Cistromic and genetic evidence that the vitamin D receptor mediates susceptibility to latitude-dependent autoimmune diseases.

The vitamin D receptor (VDR) is a ligand-activated transcription factor that regulates gene expression in many cell types, including immune cells. It requires binding of 1,25 dihydroxy vitamin D3 (1,25D3) for activation. Many autoimmune diseases show latitude-dependent prevalence and/or association with vitamin D deficiency, and vitamin D supplementation is commonly used in their clinical management. 1,25D3 is regulated by genes associated with the risk of autoimmune diseases and predominantly expressed in myeloid cells. We determined the VDR cistrome in monocytes and monocyte-derived inflammatory (DC1) and tolerogenic dendritic cells (DC2). VDR motifs were highly overrepresented in ChIP-Seq peaks in stimulated monocyte (40%), DC1 (21%) and DC2 (47%), P

Unremodeled endochondral bone is a major architectural component of the cortical bone of the rat (Rattus norvegicus).

The laboratory rat is one of the most frequently-used animal models for studying bone biology and skeletal diseases. Here we show that a substantial portion of the cortical bone of mature rats is primary endochondral bone, consisting of a disorganized arrangement of mineralized collagen fibers. We characterize the structure and mechanical properties of the cortical bone of the rat. We show that the cortical bone consists of two architecturally distinct regions. One region, consisting of well-organized circumferential lamellae (CLB), is located in the endosteal and/or the periosteal regions while another, disorganized region, is located in the more central region of the cortex. Unexpectedly, we found that the disorganized region contains many islands of highly mineralized cartilage. Micro tomography showed different structural and compositional properties of the two primary structural elements; the CLB region has lower mineral density, lower porosity, larger but fewer blood vessels and fewer lacunae. However, no difference was found in the average lacunar volume. Additionally the mean indentation modulus of the CLB region was lower than that of the disorganized region. The islands of calcified cartilage were found to be extremely stiff, with an indentation modulus of 33.4 ± 3.5GPa. We conclude that though the cortical bone of rats is in part lamellar, its architecture is markedly different from that of the cortical bone of humans, a fact that must be borne in mind when using the rat as a model animal for studies of human bone biology and disease.

Treatment of tarsal joint deformities with hinged transarticular external fixators in three young birds.

Pelvic limb deformities are common in many avian species. Three young birds, including a six-week-old Cockatoo and two three-month-old goslings, were presented with tarsal joint deformities. They were treated with an experimental prototype of a hinged linear external fixator, placed in a transarticular fashion, in order to maintain joint function during treatment. All birds had close to normal leg function at six to ten weeks postoperatively. These results suggest that the hinged external fixator may be a viable treatment option for tarsal joint deformities in young birds.

Two new classes of conopeptides inhibit the alpha1-adrenoceptor and noradrenaline transporter.

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha1-adrenoceptors (rho-TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi-MrIA and chi-MrIB from the mollusk-hunting C. marmoreus). rho-TIA and chi-MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

Assessment of Instrumental Activities of Daily Living in Older Adults with Subjective Cognitive Decline Using the Virtual Reality Functional Capacity Assessment Tool (VRFCAT).

BACKGROUND: Continuing advances in the understanding of Alzheimer's disease progression have inspired development of disease-modifying therapeutics intended for use in preclinical populations. However, identification of clinically meaningful cognitive and functional outcomes for individuals who are, by definition, asymptomatic remains a significant challenge. Clinical trials for prevention and early intervention require measures with increased sensitivity to subtle deficits in instrumental activities of daily living (IADL) that comprise the first functional declines in prodromal disease. Validation of potential endpoints is required to ensure measure sensitivity and reliability in the populations of interest. OBJECTIVES: The present research validates use of the Virtual Reality Functional Capacity Assessment Tool (VRFCAT) for performance-based assessment of IADL functioning in older adults (age 55+) with subjective cognitive decline. DESIGN: Cross-sectional validation study. SETTING: All participants were evaluated on-site at NeuroCog Trials, Durham, NC, USA. PARTICIPANTS: Participants included 245 healthy younger adults ages 20-54 (131 female), 247 healthy older adults ages 55-91 (151 female) and 61 older adults with subjective cognitive decline (SCD) ages 56-97 (45 female). MEASURES: Virtual Reality Functional Capacity Assessment Tool; Brief Assessment of Cognition App; Alzheimer's Disease Cooperative Study Prevention Instrument Project - Mail-In Cognitive Function Screening Instrument; Alzheimer's Disease Cooperative Study Instrumental Activities of Daily Living - Prevention Instrument, University of California, San Diego Performance-Based Skills Assessment - Validation of Intermediate Measures; Montreal Cognitive Assessment; Trail Making Test- Part B. RESULTS: Participants with SCD performed significantly worse than age-matched normative controls on all VRFCAT endpoints, including total completion time, errors and forced progressions (p≤0001 for all, after Bonferonni correction). Consistent with prior findings, both groups performed significantly worse than healthy younger adults (age 20-54). Participants with SCD also performed significantly worse than controls on objective cognitive measures. VRFCAT performance was strongly correlated with cognitive performance. In the SCD group, VRFCAT performance was strongly correlated with cognitive performance across nearly all tests with significant correlation coefficients ranging from 0.3 to 0.7; VRFCAT summary measures all had correlations greater than r=0.5 with MoCA performance and BAC App Verbal Memory (p<0.01 for all). CONCLUSIONS: Findings suggest the VRFCAT provides a sensitive tool for evaluation of IADL functioning in individuals with subjective cognitive decline. Strong correlations with cognition across groups suggest the VRFCAT may be uniquely suited for clinical trials in preclinical AD, as well as longitudinal investigations of the relationship between cognition and function.

