Diagnostic and economic evaluation of new biomarkersfor Alzheimer¿s disease: the research protocol of a prospective cohort study

ABSTRACT: BACKGROUND: New research criteria for the diagnosis of Alzheimer's disease (AD) have recently been developed to enable an early diagnosis of AD pathophysiologyby relying on emerging biomarkers. To enable efficient allocation of health care resources, evidence is needed to support decision makers on the adoption of emerging biomarkers in clinical practice. The research goals are to 1) assess the diagnostic test accuracy of current clinical diagnostic work-up and emerging biomarkers in MRI, PET and CSF, 2) perform a cost-consequence analysis and 3) assess long-term cost-effectiveness by an economic model.Methods/designIn a cohort design 223 consecutive patients suspected of having a primary neurodegenerative disease are approached in four academic memory clinics and followed for two years. Clinical data and data on quality of life, costs and emerging biomarkers are gathered.Diagnostic test accuracy is determined by relating the clinical practice and new research criteria diagnoses to the reference diagnosis. The clinical practice diagnosis at baseline is reflected by a consensus procedure among experts using clinical information only (no biomarkers). The diagnosis based on the new research criteria is reflected by decision rules that combine clinical and biomarker information. The reference diagnosis is determined by a consensus procedure among experts based on clinical information on the course of symptoms over a two-year time period.A decision analytic model is built combining available evidence from different resources among which (accuracy) results from the study, literature and expert opinion to assess long-term cost-effectiveness of the emerging biomarkers. DISCUSSION: Several other multi-centre trials study the relative value of new biomarkers for early evaluation of AD and related disorders. The uniqueness of this study is the assessment of resource utilization and quality of life to enable an economic evaluation. The study results are generalizable to a population of patients who are referred to a memory clinic due to their memory problems.Trial registrationNCT01450891.     

Protein-lipid interactions of bacteriophage M13 major coat protein

During the past years, remarkable progress has been made in our understanding of the replication cycle of bacteriophage M13 and the molecular details that enable phage proteins to navigate in the complex environment of the host cell. With new developments in molecular membrane biology in combination with spectroscopic techniques, we are now in a position to ask how phages carry out this delicate process on a molecular level, and what sort of protein-lipid and protein-protein interactions are involved. In this review we will focus on the molecular details of the protein-protein and protein-lipid interactions of the major coat protein (gp8) that may play a role during the infection of Escherichia coli by bacteriophage M13.     

Influence of erythrocyte shape on the rate of Ca2+-induced scrambling of phosphatidylserine

Membrane-perturbing agents that cause transformation of biconcave erythrocytes into echinocytes or stomatocytes were used to investigate the influence of erythrocyte shape on the rate of Ca(2+)-induced scrambling of phospholipids. Erythrocytes were treated with a variety of lipid-soluble compounds to induce these shape changes, followed by incubation with calcium and ionomycin to activate lipid scramblase. Prothrombinase activity of the cells was used to monitor the rate of surface exposure of phosphatidylserine, which is taken as a measure of scramblase activity. Echinocytes show an enhanced rate of scrambling, whereas stomatocytes show a reduced rate, relative to normocytes. This phenomenon appears to correlate with enhanced and diminished micro-exovesicle shedding from echinocytes and stomatocytes, respectively. It is concluded that the rate of calcium-induced phosphatidylserine exposure (rate of lipid scrambling) in erythrocytes depends for a considerable part on the cells' ability to form microvesicles.     

Conformation and orientation of the gene 9 minor coat protein of bacteriophage M13 in phospholipid bilayers

The membrane-bound state of the gene 9 minor coat protein of bacteriophage M13 was studied in model membrane systems, which varied in lipid head group and lipid acyl chain composition. By using FTIR spectroscopy and subsequent band analysis a quantitative analysis of the secondary structure of the protein was obtained. The secondary structure of the gene 9 protein predominantly consists of alpha-helical (67%) and turn (33%) structures. The turn structure is likely to be located C-terminally where it has a function in recognizing the phage DNA during bacteriophage assembly. Attenuated total reflection FTIR spectroscopy was used to determine the orientation of gene 9 protein in the membrane, revealing that the alpha-helical domain is mainly transmembrane. The conformational and orientational measurements result in two models for the gene 9 protein in the membrane: a single transmembrane helix model and a two-helix model consisting of a 15 amino acid long transmembrane helix and a 10 amino acid long helix oriented parallel to the membrane plane. Potential structural consequences for both models are discussed.     

