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PrecisionMed, Inc. provides the highest quality human biological material for research. Institutions worldwide rely on our extensive sample inventory. Below, you’ll find a list of journal articles for which PrecisionMed, Inc. provided human biological material. Browse our inventory or request a quote for the material you are looking for.

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CSF

Title
Intracellular clusterin interacts with brain isoforms of the bridging integrator 1 and with the microtubule-associated protein Tau in Alzheimer’s disease

Journal
PLoS ONE

Overview
… were obtained from commercial sources and analyzed anonymously. Human blood and cerebrospinal fluid (CSF) from clinically diagnosed Alzheimer’s disease patients and aged controls were purchased from PrecisionMed, Inc (www.precisionmed.com). CSF samples were aliquoted and stored at −80°C until analysis. Genomic DNA was extracted from blood cells using the QIAamp DNA blood midi prep (Qiagen).

Year
2014

Authors
Zhou, Y;Hayashi, I;Wong, J;Tugusheva, K;Renger, JJ;Zerbinatti, C;

Link
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103187

Abstract
Sporadic or late-onset Alzheimer’s disease (AD) is expected to affect 50% of individuals reaching 85 years of age. The most significant genetic risk factor for late-onset AD is the e4 allele of APOE gene encoding apolipoprotein E, a lipid carrier shown to modulate brain amyloid burden. Recent genome-wide association studies have uncovered additional single nucleotide polymorphisms (SNPs) linked to AD susceptibility, including those in the CLU and BIN1 genes encoding for clusterin (CLU) and the bridging integrator 1 (BIN1) proteins, respectively. Because CLU has been implicated in brain amyloid-β (Aβ) clearance in mouse models of amyloid deposition, we sought to investigate whether an AD-linked SNP in the CLU gene altered Aβ42 biomarker levels in the cerebrospinal fluid (CSF). Instead, we found that the CLU rs11136000 SNP modified CSF levels of the microtubule-associated protein Tau in AD patients. We also found that an intracellular form of CLU (iCLU) was upregulated in the brain of Tau overexpressing Tg4510 mice, but not in Tg2576 amyloid mouse model. By overexpressing iCLU and Tau in cell culture systems we discovered that iCLU was a Tau-interacting protein and that iCLU associated with brain-specific isoforms of BIN1, also recently identified as a Tau-binding protein. Through expression analysis of CLU and BIN1 variants, we found that CLU and BIN1 interacted via their coiled-coil motifs. In co-immunoprecipitation studies using human brain tissue, we showed that iCLU and the major BIN1 isoform expressed in neurons were associated with modified Tau species found in AD. Finally, we showed that expression of certain coding CLU variants linked to AD risk led to increased levels of iCLU. Together, our findings suggest that iCLU and BIN1 interaction might impact Tau function in neurons and uncover potential new mechanisms underlying the etiology of Tau pathology in AD.

Title
Interleukin-6 is elevated in the cerebrospinal fluid of suicide attempters and related to symptom severity

Journal
Biol. Psychiatry

Overview
No Diagnosis (n), 7, 2, N/A. N/A, not applicable. Control Subjects. The healthy control subjects were recruited through the Neuropsychiatric and Psychiatric Clinics at the University Hospitals in Malmo and Linkoping, Sweden, and PrecisionMed, San Diego, California.

Year
2009

Authors
Lindqvist, D;Janelidze, S;Hagell, P;Erhardt, S;Samuelsson, M;Minthon, L;Hansson, O;Björkqvist, M;Träskman-Bendz, L;Brundin, L;

Link
https://www.sciencedirect.com/science/article/pii/S0006322309001280

Abstract
Depressive disorders are associated with immune system alterations that can be detected in the blood. Cytokine concentrations in cerebrospinal fluid (CSF) and their relationship to aspects of suicidality have previously not been investigated. We measured interleukin-1beta, interleukin-6 (IL-6), interleukin-8, and tumor necrosis factor-alpha (TNF-alpha) in CSF and plasma of suicide attempters (n = 63) and healthy control subjects (n = 47). Patients were classified according to diagnosis and violent or nonviolent suicide attempt. We evaluated suicidal ideation and depressive symptoms using the Suicide Assessment Scale and the Montgomery-Asberg Depression Rating Scale (MADRS). We also analyzed the relation between cytokines and monoamine metabolites 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in CSF, as well as the integrity of the blood-brain barrier as reflected by the CSF:serum albumin ratio. IL-6 in CSF was significantly higher in suicide attempters than in healthy control subjects. Patients who performed violent suicide attempts displayed the highest IL-6. Furthermore, there was a significant positive correlation between MADRS scores and CSF IL-6 levels in all patients. IL-6 and TNF-alpha correlated significantly with 5-HIAA and HVA in CSF, but not with MHPG. Cytokine levels in plasma and CSF were not associated, and patients with increased blood-brain barrier permeability did not exhibit elevated cytokine levels. We propose a role for CSF IL-6 in the symptomatology of suicidal behavior, possibly through mechanisms involving alterations of dopamine and serotonin metabolism.

Title
Characterization of IL-17AA and IL-17FF in rheumatoid arthritis and multiple sclerosis

Journal
Bioanalysis

Overview
Samples & sample analysis. Serum samples from NHS were obtained from PrecisionMed, Inc Serum and CSF samples from 50 RRMS patients were obtained from PrecisionMed, Inc. (Table 4 & Supplementary Table 9). Normal CSF was obtained from PrecisionMed, Inc.

Year
2016

Authors
Schofield, C;Fischer, SK;Townsend, MJ;Mosesova, S;Peng, K;Setiadi, AF;Song, A;Baruch, A;

Link
https://www.future-science.com/doi/abs/10.4155/bio-2016-0207

Abstract
IL-17 is thought to play a prominent role in immune disorders. Sensitive and specific IL-17AA and IL-17FF assays were developed and used to determine levels in serum and cerebrospinal fluid (CSF) from patients with rheumatoid arthritis and relapsing remitting multiple sclerosis (RRMS). Qualified assays detected IL-17AA and IL-17FF in healthy and disease samples. Serum IL-17AA was significantly higher in rheumatoid arthritis and RRMS as compared with normal healthy subjects. IL-17AA was also elevated in RRMS CSF as compared with normal healthy subjects; although correlation was observed between serum levels of the two isoforms, no correlation was detected between serum and CSF levels. Reliable determination of IL-17 isoforms in the systemic and CNS compartments sheds light on the involvement of IL-17AA and IL-17FF in autoimmunity.

Title
Polar anionic metabolome analysis by nano-LC/MS with a metal chelating agent

Journal
Anal. Chem.

Overview
The samples were then centrifuged at 12 000 rpm for 20 min. The upper phase (MeOH and water) was used for analysis of polar anionic metabolites. Human plasma (50 μL) and cerebrospinal fluid (CSF) (100 μL), purchased from Precision Med Inc.

Year
2009

Authors
Myint, KT;Uehara, T;Aoshima, K;Oda, Y;

Link
https://pubs.acs.org/doi/abs/10.1021/ac901269h

Abstract
We have developed a practical method for the comprehensive analysis of polar anionic metabolites in biological samples with the use of a nano-LC/MS system. A polyamine-bonded polymer-based apHera NH2 column, which is compatible with ammonium carbonate buffer, effectively retained anionic polar metabolites, such as organic acids, sulfates, and phosphates, but multiply phosphorylated or carboxylated compounds showed highly distorted peak shapes on chromatograms. We found that addition of a trace amount of the metal chelating reagent ethylenediaminetetraacetic acid (EDTA) to the sample solution dramatically improved peak shapes of multiply charged anionic compounds, even though the mass spectra showed no trace of adduct ions in the absence of EDTA. The detection limits of typical polar anionic metabolites in the full-scan mode were from 0.19 to 2.81 pmol. After optimization of all the procedures from sample preparation to nano-LC/MS analysis, we applied our method to real biological samples: Hela cells, mouse brain, human cerebrospinal fluid (CSF), and human plasma. Our results indicated that phosphorylated metabolites were abundant in Hela cells and brain, while plasma and cerebrospinal fluid (CSF) mostly contained organic acids. Phosphorylated compounds might not be secreted into CSF/plasma or might be unstable in CSF/plasma. Finally, the method was used to examine the mode of action of the anticancer drug methotrexate (MTX), which inhibits purine de novo biosynthesis and thymidine biosynthesis. In addition of the expected changes of metabolite levels, we found that a previously unreported metabolite, probably a methylated uridine 5′-triphosphate (UTP), was produced by MTX-treated Hela cells.

Title
Identification of a new plasma biomarker of Alzheimer’s disease using metabolomics technology

Journal
J. Lipid Res.

Overview
(Alabaster, AL). Samples collection. Plasma and CSF samples of elderly controls and subjects diagnosed with AD, MCI, schizophrenia, and Parkinson’s disease were purchased from PrecisionMed, Inc. (San Diego, CA) and stored at −80°C until use…

Year
2012

Authors
Sato, Y;Suzuki, I;Nakamura, T;Bernier, F;Aoshima, K;Oda, Y;

Link
http://www.jlr.org/content/53/3/567.short

Abstract
We performed unbiased analysis of steroid-related compounds to identify novel Alzheimer’s disease (AD) plasma biomarkers using liquid chromatography-atmospheric pressure chemical ionization-mass spectroscopy. The analysis revealed that desmosterol was found to be decreased in AD plasma versus controls. To precisely quantify variations in desmosterol, we established an analytical method to measure desmosterol and cholesterol. Using this LC-based method, we discovered that desmosterol and the desmosterol/cholesterol ratio are significantly decreased in AD. Finally, the validation of this assay using 109 clinical samples confirmed the decrease of desmosterol in AD as well as a change in the desmosterol/cholesterol ratio in AD. Interestingly, we could also observe a difference between mild cognitive impairment and control. In addition, the decrease of desmosterol was somewhat more significant in females. Receiver operating characteristic (ROC) analysis between controls and AD, using plasma desmosterol shows a score of 0.80, indicating a good discrimination power for this marker in the two reference populations and confirms the potential usefulness of measuring plasma desmosterol levels for diagnosing AD. Further analysis showed a significant correlation of plasma desmosterol with Mini-Mental State Examination scores. Although larger sample populations will be needed to confirm this diagnostic marker sensitivity, our studies demonstrate a sensitive and accurate method of detecting plasma desmosterol concentration and suggest that plasma desmosterol could become a powerful new specific biomarker for early and easy AD diagnosis.

Title
Ethylenediaminetetraacetic acid increases identification rate of phosphoproteomics in real biological samples

Journal
J. Proteome Res.

Overview
Aldrich Co. (St. Louis, MO), respectively. Human plasma and human cerebrospinal fluid (CSF) were purchased from PrecisionMed. Inc. (San Diego, CA). Other reagents of analytical grade were used without further treatment.

Year
2010

Authors
Nakamura, T;Myint, KT;Oda, Y;

Link
https://pubs.acs.org/doi/abs/10.1021/pr900918h

Abstract
We have developed a novel approach to enhance phosphopeptide identification in liquid chromatography/mass spectrometry (LC/MS)-based phosphoproteomics. After enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO(2)) microcolumns, samples were coinjected with ethylenediaminetetraacetic acid (EDTA) into LC/MS. This procedure decreased the MS peak intensity of nonphosphorylated peptides, but not that of phosphopeptides, and as a result, the number of identified phosphopeptides was increased. EDTA appeared to have no effect on liquid chromatographic separation of phosphorylated and nonphosphorylated peptides. Although the mechanism of the positive effect of EDTA on identification of phosphopeptides is unknown, and we have never observed metal ion adduct peaks in LC/MS spectra, coinjection of EDTA seemed to enhance phosphopeptide recovery from the LC/MS system. This simple technique was successfully applied to the identification of phosphopeptides in mouse brain (2938 phosphopeptides), human plasma (127 phosphopetides), and human cerebrospinal fluid (CSF) (123 phosphopeptides). We also identified nonphosphopeptides in the same samples using a two-dimensional (2D) LC/MS-based shotgun approach. The results overall indicated that 20-25% of brain proteins were phosphorylated, while only 1-2% of proteins in plasma and CSF were phosphorylated. These ratios were almost constant throughout the range of protein expression levels. In addition, EDTA-enhanced phosphoproteomics could identify low-abundance proteins in the samples, because nonphosphoproteins corresponding to more than one-third of the identified phosphoproteins could not be identified by 2D-LC/MS. Finally, we were able to find that the newly developed approach was very effective for the phosphoproteome analysis in Alzheimer disease model mice brain.

Title
High-throughput and simultaneous quantitative analysis of homocysteine-methionine cycle metabolites and co-factors in blood plasma and cerebrospinal fluid by isotope dilution LC-MS/MS

Journal
Anal Bioanal Chem

Overview
The injection volume was 10 μL and the total run time of analysis was 13 min. Human sample collection for method validation. The human sub-cohort was supplied from PrecisionMed, Inc. (CA, USA, protocol 8009). This…

Year
2017

Authors
Guiraud, SP;Montoliu, I;Da Silva, L;Dayon, L;Galindo, AN;Corthésy, J;Kussmann, M;Martin, FP;

Link
https://link.springer.com/article/10.1007/s00216-016-0003-1

Abstract
The methionine cycle is a key pathway contributing to the regulation of human health, with well-established involvement in cardiovascular diseases and cognitive function. Changes in one-carbon cycle metabolites have also been associated with mild cognitive decline, vascular dementia, and Alzheimer’s disease. Today, there is no single analytical method to monitor both metabolites and co-factors of the methionine cycle. To address this limitation, we here report for the first time a new method for the simultaneous quantitation of 17 metabolites in the methionine cycle, which are homocysteic acid, taurine, serine, cysteine, glycine, homocysteine, riboflavin, methionine, pyridoxine, cystathionine, pyridoxamine, S-adenosylhomocysteine, S-adenosylmethionine, betaine, choline, dimethylglycine, and 5-methyltetrahydrofolic acid. This multianalyte method, developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), provides a highly accurate and precise quantitation of these 17 metabolites for both plasma and cerebrospinal fluid metabolite monitoring. The method requires a simple sample preparation, which, combined with a short chromatographic run time, ensures a high sample throughput. This analytical strategy will thus provide a novel metabolomics approach to be employed in large-scale observational and intervention studies. We expect such a robust method to be particularly relevant for broad and deep molecular phenotyping of individuals in relation to their nutritional requirements, health monitoring, and disease risk management.

Title
Cerebrospinal fluid neurofilament concentration reflects disease severity in frontotemporal degeneration

Journal
Ann. Neurol.

Overview
17 with corticobasal syndrome (CBS).23 Forty‐seven NCs had normal neurological examinations, neuropsychological testing scores, and Clinical Dementia Rating (CDR) scores of 0 (27 were evaluated at UCSF and 20 were samples purchased from PrecisionMed (San Diego)

Year
2014

Authors
Scherling, CS;Hall, T;Berisha, F;Klepac, K;Karydas, A;Coppola, G;Kramer, JH;Rabinovici, G;Ahlijanian, M;Miller, BL;Seeley, W;Grinberg, LT;Rosen, H;Meredith, J;Boxer, AL;

Link
https://onlinelibrary.wiley.com/doi/abs/10.1002/ana.24052

Abstract
Cerebrospinal fluid (CSF) neurofilament light chain (NfL) concentration is elevated in neurological disorders, including frontotemporal degeneration (FTD). We investigated the clinical correlates of elevated CSF NfL levels in FTD. CSF NfL, amyloid-β1-42 (Aβ42), tau, and phosphorylated tau concentrations were compared in 47 normal controls (NC), 8 asymptomatic gene carriers (NC2) of FTD-causing mutations, and 79 FTD (45 behavioral variant frontotemporal dementia [bvFTD], 18 progressive nonfluent aphasia [PNFA], 16 semantic dementia [SD]), 22 progressive supranuclear palsy, 50 Alzheimer disease, 6 Parkinson disease, and 17 corticobasal syndrome patients. Correlations between CSF analyte levels were performed with neuropsychological measures and the Clinical Dementia Rating scale sum of boxes (CDRsb). Voxel-based morphometry of structural magnetic resonance images determined the relationship between brain volume and CSF NfL. Mean CSF NfL concentrations were higher in bvFTD, SD, and PNFA than other groups. NfL in NC2 was similar to NC. CSF NfL, but not other CSF measures, correlated with CDRsb and neuropsychological measures in FTD, but not in other diagnostic groups. Analyses in 2 independent FTD cohorts and a group of autopsy-verified or biomarker-enriched cases confirmed the larger group analysis. In FTD, gray and white matter volume negatively correlated with CSF NfL concentration, such that individuals with the highest NfL levels exhibited the most atrophy. CSF NfL is elevated in symptomatic FTD and correlates with disease severity. This measurement may be a useful surrogate endpoint of disease severity in FTD clinical trials. Longitudinal studies of CSF NfL in FTD are warranted. © 2014 Child Neurology Society/American Neurological Association.

