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BACKGROUND Cell or tissue specific background may influence the consequences of expressing the Huntington's disease (HD) mutation. Aggregate formation is known to occur in skeletal muscle, but not heart of the R6/2 fragment HD model. OBJECTIVE We asked whether aggregate formation and the expression and subcellular localization of huntingtin species was associated with mitochondrial dysfunction. METHODS We analyzed levels of soluble HTT and HTT aggregates, as well as important fission and fusion proteins and mitochondrial respiratory chain activities, in quadriceps and heart of the R6/2 N-terminal fragment mouse model (12 weeks, 160±10 CAG repeats). RESULTS Soluble mutant HTT was present in both tissues with expression higher in cytoplasmic/mitochondrial than nuclear fractions. HTT aggregates were only detectable in R6/2 quadriceps, in association with increased levels of the pro-fission factor DRP1 and its phosphorylated active form, and decreased levels of the pro-fusion factor MFN2. In addition, respiratory chain complex activities were decreased. In heart that was without detectable HTT aggregates, we found no evidence for mitochondrial dysfunction. CONCLUSION Tissue specific factors may exist that protect the R6/2 heart from HTT aggregate formation and mitochondrial pathology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background Huntington’s disease (HD) is a progressive neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin (HTT) protein with no effective disease-modifying therapies available. Mutant HTT toxicity, associated with the misfolding of the expanded polyglutamine repeat-containing protein, affects several intracellular pathways and leads to widespread neurodegeneration. In addition, mutant HTT forms intracellular aggregates, which contribute to disease pathogenesis. One promising strategy to develop HD modifying therapies aim to repress or reduce the amount of mutant HTT expression and aggregation. However, in order to monitor the efficacy of such approaches there is the need for robust and reliable immunoassays that are able to quantitate mutant HTT in biological samples. Aims To develop immunoassays and high content histological approaches able to quantify and monitor changes in soluble HTT levels as well as in HTT aggregates in different biological samples. Methods Meso Scale Discovery (MSD), TR-FRET and Millipore Erenna platforms were used to develop several immunoassays to measure soluble and aggregated mutant HTT. High-content histological approaches using the Opera platforms in combination with image analysis algorithms were optimized to quantify mutant HTT inclusions. Results We have established and validated sensitive and robust immunoassays to be able to measure soluble and aggregated HTT levels in pre-clinical and clinical biological samples. Using the Millipore Erenna platform, we can also reliably quantify mutant HTT in cerebrospinal fluid from HD patients. Finally, a combination of high content histological analysis with custom adaptations of image analysis algorithms allows us to perform quantitation of spatial and temporal changes in HTT inclusions in tissues from HD animal models. Conclusions We have established a wide portfolio of immunoassays and imaging tools able to investigate changes in HTT levels and aggregation as well as monitor disease progression in pre-clinical and clinical samples, including assessment of CSF mutant HTT levels. These assays are valuable tools to support the therapeutic approaches aimed to lower HTT expression.

Specific glycosylated peptides of clusterin are found associated with hippocampal atrophy. The glycosylation of clusterin from human plasma was comprehensively analyzed and characterized using mass spectrometry (MS)-based glycoproteomics analysis. All six known N-glycosylation sites are covered, three in the alpha subunit (α64N, α81N and α123N) and three in the beta subunit (β64N, β127N, and β147N). More detailed structural characterization of clusterin glycopeptides was also performed, demonstrating the presence of glycosylated peptides and their corresponding glycans. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we have determined the differences in the glycoforms associated at each of the different glycosylation sites in plasma clusterin obtained from subjects of low hippocampal atrophy (n = 13) and high hippocampal atrophy (n = 14). In our pilot study, the β64N site shows the most significant regulations between clinical groups. Eight β64N glycoforms are significantly reduced in patients with high atrophy compared with those with low atrophy, which demonstrates the utility of clusterin isoforms as diagnostic and prognostic Alzheimer's disease (AD) markers. These results provide a novel and robust workflow suitable for rapid verification of specific clusterin glycoforms with utility as AD biomarkers.

