Utility of Polygenic Risk Scoring to Predict Cognitive Impairment as Measured by Preclinical Alzheimer Cognitive Composite Score.

Background: The utility of Polygenic Risk Scores (PRS) is gaining increasing attention for generating an individual genetic risk profile to predict subsequent likelihood of future onset of Alzheimer's disease (AD), especially those carry two copies of the APOE E3 allele, currently considered at neutral risk in all populations studied. Objectives: To access the performance of PRS in predicting individuals whilst pre-symptomatic or with mild cognitive impairment who are at greatest risk of progression of cognitive impairment due to Alzheimer's Disease from the Alzheimer's Disease Neuroimaging Initiative (ADNI) as measured by the Preclinical Alzheimer Cognitive Composite (PACC) score profile. Design: A longitudinal analysis of data from the ADNI study conducted across over 50 sites in the US and Canada. Setting: Multi-centre genetics study. Participants: 594 subjects either APOE E3 homozygotes or APOE E3/E4 heterozygotes who upon entry to the study were diagnosed as cognitively normal or with mild cognitive impairment. Measurements: Use of genotyping and/or whole genome sequencing data to calculate polygenic risk scores and assess its ability to predict subsequent cognitive decline as measured by PACC over 5 years. Results: Assessing both cognitively normal and mild cognitive impaired subjects using a PRS threshold of greater than 0.6, the high genetic risk participant group declined more than the low risk group over 5 years as measured by PACC score (PACC score reduced by time). Conclusions: Our findings have shown that polygenic risk score provides a promising tool to identify those with higher risk to decline over 5 years regardless of their APOE alleles according to modified PACC profile, especially its ability to identify APOE3/E3 cognitively normal individuals who are at most risk for early cognitive decline. This genotype accounts for approximately 60% of the general population and 35% of the AD population but currently would not be considered at higher risk without access to expensive or invasive biomarker testing.

Polygenic Risk Scoring is an Effective Approach to Predict Those Individuals Most Likely to Decline Cognitively Due to Alzheimer's Disease.

BACKGROUND: There is a clear need for simple and effective tests to identify individuals who are most likely to develop Alzheimer's Disease (AD) both for the purposes of clinical trial recruitment but also for improved management of patients who may be experiencing early pre-clinical symptoms or who have clinical concerns. OBJECTIVES: To predict individuals at greatest risk of progression of cognitive impairment due to Alzheimer's Disease in individuals from the Alzheimer's Disease Neuroimaging Initiative (ADNI) using a polygenic risk scoring algorithm. To compare the performance of a PRS algorithm in predicting cognitive decline against that of using the pTau/Aß1-42 ratio CSF biomarker profile. DESIGN: A longitudinal analysis of data from the Alzheimer's Disease Neuroimaging Initiative study conducted across over 50 sites in the US and Canada. SETTING: Multi-center genetics study. PARTICPANTS: 515 subjects who upon entry to the study were diagnosed as cognitively normal or with mild cognitive impairment. MEASUREMENTS: Use of genotyping and/or whole genome sequencing data to calculate polygenic risk scores and assess ability to predict subsequent cognitive decline as measured by CDR-SB and ADAS-Cog13 over 4 years. RESULTS: The overall performance for predicting those individuals who would decline by at least 15 ADAS-Cog13 points from a baseline mild cognitive impairment in 4 years was 72.8% (CI:67.9-77.7) AUC increasing to 79.1% (CI: 75.6-82.6) when also including cognitively normal participants. Assessing mild cognitive impaired subjects only and using a threshold of greater than 0.6, the high genetic risk participant group declined, on average, by 1.4 points (CDR-SB) more than the low risk group over 4 years. The performance of the PRS algorithm tested was similar to that of the pTau/Aß1-42 ratio CSF biomarker profile in predicting cognitive decline. CONCLUSION: Calculating polygenic risk scores offers a simple and effective way, using DNA extracted from a simple mouth swab, to select mild cognitively impaired patients who are most likely to decline cognitively over the next four years.

Genetic diversity of the tropical tree Terminalia amazonia (Combretaceae) in naturally fragmented populations.