Title
Identification of longitudinally dynamic biomarkers in Alzheimer’s disease cerebrospinal fluid by targeted proteomics

Journal
Mol Neurodegener

Overview
Purchased from Bioreclamation, LLC (Hicksville, NY) (pooled cynomolgus monkey and pooled human CSF used in discovery experiments), Folio Biosciences (Columbus, OH) (individual longitudinal AD only) and PrecisionMed, Inc. (San Diego, CA) (individual longitudinal Control, MCI and AD). Details on sample collection were provided by the vendors. All donors provided informed consent for use of these studies.

Year
2014

Authors
Wildsmith, KR;Schauer, SP;Smith, AM;Arnott, D;Zhu, Y;Haznedar, J;Kaur, S;Mathews, WR;Honigberg, LA;

Link
https://molecularneurodegeneration.biomedcentral.com/articles/10.1186/1750-1326-9-22

Abstract
Alzheimer’s disease (AD) is the leading cause of dementia affecting greater than 26 million people worldwide. Although cerebrospinal fluid (CSF) levels of Aβ42, tau, and p-tau181 are well established as diagnostic biomarkers of AD, there is a need for additional CSF biomarkers of neuronal function that continue to change during disease progression and could be used as pharmacodynamic measures in clinical trials. Multiple proteomic discovery experiments have reported a range of CSF biomarkers that differ between AD and control subjects. These potential biomarkers represent multiple aspects of the disease pathology. The performance of these markers has not been compared with each other, and their performance has not been evaluated longitudinally. We developed a targeted-proteomic, multiple reaction monitoring (MRM) assay for the absolute quantitation of 39 peptides corresponding to 30 proteins. We evaluated the candidate biomarkers in longitudinal CSF samples collected from aged, cognitively-normal control (n = 10), MCI (n = 5), and AD (n = 45) individuals (age > 60 years). We evaluated each biomarker for diagnostic sensitivity, longitudinal consistency, and compared with CSF Aβ42, tau, and p-tau181. Four of 28 quantifiable CSF proteins were significantly different between aged, cognitively-normal controls and AD subjects including chitinase-3-like protein 1, reproducing published results. Four CSF markers demonstrated significant longitudinal change in AD: Amyloid precursor protein, Neuronal pentraxin receptor, NrCAM and Chromogranin A. Robust correlations were observed within some subgroups of proteins including the potential disease progression markers. Using a targeted proteomics approach, we confirmed previous findings for a subset of markers, defined longitudinal performance of our panel of markers, and established a flexible proteomics method for robust multiplexed analyses.

Title
VDJServer: A Cloud-Based Analysis Portal and Data Commons for Immune Repertoire Sequences and Rearrangements

Journal
Front Immunol

Overview
… with IRB-approved protocols at UT Southwestern Medical Center, the University of Massachusetts Memorial Medical Center (UMass), Johns Hopkins University, or purchased from a commercial biorepository (PrecisionMed, Solana Beach, CA, USA). The analysis workflow included VDJPipe for preprocessing, IgBlast for V(D)J assignment, and RepSum for rearrangement annotation. VDJPipe preprocessing trimmed …

Year
2018

Authors
Christley, S;Scarborough, W;Salinas, E;Rounds, WH;Toby, IT;Fonner, JM;Levin, MK;Kim, M;Mock, SA;Jordan, C;Ostmeyer, J;Buntzman, A;Rubelt, F;Davila, ML;Monson, NL;Scheuermann, RH;Cowell, LG;

Link
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953328/

Abstract
Recent technological advances in immune repertoire sequencing have created tremendous potential for advancing our understanding of adaptive immune response dynamics in various states of health and disease. Immune repertoire sequencing produces large, highly complex data sets, however, which require specialized methods and software tools for their effective analysis and interpretation. VDJServer is a cloud-based analysis portal for immune repertoire sequence data that provide access to a suite of tools for a complete analysis workflow, including modules for preprocessing and quality control of sequence reads, V(D)J gene segment assignment, repertoire characterization, and repertoire comparison. VDJServer also provides sophisticated visualizations for exploratory analysis. It is accessible through a standard web browser via a graphical user interface designed for use by immunologists, clinicians, and bioinformatics researchers. VDJServer provides a data commons for public sharing of repertoire sequencing data, as well as private sharing of data between users. We describe the main functionality and architecture of VDJServer and demonstrate its capabilities with use cases from cancer immunology and autoimmunity. VDJServer provides a complete analysis suite for human and mouse T-cell and B-cell receptor repertoire sequencing data. The combination of its user-friendly interface and high-performance computing allows large immune repertoire sequencing projects to be analyzed with no programming or software installation required. VDJServer is a web-accessible cloud platform that provides access through a graphical user interface to a data management infrastructure, a collection of analysis tools covering all steps in an analysis, and an infrastructure for sharing data along with workflows, results, and computational provenance. VDJServer is a free, publicly available, and open-source licensed resource.

Title
Quantitative profiling of polar cationic metabolites in human cerebrospinal fluid by reversed-phase nanoliquid chromatography/mass spectrometry

Journal
Anal. Chem.

Overview
CSF Preparation. Cerebrospinal fluid samples of age-matched controls and subjects diagnosed with Alzheimer’s disease (AD) were purchased from PrecisionMed Inc., San Diego, CA. The deep-frozen CSF samples were thawed at room temperature and filtered with an Ultrafree-MC 5 000 NMWL centrifuge filter unit (Millipore Corporation, Bedford, MA) to remove proteins. Then 0.15 mL of protein-free CSF fraction was spiked with…

Year
2009

Authors
Myint, KT;Aoshima, K;Tanaka, S;Nakamura, T;Oda, Y;

Link
https://pubs.acs.org/doi/abs/10.1021/ac802259r

Abstract
Reversed-phase (RP) nanoliquid chromatography (LC)/mass spectrometry (MS) is widely used for proteome analysis, but hydrophilic metabolites are poorly retained on RP columns. We describe here the development and application of an efficient, robust, and quantitative nano-LC/MS method for cationic metabolome analysis in the positive ionization mode without any derivatization of analytes. Various stationary phases for nano-LC, coating of the internal wall of the capillary column, and various mobile phases were evaluated in terms of separation and peak shapes for 33 hydrophilic metabolites, including nonderivatized amino acids. Polar cationic compounds were strongly bound to mixed-functional RP with cation exchange mode resin, and the best separation was obtained with hydrophilic internal wall coating and a two-step trifluoroacetic acid (TFA) gradient in methanol as the mobile phase. Simple, but optimized, sample processing and the use of a high content of methanol allowed robust nano-LC/MS analysis. Our developed method was applied for biomarker discovery in Alzheimer’s disease (AD). Several hundred peaks were detected from 10 microL of cerebrospinal fluid (CSF). In a principal component analysis (PCA) plot using peak intensities without normalization, peak separation depended on the experimental date, not disease state. Therefore, constant amounts of two stable isotope-labeled amino acids, Val and Lys, were added as internal standards (ISs) to each sample before processing. These ISs were eluted in different gradient slopes in the two-step gradient, and the normalized peak ratios using the corresponding ISs gave a unique group of PCA scores which could distinguish AD CSF samples from age-matched control CSF samples.

Title
Opportunistic DNA Recombination With Epstein-Barr Virus at Sites of Control Region Rearrangements Mediating JC Virus Neurovirulence

Journal
J. Infect. Dis.

Overview
… Methods [http://jid.oxfordjournals.org/lookup/suppl/doi:10.1093/infdis/jiv755/-/DC1]. DNA FROM CEREBROSPINAL FLUID OF PATIENTS WITH MS Patients’ cerebrospinal fluid (CSF) samples were obtained from PrecisionMed. All of the patients were properly consented volunteer donors, and CSF samples were collected using institutional review board–approved protocols. All samples are from the United States.

Year
2016

Authors
Wortman, MJ;Lundberg, PS;Dagdanova, AV;Venkataraman, P;Daniel, DC;Johnson, EM;

Link
https://academic.oup.com/jid/article-abstract/213/9/1436/2459498

Abstract
We document a unique DNA recombination between polyomavirus JC (JC virus [JCV]) and Epstein-Barr virus (EBV) at sequences of JCV found infecting the brain. Archetype JCV is present in bone marrow and uroepithelial cells of most adults. During immunosuppression, JCV can infect the brain, causing a demyelinating disease, progressive multifocal leukoencephalopathy. Rearrangements in the archetype noncoding control region are necessary for neurovirulence. Two NCCR deletions and a duplication occur at sequences of homology with EBV, present latently in B cells, which may be coinfected with both viruses. Recombination between JCV and EBV occurs in B lymphoblasts at a sequence essential for JCV neurovirulence and in cerebrospinal fluid of immunosuppressed patients with multiple sclerosis, those susceptible to progressive multifocal leukoencephalopathy. Interviral recombination is a model for conferring advantages on JCV in the brain. It can alter a critical noncoding control region sequence and potentially facilitate use of EBV DNA abilities to transfer among different cell types. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Title
Autoantibodies to the adenosine triphosphate synthase play a pathogenetic role in Alzheimer’s disease

Journal
Neurobiol. Aging

Overview
Control sera from 48 healthy subjects with preserved cognitive functions were divided in two groups to match with patients’ age. We also analyzed three cerebrospinal fluids (CSF) from patients with AD provided by PrecisionMed, Inc.

Year
2012

Authors
Vacirca, D;Delunardo, F;Matarrese, P;Colasanti, T;Margutti, P;Siracusano, A;Pontecorvo, S;Capozzi, A;Sorice, M;Francia, A;Malorni, W;Ortona, E;

Link
https://www.sciencedirect.com/science/article/pii/S0197458010002277

Abstract
It has become evident that an autoimmune component could play a role in Alzheimer’s disease (AD) onset and/or progression. The aim of this study was to identify neuronal antigenic targets specifically recognized by serum autoantibodies and to investigate their cellular effects and their possible pathogenetic role. We identified, by an immunoproteomic approach using mouse brain proteins, the adenosine triphosphate (ATP) synthase β subunit as a new autoantigen in AD. Using an ELISA assay we found that serum anti-ATP synthase autoantibodies were present in 38% of patients with AD, but in no age-matched healthy subjects or in patients with Parkinson’s disease or atherosclerosis. Analytical cytology studies, using SH-SY5Y neuroblastoma cell line, showed that ATP synthase autoantibodies were capable of inducing the inhibition of ATP synthesis, alterations of mitochondrial homeostasis and cell death by apoptosis. These findings suggest that autoantibodies specific to ATP synthase can exert a pathogenetic role via a mechanism that brings into play the impairment of the extracellular ATP homeostasis and the alteration of mitochondrial function triggering cell death by apoptosis. Copyright © 2012 Elsevier Inc. All rights reserved.

Title
Altered chemokine levels in the cerebrospinal fluid and plasma of suicide attempters

Journal
Psychoneuroendocrinology

Overview
Forty-three healthy control subjects with no previous or ongoing psychiatric condition were recruited through the Neuropsychiatric and Psychiatric Clinics at the University Hospitals in Malmoe and Linkoeping, Sweden (n = 38), and PrecisionMed Inc., San Diego, CA (n = 5

Year
2013

Authors
Janelidze, S;Ventorp, F;Erhardt, S;Hansson, O;Minthon, L;Flax, J;Samuelsson, M;Traskman-Bendz, L;Brundin, L;

Link
https://www.sciencedirect.com/science/article/pii/S0306453012003265

Abstract
Chemokines constitute a class of small inflammatory proteins that control the chemotaxis of leukocytes. They are also present in the central nervous system (CNS) and contribute to diverse physiological functions, such as the regulation of cell migration, axonal growth and neuronal survival. It is to date not known whether chemokines in the CNS are affected in psychiatric disorders. In this study, chemokine levels were measured in the cerebrospinal fluid (CSF) of 137 psychiatric patients in conjunction to a suicide attempt, and 43 healthy controls. A subgroup of patients (n = 42) was followed up with blood samples 12 years after the initial CSF collection, when they did not show suicidal behavior. The follow-up chemokine levels were compared to those of psychiatric patients (n = 17) who had never attempted suicide. Ultra-sensitive chemokine multiplex immunoassay was used to quantify eotaxin-1 (CCL11), interferon gamma-induced protein-10 (IP-10, CXCL10), macrophage inflammatory protein-1β (MIP-1β, CCL4), monocyte chemotactic protein-1 (MCP-1, CCL2), MCP-4 (CCL13) and thymus and activation regulated chemokine (TARC, CCL17). Patients were diagnosed using DSM-III-R/DSM-IV, and assessed using the Comprehensive Psychopathological Rating Scale (CPRS), including subscales, and the Suicidal Intent Scale (SIS). CSF eotaxin-1, MIP-1β, MCP-1, MCP-4 and TARC were significantly lower in suicide attempters than in healthy controls. Low chemokine levels were specifically associated with psychotic symptoms and pain. In the samples collected at follow-up, TARC was significantly lower in suicide attempters compared to psychiatric patients who had never attempted suicide. We also found a positive correlation between blood TARC and brain-derived neurotrophic factor (BDNF) levels. Our study thus provides evidence of reduced chemokine levels in suicide attempters, both in the acute suicidal setting, and at long-term, compared to non-attempters. These results warrant future studies on the detailed neurobiological functions of chemokines in psychiatric patients. Copyright © 2012 Elsevier Ltd. All rights reserved.

Title
Cerebrospinal Aβ11-x and 17-x levels as indicators of mild cognitive impairment and patients’ stratification in Alzheimer’s disease

Journal
Transl Psychiatry

Overview
(Gent, Belgium). CSF samples. Human CSF samples from age-matched patients with AD (n=23) or MCI (n=23) and donors with normal cognition (controls, n=21) were provided by PrecisionMed (San Diego, CA, USA 31 ).

Year
2013

Authors
Abraham, JD;Promé, S;Salvetat, N;Rubrecht, L;Cobo, S;du Paty, E;Galéa, P;Mathieu-Dupas, E;Ranaldi, S;Caillava, C;Crémer, GA;Rieunier, F;Robert, P;Molina, F;Laune, D;Checler, F;Fareh, J;

Link
https://www.nature.com/articles/tp201358

Abstract
In the present work, the concentrations of Aβ11-x and Aβ17-x peptides (x=40 or 42), which result from the combined cleavages of β-amyloid precursor protein (AβPP) by β’/α or α/γ-secretases, respectively, were assessed in cerebrospinal fluid (CSF) samples from patients with Alzheimer’s disease (AD) or mild cognitive impairment (MCI). Specific multiplexed assays were set up using new anti-40 and anti-42 monoclonal antibodies (mAbs) for the capture of these N-truncated Aβ peptides and anti-11 or anti-17 mAbs for their detection. The specificity, sensitivity and reproducibility of such assays were assessed using synthetic peptides and human cell models. Aβ11-x and Aβ17-x were then measured in CSF samples from patients with AD (n=23), MCI (n=23) and controls with normal cognition (n=21). Aβ11-x levels were significantly lower in patients with MCI than in controls. Compared with the combined quantification of Aβ1-42, total Tau (T-Tau) and phosphorylated Tau (P-Tau; AlzBio3, Innogenetics), the association of Aβ11-40, Aβ17-40 and T-Tau improved the discrimination between MCI and controls. Furthermore, when patients with MCI were classified into two subgroups (MCI ≤1.5 or ≥2 based on their CDR-SB (Cognitive Dementia Rating-Sum of Boxes) score), the CSF Aβ17-40/Aβ11-40 ratio was significantly higher in patients with CDR-SB ≤1.5 than in controls, whereas neither Aβ1-42, T-Tau nor P-Tau allowed the detection of this subpopulation. These results need to be confirmed in a larger clinical prospective cohort.

Title
Novel one-step immunoassays to quantify α-synuclein: applications for biomarker development and high-throughput screening

Journal
J. Biol. Chem.

Overview
To prepare immunodepleted human CSF to serve as a calibration matrix, 0.5 ml of commercially available CSF from a healthy donor (PrecisionMed; supplemented with 1 % TritonX- 100, 0.2 % NP-40, and protease inhibitors) was mixed with 350 ng of hSA5.1 antibody for 1 h…

Year
2012

Authors
Bidinosti, M;Shimshek, DR;Mollenhauer, B;Marcellin, D;Schweizer, T;Lotz, GP;Schlossmacher, MG;Weiss, A;

Link
http://www.jbc.org/content/early/2012/07/27/jbc.M112.379792.short

Abstract
Familial Parkinson disease (PD) can result from α-synuclein gene multiplication, implicating the reduction of neuronal α-synuclein as a therapeutic target. Moreover, α-synuclein content in human cerebrospinal fluid (CSF) represents a PD biomarker candidate. However, capture-based assays for α-synuclein quantification in CSF (such as by ELISA) have shown discrepancies and have limited suitability for high-throughput screening. Here, we describe two sensitive, in-solution, time-resolved Förster’s resonance energy transfer (TR-FRET)-based immunoassays for total and oligomeric α-synuclein quantification. CSF analysis showed strong concordance for total α-synuclein content between two TR-FRET assays and, in agreement with a previously characterized 36 h protocol-based ELISA, demonstrated lower α-synuclein levels in PD donors. Critically, the assay suitability for high-throughput screening of siRNA constructs and small molecules aimed at reducing endogenous α-synuclein levels was established and validated. In a small-scale proof of concept compound screen using 384 well plates, signals ranged from <30 to >120% of the mean of vehicle-treated cells for molecules known to lower and increase cellular α-synuclein, respectively. Furthermore, a reverse genetic screen of a kinase-directed siRNA library identified seven genes that modulated α-synuclein protein levels (five whose knockdown increased and two that decreased cellular α-synuclein protein). This provides critical new biological insight into cellular pathways regulating α-synuclein steady-state expression that may help guide further drug discovery efforts. Moreover, we describe an inherent limitation in current α-synuclein oligomer detection methodology, a finding that will direct improvement of future assay design. Our one-step TR-FRET-based platform for α-synuclein quantification provides a novel platform with superior performance parameters for the rapid screening of large biomarker cohorts and of compound and genetic libraries, both of which are essential to the development of PD therapies.

Title
Generation and Partial Characterization of Rabbit Monoclonal Antibody to Amyloid-β Peptide 1-37 (Aβ37)

Journal
J. Alzheimers Dis.

Overview
Bank for Developmental Disabilities and Aging. Collection of CSF and plasma. CSF from 11 patients with probable AD, and 11 controls were purchased from PrecisionMed (Solana Beach, CA). Plasma from 28 patients with probable.

Year
2017

Authors
Mehta, PD;Blain, JF;Freeman, EA;Patrick, BA;Barshatzky, M;Hrdlicka, LA;Mehta, SP;Frackowiak, J;Mazur-Kolecka, B;Wegiel, J;Patzke, H;Miller, DL;

Link
https://content.iospress.com/articles/journal-of-alzheimers-disease/jad161207

Abstract
Secreted soluble amyloid-β 1-37 (Aβ37) peptide is one of the prominent Aβ forms next to Aβ40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aβ37 levels in combination with Aβ38, Aβ40, and Aβ42 to support the diagnosis of patients with probable Alzheimer’s disease (AD), and the value of antibody to Aβ37 to facilitate drug discovery studies. However, the availability of reliable and specific monoclonal antibody to Aβ37 is very limited. Our aims were: 1) to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aβ37, and 2) to determine whether the antibody detects changes in Aβ37 levels produced by a γ-secretase modulator (GSM). Our generated RabmAb to Aβ37 was found to be specific to Aβ37, since it did not react with Aβ36, Aβ38, Aβ39, Aβ40, and Aβ42 in an ELISA or immunoblotting. The epitope of the antibody was contained in the seven C-terminal residues of Aβ37. The antibody was sensitive enough to measure CSF and plasma Aβ37 levels in ELISA. Immunohistological studies showed the presence of Aβ37-positive deposits in the brain of AD, and Down syndrome persons diagnosed with AD. Our studies also showed that the antibody detected Aβ37 increases in CSF and brains of rodents following treatment with a GSM. Thus, our antibody can be widely applied to AD research, and in a panel based approach it may have potential to support the diagnosis of probable AD, and in testing the effect of GSMs to target AD.

Title
The amyloid-β oligomer count in cerebrospinal fluid is a biomarker for Alzheimer’s disease

Journal
J. Alzheimers Dis.

Overview
Channel excluded background with low fluorescence intensities from the analysis. Human CSF samples CSF samples of elderly controls and subjects diag- nosed with AD were purchased from PrecisionMed, Inc. (San Diego, CA) and stored at −80 ◦ C…

Year
2013

Authors
Wang-Dietrich, L;Funke, SA;Kühbach, K;Wang, K;Besmehn, A;Willbold, S;Cinar, Y;Bannach, O;Birkmann, E;Willbold, D;

Link
https://content.iospress.com/articles/journal-of-alzheimers-disease/jad122047

Abstract
Recent studies indicate that small amyloid-β peptide (Aβ) oligomers are the major toxic species responsible for development and progression of Alzheimer’s disease (AD). Therefore, we suggest that the number of Aβ oligomers in body fluids is the most direct and relevant biomarker for AD. Determination of the Aβ oligomer content of cerebrospinal fluid (CSF) samples from 14 AD patients and 12 age-matched controls revealed a clear distinction between both groups. All samples of the control group showed homogenously low numbers of Aβ oligomers, while the samples of the AD group exhibited significantly higher levels of Aβ oligomers. The Aβ oligomer numbers correlated with the patients’ Mini-Mental State Examination scores. This indicates that the quantity of Aβ oligomers in CSF reflects the severity of the disease and that Aβ oligomers play a crucial role in AD pathology and in turn can be used as a diagnostic biomarker.

Title
Development and advanced validation of an optimized method for the quantitation of Aβ42 in human cerebrospinal fluid

Journal
AAPS J

Overview
INNOTEST® β-AMYLOID (1-42) assay kit was obtained from Innogenetics (Ghent, Belgium) and utilizes a 21F12/3D6 sandwich pair and a Aβ 1-42 peptide solution as calibration standard. Single-subject lots of normal and AD donor CSF were obtained from Precision Med, Inc

Year
2012

Authors
Cullen, VC;Fredenburg, RA;Evans, C;Conliffe, PR;Solomon, ME;

Link
https://link.springer.com/article/10.1208/s12248-012-9360-7

Abstract
Cerebrospinal fluid (CSF) biomarkers have been extensively utilized in the diagnosis of Alzheimer’s disease (AD) and characterization of progression. One important CSF biomarker is the amyloid beta 42 (Aβ(42)) peptide, a key player in AD pathogenesis. The INNOTEST® Aβ(42) ELISA kit has been widely used but an advanced level of method development and validation has not been reported. To support a clinical trial in AD, we successfully completed a Good Laboratory Practices (GLP)-level validation of the method to establish the parameters of precision, accuracy, parallelism, selectivity, specificity, and linearity of dilution of the assay in CSF matrix, as well as CSF storage stability. Several modifications were required to optimize the assay and ensure consistent results in a clinical-trial setting. These included the use of additional calibrators, an adjusted standard curve range, a minimum required dilution (MRD) of CSF by 6-fold to avoid matrix interference and mitigation of analyte adsorption to labware by the addition of Tween-20. The optimized method displayed a quantitative range of 375-4,500 pg/mL. The inter-assay precision was ≤12.1 % CV and the inter-assay relative accuracy was ≤10.9 % absolute bias, bringing the total error of the assay to ≤23 %. The intra-assay precision of the assay at the high validation standard and below was ≤5.5 % CV; this enables sensitive detection of biomarker changes across a therapeutic regime. The INNOTEST® Aβ(42) ELISA kit, modified as reported here, may be appropriate for many applications, including regulatory agency acceptable clinical diagnosis and pharmacodynamic assessment.

Title
Quality assessment and interference detection in targeted mass spectrometry data using machine learning

Journal
Clin Proteomics

Overview
Methods. Materials and reagents. CSF samples were purchased from PrecisionMed, Inc. (San Diego, CA). All donors provided informed consent for use of these studies with institutional review board approval for human collection protocols.

Year
2018

Authors
Toghi Eshghi, S;Auger, P;Mathews, WR;

Link
https://clinicalproteomicsjournal.biomedcentral.com/articles/10.1186/s12014-018-9209-x

Abstract
Advances in the field of targeted proteomics and mass spectrometry have significantly improved assay sensitivity and multiplexing capacity. The high-throughput nature of targeted proteomics experiments has increased the rate of data production, which requires development of novel analytical tools to keep up with data processing demand. Currently, development and validation of targeted mass spectrometry assays require manual inspection of chromatographic peaks from large datasets to ensure quality, a process that is time consuming, prone to inter- and intra-operator variability and limits the efficiency of data interpretation from targeted proteomics analyses. To address this challenge, we have developed TargetedMSQC, an R package that facilitates quality control and verification of chromatographic peaks from targeted proteomics datasets. This tool calculates metrics to quantify several quality aspects of a chromatographic peak, e.g. symmetry, jaggedness and modality, co-elution and shape similarity of monitored transitions in a peak group, as well as the consistency of transitions’ ratios between endogenous analytes and isotopically labeled internal standards and consistency of retention time across multiple runs. The algorithm takes advantage of supervised machine learning to identify peaks with interference or poor chromatography based on a set of peaks that have been annotated by an expert analyst. Using TargetedMSQC to analyze targeted proteomics data reduces the time spent on manual inspection of peaks and improves both speed and accuracy of interference detection. Additionally, by allowing the analysts to customize the tool for application on different datasets, TargetedMSQC gives the users the flexibility to define the acceptable quality for specific datasets. Furthermore, automated and quantitative assessment of peak quality offers a more objective and systematic framework for high throughput analysis of targeted mass spectrometry assay datasets and is a step towards more robust and faster assay implementation.

Title
Development of a method for the determination of glycine in human cerebrospinal fluid using pre-column derivatization and LC-MS/MS

Journal
J Pharm Biomed Anal

Overview
Human cerebrospinal fluid used for measurement of glycine in healthy subjects was obtained from Bioreclamation Inc. (East Meadow, NY, USA) and PrecisionMed Human Biological Samples (San Diego, CA, USA) via lumbar puncture.

Year
2011

Authors
Wilson, SF;James, CA;Zhu, X;Davis, MT;Rose, MJ;

Link
https://www.sciencedirect.com/science/article/pii/S0731708511002688

Abstract
An LC-MS/MS method using pre-column derivatization with phenylisothiocyanate (PITC) was developed to quantify glycine in human cerebrospinal fluid (CSF) and applied to the determination of glycine in human samples collected during clinical testing. The calibration curve range for the assay was 50-10,000 ng/mL and ¹³C₂¹⁵N-glycine was used as an internal standard. Artificial CSF was used as a surrogate matrix for standards due to the presence of endogenous glycine in human CSF and this approach was validated with additional experiments involving either standard addition, or stable labeled glycine as an alternate calibration standard for endogenous glycine. Interday bias (% RE) and precision (% CV) were -4.2 and 12.3% at the LLOQ, and less than ±0.9 and 8.3% for higher concentrations, respectively. Glycine was stable in artificial CSF for at least 5h at room temperature, 55 days at -70 °C (-60 to -80 °C range), and through three freeze-thaw cycles. Copyright © 2011. Published by Elsevier B.V.

Title
Characterization of novel CSF Tau and ptau biomarkers for Alzheimer’s disease

Journal
PLoS ONE

Overview
Individual AD and age-matched CSF samples were purchased from Precision Med (Solana Beach, CA). Written and verbal consents were obtained from participants at screening and enrollment. For all patients, participants were 55 years of age, in good general health having no other neurological, psychiatric or major medical diagnosis that could contribute significantly to cognitive impairment or dementia.

Year
2013

Authors
Meredith, JE;Sankaranarayanan, S;Guss, V;Lanzetti, AJ;Berisha, F;Neely, RJ;Slemmon, JR;Portelius, E;Zetterberg, H;Blennow, K;Soares, H;Ahlijanian, M;Albright, CF;

Link
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076523

Abstract
Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.

Title
Detection of cerebrospinal fluid leakage by specific measurement of transferrin glycoforms

Journal
Electrophoresis

Overview
A two-step enzymatic reaction including neuraminidase (Sigma) and galactose oxidase (Sigma) was performed on human CSF (PrecisionMed), on pooled human serum (Innovative Research Inc.) and on individual human serum samples (BioreclaimationIVT)

Year
2015

Authors
Kwon, SJ;Zhang, F;Dordick, JS;Sonstein, WJ;Linhardt, RJ;

Link
https://onlinelibrary.wiley.com/doi/abs/10.1002/elps.201500128

Abstract
A simple and rapid detection of cerebrospinal fluid (CSF) leakage would benefit spine surgeons making critical postoperative decisions on patient care. We have assessed novel approaches to selectively determine CSF β2-transferrin (β2TF), an asialo-transferrin (aTF) biomarker, without interference from serum sialo-transferrin (sTF) in test samples. First, we performed mild periodate oxidation to selectively generate aldehyde groups in sTF for capture with magnetic hydrazide microparticles, and selective removal with a magnetic separator. Using this protocol sTF was selectively removed from mixtures of CSF and serum containing CSF aTF (β2TF) and serum sTF, respectively. Second, a two-step enzymatic method was developed with neuraminidase and galactose oxidase for generating aldehyde groups in sTF present in CSF and serum mixtures for magnetic hydrazide microparticle capture. After selectively removing sTF from mixtures of CSF and serum, ELISA could detect significant TF signal only in CSF, while the TF signal in serum was negligible. The new approach for selective removal of only sTF in test samples will be promising for the required intervention by a spine surgeon. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Title
Optimization and evaluation of surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry for protein profiling of cerebrospinal fluid

Journal
Proteome Sci

Overview
…as a screening tool for sample selection prior to more in-depth analysis. Materials and methods Cerebrospinal fluid samples Normal CSF samples obtained from consenting patients were provided by PrecisionMed Inc. (San Diego, CA). CSF samples were obtained by lumbar puncture as part of a routine clinical procedure. The samples were collected in polypropylene tubes and gently mixed to avoid…

Year
2006

Authors
Guerreiro, N;Gomez-Mancilla, B;Charmont, S;

Link
https://proteomesci.biomedcentral.com/articles/10.1186/1477-5956-4-7

Abstract
Cerebrospinal fluid (CSF) potentially carries an archive of peptides and small proteins relevant to pathological processes in the central nervous system (CNS) and surrounding brain tissue. Proteomics is especially well suited for the discovery of biomarkers of diagnostic potential in CSF for early diagnosis and discrimination of several neurodegenerative diseases. ProteinChip surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is one such approach which offers a unique platform for high throughput profiling of peptides and small proteins in CSF. In this study, we evaluated methodologies for the retention of CSF proteins < 20 kDa in size, and identify a strategy for screening small proteins and peptides in CSF. ProteinChip array types, along with sample and binding buffer conditions, and matrices were investigated. By coupling the processing of arrays to a liquid handler reproducible and reliable profiles, with mean peak coefficients of variation < 20%, were achieved for intra- and inter-assays under selected conditions. Based on peak m/z we found a high degree of overlap between the tested array surfaces. The combination of CM10 and IMAC30 arrays was sufficient to represent between 80-90% of all assigned peaks when using either sinapinic acid or alpha-Cyano-4-hydroxycinnamic acid as the energy absorbing matrices. Moreover, arrays processed with SPA consistently showed better peak resolution and higher peak number across all surfaces within the measured mass range. We intend to use CM10 and IMAC30 arrays prepared in sinapinic acid as a fast and cost-effective approach to drive decisions on sample selection prior to more in-depth discovery of diagnostic biomarkers in CSF using alternative but complementary proteomic strategies.

Title
QUICK FIRES (Concurrent Short Communications) EUROSPINE 2015

Journal
Eur Spine J

Overview
10-plex (Millipore), so that 35 protein markers were investigated. Samples of CSF from healthy volunteers were purchased (PrecisionMed) and used as controls. Among cytokines/chemokines, we found an increase in GRO-A

Year
2015

Authors
FIBROSUS, A;

Link
https://link.springer.com/content/pdf/10.1007/s00586-015-4130-8.pdf

Title
The Relationship between Different Assays for Detection and Quantification of Amyloid Beta 42 in Human Cerebrospinal Fluid

Journal
Int J Alzheimers Dis

Overview
2. Materials and Methods. 2.1. Subjects. Human cerebrospinal fluid (CSF) samples were purchased from Precision Med, Inc. (San Diego, CA), which is in compliance with all applicable rules and regulations for human sample collection and dissemination.

Year
2012

Authors
Ellis, TA;Li, J;Leblond, D;Waring, JF;

Link
https://www.hindawi.com/journals/ijad/2012/984746/abs/

Abstract
Alzheimer’s disease (AD), which is characterized by a degeneration of neurons and their synapses, is one of the most common forms of dementia. CSF levels of amyloid β(42) (Aβ(42)) have been recognized as a strong candidate to serve as an AD biomarker. There are a number of commercial assays that are routinely employed for measuring Aβ(42); however, these assays give diverse ranges for the absolute levels of CSF Aβ(42). In order to employ CSF Aβ(42) as a biomarker across multiple laboratories, studies need to be performed to understand the relationship between the different platforms. We have analyzed CSF samples from both diseased and nondiseased subjects with two different widely used assay platforms. The results showed that different values for the levels of CSF Aβ(42) were reported, depending on the assay used. Nonetheless, both assays clearly demonstrated statistically significant differences in the levels of Aβ(42) in CSF from AD relative to age-matched controls (AMC). This paper provides essential data for establishing the relationship between these assays and provides an important step towards the validation of Aβ(42) as a biomarker for AD.

Title
A candidate plasma protein classifier to identify Alzheimer’s disease

Journal
Journal of Alzheimer’s Disease

Overview
…protein biomarkers. MATERIALS AND METHODS Study design Two cohorts of samples from non-demented con- trol individuals and AD patients were purchased from PrecisionMed, Inc. (Solana Beach, CA, USA).