BACKGROUND:Four previously reported studies have tested for association of blood proteins with neocortical amyloid-β burden (NAB). If shown to be robust, these proteins could have utility as a blood test for enrichment in clinical trials of Alzheimer's disease (AD) therapeutics.OBJECTIVE:This study aimed to investigate whether previously identified blood proteins also show evidence for association with NAB in serum samples from the Australian Imaging, Biomarker and Lifestyle Flagship Study of Ageing (AIBL). The study considers candidate proteins seen in cohorts other than AIBL and candidates previously discovered in the AIBL cohort.METHODS:Our study used the SOMAscan platform for protein quantification in blood serum. Linear and logistic regressions were used to model continuous NAB and dichotomized NAB respectively using single proteins as a predictor. Multiple protein models were built using stepwise regression techniques and support vectors machines. Age and APOE ε4 carriage were used as covariates for all analysis.RESULTS:Of the 41 proteins previously reported, 15 AIBL candidates and 20 non-AIBL candidates were available for testing. Of these candidates, pancreatic polypeptide (PPY) and IgM showed a significant association with NAB. Notably, IgM was found to associate with continuous NAB across cognitively normal control subjects.CONCLUSIONS:We have further demonstrated the association of PPY and IgM with NAB, despite technical differences between studies. There are several reasons for a lack of significance for the other candidates including platform differences and the use of serum rather than plasma samples. To investigate the possibility of technical differences causing lack of further replication, further studies are required.

A blood-based protein biomarker, or set of protein biomarkers, that could predict onset and progression of Alzheimer's disease (AD) would have great utility; potentially clinically, but also for clinical trials and especially in the selection of subjects for preventative trials. We reviewed a comprehensive list of 21 published discovery or panel-based (> 100 proteins) blood proteomics studies of AD, which had identified a total of 163 candidate biomarkers. Few putative blood-based protein biomarkers replicate in independent studies but we found that some proteins do appear in multiple studies; for example, four candidate biomarkers are found to associate with AD-related phenotypes in five independent research cohorts in these 21 studies: α-1-antitrypsin, α-2-macroglobulin, apolipoprotein E, and complement C3. Using SomaLogic's SOMAscan proteomics technology, we were able to conduct a large-scale replication study for 94 of the 163 candidate biomarkers from these 21 published studies in plasma samples from 677 subjects from the AddNeuroMed (ANM) and the Alzheimer's Research UK/Maudsley BRC Dementia Case Registry at King's Health Partners (ARUK/DCR) research cohorts. Nine of the 94 previously reported candidates were found to associate with AD-related phenotypes (False Discovery Rate (FDR) q-value < 0.1). These proteins show sufficient replication to be considered for further investigation as a biomarker set. Overall, we show that there are some signs of a replicable signal in the range of proteins identified in previous studies and we are able to further replicate some of these. This suggests that AD pathology does affect the blood proteome with some consistency.

Treatment of Alzheimer's disease (AD) is significantly hampered by the lack of easily accessible biomarkers that can detect disease presence and predict disease risk reliably. Fluid biomarkers of AD currently provide indications of disease stage; however, they are not robust predictors of disease progression or treatment response, and most are measured in cerebrospinal fluid, which limits their applicability. With these aspects in mind, the aim of this article is to underscore the concerted efforts of the Blood-Based Biomarker Interest Group, an international working group of experts in the field. The points addressed include: (1) the major challenges in the development of blood-based biomarkers of AD, including patient heterogeneity, inclusion of the "right" control population, and the blood–brain barrier; (2) the need for a clear definition of the purpose of the individual markers (e.g., prognostic, diagnostic, or monitoring therapeutic efficacy); (3) a critical evaluation of the ongoing biomarker approaches; and (4) highlighting the need for standardization of preanalytical variables and analytical methodologies used by the field.