Development of the technical capabilities needed to build and position a prepolarization coil for a magnetic resonance imaging magnet.

An experiment to show that a magnetic resonance imaging (MRI) magnet could be assembled around a patient, and used as part of a prepolarization system in which substantial transient forces are applied to parts of it, is described. The paper describes the circumstances that develop as a result of the application of the large transient fields used in this type of study, and outlines the reason for the tolerances that are permissible on the alignment of the system components. It then describes a test rig used to evaluate how the various problems might be overcome, and reports on the performance achieved with this rig. On the basis of this work, it appears that a system could be developed that would allow the application of these methods in clinical MRI.

Cell-based selection of internalizing fully human antagonistic antibodies directed against FLT3 for suppression of leukemia cell growth.

FMS-like tyrosine kinase 3 (FLT3) receptor is highly expressed in an array of hematological malignancies including approximately 90% of acute myelogenous leukemia (AML). Ligand stimulation of the receptor promotes the survival and proliferation of leukemia cells. Strategies targeting FLT3 using monoclonal antibodies may therefore constitute an effective therapeutic approach for these leukemia. Towards this, we selected a naïve antibody phage display library on both recombinant FLT3 receptor protein and FLT3-expressing leukemia cells using a tailored selection scheme that was designed to isolate antagonistic phage antibodies that not only interfere with receptor/ligand binding but also trigger receptor internalization upon cell surface binding. Phage antibodies were screened first for their ability to bind to cell surface receptor and induce receptor internalization, followed by their activity in blocking ligand-receptor interaction and neutralizing ligand-stimulated receptor activation and cell proliferation. We identified three fully human antibodies, EB10, A2IN, and D4-3, which bound specifically to both soluble and cell surface-expressed FLT3. All three antibodies were shown to be internalized upon binding to cell surface-expressed receptor in a time-dependent fashion. EB10 and D4-3 blocked ligand binding to the receptor with IC(50)s of 14 and 7 nM, respectively. Further, EB10 and D4-3 inhibited FLT3 ligand-induced receptor phosphorylation and cell proliferation in EOL-1 leukemia cells. Taken together, these results suggest that both EB10 and D4-3 may represent excellent therapeutic candidates for the treatment of FLT3-expressing human leukemia, both as unmodified antibodies and as conjugates of cytotoxic agents.

Slice-push, formation of grooves and the scale effect in cutting.

Three separate aspects of cutting are investigated which complement other papers on the mechanics of separation processes presented at this interdisciplinary Theo Murphy meeting. They apply in all types of cutting whether blades are sharp or blunt, and whether the material being cut is 'hard, stiff and strong' or 'soft, compliant and weak'. The first topic discusses why it is easier to cut when there is motion along (parallel to) the blade as well motion across (perpendicular to) the cutting edge, and the analysis is applied to optimization of blade geometries to produce minimum cutting forces and hence minimum damage to cut surfaces. The second topic concerns cutting with more than one edge with particular application to the formation of grooves in surfaces by hard pointed tools. The mechanics are investigated and applied to the topic of abrasive wear by hard particles. Traditional analyses say that abrasive wear resistance increases monotonically with the hardness of the workpiece, but we show that the fracture toughness of the surface material is also important, and that behaviour is determined by the toughness-to-hardness ratio rather than hardness alone. Scaling forms the third subject. As cutting is a branch of elasto-plastic fracture mechanics, cube-square energy scaling applies in which the important length scale is (ER/k (2)), where E is Young's modulus, R is the fracture toughness and k is the shear yield strength. Whether, in cutting, material is removed as ductile ribbons, as semi-ductile discontinuous chips, or by brittle 'knocking lumps out' is shown to depend on the depth of cut relative to this characteristic length parameter. Scaling in biology is called allometry and its relationship with engineering scaling is discussed. Some speculative predictions are made in relation to the action of teeth on food.