Year
2015

Authors
Xuemeia, Z;Sergueib, L;Jeffreya, S;Chunshengb, Z;TCc, R;Jd, HD;Hongyueb, D;Russella, W;Fa, LO;

Link
https://content.iospress.com/articles/journal-of-alzheimers-disease/jad141149

Abstract
Biomarkers currently used in the aid for the diagnosis of Alzheimer’s disease (AD) are cerebrospinal fluid (CSF) protein markers and brain neuroimaging markers. These biomarkers, however, either involve semi-invasive procedures or are costly to measure. Thus, AD biomarkers from more easily accessible body fluids, such as plasma, are very enticing. Using an aptamer-based proteomic technology, we profiled 1,129 plasma proteins of AD patients and non-demented control individuals. A 5-protein classifier for AD identification was constructed in the discovery study with excellent 10-fold cross-validation performance (90.1% sensitivity, 84.2% specificity, 87.9% accuracy, and AUC as 0.94). In an independent validation study, the classifier was applied and correctly predicted AD with 100.0% sensitivity, 80.0% specificity, and 90.0% accuracy, matching or outperforming the CSF Aβ42 and tau biomarkers whose performance were assessed in individual-matched CSF samples obtained at the same visit as plasma sample collection. Moreover, the classifier also correctly predicted mild cognitive impairment, an early pre-dementia state of the disease, with 96.7% sensitivity, 80.0% specificity, and 92.5% accuracy. These studies demonstrate that plasma proteins could be used effectively and accurately to contribute to the clinical diagnosis of AD. Although additional and more diverse cohorts are needed for further validation of the robustness, including the support of postmortem diagnosis, the 5-protein classifier appears to be a promising blood test to contribute diagnosis of AD.

Title
Generation and Partial Characterization of Rabbit Monoclonal Antibody to Pyroglutamate Amyloid-β 3-42 (pE 3-Aβ)

Journal
Journal of Alzheimer’s Disease

Overview
In addition, CSF from 11 patients with probable AD, and 11 controls were purchased from PrecisionMed (Solana Beach, CA). All samples were coded, and laboratory personnel were blinded to group assignments. 2.12Amyloid

Year
2018

Authors
Mehtaa, PD;Patricka, BA;Barshatzkya, M;Mehtaa, SP;Frackowiaka, J;Mazur-Koleckaa, B;Wegiela, J;Millera, TW;

Link
https://content.iospress.com/articles/journal-of-alzheimers-disease/jad170898

Abstract
N-terminally truncated pyroglutamate amyloid-β (Aβ) peptide starting at position 3 represents a significant fraction of Aβ peptides (pE3-Aβ) in amyloid plaques of postmortem brains from patients with Alzheimer’s disease (AD) and older persons with Down syndrome (DS). Studies in transgenic mouse models of AD also showed that pE3-Aβ is a major component of plaques, and mouse monoclonal antibody to pE3-Aβ appears to be a desirable therapeutic agent for AD. Since small peptides do not typically elicit a good immune response in mice, but do so favorably in rabbits, our aims were to generate and partially characterize a rabbit monoclonal antibody (RabmAb) to pE3-Aβ. The generated RabmAb was found to be specific for pE3-Aβ, since it showed no reactivity with Aβ16, Aβ40, Aβ42, Aβ3-11, and pE11-17 Aβ peptides in an enzyme linked immunosorbent assay (ELISA). The isotype of the antibody was found to be IgG class. The antibody possesses high affinity to pE3-Aβ with dissociation constant (KD) for the antibody of 1 nM. The epitope of the antibody lies within the sequence of pE3-FRHD. In dot blotting, the optimal detection of pE3-Aβ was at an antibody concentration of 0.5 μg/ml. The threshold of pE3-Aβ detection was 2 fmol. The antibody was sensitive enough to detect 10 pg/ml of pE3-Aβ in sandwich ELISA. pE3-Aβ was detected in AD and DS brain extracts in ELISA and immunoblotting. Immunohistological studies showed immunolabeling of plaques and blood vessels in brains from patients with AD, and DS showing AD pathology. Thus, the antibody can be widely applied in AD and DS research, and therapeutic applications.

Title
Cerebrospinal fluid tau cleaved by caspase-6 reflects brain levels and cognition in aging and Alzheimer disease

Journal
J. Neuropathol. Exp. Neurol.

Overview
The postmortem CSF from AD and controls was provided by the Religious Orders Study. The multiple sclerosis (MS) and Parkinson disease (PD) CSF was obtained from Precision Med, Inc. (San Diego, CA). Multiple sclerosis and PD CSF was obtained premortem. Although the ages of the MS group (62.2 ± 6.7 years) and PD group (73.6 ± 4.3 years) were significantly younger than the AD group (89.6 ± 4.4 years; p 0.01), the PD age did not differ significantly from the NCI group used in this study.

Year
2013

Authors
Ramcharitar, J;Albrecht, S;Afonso, VM;Kaushal, V;Bennett, DA;Leblanc, AC;

Link
https://academic.oup.com/jnen/article-abstract/72/9/824/1802026

Abstract
Caspase-6 (Casp6) activation in the brain is implicated early in the pathogenesis of Alzheimer disease (AD). In view of the need for early AD diagnosis, brain Casp6 activity was investigated by measuring Tau cleaved by Casp6 (TauΔCasp6) protein in postmortem cerebrospinal fluid (CSF) of 7 non-cognitively impaired; 5 mild cognitively impaired; and 12 mild, moderate, and severe AD patients. Levels of TauΔCasp6 in CSF accurately reflected the levels of active Casp6 and TauΔCasp6 detected using immunohistochemistry in hippocampal sections from the same individuals. Levels of CSF TauΔCasp6 significantly correlated with AD severity and with lower Global Cognitive Scores; Mini-Mental State Examination scores; and episodic, semantic, and working memory scores. Regression analyses suggested that the CSF TauΔCasp6 levels combined with TauΔCasp6 brain pathology predict cognitive performance. These results indicate that CSF TauΔCasp6 levels hold promise as a novel early biomarker of AD.

Title
Quantitative Systems Pharmacology Model for Alzheimer Disease Indicates Targeting Sphingolipid Dysregulation as Potential Treatment Option

Journal
CPT Pharmacometrics Syst Pharmacol

Overview
In short, samples were prepared and measured for lipid content by a gas-LCMS/MS method. Human CSF and plasma samples were purchased at Precision Med Inc and included n = 20 young adult subjects, n = 25 age-matched (to AD) controls and n = 25 AD subjects

Year
2018

Authors
Clausznitzer, D;Pichardo-Almarza, C;Relo, AL;van Bergeijk, J;van der Kam, E;Laplanche, L;Benson, N;Nijsen, M;

Link
https://ascpt.onlinelibrary.wiley.com/doi/abs/10.1002/psp4.12351

Abstract
Alzheimer disease (AD) is a devastating neurodegenerative disorder with high unmet medical need. Drug development is hampered by limited understanding of the disease and its driving factors. Quantitative Systems Pharmacology (QSP) modeling provides a comprehensive quantitative framework to evaluate the relevance of biological mechanisms in the context of disease and to predict the efficacy of novel treatments. Here, we report a QSP model for AD with a particular focus on investigating the relevance of dysregulation of cholesterol and sphingolipids. We show that our model captures the modulation of several biomarkers in subjects with AD, as well as the response to pharmacological interventions. We evaluate the impact of targeting the sphingosine-1-phosphate 5 receptor (S1PR5) as a potential novel treatment option for AD, and model predictions increase our confidence in this novel disease pathway. Future applications for the QSP model are in validation of further targets and identification of potential treatment response biomarkers. © 2018 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.

Title
MSPrecise: A molecular diagnostic test for multiple sclerosis using next generation sequencing

Journal
Gene

Overview
By lumbar puncture in accordance with IRB-approved protocols at UT Southwestern Medical Center, the University of Massachusetts Memorial Medical Center (UMass), John Hopkins University (JHU), or purchased from a commercial biorepository (PrecisionMed, Solana Beach.

Year
2015

Authors
Rounds, WH;Salinas, EA;Wilks, TB;Levin, MK;Ligocki, AJ;Ionete, C;Pardo, CA;Vernino, S;Greenberg, BM;Bigwood, DW;Eastman, EM;Cowell, LG;Monson, NL;;

Link
https://www.sciencedirect.com/science/article/pii/S0378111915008215

Abstract
We have previously demonstrated that cerebrospinal fluid-derived B cells from early relapsing-remitting multiple sclerosis (RRMS) patients that express a VH4 gene accumulate specific replacement mutations. These mutations can be quantified as a score that identifies such patients as having or likely to convert to RRMS. Furthermore, we showed that next generation sequencing is an efficient method for obtaining the sequencing information required by this mutation scoring tool, originally developed using the less clinically viable single-cell Sanger sequencing. To determine the accuracy of MSPrecise, the diagnostic test that identifies the presence of the RRMS-enriched mutation pattern from patient cerebrospinal fluid B cells. Cerebrospinal fluid cell pellets were obtained from RRMS and other neurological disease (OND) patient cohorts. VH4 gene segments were amplified, sequenced by next generation sequencing and analyzed for mutation score. The diagnostic test showed a sensitivity of 75% on the RRMS cohort and a specificity of 88% on the OND cohort. The accuracy of the test in identifying RRMS patients or patients that will develop RRMS is 84%. MSPrecise exhibits good performance in identifying patients with RRMS irrespective of time with RRMS. Copyright © 2015. Published by Elsevier B.V.

Title
Identification of biomarkers for amyotrophic lateral sclerosis by comprehensive analysis of exosomal mRNAs in human cerebrospinal fluid

Journal
BMC Med Genomics

Overview
CSF specimens from four NH donors and four ALS patients matched by age and sex were purchased from PrecisionMed (CA, USA). All donors for the specimens in this study provided informed consent. Detailed information on specimens such as age, sex, duration of disease, and ALS Functional Rating Scale-R

Year
2019

Authors
Otake, K;Kamiguchi, H;Hirozane, Y;

Link
https://www.sciencedirect.com/science/article/pii/S0378111915008215

Abstract
Exosomes are a subset of extracellular vesicles 30-200nm in diameter secreted from cells, which contain functional mRNAs and microRNAs. Cerebrospinal fluid (CSF) is the primary source for liquid biopsy to examine diseases in central nervous system. To date, there is no available method to analyze exosomal mRNAs comprehensively in human CSF. The main purpose of this study is to established the methodology of comprehensive analysis of exosomal mRNAs in CSF by a highly sensitive next-generation sequencing. The signatures of CSF exosomal mRNAs were then compared between four normal healthy donors and four sporadic amyotrophic lateral sclerosis patients to identify disease-related biomarkers. Differentially expressed genes were identified by DESeq2. RNA sequencing from CSF exosomes was successfully performed, that was demonstrated by the high pearson’s product-moment correlation coefficient (r=0.993) in the technical replicates. Also, position coverage analysis revealed that most detected mRNAs retained their integrity throughout their full-length in CSF exosomes. In CSF exosomes from normal healthy donors, an average of 14,807 genes were detected, of which 4580 genes were commonly detected among four individuals, including neuron-enriched genes such as TUBB3 and CAMK2A. In comparison with exosomal mRNAs in CSF from four patients with amyotrophic lateral sclerosis, 543 genes were significantly changed, as represented by CUEDC2. Gene Ontology analysis and pathway analysis with these genes revealed functional enrichment of ubiquitin-proteasome pathway, oxidative stress response, and unfolded protein response. These pathways are related to pathomechanisms of amyotrophic lateral sclerosis. We successfully established the methodology of comprehensive analysis of exosomal mRNAs in human CSF. It was shown to be useful to identify disease biomarkers for central nervous system. Several genes, such as CUEDC2, in CSF exosomes were suggested to be candidate disease biomarkers for amyotrophic lateral sclerosis.

Title
Glycosaminoglycans in human cerebrospinal fluid determined by LC-MS/MS MRM

Journal
Analytic Biochemistry

Overview
In the current study we investigated a pooled CSF sample (PrecisionMed, Inc.,Solana Beach, CA) and CSF samples from three individuals collected locally following consensus guidelines for blood biobanking…

Year
2019

Authors
Yu, Y;Zhang, F;Colón, W;Linhardt, RJ;Xia, K;

Link
https://www.sciencedirect.com/science/article/pii/S0378111915008215

Abstract
Glycosaminoglycans (GAGs) were recovered from human cerebral spinal fluid (CSF) and after their conversion to disaccharides using polysaccharide lyases were analyzed by liquid chromatography tandem mass spectrometry using multiple reaction monitoring. CSF showed ng/mL levels of heparan sulfate, chondroitin sulfates and hyaluronan. The amounts and disaccharide composition of these GAGs differed from those found in human plasma. This approach may offer a new method for the discovery of biomarkers for diseases of the central nervous system.                 Copyright © 2018. Published by Elsevier Inc.

Title
Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

Journal
Sci Adv

Overview
Protocols were obtained from the University of California Davis Alzheimer’s Disease Center, the University of California San Francisco (UCSF) Neurosurgery Tissue Bank, ProteoGenex (Culver City, CA), and PrecisionMed (Solana Beach, CA).

Year
2019

Authors
Dominy, SS;Lynch, C;Ermini, F;Benedyk, M;Marczyk, A;Konradi, A;Nguyen, M;Haditsch, U;Raha, D;Griffin, C;Holsinger, LJ;Arastu-Kapur, S;Kaba, S;Lee, A;Ryder, MI;Potempa, B;Mydel, P;Hellvard, A;Adamowicz, K;Hasturk, H;Walker, GD;Reynolds, EC;Faull, RLM;Curtis, MA;Dragunow, M;Potempa, J;

Link
http://advances.sciencemag.org/content/5/1/eaau3333

Abstract
Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer’s disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer’s patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aβ1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aβ1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer’s disease.

PLASMA

Title
Polar anionic metabolome analysis by nano-LC/MS with a metal chelating agent

Journal
Anal. Chem.

Overview
The samples were then centrifuged at 12 000 rpm for 20 min. The upper phase (MeOH and water) was used for analysis of polar anionic metabolites. Human plasma (50 μL) and cerebrospinal fluid (CSF) (100 μL), purchased from Precision Med Inc.

Year
2009

Authors
Myint, KT;Uehara, T;Aoshima, K;Oda, Y;

Link
https://pubs.acs.org/doi/abs/10.1021/ac901269h

Abstract
We have developed a practical method for the comprehensive analysis of polar anionic metabolites in biological samples with the use of a nano-LC/MS system. A polyamine-bonded polymer-based apHera NH2 column, which is compatible with ammonium carbonate buffer, effectively retained anionic polar metabolites, such as organic acids, sulfates, and phosphates, but multiply phosphorylated or carboxylated compounds showed highly distorted peak shapes on chromatograms. We found that addition of a trace amount of the metal chelating reagent ethylenediaminetetraacetic acid (EDTA) to the sample solution dramatically improved peak shapes of multiply charged anionic compounds, even though the mass spectra showed no trace of adduct ions in the absence of EDTA. The detection limits of typical polar anionic metabolites in the full-scan mode were from 0.19 to 2.81 pmol. After optimization of all the procedures from sample preparation to nano-LC/MS analysis, we applied our method to real biological samples: Hela cells, mouse brain, human cerebrospinal fluid (CSF), and human plasma. Our results indicated that phosphorylated metabolites were abundant in Hela cells and brain, while plasma and cerebrospinal fluid (CSF) mostly contained organic acids. Phosphorylated compounds might not be secreted into CSF/plasma or might be unstable in CSF/plasma. Finally, the method was used to examine the mode of action of the anticancer drug methotrexate (MTX), which inhibits purine de novo biosynthesis and thymidine biosynthesis. In addition of the expected changes of metabolite levels, we found that a previously unreported metabolite, probably a methylated uridine 5′-triphosphate (UTP), was produced by MTX-treated Hela cells.

Title
MSPrecise: A molecular diagnostic test for multiple sclerosis using next generation sequencing

Journal
Gene

Overview
By lumbar puncture in accordance with IRB-approved protocols at UT Southwestern Medical Center, the University of Massachusetts Memorial Medical Center (UMass), John Hopkins University (JHU), or purchased from a commercial biorepository (PrecisionMed, Solana Beach.

Year
2015

Authors
Rounds, WH;Salinas, EA;Wilks, TB;Levin, MK;Ligocki, AJ;Ionete, C;Pardo, CA;Vernino, S;Greenberg, BM;Bigwood, DW;Eastman, EM;Cowell, LG;Monson, NL;;

Link
https://www.sciencedirect.com/science/article/pii/S0378111915008215

Abstract
We have previously demonstrated that cerebrospinal fluid-derived B cells from early relapsing-remitting multiple sclerosis (RRMS) patients that express a VH4 gene accumulate specific replacement mutations. These mutations can be quantified as a score that identifies such patients as having or likely to convert to RRMS. Furthermore, we showed that next generation sequencing is an efficient method for obtaining the sequencing information required by this mutation scoring tool, originally developed using the less clinically viable single-cell Sanger sequencing. To determine the accuracy of MSPrecise, the diagnostic test that identifies the presence of the RRMS-enriched mutation pattern from patient cerebrospinal fluid B cells. Cerebrospinal fluid cell pellets were obtained from RRMS and other neurological disease (OND) patient cohorts. VH4 gene segments were amplified, sequenced by next generation sequencing and analyzed for mutation score. The diagnostic test showed a sensitivity of 75% on the RRMS cohort and a specificity of 88% on the OND cohort. The accuracy of the test in identifying RRMS patients or patients that will develop RRMS is 84%. MSPrecise exhibits good performance in identifying patients with RRMS irrespective of time with RRMS. Copyright © 2015. Published by Elsevier B.V.

