Cerebrospinal fluid markers including trefoil factor 3 are associated with neurodegeneration in amyloid-positive individuals.

We aimed to identify cerebrospinal fluid (CSF) biomarkers associated with neurodegeneration in individuals with and without CSF evidence of Alzheimer pathology. We investigated 287 Alzheimer's Disease Neuroimaging Initiative (ADNI) subjects (age=74.9±6.9; 22/48/30% with Alzheimer's disease/mild cognitive impairment/controls) with CSF multiplex analyte data and serial volumetric MRI. We calculated brain and hippocampal atrophy rates, ventricular expansion and Mini Mental State Examination decline. We used false discovery rate corrected regression analyses to assess associations between CSF variables and atrophy rates in individuals with and without amyloid pathology, adjusting in stages for tau, baseline volume, p-tau, age, sex, ApoE4 status and diagnosis. Analytes showing statistically significant independent relationships were entered into reverse stepwise analyses. Adjusting for tau, baseline volume, p-tau, age, sex and ApoE4, 4/83 analytes were significantly independently associated with brain atrophy rate, 1/83 with ventricular expansion and 2/83 with hippocampal atrophy. The strongest CSF predictor for the three atrophy measures was low trefoil factor 3 (TFF3). High cystatin C (CysC) was associated with higher whole brain atrophy and hippocampal atrophy rates. Lower levels of vascular endothelial growth factor and chromogranin A (CrA) were associated with higher whole brain atrophy. In exploratory reverse stepwise analyses, lower TFF3 was associated with higher rates of whole brain, hippocampal atrophy and ventricular expansion. Lower levels of CrA were associated with higher whole brain atrophy rate. The relationship between low TFF3 and increased hippocampal atrophy rate remained after adjustment for diagnosis. We identified a series of CSF markers that are independently associated with rate of neurodegeneration in amyloid-positive individuals. TFF3, a substrate for NOTCH processing may be an important biomarker of neurodegeneration across the Alzheimer spectrum

Enhancing Lentiviral and Alpharetroviral Transduction of Human Hematopoietic Stem Cells for Clinical Application

Ex vivo retroviral gene transfer into CD34+ hematopoietic stem and progenitor cells (HSPCs) has demonstrated remarkable clinical success in gene therapy for monogenic hematopoietic disorders. However, little attention has been paid to enhancement of culture and transduction conditions to achieve reliable effects across patient and disease contexts and to maximize potential vector usage and reduce treatment cost. We systematically tested three HSPC culture media manufactured to cGMP and eight previously described transduction enhancers (TEs) to develop a state-of-the-art clinically applicable protocol. Six TEs enhanced lentiviral (LV) and five TEs facilitated alpharetroviral (ARV) CD34+ HSPC transduction when used alone. Combinatorial TE application tested with LV vectors yielded more potent effects, with up to a 5.6-fold increase in total expression of a reporter gene and up to a 3.8-fold increase in VCN. Application of one of the most promising combinations, the poloxamer LentiBOOST and protamine sulfate, for GMP-compliant manufacturing of a clinical-grade advanced therapy medicinal product (ATMP) increased total VCN by over 6-fold, with no major changes in global gene expression profiles or inadvertent loss of CD34+CD90+ HSPC populations. Application of these defined culture and transduction conditions is likely to significantly improve ex vivo gene therapy manufacturing protocols for HSPCs and downstream clinical efficacy

The Functional DRD3 Ser9Gly Polymorphism (rs6280) Is Pleiotropic, Affecting Reward as Well as Movement

Abnormalities of motivation and behavior in the context of reward are a fundamental component of addiction and mood disorders. Here we test the effect of a functional missense mutation in the dopamine 3 receptor (DRD3) gene (ser9gly, rs6280) on reward-associated dopamine (DA) release in the striatum. Twenty-six healthy controls (HCs) and 10 unmedicated subjects with major depressive disorder (MDD) completed two positron emission tomography (PET) scans with [11C]raclopride using the bolus plus constant infusion method. On one occasion subjects completed a sensorimotor task (control condition) and on another occasion subjects completed a gambling task (reward condition). A linear regression analysis controlling for age, sex, diagnosis, and self-reported anhedonia indicated that during receipt of unpredictable monetary reward the glycine allele was associated with a greater reduction in D2/3 receptor binding (i.e., increased reward-related DA release) in the middle (anterior) caudate (p<0.01) and the ventral striatum (p<0.05). The possible functional effect of the ser9gly polymorphism on DA release is consistent with previous work demonstrating that the glycine allele yields D3 autoreceptors that have a higher affinity for DA and display more robust intracellular signaling. Preclinical evidence indicates that chronic stress and aversive stimulation induce activation of the DA system, raising the possibility that the glycine allele, by virtue of its facilitatory effect on striatal DA release, increases susceptibility to hyperdopaminergic responses that have previously been associated with stress, addiction, and psychosis

Successful Protein Extraction from Over-Fixed and Long-Term Stored Formalin-Fixed Tissues

One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease

Differential cross sections and spin density matrix elements for the reaction gamma p -> p omega