Effect of acyl chain length on the structure and motion of gramicidin A in lipid bilayers.

The transmembrane ion transport properties of gramicidin A have previously been shown to dependent on the nature of its lipid environment. Solid-state NMR spectroscopic studies of 13C-labelled analogues of gramicidin in oriented multilayers of phosphatidylcholine have shown that variation of the lipid hydrocarbon chain length has no effect on the structure or orientation of the peptide backbone.

Solid-state 13C-NMR studies of the effects of sodium ions on the gramicidin A ion channel.

End-to-end helical dimers of gramicidin A form transmembrane pores in lipid bilayers, through which monovalent ions may pass. The groups within the peptide that interact with these ions have been studied by application of solid-state spectroscopic methods to a series of gramicidin A analogues synthesized with 13C in selected peptide carbonyl groups. The resonances of D-Leu10, D-Leu12 and D-Leu14 analogues were perturbed in the presence of 0.16 M sodium ions, whereas the resonances of the carbonyls of Gly2, Ala3, D-Leu4 and Val7, which are closer to the formylated N-terminal end of the peptide, were unaffected. The observed changes in chemical shift anisotropy are indicative of a change in orientation of the abovementioned leucine carbonyls.

Solution structure of the third immunoglobulin domain of the neural cell adhesion molecule N-CAM: can solution studies define the mechanism of homophilic binding?

Homophilic binding of the neural cell adhesion molecule (N-CAM) mediates the calcium-independent cell-cell adhesion that is involved in neuronal development. Two hypotheses have been advanced for the mechanism of homophilic binding. Cell-based experiments have implicated each of the five extracellular immunoglobulin (Ig) domains of N-CAM in the homophilic adhesion interaction, and have predicted that the third domain (Ig III) self-associates. The alternative hypothesis is based on solution observations, which implicate a specific antiparallel interaction between the first two Ig domains (Ig I and Ig II). In order to test these hypotheses, we have determined a high-resolution solution structure of recombinant Ig III (sequence derived from chicken N-CAM) and examined the aggregation behavior of isolated Ig domains in solution. The structure shows that Ig III adopts a canonical Ig fold, in which the beta strands ABED and A'GFCC' form two beta sheets that are linked by a disulfide bond. In contrast to the demonstrated aggregation of Ig III on solid supports, we were unable to demonstrate self-association of Ig III under any of a variety of solution conditions. The structure shows that the surface of Ig III is dominated by two large acidic patches, which may explain our failure to observe self-association in solution. To evaluate the involvement of the Ig I-Ig II interaction in cell-cell adhesion, we designed a point mutation in Ig I (F19S) that proved sufficient to abrogate the Ig I-Ig II interaction seen in solution. However, the introduction of this mutation into full-length N-CAM expressed in COS-7 cells failed to affect N-CAM-mediated cell-cell adhesion. The inability to observe Ig III self-association in solution, combined with the failure of the F19S mutation to affect N-CAM-mediated cell-cell adhesion, suggests that, although solution studies can give important insights into the structures of individual domains, the interactions observed in solution between the domains may not be representative of the interactions that occur on the cell surface.

Assessment of Age-Related Differences in Functional Capacity Using the Virtual Reality Functional Capacity Assessment Tool (VRFCAT).

Clinical trials for primary prevention and early intervention in preclinical AD require measures of functional capacity with improved sensitivity to deficits in healthier, non-demented individuals. To this end, the Virtual Reality Functional Capacity Assessment Tool (VRFCAT) was developed as a direct performance-based assessment of functional capacity that is sensitive to changes in function across multiple populations. Using a realistic virtual reality environment, the VRFCAT assesses a subject's ability to complete instrumental activities associated with a shopping trip. The present investigation represents an initial evaluation of the VRFCAT as a potential co-primary measure of functional capacity in healthy aging and preclinical MCI/AD by examining test-retest reliability and associations with cognitive performance in healthy young and older adults. The VRFCAT was compared and contrasted with the UPSA-2-VIM, a traditional performance-based assessment utilizing physical props. Results demonstrated strong age-related differences in performance on each VRFCAT outcome measure, including total completion time, total errors, and total forced progressions. VRFCAT performance showed strong correlations with cognitive performance across both age groups. VRFCAT Total Time demonstrated good test-retest reliability (ICC=.80 in young adults; ICC=.64 in older adults) and insignificant practice effects, indicating the measure is suitable for repeated testing in healthy populations. Taken together, these results provide preliminary support for the VRFCAT as a potential measure of functionally relevant change in primary prevention and preclinical AD/MCI trials.

NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor.

The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB(1-2)) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB(1-2) has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and H alpha chemical shifts and the low phi and psi angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB(1-2) structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.

Conformation of sarafotoxin-6b in aqueous solution determined by NMR spectroscopy and distance geometry.

The solution structure of sarafotoxin-6b in water has been determined using high-resolution NMR spectroscopy. 127 proton-proton distance measurements and three phi dihedral angle constraints derived from NMR spectra were used to calculate the solution structure using a combination of distance geometry and restrained molecular dynamics. The major structural feature of the resulting family of five structures was a right-handed alpha-helix extending from K9 to Q17. In contrast, the C-terminal region of the peptide appears not to adopt a preferred conformation in aqueous solution. The present structure is compared with those previously determined for endothelin peptides in non-aqueous solvents.

Association between the first two immunoglobulin-like domains of the neural cell adhesion molecule N-CAM.

The extracellular domain of N-CAM contains five immunoglobulin-like (Ig) and two fibronectin type III-like domains and facilitates cell-cell binding through multiple, weak interdomain interactions. NMR spectroscopy indicated that the two N-terminal Ig-like domains from chicken N-CAM (Ig I and Ig II) interact with millimolar affinity. Physico-chemical studies show that this interaction is significantly amplified when the domains are covalently linked, consistent with an antiparallel domain arrangement. The binding of the two individual domains and the dimerization of the concatenated protein were essentially independent of salt, up to a concentration of 200 mM. The residues in Ig I involved in the interaction map to the BED strands of the beta sandwich, and delineate a largely hydrophobic patch.

Kinetics of 4-aminobutyrate:2-oxoglutarate aminotransferase from Nippostrongylus brasiliensis.

A gamma-aminobutyric acid transferase (4-aminobutyrate:2-oxoglutarate aminotransferase; EC 2.6.1.19) preparation from Nippostrongylus brasiliensis was found to contain only one peak of enzyme activity with a highly basic pI of 10.5 when analysed by isoelectric focusing and chromatofocusing. This material was used in kinetic studies to demonstrate that the parasite enzyme reaction mechanism conforms to the usual binary, non-sequential ('Bi Bi Ping Pong') type found with aminotransferases. The Km for 4-aminobutyrate was 0.33 mM, the Km for 2-oxoglutarate was 0.57 mM and Ki for glutamate was 0.35 mM. In holoenzyme reconstitution experiments with the cofactor, pyridoxal 5-phosphate, the KD was 1.54 microM. The values are comparable to those reported for other tissues. Only 2-oxoglutarate could function as the keto acid substrate whereas several amino acids besides 4-aminobutyrate (beta-alanine, alpha-L-alanine, L-aspartate and L-arginine) could apparently act as substrate although the possible presence of other amino acid:2-oxoglutarate aminotransferases was not excluded. In preliminary studies on the usefulness of conventional substrate analogues as parasite gamma-aminobutyric acid transferase inhibitors only canaline was effective.

Inhibition of acetylcholinesterase secretion from Nippostrongylus brasiliensis by benzimidazole anthelmintics.

Treatment of rats infected with Nippostrongylus brasiliensis with a single, oral therapeutic dose of the anthelmintic benzimidazole carbamates oxfendazole or mebendazole resulted, 24 hr later, in a marked reduction (60-90%) in the secretion of a low molecular weight acetylcholinesterase from the parasites when they were incubated in vitro. This effect coincided with the expulsion of parasites from the host as a result of the therapy. When parasites were incubated in vitro with 0.1 mM oxfendazole, mebendazole, flubendazole, parbendazole, cambendazole or thiabendazole a similar effect was observed; with oxfendazole and mebendazole the effect was apparent within 1 hr and lasted for at least 4 hr after removal to fresh, drug-free medium. Whether treated in the host or in vitro the reduction in secretion was balanced by an equivalent rise in acetylcholinesterase activity within the parasites. It is suggested that the inhibition of protein secretion may be a specific manifestation of a general effect of these compounds on microtubule function.

The nucleotidase of Boophilus microplus and its relationship to enzymes from the rat and Escherichia coli.

Boophilus microplus contains a nucleotidase-like enzyme which is able to hydrolyze a range of nucleoside 5'-mono-, di- and triphosphates to the nucleoside. Its relationship to several other nucleotide hydrolyzing enzymes has been explored. Limited peptide sequencing shows similarities to both mammalian nucleotidases and the Escherichia coli uridine diphosphate sugar hydrolase. The tick enzyme also hydrolyzes UDP-glucose, though by a mechanism different to that of the bacterial enzyme. On the other hand, it resembles the mammalian nucleotidases in that there is evidence that it is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor.