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The core cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers amyloid beta (Aβ42 and Aβ40), total tau, and phosphorylated tau, have been extensively clinically validated, with very high diagnostic performance for AD, including the early phases of the disease. However, between-center differences in pre-analytical procedures may contribute to variability in measurements across laboratories. To resolve this issue, a workgroup was led by the Alzheimer's Association with experts from both academia and industry. The aim of the group was to develop a simplified and standardized pre-analytical protocol for CSF collection and handling before analysis for routine clinical use, and ultimately to ensure high diagnostic performance and minimize patient misclassification rates. Widespread application of the protocol would help minimize variability in measurements, which would facilitate the implementation of unified cut-off levels across laboratories, and foster the use of CSF biomarkers in AD diagnostics for the benefit of the patients.

α-Synuclein is the major component of Lewy bodies and a candidate biomarker for neurodegenerative diseases in which Lewy bodies are common, including Parkinson's disease and dementia with Lewy bodies. A large body of literature suggests that these disorders are characterized by reduced concentrations of α-synuclein in cerebrospinal fluid (CSF), with overlapping concentrations compared to healthy controls and variability across studies. Several reasons can account for this variability, including technical ones, such as inter-assay and inter-laboratory variation (reproducibility). We compared four immunochemical methods for the quantification of α-synuclein concentration in 50 unique CSF samples. All methods were designed to capture most of the existing α-synuclein forms in CSF (‘total’ α-synuclein). Each of the four methods showed high analytical precision, excellent correlation between laboratories (R2 0.83–0.99), and good correlation with each other (R2 0.64–0.93), although the slopes of the regression lines were different between the four immunoassays. The use of common reference CSF samples decreased the differences in α-synuclein concentration between detection methods and technologies. Pilot data on an immunoprecipitation mass spectrometry (IP-MS) method is also presented. Our results suggest that the four immunochemical methods and the IP-MS method measure similar forms of α-synuclein and that a common reference material would allow harmonization of results between immunoassays. (Figure presented.).

Background:The core Alzheimer’s disease cerebrospinal fluid (CSF) biomarkers total tau (T-tau), phosphorylated tau (P-tau), β-amyloid 1-42 (Aβ42) and β-amyloid 1-40 (Aβ40) are increasing in importance and are now part of the research criteria for the diagnosis of the disease. The main aim of this study is to evaluate whether a set of certified reference materials (CRMs) are commutable for Aβ42 and to serve as a feasibility study for the other markers. This property is a prerequisite for the establishment of CRMs which will then be used by manufacturers to calibrate their assays against. Once the preanalytical factors have been standardized and proper selection criteria are available for subject cohorts this harmonization between methods will allow for universal cut-offs to be determined.Methods:Thirty-four individual CSF samples and three different CRMs where analyzed for T-tau, P-tau, Aβ42 and Aβ40, using up to seven different commercially available methods. For Aβ40 and Aβ42 a mass spectrometry-based procedure was also employed.Results:There were strong pairwise correlations between the different methods (Spearman’s ρ>0.92) for all investigated analytes and the CRMs were not distinguishable from the individual samples.Conclusions:This study shows that the CRMs are commutable for the different assays for Aβ42. For the other analytes the results show that it would be feasible to also produce CRMs for these. However, additional studies are needed as the concentration interval for the CRMs were selected based on Aβ42 concentrations only and did in general not cover satisfactory large concentration intervals for the other analytes.