Increasingly, clinical trials for Alzheimer’s disease (AD) are being conducted earlier in the disease phase and with biomarker confirmation using in vivo amyloid PET imaging or CSF tau and Aβ measures to quantify pathology. However, making such a pre-clinical AD diagnosis is relatively costly and the screening failure rate is likely to be high. Having a blood-based marker that would reduce such costs and accelerate clinical trials through identifying potential participants with likely pre-clinical AD would be a substantial advance. In order to seek such a candidate biomarker, discovery phase proteomic analyses using 2DGE and gel-free LC-MS/MS for high and low molecular weight analytes were conducted on longitudinal plasma samples collected over a 12-year period from non-demented older individuals who exhibited a range of 11C-PiB PET measures of amyloid load. We then sought to extend our discovery findings by investigating whether our candidate biomarkers were also associated with brain amyloid burden in disease, in an independent cohort. Seven plasma proteins, including A2M, Apo-A1, and multiple complement proteins, were identified as pre-clinical biomarkers of amyloid burden and were consistent across three time points (p < 0.05). Five of these proteins also correlated with brain amyloid measures at different stages of the disease (q < 0.1). Here we show that it is possible to detect a plasma based biomarker signature indicative of AD pathology at a stage long before the onset of clinical disease manifestation. As in previous studies, acute phase reactants and inflammatory markers dominate this signature.

BackgroundThe study aimed to validate previously discovered plasma biomarkers associated with AD, using a design based on imaging measures as surrogate for disease severity and assess their prognostic value in predicting conversion to dementia.MethodsThree multicenter cohorts of cognitively healthy elderly, mild cognitive impairment (MCI), and AD participants with standardized clinical assessments and structural neuroimaging measures were used. Twenty-six candidate proteins were quantified in 1148 subjects using multiplex (xMAP) assays.ResultsSixteen proteins correlated with disease severity and cognitive decline. Strongest associations were in the MCI group with a panel of 10 proteins predicting progression to AD (accuracy 87%, sensitivity 85%, and specificity 88%).ConclusionsWe have identified 10 plasma proteins strongly associated with disease severity and disease progression. Such markers may be useful for patient selection for clinical trials and assessment of patients with predisease subjective memory complaints.

BIOMARKERS RELATING TO CSF MEASURES OF ALZHEIMER’S DISEASE PATHOLOGY Alison L. Baird, Nicholas Ashton, Sarah Westwood, Malcolm Ward, Chantal Bazenet, Madhav Thambisetty, Philip Scheltens, Charlotte Teunissen, Simon Lovestone, Kings College London, London, United Kingdom; Kings College London, Institute of Psychiatry, London, United Kingdom; King’s College London, London, United Kingdom; 4 National Institute on Aging, Baltimore, Maryland, United States; 5 VU University Medical Centre, Amsterdam, Netherlands. Contact e-mail: alison.baird@kcl.ac.uk

Background: Studies in humans have shown the potential role of alterations in DNA methylation in the pathogenesis of Late-Onset Alzheimer’s disease (LOAD). The epigenetic regulation of the genes involved in the Alzheimer’s pathogenesis may help to explain the complexity of the LOAD. Methods: DNAwas extracted from peripheral blood mononuclear cells of 100 Colombian AD patients (mean age 77.56 5 years) and 100 healthy subjects (mean age 75.36 5,5years). DNAwas bisulfite treated using EZ DNAMethylation Direct Kit (Zymo-research). Primers were manually designed for the 5’ region of the CpG Island in the APP promoter and APOE CpG Island on exon 4. The APP andAPOEMS-HRMswere performed in a CFX 96 device (BioRad), using 14 ng of bisulfite treated DNA. The quantitative results were obtained by interpolation of the first derived RFU normalized data, generated by the linear regression of the standard curve. Results: The correlation coefficient of the linear regression of the standard curves was r1⁄4 0.96 for APP gen and 0.898 for APOE gen The inter-assay and intra-assay reproducibilities were verified, having a coefficient of variance less than 0.5 between replicates. In a preliminary analysis we did not find differences in APP methylation levels between AD patients and control subjects (mean 2.14% +1.15 vs 2.05 +0.96). For APOE the difference was not significant inmethylation levels (patients vs. Controls 73.01 vs 70.9)Conclusions:The present study tested changes in DNA methylation of in blood samples from Colombian LOAD patients, which showed no significant differences in comparison with control samples for APP and APOE genes. This is the first report of the analysis of APP and APOE DNA methylation levels in LOAD using the quantitative and cost-effective in Latin American AD patients. It would be important to analyze, in further studies, the methylation levels for additional candidate regions in several tissues from AD patients.