The effect of long-term fragmentation on the genetic diversity of populations of the neotropical tree species, Terminalia amazonia, was studied using random amplified polymorphic DNA (RAPD) analysis. Leaf material from 104 trees was collected from three naturally fragmented gallery forest patches and three plots in nearby continuous forest in the Mountain Pine Ridge, Belize. In total, 30 RAPD bands generated by five decamer primers were used to compare the genetic diversity of the six populations in the two groups. Genetic variation within the populations (H0), as estimated by the Shannon diversity index, ranged from 0.32 to 0.38, with an overall diversity of 0.38 (Hspecies). Analysis of molecular variation revealed that most (94.4%, P<0.001) of the variation was attributable to differences among individuals within populations. Population differentiation was significantly (P=0.038) lower among the fragmented populations than among continuous forest populations. On average, the fragmented populations also had slightly, but statistically significant (P=0.046) lower levels of genetic diversity. However, one gallery forest site had a higher level of genetic diversity than two of the continuous forest sites. We suggest that the long-term effect of fragmentation on the genetic diversity of tropical trees will depend upon the amount of local forest cover in proximity to the fragmented populations.

Biochemical responses in cultured cells following exposure to (89)SrCl(2): potential relevance to the mechanism of action in pain palliation.

(89)SrCl(2) is currently used as a systemic radioactive palliative treatment for painful osseous metastases associated with an osteoblastic reaction in bone. However, the biological mechanism by which (89)SrCl(2) mediates pain palliation remains unclear. In this study, attempts were made to elucidate the mechanisms by which (89)SrCl(2) might influence pain at these sites. Both the direct radiotoxic effects of (89)SrCl(2) on cell viability and its influence on cellular biosynthetic activity were investigated. The direct radiotoxic effects of (89)SrCl(2) and X-rays were compared using the prostate carcinoma cell line, PC-3. Comparable effects upon PC-3 cell viability were seen in response to exposure to an equivalent dose given by (89)SrCl(2) and X-rays (2 Gy). Experiments to investigate the indirect action of (89)SrCl(2) exposure employed the MC3T3-E1 cell line and focused on their production of Prostaglandin E(2) (PGE(2)) and interleukin-6 (IL-6). Exposure of the MC3T3-E1 cell line to (89)SrCl(2) resulted in an increased production of PGE(2) in a concentration-dependent manner. No increased PGE(2) production was seen by the MC3T3-E1 cells in response to X-ray exposure either in the presence or absence of SrCl(2). IL-6 was produced by the MC3T3-E1 cells in response to (89)SrCl(2) exposure via a PGE(2)-mediated pathway. This study demonstrates the release of potent biochemical modifiers of bone turnover in response to the systemically applied radiotherapeutic (89)SrCl(2). This strongly suggests the mechanism of pain palliation by (89)SrCl(2) is likely to result from a complex interaction of direct and indirect radiation-induced effects.

Estrogen-derived steroidal metal complexes: agents for cellular delivery of metal centers to estrogen receptor-positive cells.

Targeted cellular delivery of drugs to specific tissues is an important goal in biomedical chemistry. Achieving this requires harnessing and applying molecular-level recognition events prevalent in (or specific to) the desired tissue type. Tissues rich in estrogen receptors (ERs), which include many types of breast cancer, accumulate molecules that have high binding affinities for these receptors. Therefore, molecules that (i) bind to the ER, (ii) have favorable cellular transport properties, and (iii) contain a second functionality (such as a center that may be used for diagnostic imaging or medical therapy) are exciting synthetic targets in the field of drug delivery. To this end, we have prepared a range of metallo-estrogens based on 17alpha-ethynylestradiol and examined their binding to the ER both as isolated receptor and in whole cell assays (ER positive MCF-7 cells). Estrogens functionalized with metal binding units are prepared by palladium-catalyzed cross-coupling reactions and a wide range of metal centers introduced readily. All the compounds prepared and tested exhibit effective binding to the estrogen receptor and are delivered across the cell membrane into MCF-7 cells. In the whole cell assays, despite their monocationic nature, the palladium and platinum complexes prepared exhibit similar (and even enhanced) receptor binding affinities compared to their corresponding neutral free ligands. It is unprecedented for a higher ER binding affinity to be observed for a cationic complex than for its metal-free ligand.

Biologic mechanisms of 89SrCl2 incorporation into type I collagen during bone mineralization.