Parkinson's disease biomarkers are needed to increase diagnostic accuracy, to objectively monitor disease progression and to assess therapeutic efficacy as well as target engagement when evaluating novel drug and therapeutic strategies. This article summarizes perianalytical considerations for biomarker studies (based on immunoassays) in Parkinson's disease, with emphasis on quantifying total α-synuclein protein in biological fluids. Current knowledge and pitfalls are discussed, and selected perianalytical variables are presented systematically, including different temperature of sample collection and types of collection tubes, gradient sampling, the addition of detergent, aliquot volume, the freezing time, and the different thawing methods. We also discuss analytical confounders. We identify gaps in the knowledge and delineate specific areas that require further investigation, such as the need to identify posttranslational modifications of α-synuclein and antibody-independent reference methods for quantification, as well as the analysis of potential confounders, such as comorbidities, medication, and phenotypes of Parkinson's disease in larger cohorts. This review could be used as a guideline for future Parkinson's disease biomarker studies and will require regular updating as more information arises in this growing field, including new technical developments as they become available. In addition to reviewing best practices, we also identify the current technical limitations and gaps in the knowledge that should be addressed to enable accurate and quantitative assessment of α-synuclein levels in the clinical setting. © 2017 International Parkinson and Movement Disorder Society.

Background: The cerebrospinal fluid (CSF) amyloid-β (Aβ42) peptide is an important biomarker for Alzheimer’s disease (AD). Variability in measured Aβ42 concentrations at different laboratories may be overcome by standardization and establishing traceability to a reference system. Candidate certified reference materials (CRMs) are validated herein for this purpose. Methods: Commutability of 16 candidate CRM formats was assessed across five CSF Aβ42 immunoassays and one mass spectrometry (MS) method in a set of 48 individual clinical CSF samples. Promising candidate CRM formats (neat CSF and CSF spiked with Aβ42) were identified and subjected to validation across eight (Elecsys, EUROIMMUN, IBL, INNO-BIA AlzBio3, INNOTEST, MSD, Simoa, and Saladax) immunoassays and the MS method in 32 individual CSF samples. Commutability was evaluated by Passing-Bablok regression and the candidate CRM termed commutable when found within the prediction interval (PI). The relative distance to the regression line was assessed. Results: The neat CSF candidate CRM format was commutable for almost all method comparisons, except for the Simoa/MSD, Simoa/MS and MS/IBL where it was found just outside the 95% PI. However, the neat CSF was found within 5% relative distance to the regression line for MS/IBL, between 5% and 10% for Simoa/MS and between 10% and 15% for Simoa/MSD comparisons. Conclusions: The neat CSF candidate CRM format was commutable for 33 of 36 method comparisons, only one comparison more than expected given the 95% PI acceptance limit. We conclude that the neat CSF candidate CRM can be used for value assignment of the kit calibrators for the different Aβ42 methods.

Alzheimer’s disease (AD) drug development is burdened with the current requirement to conduct large, lengthy, and costly trials to overcome uncertainty in patient progression and effect size on treatment outcome measures. There is an urgent need for the discovery, development, and implementation of novel, objectively measured biomarkers for AD that would aid selection of the appropriate subpopulation of patients in clinical trials, and presumably, improve the likelihood of successfully evaluating innovative treatment options. Amyloid deposition and tau in the brain, which are most commonly assessed either in cerebrospinal fluid (CSF) or by molecular imaging, are consistently and widely accepted. Nonetheless, a clear gap still exists in the accurate identification of subjects that truly have the hallmarks of AD. The Coalition Against Major Diseases (CAMD), one of 12 consortia of the Critical Path Institute (C-Path), aims to streamline drug development for AD and related dementias by advancing regulatory approved drug development tools for clinical trials through precompetitive data sharing and adoption of consensus clinical data standards. This report focuses on the regulatory process for biomarker qualification, briefly comments on how it contrasts with approval or clearance of companion diagnostics, details the qualifications currently available to the field of AD, and highlights the current challenges facing the landscape of CSF biomarkers qualified as hallmarks of AD. Finally, it recommends actions to accelerate regulatory qualification of CSF biomarkers that would, in turn, improve the efficiency of AD therapeutic development.