Localization and rearrangement modulation of the N-terminal arm of the membrane-bound major coat protein of bacteriophage M13

During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17-24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane.     

Aggregation-related conformational change of the membrane-associated coat protein of bacteriophage M13

The state of the coat protein of bacteriophage M13, reconstituted into amphiphilic media, has been investigated. The in situ conformation of the coat protein has been determined by using circular dichroism. Minimum numbers for the protein aggregation in the system have been determined after disruption of the lipid-protein system and subsequent uptake of the protein in cholate micelles. The aggregational state and conformation of the protein were affected by (1) the method of coat protein isolation (phenol extraction vs cholate isolation), (2) the nature of amphiphiles used (variation in phospholipid headgroups and acyl chains), and (3) the ratio of amphiphiles and protein. Under all conditions, phenol-extracted coat protein was in a predominantly beta-structure and in a highly aggregated polymeric form. Cholate-isolated coat protein was initially oligomeric and contained a substantial amount of alpha-helix. Below an aggregation number of 20, this protein showed a reversible aggregation with no change in conformation. Upon further aggregation, a conformational change was observed, and aggregation was irreversible, resulting in predominantly beta-structured coat protein polymers. This effect was observed upon uptake in phospholipids at low lipid to protein molar ratios (L/P ratios) and with phosphatidylcholines (PC) and phosphatidic acids (PA) containing saturated acyl chains. After reconstitution in phospholipids with unsaturated acyl chains and with phosphatidylglycerols (PG) at high L/P ratios, the original alpha-helix-containing state of the coat protein was maintained. Cross-linking experiments demonstrated that the beta-polymers are able to form reversible superaggregates within the vesicle system. An aggregation-related conformational change mechanism for the coat protein in phospholipid systems is proposed.     

Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins

Internal eliminated sequences (IESs) often interrupt ciliate genes in the silent germline nucleus but are exactly excised and eliminated from the developing somatic nucleus from which genes are then expressed. Some long IESs are transposons, supporting the hypothesis that short IESs are ancient transposon relics. In light of that hypothesis and to explore the evolutionary history of a collection of IESs, we have compared various alleles of a particular locus (the 81 locus) of the ciliated protozoa Oxytricha trifallax and O. fallax. Three short IESs that interrupt two genes of the locus are found in alleles from both species, and thus must be relatively ancient, consistent with the hypothesis that short IESs are transposon relics. In contrast, TBE1 transposon interruptions of the locus are allele-specific and probably the results of recent transpositions. These IESs (and the TBE1s) are precisely excised from the DNA of the developing somatic macronucleus. Each IES interrupts a highly conserved sequence. A few nucleotides at the ends of each IES are also conserved, suggesting that they interact critically with IES excision machinery. However, most IES nucleotide positions have evolved at high rates, showing little or no selective constraint for function. Nonetheless, the length of each IES has been maintained (+/- 3 bp). While one IES is approximately 33 bp long, three other IESs have very similar sizes, approximately 70 bp long. Two IESs are surrounded by direct repeats of the sequence TTCTT. No other sequence similarities were found between any of the four IESs. However, the ends of one IES do match the inverted terminal repeat consensus sequence of the "TA" IESs of Paramecium. Three O. trifallax alleles appear to have been recipients in recent conversion events that could have been provoked by double-strand breaks associated with IES ends subsequent to IES transposition. Our findings support the hypothesis that short IESs evolved from ancient transposons that have lost most of their sequences, except those necessary for precise excision during macronuclear development.      

Two groups control light-induced schiff base deprotonation and the proton affinity of asp(85) in the Arg(82)His mutant of bacteriorhodopsin

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.     

A Machine for Sawing 80-Micrometer Slices of Carious Enamel

Design and construction of a machine that cats 80-μm slices of sound and carious dental enamel and other calcified tissues is described. These slices can be used for quantitative microradiographic studies. Preparation takes minutes. Thickness for a given slice is uniform within 2 μn, mean thickness is within 4 μm of the intended value and roughness is about 0.1 μm. Commercial components have been used where possible.' Information is provided to permit purchase of the components of the machine and its construction in the average university workshop.     

Viruses: incredible nanomachines. New advances with filamentous phages

During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into account.