High-statistics differential cross sections and spin density matrix elements for the reaction gamma p -> p omega have been measured using the CLAS at Jefferson Lab for center-of-mass (CM) energies from threshold up to 2.84 GeV. Results are reported in 112 10-MeV wide CM energy bins, each subdivided into cos(theta_CM) bins of width 0.1. These are the most precise and extensive omega photoproduction measurements to date. A number of prominent structures are clearly present in the data. Many of these have not previously been observed due to limited statistics in earlier measurements

Exclusive ρ0\rho^0 electroproduction on the proton at CLAS

The $e p\to e^\prime p \rho^0$ reaction has been measured, using the 5.754 GeV electron beam of Jefferson Lab and the CLAS detector. This represents the largest ever set of data for this reaction in the valence region. Integrated and differential cross sections are presented. The $W$, $Q^2$ and $t$ dependences of the cross section are compared to theoretical calculations based on $t$-channel meson-exchange Regge theory on the one hand and on quark handbag diagrams related to Generalized Parton Distributions (GPDs) on the other hand. The Regge approach can describe at the $\approx$ 30% level most of the features of the present data while the two GPD calculations that are presented in this article which succesfully reproduce the high energy data strongly underestimate the present data. The question is then raised whether this discrepancy originates from an incomplete or inexact way of modelling the GPDs or the associated hard scattering amplitude or whether the GPD formalism is simply inapplicable in this region due to higher-twists contributions, incalculable at present.Comment: 29 pages, 29 figure

Effect of solution saturation state and temperature on diopside dissolution

Steady-state dissolution rates of diopside are measured as a function of solution saturation state using a titanium flow-through reactor at pH 7.5 and temperature ranging from 125 to 175°C. Diopside dissolved stoichiometrically under all experimental conditions and rates were not dependent on sample history. At each temperature, rates continuously decreased by two orders of magnitude as equilibrium was approached and did not exhibit a dissolution plateau of constant rates at high degrees of undersaturation. The variation of diopside dissolution rates with solution saturation can be described equally well with a ion exchange model based on transition state theory or pit nucleation model based on crystal growth/dissolution theory from 125 to 175°C. At 175°C, both models over predict dissolution rates by two orders of magnitude indicating that a secondary phase precipitated in the experiments. The ion exchange model assumes the formation of a Si-rich, Mg-deficient precursor complex. Lack of dependence of rates on steady-state aqueous calcium concentration supports the formation of such a complex, which is formed by exchange of protons for magnesium ions at the surface. Fit to the experimental data yields [Formula: see text] where the Mg-H exchange coefficient, n = 1.39, the apparent activation energy, E(a )= 332 kJ mol(-1), and the apparent rate constant, k = 10(41.2 )mol diopside cm(-2 )s(-1). Fits to the data with the pit nucleation model suggest that diopside dissolution proceeds through retreat of steps developed by nucleation of pits created homogeneously at the mineral surface or at defect sites, where homogeneous nucleation occurs at lower degrees of saturation than defect-assisted nucleation. Rate expressions for each mechanism (i) were fit to [Formula: see text] where the step edge energy (α) for homogeneously nucleated pits were higher (275 to 65 mJ m(-2)) than the pits nucleated at defects (39 to 65 mJ m(-2)) and the activation energy associated with the temperature dependence of site density and the kinetic coefficient for homogeneously nucleated pits (E(b-homogeneous )= 2.59 × 10(-16 )mJ K(-1)) were lower than the pits nucleated at defects (E(b-defect assisted )= 8.44 × 10(-16 )mJ K(-1))

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Cytotoxic targeting of F9 teratocarcinoma tumours with anti-ED-B fibronectin scFv antibody modified liposomes

We prepared small unilamellar liposomes derivatised with single chain antibody fragments specific for the ED-B domain of B-fibronectin. This extracellular matrix associated protein is expressed around newly forming blood vessels in the vicinity of many types of tumours. The single chain antibody fragments were functionalised by introduction of C-terminal cysteines and linked to liposomes via maleimide groups located at the terminal ends of poly(ethylene glycol) modified phospholipids. The properties of these anti-ED-B single chain antibody fragments-liposomes were analysed in vitro on ED-B fibronectin expressing Caco-2 cells and in vivo by studying their biodistribution and their therapeutic potential in mice bearing subcutanous F9 teratocarcinoma tumours. Radioactively labelled (114mIndium) single chain antibody fragments-liposomes accumulated in the tumours at 2–3-fold higher concentrations during the first 2 h after i.v. injection compared to unmodified liposomes. After 6–24 h both liposome types were found in similar amounts (8–10% injected dose g−1) in the tumours. Animals treated i.v. with single chain antibody fragments-liposomes containing the new cytotoxic agent 2′-deoxy-5-fluorouridylyl-N4-octadecyl-1-β-D-arabinofuranosylcytosine (30 mg kg-1 per dose, five times every 24 h) showed a reduction of tumour growth by 62–90% determined on days 5 and 8, respectively, compared to animals receiving control liposomes. Histological analysis revealed a marked reduction of F9 tumour cells and excessive deposition of fibronectin in the extracellular matrix after treatment with single chain antibody fragments-2-dioxy-5-fluorouridylyl-N4-octadecyl-1-β-D-arabinofuranosylcytosine-liposomes. Single chain antibody fragments-liposomes targeted to ED-B fibronectin positive tumours therefore represent a promising and versatile novel drug delivery system for the treatment of tumours