Item does not contain fulltext Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid beta42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid beta42. 01 augustus 2012

A novel platform for the electronic detection of nucleic acids on microarrays is introduced and shown to perform well as a selective detection system for applications in molecular diagnostics. A gold electrode in a printed circuit board is coated with a self-assembled monolayer (SAM) containing DNA capture probes. Unlabeled nucleic acid targets are immobilized on the surface of the SAM through sequence-specific hybridization with the DNA capture probe. A separate signaling probe, containing ferrocene-modified nucleotides and complementary to the target in the region adjoining the capture probe binding site, is held in close proximity to the SAM in a sandwich complex. The SAM allows electron transfer between the immobilized ferrocenes and the gold, while insulating the electrode from soluble redox species, including unbound signaling probes. Here, we demonstrate sequence-specific detection of amplicons after simple dilution of the reaction product into hybridization buffer. In addition, single nucleotide polymorphism discrimination is shown. A genotyping chip for the C282Y single nucleotide polymorphism associated with hereditary hemochromatosis is used to confirm the genotype of six patients' DNA. In addition, a gene expression-monitoring chip is described that surveys five genes that are differentially regulated in the cellular apoptosis response. Finally, custom modification of individual electrodes through sequence-specific hybridization demonstrates the potential of this system for infectious disease diagnostics. The versatility of the electronic detection platform makes it suitable for multiple applications in diagnostics and pharmacogenetics.

Background: A bioelectronic detection platform has recently been developed that facilitates the detection and characterization of nucleic acids. The DNA chip platform is compatible with homogeneous assays because separate labeling and wash steps are not required. A one-step, bioelectronic detection assay was developed to genotype patient samples with respect to the H63D polymorphism of the Hfe gene, associated with hereditary hemochromatosis. Methods and Results: Electrode arrays were modified with DNA capture probes that were perfectly matched to the wild-type or mutant allele of H63D. Amplicons containing the polymorphic site were hybridized with the capture probes on the electrode arrays in the presence of electronically labeled reporter (signaling) probes. Voltammetric analysis of the electrode arrays was conducted first at ambient temperature and then at elevated temperature. The electronic signal was preferentially diminished at elevated temperature from electrodes that hybridized with mismatched target amplicons. Conclusion: An assay for bioelectronic genotyping of the H63D polymorphism was developed and used with six patient specimens to show the feasibility of this system as a model for point mutation detection.

Ribosomal DNA (rDNA) replication origins of Saccharomyces cerevisiae are known to function inefficiently, both in the context of the tandem rDNA repeats in the chromosome and as single copy autonomously replicating sequences (ARSs) in plasmids. Here we examined components of the rDNA ARS that might contribute to inefficient extrachromosomal replication. Like the efficient H4 ARS, the rDNA ARS requires a match to the 11 bp ARS consensus sequence (ACS) and a broad non-conserved region that may contain multiple elements, including a DNA unwinding element (DUE). Using a single-strand-specific nuclease hypersensitivity assay and by determining the superhelical density required for stable DNA unwinding, we found that the DNA of the rDNA ARS is not as easily unwound as the H4 ARS. Unwinding of the rDNA ARS required additional energy, similar to the unwinding of mutations in the H4 ARS that stabilize the double helix in the DUE region and impair replication. In vivo extrachromosomal replication of the rDNA ARS was cold sensitive, like H4 ARS mutants that require additional energy to unwind the DUE region but unlike the easily unwound, wild-type H4 ARS. Impairment of replication function at reduced temperature suggests that the elevated energy requirement for DNA unwinding inherent in the wild-type rDNA ARS contributes to inefficient replication function. We also examined the essential ACS match in the rDNA ARS, which is known to be imperfect at one position. A point mutation in the essential ACS that corrects the imperfect match increased the efficiency of extrachromosomal replication. Our results reveal that the essential ACS element and DNA unwinding in the rDNA ARS are naturally impaired, suggesting that inefficient function of the rDNA replication origin has a biological purpose.

Background: The cerebrospinal fluid (CSF) biomarkers amyloid beta 1-42, total tau, and phosphorylated tau are used increasingly for Alzheimer's disease (AD) research and patient management. However, there are large variations in biomarker measurements among and within laboratories. Methods: Data from the first nine rounds of the Alzheimer's Association quality control program was used to define the extent and sources of analytical variability. In each round, three CSF samples prepared at the Clinical Neurochemistry Laboratory (Molndal, Sweden) were analyzed by single-analyte enzyme-linked immunosorbent assay (ELISA), a multiplexing xMAP assay, or an immunoassay with electrochemoluminescence detection. Results: A total of 84 laboratories participated. Coefficients of variation (CVs) between laboratories were around 20% to 30%; within-run CVs, less than 5% to 10%; and longitudinal within-laboratory CVs, 5% to 19%. Interestingly, longitudinal within-laboratory CV differed between biomarkers at individual laboratories, suggesting that a component of it was assay dependent. Variability between kit lots and between laboratories both had a major influence on amyloid beta 1-42 measurements, but for total tau and phosphorylated tau, between-kit lot effects were much less than between-laboratory effects. Despite the measurement variability, the between-laboratory consistency in classification of samples (using prehoc-derived cutoffs for AD) was high (>90% in 15 of 18 samples for ELISA and in 12 of 18 samples for xMAP). Conclusions: The overall variability remains too high to allow assignment of universal biomarker cutoff values for a specific intended use. Each laboratory must ensure longitudinal stability in its measurements and use internally qualified cutoff levels. Further standardization of laboratory procedures and improvement of kit performance will likely increase the usefulness of CSF AD biomarkers for researchers and clinicians. (C) 2013 The Alzheimer's Association. All rights reserved.

Recognizing that international collaboration is critical for the acceleration of biomarker standardization efforts and the efficient development of improved diagnosis and therapy, the Alzheimer's Association created the Global Biomarkers Standardization Consortium (GBSC) in 2010. The consortium brings together representatives of academic centers, industry, and the regulatory community with the common goal of developing internationally accepted common reference standards and reference methods for the assessment of cerebrospinal fluid (CSF) amyloid β42 (Aβ42) and tau biomarkers. Such standards are essential to ensure that analytical measurements are reproducible and consistent across multiple laboratories and across multiple kit manufacturers. Analytical harmonization for CSF Aβ42 and tau will help reduce confusion in the AD community regarding the absolute values associated with the clinical interpretation of CSF biomarker results and enable worldwide comparison of CSF biomarker results across AD clinical studies.

Contains fulltext : 98400.pdf (Publisher’s version ) (Closed access) BACKGROUND: The cerebrospinal fluid (CSF) biomarkers amyloid beta (Abeta)-42, total-tau (T-tau), and phosphorylated-tau (P-tau) demonstrate good diagnostic accuracy for Alzheimer's disease (AD). However, there are large variations in biomarker measurements between studies, and between and within laboratories. The Alzheimer's Association has initiated a global quality control program to estimate and monitor variability of measurements, quantify batch-to-batch assay variations, and identify sources of variability. In this article, we present the results from the first two rounds of the program. METHODS: The program is open for laboratories using commercially available kits for Abeta, T-tau, or P-tau. CSF samples (aliquots of pooled CSF) are sent for analysis several times a year from the Clinical Neurochemistry Laboratory at the Molndal campus of the University of Gothenburg, Sweden. Each round consists of three quality control samples. RESULTS: Forty laboratories participated. Twenty-six used INNOTEST enzyme-linked immunosorbent assay kits, 14 used Luminex xMAP with the INNO-BIA AlzBio3 kit (both measure Abeta-(1-42), P-tau(181P), and T-tau), and 5 used Meso Scale Discovery with the Abeta triplex (AbetaN-42, AbetaN-40, and AbetaN-38) or T-tau kits. The total coefficients of variation between the laboratories were 13% to 36%. Five laboratories analyzed the samples six times on different occasions. Within-laboratory precisions differed considerably between biomarkers within individual laboratories. CONCLUSIONS: Measurements of CSF AD biomarkers show large between-laboratory variability, likely caused by factors related to analytical procedures and the analytical kits. Standardization of laboratory procedures and efforts by kit vendors to increase kit performance might lower variability, and will likely increase the usefulness of CSF AD biomarkers.

We performed a correlative study to assess HER-2/neu oncogene amplification and protein overexpression. Gene amplification was determined by simultaneous hybridization of human breast tumor DNA with probes for the oncogene and a single copy reference gene, lmmunohistochemistry with a murine anti–HER-2/neu antibody was done on different portions of the same tumors. It has been demonstrated that the extent of gene amplification plays a role in disease outcome in breast cancer patients. The results of our investigation allowed this controversial, but sometimes useful, prognostic indicator to be used by a hospital’s medical staff.

Autonomously replicating sequence (ARS) elements function as plasmid replication origins. Our studies of the H4 ARS and ARS307 have established the requirement for a DNA unwinding element (DUE), a broad easily-unwound sequence 3' to the essential consensus that likely facilitates opening of the origin. In this report, we examine the intrinsic ease of unwinding a variety of ARS elements using (1) a single-strand-specific nuclease to probe for DNA unwinding in a negatively-supercoiled plasmid, and (2) a computer program that calculates DNA helical stability from the nucleotide sequence. ARS elements that are associated with replication origins on chromosome III are nuclease hypersensitive, and the helical stability minima correctly predict the location and hierarchy of the hypersensitive sites. All well-studied ARS elements in which the essential consensus sequence has been identified by mutational analysis contain a 100-bp region of low helical stability immediately 3' to the consensus, as do ARS elements created by mutation within the prokaryotic M13 vector. The level of helical stability is, in all cases, below that of ARS307 derivatives inactivated by mutations in the DUE. Our findings indicate that the ease of DNA unwinding at the broad region directly 3' to the ARS consensus is a conserved property of yeast replication origins.

We used a sensitive and specific beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) assay to determine the relationship between BACE1 activity in cerebrospinal fluid (CSF) and markers of APP metabolism and axonal degeneration in early and late stages of Alzheimer disease (AD).To assess CSF BACE1 activity in AD.Case-control and longitudinal follow-up study.Specialized memory clinic. Patients Eighty-seven subjects with AD, 33 cognitively normal control subjects, and 113 subjects with mild cognitive impairment (MCI), who were followed up for 3 to 6 years.Cerebrospinal fluid BACE1 activity in relation to diagnosis and CSF levels of secreted APP and amyloid beta protein (Abeta) isoforms and the axonal degeneration marker total tau.Subjects with AD had higher CSF BACE1 activity (median, 30 pM [range, 11-96 pM]) than controls (median, 23 pM [range, 8-43 pM]) (P =.02). Subjects with MCI who progressed to AD during the follow-up period had higher baseline BACE1 activity (median, 35 pM [range, 18-71 pM]) than subjects with MCI who remained stable (median, 29 pM [range, 14-83 pM]) (P.001) and subjects with MCI who developed other forms of dementia (median, 20 pM [range, 10-56 pM]) (P.001). BACE1 activity correlated positively with CSF levels of secreted APP isoforms and Abeta(40) in the AD and control groups and in all MCI subgroups (P.05) except the MCI subgroup that developed AD. Strong positive correlations were found between CSF BACE1 activity and total tau levels in all MCI subgroups (ror= 0.57, Por= .009).Elevated BACE1 activity may contribute to the amyloidogenic process in sporadic AD and is associated with the intensity of axonal degeneration.

Yeast replication origins contain a DNA sequence element whose biological activity correlates with hypersensitivity to single-strand-specific nucleases in negatively supercoiled plasmids. By using two-dimensional gel electrophoresis of plasmid topoisomers, we demonstrate that thermodynamically stable origin unwinding accounts for the nuclease hypersensitivity and, furthermore, that increased thermal energy facilitates stable origin unwinding in vitro. In living cells, increased thermal energy can suppress origin mutations that raise the free-energy cost for unwinding the nuclease-hypersensitive element. Specifically, mutational defects in autonomously replicating sequence (ARS)-mediated plasmid replication are less severe in cells grown at 30 degrees C as compared to 23 degrees C. Our findings indicate that the energetics of DNA unwinding at the nuclease-hypersensitive element are biologically important. We call the nuclease-hypersensitive sequence the DNA unwinding element (DUE) and propose that it serves as the entry site for yeast replication enzymes into the DNA helix.