UNLABELLED: 89SrCl2 is currently used as a palliative treatment for painful osseous metastases associated with an osteoblastic reaction in bone. However, the underlying biologic mechanism by which 89SrCl2 accumulates at these lesions and mediates palliation remains unclear. The aim of this study was therefore to elucidate this mechanism. METHODS: An in vitro cell biologic model, incorporating the MC3T3-E1 murine osteoblast cell line, was established to replicate the process of collagen production and mineralization. Experiments were performed to investigate the cellular association of 89SrCl2 and 45CaCl2 with both MC3T3-E1 cells and the PC-3 human prostate adenocarcinoma cell line. RESULTS: No evidence of intracellular localization of 89SrCl2 or 45CaCl2 was found for either cell line. Localization of radiolabel was seen to be associated with MC3T3-E1 cells but only in cultures that had undergone both differentiation and mineralization. The association of 89SrCl2 was inhibited by the alkaline phosphatase inhibitor levamisole, and extracellular localization of 89SrCl2 was confirmed by microautoradiography. CONCLUSION: 89SrCl2 acts as a calcium mimic and, as such, becomes associated with the collagen matrix produced by the MC3T3-E1 cells during collagen mineralization.

Development of a high-volume in situ mRNA hybridization assay for the quantification of gene expression utilizing scintillating microplates.

A high-volume plate-based in situ hybridization assay has been developed, utilizing Amersham Cytostar-T scintillating microplates. This assay reliably detects specific mRNA transcripts at the level of 10-20 copies per cell. Radiolabeled antisense riboprobes specific for c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as well as nonhomologous vector-derived control probes were used to compare mRNA levels in quiesced rat A10 smooth muscle cells after stimulation with platelet-derived growth factor (PDGF). Maximal c-fos induction occurred following stimulation of A10 cells with 30 ng/ml of PDGF, corresponding to a signal from the c-fos probe of 700 cpm. The nonhomologous control background of 50 cpm and the GAPDH signals of 1700 cpm were independent of stimulation with PDGF or serum. Using PDGF, at 30 ng/ml, quiesced cells were stimulated at various times to provide an induction time course for c-fos mRNA which peaked at 30 min and returned to basal levels within 2 h. Comparison with parallel Northern blotting experiments showed this in situ assay to be at least 20-fold more sensitive and much more rapid to perform.

Distribution of epitopes within the amino acid sequence of the Epstein-Barr virus major envelope glycoprotein, gp340, recognized by hyperimmune rabbit sera.

Epstein-Barr virus (EBV) is a major human pathogen for which the development of an effective vaccine remains an important goal. Rabbits were immunized with one of a set of 10 fusion proteins representing protein fragments from the EBV receptor-binding ligand and candidate subunit vaccine gp340. Sera from recipients of fragments from the amino-terminal half of the polypeptide chain bound gp340 in Western blot assays and ELISA but were not virus-neutralizing. The fine epitope specificity of these sera, and of EBV-neutralizing rabbit sera raised against whole EBV and gp340-containing immune-stimulating complexes, were assessed in a peptide ELISA. All but two of these sera bound peptides located between positions 236 and 327 in the 907 amino acids of the gp340 polypeptide chain. Among these it was possible to identify regions containing candidate virus-neutralizing B cell epitopes. The use of a gp340 fusion protein affinity column to isolate antibodies from EBV-neutralizing rabbit sera specific for this region suggests the presence of both continuous and discontinuous B cell epitopes with potential roles in EBV neutralization.

Mapping of B-cell epitopes on the polypeptide chain of the Epstein-Barr virus major envelope glycoprotein and candidate vaccine molecule gp340.

The Epstein-Barr virus (EBV) major envelope glycoprotein gp340 is the subject of current efforts to develop an EBV subunit vaccine. The importance of gp340-specific humoral immunity has been highlighted by studies of natural infection in humans and gp340 immunization of experimental animals. The former studies have demonstrated the presence of gp340-specific serum antibodies which mediate EBV neutralization, complement fixation, and antibody-dependent cellular cytotoxicity. The latter studies have often shown a correlation between the induction of gp340-specific EBV-neutralizing antibodies and protection from virus challenge. We have used a series of bacterial beta-galactosidase-gp340 fusion proteins and overlapping synthetic peptides from the gp340 open reading frame to map the positions of B-cell epitopes within the gp340 primary amino acid sequence. The data reported here indicate the presence of B-cell epitopes within the carboxy-terminal third of the gp340 polypeptide chain. These epitopes could not be detected with a peptide enzyme-linked immunosorbent assay, thereby suggesting that they are discontinuous. Affinity purification of antibodies with a gp340 fusion protein from the carboxy terminus of the gp340 polypeptide chain has been used to show that these antibodies are not EBV neutralizing in vitro. The consequences of these findings for future EBV vaccine development are considered.