includes jumps in equity prices. The model is fitted to data on the S&P 500 Index and the prices of vanilla options written on the index, for the period 1990 to 2011. The method is able to estimate both the parameters of the physical measure (associated with the index) and the parameters of the risk-neutral measure (associated with the options), including the volatility and jump risk premia. The estimation is implemented using a particle filter whose efficacy is demonstrated under simulation. The computational load of this estimation method, which previously has been prohibitive, is managed by the effective use of parallel computing using graphics processing units (GPUs). The empirical results indicate that the parameters of the models are reliably estimated and consistent with values reported in previous work. In particular, both the volatility risk premium and the jump risk premium are found to be significant.

Molecular Origin of the Intrinsic Bending Force for Helical Morphology Observed in Chiral Amphiphilic Assemblies: Concentration and Size Dependence

The formation of the helical morphology in monolayers and bilayers of chiral amphiphilic assemblies is believed to be driven at least partly by the interactions at the chiral centers of the amphiphiles. However, a detailed microscopic understanding of these interactions and their relation with the helix formation is still not clear. In this article a study of the molecular origin of the chirality-driven helix formation is presented by calculating, for the first time, the effective pair potential between a pair of chiral molecules. This effective potential depends on the relative sizes of the groups attached to the two chiral centers, on the orientation of the amphiphile molecules, and also on the distance between them. We find that for the mirror-image isomers (in the racemic modification) the minimum energy conformation is a nearly parallel alignment of the molecules. On the other hand, the same for a pair of molecules of one kind of enantiomer favors a tilt angle between them, thus leading to the formation of a helical morphology of the aggregate. The tilt angle is determined by the size of the groups attached to the chiral centers of the pair of molecules considered and in many cases predicted it to be close to 45 degrees. The present study, therefore, provides a molecular origin of the intrinsic bending force, suggested by Helfrich (J. Chem. Phys. 1986, 85, 1085-1087), to be responsible for the formation of helical structure. This effective potential may explain many of the existing experimental results, such as the size and the concentration dependence of the formation of helical morphology. It is further found that the elastic forces can significantly modify the pitch predicted by the chiral interactions alone and that the modified real pitch is close to the experimentally observed value. The present study is expected to provide a starting point for future microscopic studies.

Chiral molecular self-assembly of phospholipid tubules: A circular dichroism study

We report on spectroscopic studies of the chiral structure in phospholipid tubules formed in mixtures of alcohol and water. Synthetic phospholipids containing diacetylenic moieties in the acyl chains self-assemble into hollow, cylindrical tubules in appropriate conditions. Circular dichroism provides a direct measure of chirality of the molecular structure. We find that the CD spectra of tubules formed in mixtures of alcohol and water depends strongly on the alcohol used and the lipid concentration. The relative spectral intensity of different circular dichroism bands correlates with the number of bilayers observed using microscopy. The results provide experimental evidence that tubule formation is based on chiral packing of the lipid molecules and that interbilayer interactions are important to the tubule structure