Terminal differentiation is often coupled with irreversible loss of proliferative potential. The CCAAT enhancer binding protein alpha (C/EBPalpha) preferentially accumulates in postmitotic, differentiated 3T3-L1 adipocytes but declines during tumor necrosis factor alpha (TNFalpha)-induced dedifferentiation. We have discovered that this decline in C/EBPalpha correlates with an increased mitotic growth potential. In order to further investigate the antimitotic activity of C/EBPalpha, we introduced antisense C/EBPalpha RNA into 3T3-L1 cells to block endogenous C/EBPalpha expression. When treated according to the standard differentiation protocol, stable cells lines harboring antisense C/EBPalpha RNA did not differentiate into fat-laden adipocytes, consistent with previous findings (Lin F, Lane MD, Genes Dev 1992;6:533-544). We found that these undifferentiated cells expressing antisense-C/EBPalpha can reenter the cell cycle after mitogenic stimulation at a time in development when parental 3T3-L1 cells cannot. Moreover, the expression profiles of the growth-arrest-associated genes gas1 and gas2 revealed that the antisense C/EBPalpha-expressing cells withdrew from the cell cycle after the period of clonal expansion but failed to progress to the state of least proliferative potential characteristic of terminally differentiated adipocytes.

The CCAAT/enhancer-binding protein-alpha (C/EBP alpha) drives the differentiation of murine 3T3-L1 cells to adipocytes through transcriptional activation of phenotype-associated genes via proximal promoter elements. In addition, C/EBP alpha suppresses mitotic growth. We report here that C/EBP alpha directly regulates gadd45 through a C/EBP-binding site in the proximal promoter. A 3 basepair substitution, directed at the most conserved residues of the sequence, reduced C/EBP alpha-mediated transactivation and impaired binding of C/EBP alpha in adipocyte nuclear extracts. We also found that c-Myc antagonized C/EBP alpha-mediated transactivation of gadd45. Analysis of systematically altered forms of C/EBP alpha revealed that c-Myc antagonism targeted the antimitotic, transcriptional activation domain of C/EBP alpha. In addition, we localized the regulatory sequences in the gadd45 promoter that are required for c-Myc antagonism of C/EBP alpha transactivation. Our findings reveal that C/EBP alpha coordinates cellular differentiation and mitotic growth arrest through direct, coordinate regulation of phenotype-associated and growth-arrest-associated genes. In addition, our findings reveal that the reciprocal relation between C/EBP alpha and c-Myc in 3T3-L1 cells includes antagonistic transcriptional control of a growth-arrest-associated gene.

A Southern blot-based assay is presented that increases the simplicity and accuracy of the HER-2/ neu gene copy number assignment in breast cancer DNA. Genomic DNA was hybridized simultaneously with probes corresponding to portions of HER-2/ neu and a single-copy gene, myeloperoxidase. A unique restriction fragment was detected for each gene. The use of DNA probes of similar mass and equal specific activities resulted in a ratio of band intensities on the resultant autoradiograph that reported the ratio of gene copy numbers directly. Patient samples containing amplified levels of the HER-2/ neu gene were identified by simple visual inspection of a single autoradiograph. Analysis of breast cancer samples alongside the cell line DNAs, representing a range of HER-2/ neu gene copy numbers, permits visual quantitation of the tumors’ gene copy numbers. The authors show that the HER-2/ neu gene copy number can be determined accurately in marginally degraded DNA, a feature of some clinical samples.