Title
Identification of a new plasma biomarker of Alzheimer’s disease using metabolomics technology

Journal
J. Lipid Res.

Overview
(Alabaster, AL). Samples collection. Plasma and CSF samples of elderly controls and subjects diagnosed with AD, MCI, schizophrenia, and Parkinson’s disease were purchased from PrecisionMed, Inc. (San Diego, CA) and stored at −80°C until use…

Year
2012

Authors
Sato, Y;Suzuki, I;Nakamura, T;Bernier, F;Aoshima, K;Oda, Y;

Link
http://www.jlr.org/content/53/3/567.short

Abstract
We performed unbiased analysis of steroid-related compounds to identify novel Alzheimer’s disease (AD) plasma biomarkers using liquid chromatography-atmospheric pressure chemical ionization-mass spectroscopy. The analysis revealed that desmosterol was found to be decreased in AD plasma versus controls. To precisely quantify variations in desmosterol, we established an analytical method to measure desmosterol and cholesterol. Using this LC-based method, we discovered that desmosterol and the desmosterol/cholesterol ratio are significantly decreased in AD. Finally, the validation of this assay using 109 clinical samples confirmed the decrease of desmosterol in AD as well as a change in the desmosterol/cholesterol ratio in AD. Interestingly, we could also observe a difference between mild cognitive impairment and control. In addition, the decrease of desmosterol was somewhat more significant in females. Receiver operating characteristic (ROC) analysis between controls and AD, using plasma desmosterol shows a score of 0.80, indicating a good discrimination power for this marker in the two reference populations and confirms the potential usefulness of measuring plasma desmosterol levels for diagnosing AD. Further analysis showed a significant correlation of plasma desmosterol with Mini-Mental State Examination scores. Although larger sample populations will be needed to confirm this diagnostic marker sensitivity, our studies demonstrate a sensitive and accurate method of detecting plasma desmosterol concentration and suggest that plasma desmosterol could become a powerful new specific biomarker for early and easy AD diagnosis.

Title
Ethylenediaminetetraacetic acid increases identification rate of phosphoproteomics in real biological samples

Journal
J. Proteome Res.

Overview
Aldrich Co. (St. Louis, MO), respectively. Human plasma and human cerebrospinal fluid (CSF) were purchased from PrecisionMed. Inc. (San Diego, CA). Other reagents of analytical grade were used without further treatment.

Year
2010

Authors
Nakamura, T;Myint, KT;Oda, Y;

Link
https://pubs.acs.org/doi/abs/10.1021/pr900918h

Abstract
We have developed a novel approach to enhance phosphopeptide identification in liquid chromatography/mass spectrometry (LC/MS)-based phosphoproteomics. After enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO(2)) microcolumns, samples were coinjected with ethylenediaminetetraacetic acid (EDTA) into LC/MS. This procedure decreased the MS peak intensity of nonphosphorylated peptides, but not that of phosphopeptides, and as a result, the number of identified phosphopeptides was increased. EDTA appeared to have no effect on liquid chromatographic separation of phosphorylated and nonphosphorylated peptides. Although the mechanism of the positive effect of EDTA on identification of phosphopeptides is unknown, and we have never observed metal ion adduct peaks in LC/MS spectra, coinjection of EDTA seemed to enhance phosphopeptide recovery from the LC/MS system. This simple technique was successfully applied to the identification of phosphopeptides in mouse brain (2938 phosphopeptides), human plasma (127 phosphopetides), and human cerebrospinal fluid (CSF) (123 phosphopeptides). We also identified nonphosphopeptides in the same samples using a two-dimensional (2D) LC/MS-based shotgun approach. The results overall indicated that 20-25% of brain proteins were phosphorylated, while only 1-2% of proteins in plasma and CSF were phosphorylated. These ratios were almost constant throughout the range of protein expression levels. In addition, EDTA-enhanced phosphoproteomics could identify low-abundance proteins in the samples, because nonphosphoproteins corresponding to more than one-third of the identified phosphoproteins could not be identified by 2D-LC/MS. Finally, we were able to find that the newly developed approach was very effective for the phosphoproteome analysis in Alzheimer disease model mice brain.

Title
High-throughput and simultaneous quantitative analysis of homocysteine-methionine cycle metabolites and co-factors in blood plasma and cerebrospinal fluid by isotope dilution LC-MS/MS

Journal
Anal Bioanal Chem

Overview
The injection volume was 10 μL and the total run time of analysis was 13 min. Human sample collection for method validation. The human sub-cohort was supplied from PrecisionMed, Inc. (CA, USA, protocol 8009). This…

Year
2017

Authors
Guiraud, SP;Montoliu, I;Da Silva, L;Dayon, L;Galindo, AN;Corthésy, J;Kussmann, M;Martin, FP;

Link
https://link.springer.com/article/10.1007/s00216-016-0003-1

Abstract
The methionine cycle is a key pathway contributing to the regulation of human health, with well-established involvement in cardiovascular diseases and cognitive function. Changes in one-carbon cycle metabolites have also been associated with mild cognitive decline, vascular dementia, and Alzheimer’s disease. Today, there is no single analytical method to monitor both metabolites and co-factors of the methionine cycle. To address this limitation, we here report for the first time a new method for the simultaneous quantitation of 17 metabolites in the methionine cycle, which are homocysteic acid, taurine, serine, cysteine, glycine, homocysteine, riboflavin, methionine, pyridoxine, cystathionine, pyridoxamine, S-adenosylhomocysteine, S-adenosylmethionine, betaine, choline, dimethylglycine, and 5-methyltetrahydrofolic acid. This multianalyte method, developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), provides a highly accurate and precise quantitation of these 17 metabolites for both plasma and cerebrospinal fluid metabolite monitoring. The method requires a simple sample preparation, which, combined with a short chromatographic run time, ensures a high sample throughput. This analytical strategy will thus provide a novel metabolomics approach to be employed in large-scale observational and intervention studies. We expect such a robust method to be particularly relevant for broad and deep molecular phenotyping of individuals in relation to their nutritional requirements, health monitoring, and disease risk management.

Title
TNT003, an inhibitor of the serine protease C1s, prevents complement activation induced by cold agglutinins

Journal
Blood

Overview
Western Institutional Review Board. Verification of CAD diagnosis was obtained from patients’ physicians. Samples were collected by PrecisionMed Inc. (Solana Beach, CA) in accordance with the Declaration of Helsinki.

Year
2014

Authors
Shi, J;Rose, EL;Singh, A;Hussain, S;Stagliano, NE;Parry, GC;Panicker, S;

Link
http://www.bloodjournal.org/content/early/2014/04/02/blood-2014-02-556027.short

Abstract
Activation of the classical pathway (CP) of complement is often associated with autoimmune disorders in which disease pathology is linked to the presence of an autoantibody. One such disorder is cold agglutinin disease (CAD), an autoimmune hemolytic anemia in which autoantibodies (cold agglutinins) bind to red blood cells (RBCs) at low temperatures. Anemia occurs as a result of autoantibody-mediated CP activation on the surface of the erythrocyte, leading to the deposition of complement opsonins that drive extravascular hemolysis in the liver. Here we test the effects of TNT003, a mouse monoclonal antibody targeting the CP-specific serine protease C1s, on CP activity induced by cold agglutinins on human RBCs. We collected 40 individual CAD patient samples and showed that TNT003 prevented cold agglutinin-mediated deposition of complement opsonins that promote phagocytosis of RBCs. Furthermore, we show that by preventing CP activation, TNT003 also prevents cold agglutinin-driven generation of anaphylatoxins. Finally, we provide evidence that CP activity in CAD patients terminates prior to activation of the terminal cascade, supporting the hypothesis that the primary route of RBC destruction in these patients occurs via extravascular hemolysis. Our results support the development of a CP inhibitor for the treatment of CAD. © 2014 by The American Society of Hematology.

Title
Quantitative and wide-ranging profiling of phospholipids in human plasma by two-dimensional liquid chromatography/mass spectrometry

Journal
Anal. Chem.

Overview
Human plasma samples of age-matched controls and subjects diagnosed with AD and mild cognitive impairment (MCI) were purchased from PrecisionMed Inc., San Diego, CA and stored at −80 °C until sample preparation.

Year
2010

Authors
Sato, Y;Nakamura, T;Aoshima, K;Oda, Y;;

Link
https://pubs.acs.org/doi/abs/10.1021/ac102211r

Abstract
Normal-phase or reverse-phase liquid chromatography has been used in phospholipidomics for lipid separation prior to mass spectrometry analysis. However, separation using a single separation mode is often inadequate, as high-abundance phospholipids can mask large numbers of low-abundance lipids of interest. In order to detect and quantify low-abundance phospholipids, we present a novel two-dimensional (2D) approach for sensitive and quantitative global analysis of phospholipids. The methodology monitors individual glycerolipids and phospholipids through the use of a new quantitative normal-phase, solid-phase extraction procedure, followed by molecular characterization and relative quantification using an ion-trap Orbitrap equipped with a reverse-phase liquid chromatograph, with data processing by MS++ software. The CV (%) of the peak area of each lipid standard was less than 15% with this extraction method. When the method was applied to a liver sample, we could detect more phosphatidylserine (PS) compared to the previous method. Finally, our developed method was applied to Alzheimer’s disease (AD) plasma samples. Several hundred peaks were detected from a 60 μL plasma sample. A partial-least-squares discriminant analysis (PLS-DA) plot using peak area ratio gave a unique group of PLS scores which could distinguish plasma samples of Alzheimer’s disease (AD) patients from those of age-matched healthy controls.

Title
Characterization of nitrotyrosine as a biomarker for arthritis and joint injury

Journal
Osteoarthr. Cartil.

Overview
6-month period. Control plasma samples (n = 21) from an age-matched cohort of healthy volunteers were obtained from PrecisionMed (San Diego, CA, USA). Disease activity score (DAS) of 28 joints. The disease activity score…

Year
2013

Authors
Misko, TP;Radabaugh, MR;Highkin, M;Abrams, M;Friese, O;Gallavan, R;Bramson, C;Hellio Le Graverand, MP;Lohmander, LS;Roman, D;

Link
https://www.sciencedirect.com/science/article/pii/S1063458412009673

Abstract
To characterize the utility of nitrotyrosine (NT) as a biomarker for arthritis and joint injury. Synovial fluid, plasma, and urine from patients diagnosed with osteoarthritis (OA), rheumatoid arthritis (RA), anterior cruciate ligament (ACL) injury, meniscus injury and pseudogout, and knee-healthy volunteers were analyzed for concentrations of NT, nitrate and nitrite (NO(x)), matrix metalloproteinase (MMP)-3, MMP-1, MMP-9, more than 40 chemokines and cytokines. In OA, plasma and synovial fluid NT were increased versus healthy volunteers. Synovial fluid to plasma NT ratios were elevated in OA patients. Synovial fluid from patients with ACL and meniscus injury and pseudogout had increased levels of NT (P < 0.001). In these samples, NT levels significantly correlated with ARGS-aggrecan neoepitope generated by aggrecanase cleavage of aggrecan (P ≤ 0.001), cross-linked C-telopeptides of type II collagen (P < 0.001), MMP-1 (P = 0.008), and MMP-3 (P ≤ 0.001). In RA, plasma NT decreased following 6 months of anti-tumor necrosis factor (TNF) treatment. For every 1.1% change in log(10) NT, there was a 1.0% change in the log(10) disease activity scores (DAS28-3 CRP). Both predicted and observed DAS28-3 CRP showed a robust linear relationship with NT. RA plasma NT positively correlated with CRP, MMP-3 and interferon γ-induced protein 10. NT may serve as a useful biomarker for arthritis and joint injury. In RA, NT is highly correlated with several biomarkers and clinical correlates of disease activity and responds to anti-TNF therapy. Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Title
Plasma lysosphingomyelin demonstrates great potential as a diagnostic biomarker for Niemann-Pick disease type C in a retrospective study

Journal
PLoS ONE

Overview
Plasma samples for the control group came from Tissue Solutions (Glasgow, UK), The Geneticist (Glendale, Co, USA) and PrecisionMed Inc (Solana Beach, Ca, USA). Solvents were from Sigma Aldrich (Buchs, Switzerland) and were of HPLC grade. Assay validation

Year
2014

Authors
Welford, RW;Garzotti, M;Marques Lourenço, C;Mengel, E;Marquardt, T;Reunert, J;Amraoui, Y;Kolb, SA;Morand, O;Groenen, P;

Link
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114669

Abstract
Niemann-Pick disease type C (NP-C) is a devastating, neurovisceral lysosomal storage disorder which is characterised by variable manifestation of visceral signs, progressive neuropsychiatric deterioration and premature death, caused by mutations in the NPC1 and NPC2 genes. Due to the complexity of diagnosis and the availability of an approved therapy in the EU, improved detection of NP-C may have a huge impact on future disease management. At the cellular level dysfunction or deficiency of either the NPC1 or NPC2 protein leads to a complex intracellular endosomal/lysosomal trafficking defect, and organ specific patterns of sphingolipid accumulation. Lysosphingolipids have been shown to be excellent biomarkers of sphingolipidosis in several enzyme deficient lysosomal storage disorders. Additionally, in a recent study the lysosphingolipids, lysosphingomyelin (SPC) and glucosylsphingosine (GlcSph), appeared to be elevated in the plasma of three adult NP-C patients. In order to investigate the clinical utility of SPC and GlcSph as diagnostic markers, an in-depth fit for purpose biomarker assay validation for measurement of these biomarkers in plasma by liquid chromatography-tandem mass spectrometry was performed. Plasma SPC and GlcSph are stable and can be measured accurately, precisely and reproducibly. In a retrospective analysis of 57 NP-C patients and 70 control subjects, median plasma SPC and GlcSph were significantly elevated in NP-C by 2.8-fold and 1.4-fold respectively. For miglustat-naïve NP-C patients, aged 2-50 years, the area under the ROC curve was 0.999 for SPC and 0.776 for GlcSph. Plasma GlcSph did not correlate with SPC levels in NP-C patients. The data indicate excellent potential for the use of lysosphingomyelin in NP-C diagnosis, where it could be used to identify NP-C patients for confirmatory genetic testing.

Title
Increased levels of hyper-stable protein aggregates in plasma of older adults

Journal
Age (Dordr)

Overview
Material and methods. Sample source. Plasma was purchased from Bioreclamation, Inc (Westbury, NY) (individual and pooled control human plasma), and PrecisionMed, Inc Most plasma samples were obtained commercially from two different companies, PrecisionMed, Inc

Year
2016

Authors
Xia, K;Trasatti, H;Wymer, JP;Colón, W;

Link
https://link.springer.com/article/10.1007/s11357-016-9919-9

Abstract
Proteins that misfold into hyper-stable/degradation-resistant species during aging may accumulate and disrupt protein homeostasis (i.e., proteostasis), thereby posing a survival risk to any organism. Using the method diagonal two-dimensional (D2D) SDS-PAGE, which separates hyper-stable SDS-resistant proteins at a proteomics level, we analyzed the plasma of healthy young (<30 years) and older (60-80 years) adults. We discovered the presence of soluble SDS-resistant protein aggregates in the plasma of older adults, but found significantly lower levels in the plasma of young adults. We identified the inflammation-related chaperone protein haptoglobin as the main component of the hyper-stable aggregates. This observation is consistent with the growing link between accumulations of protein aggregates and aging across many organisms. It is plausible higher amounts of SDS-resistant protein aggregates in the plasma of older adults may reflect a compromise in proteostasis that may potentially indicate cellular aging and/or disease risk. The results of this study have implications for further understanding the link between aging and the accumulation of protein aggregates, as well as potential for the development of aging-related biomarkers. More broadly, this novel application of D2D SDS-PAGE may be used to identify, quantify, and characterize the degradation-resistant protein aggregates in human plasma or any biological system.

Title
Abnormal clotting of the intrinsic/contact pathway in Alzheimer disease patients is related to cognitive ability

Journal
Blood Adv

Overview
… the possibility of novel therapies targeted to this system. MATERIAL AND METHODS HUMAN PLASMA AND CSF Plasma from AD patients and ND controls was purchased from a commercial biobank vendor (PrecisionMed Inc., San Diego, CA). Plasma was thawed at 37°C for 3 minutes before performing coagulation assays. None of the donors used for this study were taking anticoagulants. ND controls had …

Year
2018

Authors
Suidan, GL;Singh, PK;Patel-Hett, S;Chen, ZL;Volfson, D;Yamamoto-Imoto, H;Norris, EH;Bell, RD;Strickland, S;

Link
http://www.bloodadvances.org/content/2/9/954.abstract

Abstract
Alzheimer disease (AD) is a neurodegenerative disorder characterized by extracellular β-amyloid (Aβ) deposition. Although peripheral inflammation and cerebrovascular pathology are reported in AD, there is a lack of plasma biomarkers in this field. Because the contact system is triggered in patient plasma, we hypothesized that the hemostasis profile could be a novel biomarker in AD. Here, we assessed the clotting profile in plasma from AD patients and age-matched controls. Utilizing clinically relevant assays, thromboelastography and activated partial thromboplastin time, we found impaired clot initiation and formation rate in AD patient plasma. These coagulation end points correlated with cerebrospinal fluid neurofilament-light levels and cognition and were more profound in younger AD patients. Ex vivo intrinsic clotting of plasma from AD mice expressing human amyloid precursor protein (APP) was also delayed in an age-dependent manner, suggesting that this phenotype is related to APP, the parent protein of Aβ. Further analysis of coagulation factors in human plasma indicated that endogenous inhibitor(s) of factors XII and XI in AD plasma contribute to this delayed clotting. Together, these data suggest that delayed clotting in young AD patients is a novel biomarker and that therapies aimed to correct this phenotype might be beneficial in this patient population. Follow-up studies in additional AD patient cohorts are warranted to further evaluate these findings. © 2018 by The American Society of Hematology.