Background:Non-invasive diagnostic markers are needed to ensure that current and future Alzheimer’s disease (AD) therapies are targeted to the right patient population. Previously, we identified plasma desmosterol as a candidate marker, with decreased levels observed (p value< 0.05, fold change1⁄4 0.36) in AD plasma samples versus controls plasma (Control/AD1⁄4 10/10). In this study, to validate this new plasma biomarker, we performed the assay using a larger commercially available Caucasian sample set and a large Asian cohort. In addition, we clarified if there are correlations between plasma desmosteroland well-established CSF biomarkers, like phosphorus Tau and Ab. Methods:We performed a validation study using a larger Caucasian sample set (Cohort 1: 109 patients, Control, MCI and AD) and a large Asian cohort (Cohort 2: 401 patients, AD and control) with LCMS. And the measurement of total Tau, phosphorus Tau (P181), Ab40 and42 were also performed using CSF from same patients. Results: At first, we performed the assay using a larger Caucasian sample set (Cohort 1: Control/MCI/AD1⁄4 42/26/41). This study validated our previous finding of significant decrease of both plasma desmosterol (p value < 0.001, fold change: 0.6) in AD and MCI patients (relative to Control), demonstrating their potential value as circulating biomarkers. The AUC of ROCcurve using plasma desmosterol was found to be 0.80, indicating high discriminating power of this marker in the two reference populations, confirming the validity of this potential biomarker in AD. Further analysis also showed significant correlations of plasma desmosterol with CSFAb 42/Ab 40 and Mini-Mental State Examination (MMSE) scores, suggesting that plasma desmosterolmight be reflective of disease progression. Next we performed a follow-up study using a large Asian cohort (Cohort 2: Control/AD1⁄4 201/200). Data analysis showed that desmosterol level in plasmawas found to be significantly different betweenADand control groups with p-values 2.3E-14 and comparableAUCofROCcurve.Moreover, like for cohort 1, high correlation between plasma desmosterol level andMMSE score was observed. Conclusions: Our study results validate the utility of desmosterol as a novel non-invasive AD biomarker candidate that should be further investigated in other dementia types.

Following binding of platelet-derived growth factor (PDGF), the PDGF alpha receptor (alphaPDGFR) becomes tyrosine phosphorylated and associates with a number of signal transduction molecules, including phospholipase Cgamma-1 (PLCgamma-1), phosphatidylinositol 3-kinase (PI3K), the phosphotyrosine phosphatase SHP-2, Grb2, and Src. Here, we present data identifying a novel phosphorylation site in the kinase insert domain of the alphaPDGFR at tyrosine (Y) 720. We replaced this residue with phenylalanine and expressed the mutated receptor (F720) in Patch fibroblasts that do not express the alphaPDGFR. Characterization of the F720 mutant indicated that binding of two proteins, SHP-2 and Grb2, was severely impaired, whereas PLCgamma-1 and PI3K associated to wild-type levels. In addition, mutating Y720 to phenylalanine dramatically reduced PDGF-dependent tyrosine phosphorylation of SHP-2. Since Y720 was required for recruitment of two proteins, we investigated the mechanism by which these two proteins associated with the alphaPDGFR. SHP-2 bound the alphaPDGFR directly, whereas Grb2 associated indirectly, most probably via SHP-2, as Grb2 and SHP-2 coimmunoprecipitated when SHP-2 was tyrosine phosphorylated. We also compared the ability of the wild-type and F720 alphaPDGFRs to mediate a number of downstream events. Preventing the alphaPDGFR from recruiting SHP-2 and Grb2 did not compromise PDGF-AA-induced activation of Ras, initiation of DNA synthesis, or growth of cells in soft agar. We conclude that phosphorylation of the alphaPDGFR at Y720 is required for association of SHP-2 and Grb2 and tyrosine phosphorylation of SHP-2; however, these events are not required for the alphaPDGFR to activate Ras or initiate a proliferative response. In addition, these findings reveal that while SHP-2 binds to both of the receptors, it binds in different locations: to the carboxy terminus of the betaPDGFR but to the kinase insert of the alphaPDGFR.