In an effort to identify protein factors that play a regulatory role in the differentiation of adipocytes, we have isolated two genes that encode polypeptides related to CCAAT/enhancer-binding protein (C/EBP; hereafter termed C/EBP alpha). The proteins encoded by these C/EBP-related genes, termed C/EBP beta and C/EBP delta, exhibit similar DNA-binding specificities and affinities compared with C/EBP alpha. Furthermore, C/EBP beta and C/EBP delta readily form heterodimers with one another as well as with C/EBP alpha. The transcriptional activating capacity of these two newly identified C/EBP isoforms was demonstrated by transient transfection experiments in which expression vectors encoding C/EBP beta and C/EBP delta were observed to induce transcription from the promoter of the serum albumin gene in cultured hepatoma cells. The mRNAs encoding C/EBP beta and C/EBP delta were detected in a number of tissues, most of which corresponded to sites of expression of C/EBP alpha. The expression pattern of C/EBP beta and C/EBP delta during adipose conversion of 3T3-L1 cells was examined by Western and Northern blotting assays. In contrast to the expression profile of the gene encoding C/EBP alpha, whose product is not detectable until the late phase of adipocyte differentiation, the c/ebp beta and c/ebp delta genes were actively expressed very early during adipocyte differentiation. Moreover, transcription of the c/ebp beta and c/ebp delta genes was observed to be induced directly by adipogenic hormones. The accumulation of C/EBP beta and C/EBP delta reached a maximal level during the first 2 days of differentiation and declined sharply before the onset of C/EBP alpha accumulation. The temporal pattern of expression of these three C/EBP isoforms during adipocyte differentiation may reflect the underpinnings of a regulatory cascade that controls the process of terminal cell differentiation.

The CCAAT-enhancer binding protein (C/EBP) has now been found to promote the terminal differentiation of adipocytes. During the normal course of adipogenesis, C/EBP expression is restricted to a terminal phase wherein proliferative growth is arrested, and specialized cell phenotype is first manifested. A conditional form of C/EBP was developed, making it feasible to test its capacity to regulate the differentiation of cultured adipocytes. Premature expression of C/EBP in adipoblasts caused a direct cessation of mitotic growth. Moreover, when abetted by the effects of three adipogenic hormones, C/EBP promoted terminal cell differentiation. Since C/EBP is expressed in a variety of tissues, it may have a fundamental role in regulating the balance between cell growth and differentiation in higher animals.

We have localized the DNA sequence that facilitates unwinding of a yeast replication origin, the H4 ARS. The readily unwound sequence lies adjacent to the previously characterized consensus core sequence of the ARS. Unwinding is detected through the formation of a single-strand-specific nuclease hypersensitive site in H4 ARS mutant derivatives present on super-coiled plasmids. Linker-scanning and linker-deletion derivatives exhibit wild-type nuclease hypersensitivity and ARS function, while large external deletions reduce or eliminate nuclease detectable unwinding and origin function. ARS unwinding and origin function can be rescued in the deletion mutants by inserting a biologically unrelated sequence with DNA unwinding properties similar to a functional ARS. The data clarify the nature of DNA sequence requirements in the ARS by suggesting that small substitutions, insertions, and deletions are tolerated in the region flanking the consensus core sequence because they do not significantly alter the unwinding properties of the region.

The lack of readily available biomarkers is a significant hindrance toward progressing to effective therapeutic and preventative strategies for Alzheimer's disease (AD). Blood-based biomarkers have potential to overcome access and cost barriers and greatly facilitate advanced neuroimaging and cerebrospinal fluid biomarker approaches. Despite the fact that preanalytical processing is the largest source of variability in laboratory testing, there are no currently available standardized preanalytical guidelines. The current international working group provides the initial starting point for such guidelines for standardized operating procedures (SOPs). It is anticipated that these guidelines will be updated as additional research findings become available. The statement provides (1) a synopsis of selected preanalytical methods utilized in many international AD cohort studies, (2) initial draft guidelines/SOPs for preanalytical methods, and (3) a list of required methodological information and protocols to be made available for publications in the field to foster cross-validation across cohorts and laboratories.

DNA fiber autoradiographic studies carried out over 20 years ago first suggested that eukaryotic DNA replication begins at discrete sites spaced at irregular intervals along chromosomal DNA molecules [1]. The discrete initiation sites were called 'origins'. Later on, the DNA replicated from a single origin was termed a 'replicon' (reviewed iri Ref. 2). At the time, it was impossible to determine whether these origins and replicons corresponded to specific nucleotide sequences or were randomly located (with different origins being used in different cells). In the absence of accurate