Title
Reduced plasma desmosterol-to-cholesterol ratio and longitudinal cognitive decline in Alzheimer’s disease

Journal
Alzheimers Dement (Amst)

Overview
…whose blood was drawn at two different time points (Table 2). Additional longitudinal plasma samples of 30 subjects at least at 3 different time points were obtained from Uppsala University Hospital (AD, n = 6; MCI, n = 6; control, n = 2) or purchased from PrecisionMed, Inc.

Year
2015

Authors
Sato, Y;Bernier, F;Yamanaka, Y;Aoshima, K;Oda, Y;Ingelsson, M;Lannfelt, L;Miyashita, A;Kuwano, R;Ikeuchi, T;

Link
https://www.sciencedirect.com/science/article/pii/S2352872915000135

Abstract
We here examined whether plasma desmosterol-to-cholesterol ratio (DES/CHO) is decreased in patients with Alzheimer’s disease (AD) and investigated the association between plasma DES/CHO and longitudinal cognitive decline. Plasma DES/CHO of AD patients and age-matched controls in a Japanese cross-sectional cohort was determined. Plasma DES/CHO at baseline and follow-up visits was assessed in relation to cognitive decline in Japanese and Swedish longitudinal cohorts. Plasma DES/CHO was significantly reduced in Japanese AD patients and significantly correlated with Mini-Mental State Examination (MMSE) score. The longitudinal analysis revealed that plasma DES/CHO in AD patients shows a significant decrease at follow-up intervals. The decline in plasma DES/CHO is larger in the AD group with rapid progression than in that with slow progression. The changes in plasma DES/CHO significantly correlated with changes in the MMSE score. Plasma DES/CHO is decreased in AD patients and may serve as a longitudinal surrogate marker associated with cognitive decline.

Title
Low IL-8 is associated with anxiety in suicidal patients: genetic variation and decreased protein levels

Journal
Acta Psychiatr Scand

Overview
Healthy control subjects were recruited through the Neuropsychiatric and Psychiatric Clinics at the University Hospitals in Malmoe and Linkoeping, Sweden (n = 38) and PrecisionMed, San Diego, California (n = 8) between 2001 and 2005. Physical examination…

Year
2015

Authors
Janelidze, S;Suchankova, P;Ekman, A;Erhardt, S;Sellgren, C;Samuelsson, M;Westrin, A;Minthon, L;Hansson, O;Träskman-Bendz, L;Brundin, L;

Link
https://onlinelibrary.wiley.com/doi/abs/10.1111/acps.12339

Abstract
Recent studies indicate that inflammation may play a role in the pathophysiology of suicidality. Interleukin-8 (IL-8) is a chemokine that in addition to its function in the immune system also exert neuroprotective properties. The involvement of this chemokine in neuropsychiatric conditions is incompletely known. We measured plasma and cerebrospinal fluid (CSF) IL-8, as well as the genotype frequency of a single nucleotide polymorphism (-251A/T, rs4073) in the promoter region of the IL8 gene, in suicide attempters (n=206) and healthy controls (n=578). Plasma and CSF levels of IL-8 were significantly lower in suicide attempters with anxiety than in healthy controls. IL-8 in both plasma and CSF correlated negatively with symptoms of anxiety. Compared with the population-based cohort, the IL-8-251T allele was more prevalent among female suicide attempters. Furthermore, suicide attempters carrying this allele showed more severe anxiety. This correlative study warrants further mechanistic studies on the effects of IL-8 in the central nervous system. We suggest that IL-8 might be involved in the biological mechanisms mediating resilience to anxiety. Thus, our findings highlight the chemokine IL-8 as a potential target for future development of anti-anxiety treatments and suicide prevention. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Title
Quantitative Systems Pharmacology Model for Alzheimer Disease Indicates Targeting Sphingolipid Dysregulation as Potential Treatment Option

Journal
CPT Pharmacometrics Syst Pharmacol

Overview
In short, samples were prepared and measured for lipid content by a gas-LCMS/MS method. Human CSF and plasma samples were purchased at Precision Med Inc and included n = 20 young adult subjects, n = 25 age-matched (to AD) controls and n = 25 AD subjects.

Year
2018

Authors
Clausznitzer, D;Pichardo-Almarza, C;Relo, AL;van Bergeijk, J;van der Kam, E;Laplanche, L;Benson, N;Nijsen, M;

Link
https://ascpt.onlinelibrary.wiley.com/doi/abs/10.1002/psp4.12351

Abstract
Alzheimer disease (AD) is a devastating neurodegenerative disorder with high unmet medical need. Drug development is hampered by limited understanding of the disease and its driving factors. Quantitative Systems Pharmacology (QSP) modeling provides a comprehensive quantitative framework to evaluate the relevance of biological mechanisms in the context of disease and to predict the efficacy of novel treatments. Here, we report a QSP model for AD with a particular focus on investigating the relevance of dysregulation of cholesterol and sphingolipids. We show that our model captures the modulation of several biomarkers in subjects with AD, as well as the response to pharmacological interventions. We evaluate the impact of targeting the sphingosine-1-phosphate 5 receptor (S1PR5) as a potential novel treatment option for AD, and model predictions increase our confidence in this novel disease pathway. Future applications for the QSP model are in validation of further targets and identification of potential treatment response biomarkers. © 2018 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.

SERUM

Title
Simultaneous Measurement of 3-Chlorotyrosine and 3,5-Dichlorotyrosine in Whole Blood, Serum and Plasma by Isotope Dilution HPLC-MS-MS

Journal
J Anal Toxicol

Overview
A convenience set of 100 serum and 100 blood samples was purchased from Tennessee Blood Services to assess background levels of each analyte in volunteers with no reported inflammatory disease (healthy). A total of 175 serum and blood samples collected from patients with declared disease states were purchased from BioreclamationIVT (Baltimore, MD), Bioserve (Beltsville, MD), Precision-Med (Solana Beach, CA), and Discovery Life Sciences (Los Osos, CA). Thirty-five samples for each of the following disease states were purchased: rheumatoid arthritis (RA), atherosclerosis (ATH), cystic fibrosis (CF), cardiovascular disease (CVD), and inflammatory bowel disease (IBD). This study used de-identified serum, plasma, and blood acquired from commercial sources, and thus, the work was determined not human subjects research as specified in 45 CFR 46.102(f).

Year
2016

Authors
Crow, BS;Quiñones-González, J;Pantazides, BG;Perez, JW;Winkeljohn, WR;Garton, JW;Thomas, JD;Blake, TA;Johnson, RC;

Link
https://academic.oup.com/jat/article-abstract/40/4/264/1750454

Abstract
Chlorine is a public health concern and potential threat due to its high reactivity, ease and scale of production, widespread industrial use, bulk transportation, massive stockpiles and history as a chemical weapon. This work describes a new, sensitive and rapid stable isotope dilution method for the retrospective detection and quantitation of two chlorine adducts. The biomarkers 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr) were isolated from the pronase digest of chlorine exposed whole blood, serum or plasma by solid-phase extraction (SPE), separated by reversed-phase HPLC and detected by tandem mass spectrometry (MS-MS). The calibration range is 2.50-1,000 ng/mL (R2 ≥ 0.998) with a lowest reportable limit (LRL) of 2.50 ng/mL for both analytes, an accuracy of ≥93% and an LOD of 0.443 ng/mL for Cl-Tyr and 0.396 ng/mL for Cl2-Tyr. Inter- and intra-day precision of quality control samples had coefficients of variation of ≤10% and ≤7.0%, respectively. Blood and serum samples from 200 healthy individuals and 175 individuals with chronic inflammatory disease were analyzed using this method to assess background levels of chlorinated tyrosine adducts. Results from patients with no known inflammatory disease history (healthy) showed baseline levels of <LRL-4.26 ng/mL Cl-Tyr and <LRL Cl2-Tyr. Patients with inflammatory disease had baseline levels of <LRL-15.4 ng/mL Cl-Tyr and <LRL-5.22 ng/mL Cl2-Tyr. Blood exposed to 2.02 ppm chlorine gas for 15 min produced 941 ng/mL Cl-Tyr and 223 ng/mL Cl2-Tyr. This high-throughput method has been developed and analytically validated for the diagnosis of human exposure to chlorine. Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Title
Characterization of IL-17AA and IL-17FF in rheumatoid arthritis and multiple sclerosis

Journal
Bioanalysis

Overview
Samples & sample analysis. Serum samples from NHS were obtained from PrecisionMed, Inc Serum and CSF samples from 50 RRMS patients were obtained from PrecisionMed, Inc. (Table 4 & Supplementary Table 9). Normal CSF was obtained from PrecisionMed, Inc.

Year
2016

Authors
Schofield, C;Fischer, SK;Townsend, MJ;Mosesova, S;Peng, K;Setiadi, AF;Song, A;Baruch, A;

Link
https://www.future-science.com/doi/abs/10.4155/bio-2016-0207

Abstract
IL-17 is thought to play a prominent role in immune disorders. Sensitive and specific IL-17AA and IL-17FF assays were developed and used to determine levels in serum and cerebrospinal fluid (CSF) from patients with rheumatoid arthritis and relapsing remitting multiple sclerosis (RRMS). Qualified assays detected IL-17AA and IL-17FF in healthy and disease samples. Serum IL-17AA was significantly higher in rheumatoid arthritis and RRMS as compared with normal healthy subjects. IL-17AA was also elevated in RRMS CSF as compared with normal healthy subjects; although correlation was observed between serum levels of the two isoforms, no correlation was detected between serum and CSF levels. Reliable determination of IL-17 isoforms in the systemic and CNS compartments sheds light on the involvement of IL-17AA and IL-17FF in autoimmunity.

Title
Validation of a second-generation multivariate index assay for malignancy risk of adnexal masses

Journal
Am. J. Obstet. Gynecol.

Overview
Serum samples were shipped on dry ice to an archive site (PrecisionMed Inc, Solana Beach, CA) where they were thawed and aliquoted, then frozen and stored at -65 to -85°C. All aliquots were thawed only once and consumed entirely during testing…

Year
2016

Authors
Coleman, RL;Herzog, TJ;Chan, DW;Munroe, DG;Pappas, TC;Smith, A;Zhang, Z;Wolf, J;

Link
https://www.sciencedirect.com/science/article/pii/S0002937816004658

Abstract
Women with adnexal mass suspected of ovarian malignancy are likely to benefit from consultation with a gynecologic oncologist, but imaging and biomarker tools to ensure this referral show low sensitivity and may miss cancer at critical stages. The multivariate index assay (MIA) was designed to improve the detection of ovarian cancer among women undergoing surgery for a pelvic mass. To improve the prediction of benign masses, we undertook the redesign and validation of a second-generation MIA (MIA2G). MIA2G was developed using banked serum samples from a previously published prospective, multisite registry of patients who underwent surgery to remove an adnexal mass. Clinical validity was then established using banked serum samples from the OVA500 trial, a second prospective cohort of adnexal surgery patients. Based on the final pathology results of the OVA500 trial, this intended-use population for MIA2G testing was high risk, with an observed cancer prevalence of 18.7% (92/493). Coded samples were assayed for MIA2G biomarkers by an external clinical laboratory. Then MIA2G results were calculated and submitted to a clinical statistics contract organization for decoding and comparison to MIA results for each subject. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, among other measures, and stratified by menopausal status, stage, and histologic subtype. Three MIA markers (cancer antigen 125, transferrin, and apolipoprotein A-1) and 2 new biomarkers (follicle-stimulating hormone and human epididymis protein 4) were included in MIA2G. A single cut-off separated high and low risk of malignancy regardless of patient menopausal status, eliminating potential for confusion or error. MIA2G specificity (69%, 277/401 [n/N]; 95% confidence interval [CI], 64.4-73.4%) and PPV (40%, 84/208; 95% CI, 33.9-47.2%) were significantly improved over MIA (specificity, 54%, 215/401; 95% CI, 48.7-58.4%, and PPV, 31%, 85/271; 95% CI, 26.1-37.1%, respectively) in this cohort. Sensitivity and NPV were not significantly different between the 2 tests. When combined with physician assessment, MIA2G correctly identified 75% of the malignancies missed by physician assessment alone. MIA2G specificity and PPV were significantly improved compared with MIA, while sensitivity and NPV were unchanged. The second-generation test significantly improved the predicted efficiency of triage vs MIA without sacrificing high sensitivity and NPV, which are essential for effectiveness. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Title
Performance of the American College of Obstetricians and Gynecologists’ ovarian tumor referral guidelines with a multivariate index assay.

Journal
Obstet Gynecol

Overview
The serum specimens for each patient were pooled and aliquots stored at −65°C to −85°C. The specimens were shipped frozen for storage to PrecisionMed International. Biomarker measurements were performed at Quest Diagnostics, Inc. and validated at Johns Hopkins Medical Institutions and Specialty Laboratories. Validation results were submitted to the U.S. Food and Drug Administration. All testing sites were blinded to the clinical and pathologic data. Data analysis was performed by Applied Clinical Intelligence.

Year
2011

Authors
Timmerman, D;Van Calster, B;Vergote, I;Van Hoorde, K;Van Gorp, T;Valentin, L;Bourne, T;

Link
https://journals.lww.com/greenjournal/Abstract/2011/06000/Performance_of_the_American_College_of.7.aspx

Title
Effectiveness of a multivariate index assay in the preoperative assessment of ovarian tumors

Journal
Obstet Gynecol

Overview
Specimens for each patient were pooled before aliquoting, stored at -65°C to -85°C, and shipped frozen to PrecisionMed International for storage. Biomarker measurements were performed by Quest Diagnostics, Inc., and blinded validation testing was done at Johns Hopkins Medical Institutions and Specialty Laboratories.

Year
2011

Authors
Ueland, FR;Desimone, CP;Seamon, LG;Miller, RA;Goodrich, S;Podzielinski, I;Sokoll, L;Smith, A;van Nagell, JR;Zhang, Z;

Link
https://journals.lww.com/greenjournal/Abstract/2011/06000/Effectiveness_of_a_Multivariate_Index_Assay_in_the.6.aspx

Abstract
To compare the effectiveness of physician assessment with a new multivariate index assay in identifying high-risk ovarian tumors. The multivariate index assay was evaluated in women scheduled for surgery for an ovarian tumor in a prospective, multi-institutional trial involving 27 primary- care and specialty sites throughout the United States. Preoperative serum was collected, and results for the multivariate index assay, physician assessment, and CA 125 were correlated with surgical pathology. Physician assessment was documented by each physician before surgery. CA 125 cutoffs were chosen in accordance with the referral guidelines of the American College of Obstetricians and Gynecologists. The study enrolled 590 women, with 524 evaluable for the multivariate index assay and CA 125, and 516 for physician assessment. Fifty-three percent were enrolled by nongynecologic oncologists. There were 161 malignancies and 363 benign ovarian tumors. Physician assessment plus the multivariate index assay correctly identified malignancies missed by physician assessment in 70% of nongynecologic oncologists, and 95% of gynecologic oncologists. The multivariate index assay also detected 76% of malignancies missed by CA 125. Physician assessment plus the multivariate index assay identified 86% of malignancies missed by CA 125, including all advanced cancers. The performance of the multivariate index assay was consistent in early- and late-stage cancers. The multivariate index assay demonstrated higher sensitivity and lower specificity compared with physician assessment and CA 125 in detecting ovarian malignancies.