Although the mechanism of Aβ action in the pathogenesis of Alzheimer's disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aβ neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aβ/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aβ toxicity and DKK1 upregulation and, conversely, Aβ increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aβ mediates neurotoxicity, we measured the effects of Aβ and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt-planar cell polarity (PCP)-c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aβ neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aβ-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt-PCP-JNK pathway is active in an Aβ-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aβ induces a clusterin/p53/Dkk1/wnt-PCP-JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aβ in neurodegenerative diseases.Molecular Psychiatry advance online publication, 20 November 2012; doi:10.1038/mp.2012.163., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Alzheimer’s disease is a complex age-dependent neurodegenerative disease where definitive diagnosis is only possible after autopsy and where there is a long prodromal or preclinical phase. Biomarkers for both early diagnosis and prediction of disease progression are needed and extensive efforts to discover them have been undertaken. In this article, we have attempted to summarize the findings of current studies using proteomics and metabolomics approaches. We are also discussing how the use of emerging technologies and better study designs can support the identification of the much-needed Alzheimer's disease plasma biomarkers.

■ ACKNOWLEDGMENTS We formally acknowledge the scientific contribution of our colleagues at Proteome Science plc and collaborators at the Institute of Psychiatry, King’s College London. We are grateful for grant funding from the Alzheimer’s Research U.K. and the NIHR Biomedical Research Centre for Mental Health and Biomedical Research Unit for Dementia at the South London and Maudsley NHS Foundation Trust and Kings College London. AddNeuroMed was funded through the EU FP6 program.

We believe this is the first study to investigate associations between blood metabolites and neocortical amyloid burden (NAB) in the search for a blood-based biomarker for Alzheimer’s disease (AD). Further, we present the first multi-modal analysis of blood markers in this field. We used blood plasma samples from 91 subjects enrolled in the University of California, San Francisco Alzheimer’s Disease Research Centre. Non-targeted metabolomic analysis was used to look for associations with NAB using both single and multiple metabolic feature models. Five metabolic features identified subjects with high NAB, with 72% accuracy. We were able to putatively identify four metabolites from this panel and improve the model further by adding fibrinogen gamma chain protein measures (accuracy=79%). One of the five metabolic features was studied in the Alzheimer’s Disease Neuroimaging Initiative cohort, but results were inconclusive. If replicated in larger, independent studies, these metabolic features and proteins could form the basis of a blood test with potential for enrichment of amyloid pathology in anti-amyloid trials.

Previous studies have shown that Src is required for platelet-derived growth factor (PDGF)-dependent cell cycle progression in fibroblasts. Since fibroblasts usually express both PDGF receptors (PDGFRs), these findings suggested that Src was mandatory for signal relay by both the alpha and betaPDGFRs. In this study, we have focused on the role of Src in signal relay by the alphaPDGFR. In response to stimulation with PDGF-AA, which selectively engages the alphaPDGFR, Src family members (Src) associated with the alphaPDGFR and Src kinase were activated. A mutant receptor, in which tyrosines 572 and 574 were replaced with phenylalanine (F72/74), failed to efficiently associate with Src or activate Src. The wild type (WT) and F72/74 receptors induced the expression of c-myc and c-fos to comparable levels. Furthermore, an equivalent extent of PDGF-dependent soft agar growth was observed in cells expressing the WT or the F72/74 alphaPDGFR. Comparing the ability of these two receptors to initiate tyrosine phosphorylation of signaling molecules indicated that both receptors mediated phosphorylation of the receptor itself, phospholipase Cgamma 1, and SHP-2 to similar levels. In contrast, the F72/74 receptor triggered phosphorylation of Shc to 1 and 20% of the WT levels for the 55- and 46-kDa Shc isoforms, respectively. These findings indicate that after exposure of cells to PDGF-AA, Src stably associates with the alphaPDGFR, and Src activity is increased. Furthermore, Src is required for the PDGF-dependent phosphorylation of signaling molecules such as Shc. Finally, activation of Src during the G0/G1 transition does not appear to be required for latter cell cycle events such as induction of c-myc or cell proliferation.

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.

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.