Title
MDDScore: confirmation of a blood test to aid in the diagnosis of major depressive disorder

Journal
J Clin Psychiatry

Overview
Non-MDD samples (n = 30) were also obtained from a commercial source (PrecisionMed; San Diego, California). To qualify All subject- related clinical data were stored at PrecisionMed. Serum samples were collected…

Year
2015

Authors
Bilello, JA;Thurmond, LM;Smith, KM;Pi, B;Rubin, R;Wright, SM;Taub, F;Henry, ME;Shelton, RC;Papakostas, GI;

Link
https://pdfs.semanticscholar.org/bb52/fd02716561817d744ac58de9f2d18636d4b3.pdf

Abstract
Previously, a biomarker panel was developed for use as an aid to major depressive disorder (MDD) diagnosis; it consisted of 9 biomarkers associated with the neurotrophic, metabolic, inflammatory, and hypothalamic-pituitary-adrenal axis pathways. This panel and associated algorithm produced good clinical sensitivity and specificity (92% and 81%, respectively) in differentiating MDD patients from individuals without MDD. To further validate the panel, we performed a prospective study using a larger set of new prospectively acquired MDD patients and a similarly collected population of nondepressed subjects. The addition of gender and body mass index (BMI) effects to the algorithm was also evaluated. Blood samples were obtained from MDD patients (n = 68) clinically evaluated at multiple sites in 2011 and 2012 using standard psychiatric assessment tools and structured clinical interviews according to DSM-IV criteria. Blood samples (n = 86) from nondepressed subjects were obtained as controls. MDD and nondepressed samples were randomized into independent training (n = 102) and validation sets (n = 52). Analytes in sera were quantified by immunoassay. Training set biomarker data were used to develop a logistic regression model that included gender and BMI in a manner that allowed for their interaction with the biochemical analytes. For the training set, the sensitivity and specificity of the test (with 95% CI) were 93% (0.80-0.98) and 95% (0.85-0.99), respectively. This method (designated the MDDScore) was then applied to the independent validation set and had a sensitivity and specificity of 96% (0.77-0.98) and 86% (0.66-0.95), respectively. The overall accuracy for the training set was 94%; the validation set accuracy was 91%. Examination of a randomized independent set of samples confirms the ability of the previously established biomarker panel to identify persons with MDD; the accuracy was over 90%. The improved model that adds gender and BMI to the previously established panel of 9 biomarkers is robust and simple; it provides the most rigorously tested, objective diagnostic test for MDD to date. © Copyright 2015 Physicians Postgraduate Press, Inc.

Title
Circulating plasmalogen levels and Alzheimer Disease Assessment Scale-Cognitive scores in Alzheimer patients

Journal
J Psychiatry Neurosci

Overview
P. Wood — Phreedom Pharma; Mankidy, Ritchie, Heath, J. Wood, Goodenowe — Phenomenome Discoveries, Saskatoon, Sask.; Flax — PrecisionMed Inc., San Diego, Calif Dr. Flax is employed by and owns stock in PrecisionMed, Inc. None declared for Dr. Mankidy.

Year
2010

Authors
Wood, PL;Mankidy, R;Ritchie, S;Heath, D;Wood, JA;Flax, J;Goodenowe, DB;

Link
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2799506/

Abstract
Plasmalogens, which are key structural phospholipids in brain membranes, are decreased in the brain and serum of patients with Alzheimer disease (AD). We performed this pilot study to evaluate the relation between the levels of circulating plasmalogens and Alzheimer Disease Assessment Scale-Cognitive (ADAS-Cog) scores in patients with AD. We evaluated participants’ ADAS-Cog scores and serum plasmalogen levels. For the 40 included AD patients with an ADAS-Cog score between 20 and 46, were tested their ADAS-Cog score 1 year later. The levels of docosahexaenoic acid plasmalogen were measured by use of liquid chromatography-tandem mass spectrometry. We found that the ADAS-Cog score increased significantly in AD patients with circulating plasmalogen levels that were <or= 75% of that of age-matched controls at entry into the study. There was no change in score among participants with normal serum plasmalogen levels at baseline (> 75%). This was a pilot study with 40 patients, and the results require validation in a larger population. Our study demonstrates that decreased levels of plasmalogen precursors in the central nervous system correlate with functional decline (as measured by ADAS-Cog scores) in AD patients. The use of both ADAS-Cog and serum plasmalogen data may be a more accurate way of predicting cognitive decline in AD patients, and may be used to decrease the risk of including patients with no cognitive decline in the placebo arm of a drug trial.

Title
Next-generation sequencing identifies altered whole blood microRNAs in neuromyelitis optica spectrum disorder which may permit discrimination from multiple sclerosis

Journal
J Neuroinflammation

Overview
…[29], which were purchased from PrecisionMed (San Diego, CA, USA), all patients with NMOSD and CIS/RRMS and healthy controls were recruited at the Department of Neurology and NeuroCure Clinical Research Center, Charite—Universitatsmedizin Berlin.

Year
2015

Authors
Keller, A;Leidinger, P;Meese, E;Haas, J;Backes, C;Rasche, L;Behrens, JR;Pfuhl, C;Wakonig, K;Gieß, RM;Jarius, S;Meder, B;Bellmann-Strobl, J;Paul, F;Pache, FC;Ruprecht, K;

Link
https://jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-015-0418-1

Abstract
Neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) have a similar clinical phenotype but represent distinct diseases, requiring different therapies. MicroRNAs (miRNAs) are short non-coding RNAs whose expression profiles can serve as diagnostic biomarkers and which may be involved in the pathophysiology of neuroinflammatory diseases. Here, we analyzed miRNA profiles in serum and whole blood of patients with NMOSD and clinically isolated syndrome (CIS)/relapsing-remitting MS (RRMS) as well as healthy controls by next-generation sequencing (NGS). MiRNA expression profiles were determined by NGS in sera of patients with aquaporin-4 antibody-positive NMOSD (n = 20), CIS/RRMS (n = 20), and healthy controls (n = 20) and in whole blood of patients with NMOSD (n = 11), CIS/RRMS (n = 60), and healthy controls (n = 43). Differentially expressed miRNAs were calculated by analysis of variance and t tests. All significance values were corrected for multiple testing. Selected miRNAs were validated in whole blood of patients with NMOSD (n = 18) and CIS/RRMS (n = 19) by quantitative real-time polymerase chain reaction (qRT-PCR). None of 261 miRNAs detected in serum but 178 of 416 miRNAs detected in whole blood showed significantly different expression levels among the three groups. Pairwise comparisons revealed 115 (NMOSD vs. CIS/RRMS), 141 (NMOSD vs. healthy controls), and 44 (CIS/RRMS vs. healthy controls) miRNAs in whole blood with significantly different expression levels. qRT-PCR confirmed different expression levels in whole blood of patients with NMOSD and CIS/RRMS for 9 out of 10 exemplarily chosen miRNAs. In silico enrichment analysis demonstrated an accumulation of altered miRNAs in NMOSD in particular in CD15(+) cells (i.e., neutrophils and eosinophils). This study identifies a set of miRNAs in whole blood, which may have the potential to discriminate NMOSD from CIS/RRMS and healthy controls. In contrast, miRNA profiles in serum do not appear to be promising diagnostic biomarkers for NMOSD. Enrichment of altered miRNAs in CD15(+) neutrophils and eosinophils, which were previously implicated in the pathophysiology of NMOSD, suggests that miRNAs could be involved in the regulation of these cells in NMOSD.

Title
Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer’s disease and dementia

Journal
J. Lipid Res.

Overview
TABLE 4. Summary of clinical data. Cognitive normal subjects (PrecisionMed). Subjects were confirmed to Examination score ⩾ 28. All subjects were of Caucasian descent. Probable DAT subjects (PrecisionMed).

Year
2007

Authors
Goodenowe, DB;Cook, LL;Liu, J;Lu, Y;Jayasinghe, DA;Ahiahonu, PW;Heath, D;Yamazaki, Y;Flax, J;Krenitsky, KF;Sparks, DL;Lerner, A;Friedland, RP;Kudo, T;Kamino, K;Morihara, T;Takeda, M;Wood, PL;

Link
http://www.jlr.org/content/48/11/2485.short

Abstract
Although dementia of the Alzheimer’s type (DAT) is the most common form of dementia, the severity of dementia is only weakly correlated with DAT pathology. In contrast, postmortem measurements of cholinergic function and membrane ethanolamine plasmalogen (PlsEtn) content in the cortex and hippocampus correlate with the severity of dementia in DAT. Currently, the largest risk factor for DAT is age. Because the synthesis of PlsEtn occurs via a single nonredundant peroxisomal pathway that has been shown to decrease with age and PlsEtn is decreased in the DAT brain, we investigated potential relationships between serum PlsEtn levels, dementia severity, and DAT pathology. In total, serum PlsEtn levels were measured in five independent population collections comprising >400 clinically demented and >350 nondemented subjects. Circulating PlsEtn levels were observed to be significantly decreased in serum from clinically and pathologically diagnosed DAT subjects at all stages of dementia, and the severity of this decrease correlated with the severity of dementia. Furthermore, a linear regression model predicted that serum PlsEtn levels decrease years before clinical symptoms. The putative roles that PlsEtn biochemistry play in the etiology of cholinergic degeneration, amyloid accumulation, and dementia are discussed.

Title
Anti-ATP synthase autoantibodies from patients with Alzheimer’s disease reduce extracellular HDL level

Journal
J. Alzheimers Dis.

Overview
8 women, 3 men; mean age 73 years, range 60-80 years) admitted to the Neurological Sciences Depart- ment of the University of Rome “Sapienza” resulted IgG positive to ATP synthase in ELISA [7]. We also analyzed 3 CSF from patients with AD provided by PrecisionMed Inc.

Year
2011

Authors
Vacirca, D;Barbati, C;Scazzocchio, B;Masella, R;Rosano, G;Malorni, W;Ortona, E;

Link
https://content.iospress.com/articles/journal-of-alzheimers-disease/jad110350

Abstract
Aside from being an integral protein involved in the synthesis and hydrolysis of ATP, Ecto-F1-ATPase plays a role in cholesterol homeostasis. We demonstrated the presence of autoantibodies to ecto-F1-ATPase (ASabs) in sera and cerebrospinal fluids from patients with Alzheimer’s disease (AD). Herein we show that ASabs, unlike irrelevant antibodies, can increase cellular uptake of HDL, a risk factor for the development of AD, via a mechanism involving the prototypical function of ecto-F1-ATPase: the generation of ADP due to the hydrolysis of ATP. Piceatannol, a specific inhibitor ecto-F1-ATPase, completely hindered these effects. We hypothesize that ASabs could exert a pathogenetic role in AD.

Title
Natural IgG autoantibodies are abundant and ubiquitous in human sera, and their number is influenced by age, gender, and disease

Journal
PLoS ONE

Overview
(Detroit, MI). Parkinson’s disease (PD) and Alzheimer’s disease (AD) serum samples were obtained from Analytical Biological Systems, Inc. (Wilmington, DE), ProteoGenex (Culver City, CA) and PrecisionMed (Solana Beach, CA).

Year
2013

Authors
Nagele, EP;Han, M;Acharya, NK;DeMarshall, C;Kosciuk, MC;Nagele, RG;;

Link
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060726

Abstract
The presence of self-reactive IgG autoantibodies in human sera is largely thought to represent a breakdown in central tolerance and is typically regarded as a harbinger of autoimmune pathology. In the present study, immune-response profiling of human serum from 166 individuals via human protein microarrays demonstrates that IgG autoantibodies are abundant in all human serum, usually numbering in the thousands. These IgG autoantibodies bind to human antigens from organs and tissues all over the body and their serum diversity is strongly influenced by age, gender, and the presence of specific diseases. We also found that serum IgG autoantibody profiles are unique to an individual and remarkably stable over time. Similar profiles exist in rat and swine, suggesting conservation of this immunological feature among mammals. The number, diversity, and apparent evolutionary conservation of autoantibody profiles suggest that IgG autoantibodies have some important, as yet unrecognized, physiological function. We propose that IgG autoantibodies have evolved as an adaptive mechanism for debris-clearance, a function consistent with their apparent utility as diagnostic indicators of disease as already established for Alzheimer’s and Parkinson’s diseases.

Title
The diagnosis of depression: current and emerging methods

Journal
Comprehensive psychiatry

Overview
…from healthy subjects. A training set of 50 serum samples from patients with MDD and 20 healthy volunteers was obtained from a biobank (PrecisionMed, San Diego, CA) and were maintained frozen at −80°C until testing.

Year
2013

Authors
Smith, KM;Renshaw, PF;Bilello, J;

Link
https://www.sciencedirect.com/science/article/pii/S0010440X12001186

Abstract
This article evaluates the technical basis for and clinical performance of these various instruments and methods to diagnosis depression in clinical settings. Traditional tools include physician-administered or patient self-administered interview tools that have reasonable clinical accuracy depending on the threshold score and may lead to a full diagnostic evaluation for high-risk patients. In addition, older laboratory methods such as the dexamethasone test have contributed to the diagnosis of depression over a long period. Newer diagnostic methods such as genomics, proteomics, and metabolomics are technically sophisticated and objective and are beginning to emerge in psychiatry. Although promising, further evaluation of these methods is needed to fully demonstrate their clinical value and accuracy.

Title
Patterns of chronic inflammation in extensively treated patients with arachnoiditis and chronic intractable pain

Journal
Postgrad Med

Overview
A series of serum samples from normal subjects (n = 31) with no history of neurologic disease, mood disorders, or chronic pain were obtained from a commercial source (PrecisionMed, San Diego, CA).

Year
2017

Authors
Bilello, JA;Tennant, FS;

Link
https://www.tandfonline.com/doi/abs/10.1080/00325481.2017.1270155

Abstract
To use biomarkers to gain insight into and gauge the residual (post-treatment) level of inflammation in two groups of intensively treated patients with severe chronic pain. Three study groups were analyzed, and included: (i) patients (n = 90) with chronic intractable pain (CIP), (ii) patients (n = 26) with chronic pain and MRI-documented arachnoiditis (ARC) and (iii) normal subjects without a diagnosis of chronic pain (n = 86). We determined and compared the serum concentrations of Alpha-1 Antitrypsin (A1AT), Myeloperoxidase (MPO) and soluble Tumor Necrosis Factor receptor type 2 (sTNFR2) in each of the patient populations studied. Patients treated for ARC or CIP had higher serum levels of A1AT and MPO than normal untreated subjects without a diagnosis of chronic pain. ARC patients had an A1AT mean serum concentration of 167.9 ± 41.9 mg/dL as compared to 148.9 ± 35.2 mg/dL for normal subjects (p = 0.023). CIP patients had the highest mean serum A1AT level 183.6 ± 39.2 mg/dL with p values of <0.0001 or 0.08 when compared to normal subjects or ARC patients respectively. ARC patients had an MPO mean serum concentration of 344.6 ± 227.9 ng/mL as compared to 188.2 ± 107.5 ng/mL for normal subjects (p = < 0.0001). CIP patients had a similar mean serum MPO level of 352.3 ± 164 ng/mL with p values of <0.0001 or 0.85 when compared to normal subjects or ARC patients respectively. In addition, we noted a difference in the pattern of MPO expression in patients with ARC in that 34% had levels of MPO at normal or below and 31% had levels 2-fold or greater than normal. This data supports the concept that in centralized pain, sites of neuroinflammation elaborate MPO and other inflammatory factors which may not be completely cleared from the system despite extensive and complex treatment regimens.

Title
A possible serologic biomarker for maternal immune activation-associated neurodevelopmental disorders found in the rat models

Journal
Neurosci. Res.

Overview
2.9. Human sera sample. Anonymized sera of sporadic schizophrenia patients and healthy controls were purchased from PrecisionMed. Inc. (CA, USA; see details at http://www.precisionmed.com/) and stored at −80 °C until use.

Year
2016

Authors
Oh-Nishi, A;Koga, K;Maeda, T;Suhara, T;

Link
https://www.sciencedirect.com/science/article/pii/S0168010216300943

Abstract
Epidemiological studies have shown that maternal infection during early pregnancy increases the risk of neurodevelopmental disorders (i.e., schizophrenia or autism) in offspring. Recently, diagnostic/stratification biomarkers for the maternal immune activation background in patients with neurodevelopmental disorders have been energetically searched for in the patient blood. Here, we report a novel serologic marker candidate for the disorders found in the maternal immune activation (MIA) rat model. Serum proteome analysis of the MIA rat showed that the immunoglobulin (Ig) light chain is reproducibly augmented. The Ig light chain in sera takes two forms – free form or bound to the Ig heavy chain. Only the former is an inflammatory disease marker, but pro-inflammatory cytokine levels in the sera of the MIA rats were below detectable limits of the ELISA protocol we used. We thereby carried out serum assays of Ig light chains and pro-inflammatory cytokines of commercially available schizophrenia patient sera for research. Although the number of samples was limited, we found augmentation of free Ig light chains but not pro-inflammatory cytokines in sporadic schizophrenia patient sera. Our findings suggest that Ig light chain assay of the schizophrenia/autism patient sera would be worthy to be validated in larger scale. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Title
Libby amphibole-induced mesothelial cell autoantibodies bind to surface plasminogen and alter collagen matrix remodeling

Journal
Physiol Rep

Overview
PAGE stained with Coomassie Blue. Normal human serum samples were obtained from Precision Med (Solana Beach, CA), in order to test for antibodies to (PLG) in sera from the general population. Age and sex demographic…

Year
2016

Authors
Hanson, R;Evilia, C;Gilmer, J;Woods, L;Black, B;Flores, R;Pfau, JC;

Link
https://onlinelibrary.wiley.com/doi/abs/10.14814/phy2.12881

Abstract
Lamellar pleural thickening (LPT) is a fibrotic disease induced by exposure to Libby amphibole (LA) asbestos that causes widespread scarring around the lung, resulting in deterioration of pulmonary function. Investigating the effects of autoantibodies to mesothelial cells (MCAA) present in the study populations has been a major part of the effort to understand the mechanism of pathogenesis. It has been shown in vitro that human mesothelial cells (Met5a) exposed to MCAA increase collagen deposition into the extracellular matrix (ECM). In this study, we sought to further elucidate how MCAA drive increased collagen deposition by identifying the protein targets bound by MCAA on the cellular surface using biotinylation to label and isolate surface proteins. Isolated surface protein fractions were identified as containing MCAA targets using ELISA The fractions that demonstrated binding by MCAA were then analyzed by tandem mass spectrometry (MS/MS) and MASCOT analysis. The most promising result from the MASCOT analysis, plasminogen (PLG), was tested for MCAA binding using purified human PLG in an ELISA We report that serum containing MCAA bound at an optical density (OD) 3 times greater than that of controls, and LA-exposed subjects had a high frequency of positive tests for anti-PLG autoantibodies. This work implicates the involvement of the plasminogen/plasmin system in the mechanism of excess collagen deposition in Met5a cells exposed to MCAA Elucidating this mechanism could contribute to the understanding of LPT. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Title
Serum phosphatidylcholine changes in Alzheimer’s disease and dementia are due to impaired peroxisomal beta oxidation

Journal
Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association

Overview
4; 20:5; 22:6) fatty acid combinations and three PlsEtn and PtdEtn species (16:0/22:4, 18:0/20:5, 16:0/22:6) were measured in serum from 572 cognitively assessed subjects [MMSE (2-30), female/male (291/281); NCI (156); MCI (208); AD (208)] obtained from Precision Med.

Year
2015

Authors
Senanayake, V;Mochizuki, A;Jayasinghe, D;Chitou, B;Smith, T;Flax, J;Goodenowe, DB;

Link
https://www.alzheimersanddementia.com/article/S1552-5260(15)02755-7/abstract

Abstract
Background Serum phosphatidylcholine (PtdCho), ethanolamine plasmalogen (PlsEtn) and phosphadidylethanolamine (PtdEtn) levels have been reported to be associated with Alzheimer’s disease (AD) and dementia. We hypothesized that impaired peroxisomal beta-oxidation (Px-β-Ox) is the underlying causal mechanism for these changes. To test this hypothesis, we analysed PtdCho, PtdEtn and PlsEtn levels using species-specific tandem mass spectrometry technology in subjects with no cognitive impairment (NCI), mild cognitive impairment (MCI), and AD. The associations between cognition and PtdCho, PlsEtn, and PtdEtn species containing fatty acids involved in Px-β-Ox pathways were determined. Methods The levels of 24 PtdCho species comprising of three sn-1 (16:0; 18:0; 18:1) and eight sn-2 (18:0; 18:1; 18:2; 18:3; 20:4; 22:4; 20:5; 22:6) fatty acid combinations and three PlsEtn and PtdEtn species (16:0/22:4, 18:0/20:5, 16:0/22:6) were measured in serum from 572 cognitively assessed subjects [MMSE (2-30), female/male (291/281); NCI (156); MCI (208); AD (208)] obtained from Precision Med Inc. The effect of Px-β-Ox was assessed within each phospholipid pool by calculating the average of three Px-β-Ox substrate/product ratios (20:5/22:4; 22:6/22:4; 20:5/22:6, POX). In addition, the average PlsEtn/PtdEtn ratio of species containing either 22:6 or 20:5) was determined. PtdCho, PlsEtn, and PtdEtn species were assayed using stable isotope tandem mass spectrometry. Results Only PtdCho species with arachidonic acid (20:4, Coef:0.841, p=3.0e-2), adrenic acid (22:4, Coef:1.723, p=9.3e-9), or eicosapentaenoic acid (20:5, Coef:-0.673, p=4.4e-4) at the sn-2 position were associated with cognition. The association of POX with cognition was similar in PtdCho (Coef: -1.37, p=3.7e-9), PtdEtn (Coef: -1.44, p=6.8e-7), and PlsEtn (Coef: -1.17, p=1.9e-8). PtdCho_POX was strongly associated with both PtdEtn_POX (Coef: 1.07, p=4.7e-167) and PlsEtn_POX (Coef: 0.829, p=6.3e-238). The average PlsEtn/PtdEtn ratio was associated with cognition (Coef:-0.987, p=2.7e-4) and PlsEtn_POX (Coef: 0.193, p=1.7e-9). Conclusions Serum PtdCho changes associated with AD and dementia are also present in PtdEtn and PlsEtn phospholipids. A relative decrease in PlsEtn (versus PtdEtn) is associated with decreased cognition. These data indicate that impaired Px-β-Ox is the primary biochemical mechanism underlying the observed associations between PtdCho, PtdEtn, and PlsEtn phospholipids and AD and dementia.

DNA

Title
Characterization of whole genome amplified (WGA) DNA for use in genotyping assay development

Journal
BMC Genomics

Overview
Caucasian. F. 20. 1xG542X. Cystic Fibrosis. 1.83. 1x3659delC. DNA samples were purchased from PrecisionMed, Inc. and dissolved in TE buffer (10 mM Tris, 1 mM EDTA at pH 8.0). There are different mutations in the two patients with cystic fibrosis (CF1 and CF2). Figure 1

Year
2012

Authors
Han, T;Chang, CW;Kwekel, JC;Chen, Y;Ge, Y;Martinez-Murillo, F;Roscoe, D;Težak, Z;Philip, R;Bijwaard, K;Fuscoe, JC;

Link
https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-13-217

Abstract
Genotyping assays often require substantial amounts of DNA. To overcome the problem of limiting amounts of available DNA, Whole Genome Amplification (WGA) methods have been developed. The multiple displacement amplification (MDA) method using Φ29 polymerase has become the preferred choice due to its high processivity and low error rate. However, the uniformity and fidelity of the amplification process across the genome has not been extensively characterized. To assess amplification uniformity, we used array-based comparative genomic hybridization (aCGH) to evaluate DNA copy number variations (CNVs) in DNAs amplified by two MDA kits: GenomiPhi and REPLI-g. The Agilent Human CGH array containing nearly one million probes was used in this study together with DNAs from a normal subject and 2 cystic fibrosis (CF) patients. Each DNA sample was amplified 4 independent times and compared to its native unamplified DNA. Komogorov distances and Phi correlations showed a high consistency within each sample group. Less than 2% of the probes showed more than 2-fold CNV introduced by the amplification process. The two amplification kits, REPLI-g and GenomiPhi, generate very similar amplified DNA samples despite the differences between the unamplified and amplified DNA samples. The results from aCGH analysis indicated that there were no obvious CNVs in the CFTR gene region due to WGA when compared to unamplified DNA. This was confirmed by quantitative real-time PCR copy number assays at 10 locations within the CFTR gene. DNA sequencing analysis of a 2-kb region within the CFTR gene showed no mutations introduced by WGA. The relatively high uniformity and consistency of the WGA process, coupled with the low replication error rate, suggests that WGA DNA may be suitable for accurate genotyping. Regions of the genome that were consistently under-amplified were found to contain higher than average GC content. Because of the consistent differences between the WGA DNA and the native unamplified DNA, characterization of the genomic region of interest, as described here, will be necessary to ensure the reliability of genotyping results from WGA DNA.

Title
A common copy number variation (CNV) polymorphism in the CNTNAP4 gene: association with aging in females

Journal
PLoS ONE

Overview
Materials and Methods. Genomic DNA. DNA samples for the discovery study were purchased from Coriell Institute (Camden, NJ) and PrecisionMed Laboratories (San Diego, CA). The samples originated from neurologically normal North American Caucasians over 50 years old.

Year
2013

Authors
Iakoubov, L;Mossakowska, M;Szwed, M;Duan, Z;Sesti, F;Puzianowska-Kuznicka, M;

Link
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079790

Abstract
Aging is a biological process strongly determined by genetics. However, only a few single nucleotide polymorphisms (SNPs) have been reported to be consistently associated with aging. While investigating whether copy number variations (CNVs) could fill this gap, we focused on CNVs that have not been studied in previous SNP-based searches via tagging SNPs. TaqMan qPCR assays were developed to quantify 20 common CNVs in 222 senior American Caucasians in order to reveal possible association with longevity. The replication study was comprised of 1283 community-dwelling senior European Caucasians. Replicated CNVs were further investigated for association with healthy aging and aging-related diseases, while association with longevity was additionally tested in Caenorhabditis elegans. In the discovery study of ≥80 vs.<80 years old seniors, a homozygous intronic CNV deletion in the CNTNAP4 gene was inversely associated with survival to the age of 80 (OR=0.51, 95%CI 0.29-0.87, p=0.015 before correction for multiple testing). After stratification by sex, association remained significant in females (OR=0.41, 95%CI 0.21-0.77, p=0.007), but not in males (OR=0.97, 95%CI 0.33-2.79, p=1). The finding was validated in a replication study (OR=0.66, 95%CI 0.48-0.90, p=0.011 for females). CNTNAP4 association with longevity was supported by a marked 25% lifespan change in C. elegans after knocking down the ortholog gene. An inverse association of the CNV del/del variant with female healthy aging was observed (OR=0.39, 95%CI 0.19-0.76, p=0.006). A corresponding positive association with aging-related diseases was revealed for cognitive impairment (OR=2.17, 95%CI 1.11-4.22, p=0.024) and, in independent studies, for Alzheimer’s (OR=4.07, 95%CI 1.17-14.14, p=0.036) and Parkinson’s (OR=1.59, 95%CI 1.03-2.42, p=0.041) diseases. This is the first demonstration for association of the CNTNAP4 gene and one of its intronic CNV polymorphisms with aging. Association with particular aging-related diseases awaits replication and independent validation.

RNA

Title
Toward an Alzheimer’s disease diagnosis via high-resolution blood gene expression

Journal
Alzheimers Dement

Overview
Eighty AD patients (33 males and 47 females) were recruited through three Tunisian clinical centers (n = 40) using Diagnostic and Statistical Manual (DSM-IV) criteria, or accrued from a United States Clinical Research Organisation (CRO) (Precision Med, San Diego, CA) (n = 40

Year
2010

Authors
Fehlbaum-Beurdeley, P;Jarrige-Le Prado, AC;Pallares, D;Carrière, J;Guihal, C;Soucaille, C;Rouet, F;Drouin, D;Sol, O;Jordan, H;Wu, D;Lei, L;Einstein, R;Schweighoffer, F;Bracco, L;

Link
https://www.sciencedirect.com/science/article/pii/S1552526009020895

Abstract
There is a significant need for reliable molecular biomarkers to aid in Alzheimer’s disease (AD) clinical diagnosis. We performed a genome-wide investigation of the human transcriptome, taking into account the discriminatory power of splice variations from the blood of 80 AD patients and 70 nondemented control (NDC) individuals. We characterized a blood RNA signature composed of 170 oligonucleotide probe sets associated with 133 genes that can correctly distinguish AD patients from NDC with a sensitivity of 100% and specificity of 96%. Functionally, this signature highlights genes involved in pathways that were associated with macrophages and lymphocytes within AD patients: Transforming growth factor (TGF-beta) signaling, oxidative stress, innate immunity and inflammation, cholesterol homeostasis, and lipid-raft perturbation, whereas other genes may also provide new insights in the biology of AD. This study provides proof-of-concept that whole-blood profiling can generate an AD-associated classification signature via the specific relative expression of biologically relevant RNAs. Such a signature will need to be validated with extended patient cohorts, and evaluated to learn whether it can differentiate AD from others types of dementia. 2010 The Alzheimer’s Association. Published by Elsevier Inc. All rights reserved.

Title
A predictive microarray-based biomarker for early detection of Alzheimer’s disease intended for clinical diagnostic application

Journal
Biomarkers

Overview
below. Set A – 40 AD patient samples and 37 CT subject samples (provided by PrecisionMed Inc., San Diego, CA). Set B – 50 AD patient samples, 50 non-AD demented patient samples and 50 CT subject samples from a clinical

Year
2013

Authors
Calciano, M;Lemarié, JC;Blondiaux, E;Einstein, R;Fehlbaum-Beurdeley, P;

Link
https://www.tandfonline.com/doi/abs/10.3109/1354750X.2013.773083

Abstract
Microarray-based signatures for clinical application are often plagued by processing variability or batch effects that compromise the robustness of the test performance. A splice variant array-based signature for early detection of Alzheimer’s disease (AD) was developed using 315 AD or normal subjects processed in three disparate microarray batches. A modified top scoring pair classifier using the signature, is robust to batch effects and outperforms other common classifiers, with sensitivity and specificity of 88.3% (95% CI:81.2%, 93.4%) and 88.9% (95% CI:65.3%, 98.6%), respectively, on an independent cohort. This splice-variant array-based signature shows promise for clinical diagnostic use in AD.

Title
MicroRNA-Seq Data Analysis Pipeline to Identify Blood Biomarkers for Alzheimer’s Disease from Public Data

Journal
Biomark Insights

Overview
… 22 samples of NC subjects, composed of 11 males and 11 females with average age 67.1 ± 7.5 years and MMSE score 29.3 ± 1.2. They obtained the samples mostly from the Biorepository and Tissue Bank PrecisionMed (www.precisionmed.com). Total RNA including miRNA was isolated from the blood by the PAXgene Blood miRNA Kit (Qiagen, Valencia, CA, USA). A total of 200 ng of RNA was processed to …

Year
2015

Authors
Satoh, J;Kino, Y;Niida, S;

Link
http://journals.sagepub.com/doi/abs/10.4137/BMI.S25132

Abstract
Alzheimer’s disease (AD) is the most common cause of dementia with no curative therapy currently available. Establishment of sensitive and non-invasive biomarkers that promote an early diagnosis of AD is crucial for the effective administration of disease-modifying drugs. MicroRNAs (miRNAs) mediate posttranscriptional repression of numerous target genes. Aberrant regulation of miRNA expression is implicated in AD pathogenesis, and circulating miRNAs serve as potential biomarkers for AD. However, data analysis of numerous AD-specific miRNAs derived from small RNA-sequencing (RNA-Seq) is most often laborious. To identify circulating miRNA biomarkers for AD, we reanalyzed a publicly available small RNA-Seq dataset, composed of blood samples derived from 48 AD patients and 22 normal control (NC) subjects, by a simple web-based miRNA data analysis pipeline that combines omiRas and DIANA miRPath. By using omiRas, we identified 27 miRNAs expressed differentially between both groups, including upregulation in AD of miR-26b-3p, miR-28-3p, miR-30c-5p, miR-30d-5p, miR-148b-5p, miR-151a-3p, miR-186-5p, miR-425-5p, miR-550a-5p, miR-1468, miR-4781-3p, miR-5001-3p, and miR-6513-3p and downregulation in AD of let-7a-5p, let-7e-5p, let-7f-5p, let-7g-5p, miR-15a-5p, miR-17-3p, miR-29b-3p, miR-98-5p, miR-144-5p, miR-148a-3p, miR-502-3p, miR-660-5p, miR-1294, and miR-3200-3p. DIANA miRPath indicated that miRNA-regulated pathways potentially downregulated in AD are linked with neuronal synaptic functions, while those upregulated in AD are implicated in cell survival and cellular communication. The simple web-based miRNA data analysis pipeline helps us to effortlessly identify candidates for miRNA biomarkers and pathways of AD from the complex small RNA-Seq data.

Title
Molecular Diagnostics of Ageing and Tackling Age-related Disease

Journal
Trends Pharmacol. Sci.

Overview
…same controls and patient samples. The samples originated either from the same USA commercial supplier (PrecisionMed CA), Melbourne case-control samples or the RosKamp memory clinic. While the reported specificity…

Year
2017

Authors
Timmons, JA;

Link
https://www.sciencedirect.com/science/article/pii/S0165614716301638

Abstract
As average life expectancy increases there is a greater focus on health-span and, in particular, how to treat or prevent chronic age-associated diseases. Therapies which were able to control ‘biological age’ with the aim of postponing chronic and costly diseases of old age require an entirely new approach to drug development. Molecular technologies and machine-learning methods have already yielded diagnostics that help guide cancer treatment and cardiovascular procedures. Discovery of valid and clinically informative diagnostics of human biological age (combined with disease-specific biomarkers) has the potential to alter current drug-discovery strategies, aid clinical trial recruitment and maximize healthy ageing. I will review some basic principles that govern the development of ‘ageing’ diagnostics, how such assays could be used during the drug-discovery or development process. Important logistical and statistical considerations are illustrated by reviewing recent biomarker activity in the field of Alzheimer’s disease, as dementia represents the most pressing of priorities for the pharmaceutical industry, as well as the chronic disease in humans most associated with age. Copyright © 2016 Elsevier Ltd. All rights reserved.

Title
A blood based 12-miRNA signature of Alzheimer disease patients

Journal
Genome Biol.

Overview
Patient blood was obtained from the SAMPLE (Serial Alzheimer diseaseand MCI Prospective Longitudinal Evaluation) Registry of PrecisionMed (San Diego, CA, USA) and blood from age-matched healthy donors from the ACE (Aging Cognition Evaluation) Registry.

Year
2013

Authors
Leidinger, P;Backes, C;Deutscher, S;Schmitt, K;Mueller, SC;Frese, K;Haas, J;Ruprecht, K;Paul, F;Stähler, C;Lang, CJ;Meder, B;Bartfai, T;Meese, E;Keller, A;

Link
https://genomebiology.biomedcentral.com/articles/10.1186/gb-2013-14-7-r78

Abstract
Alzheimer disease (AD) is the most common form of dementia but the identification of reliable, early and non-invasive biomarkers remains a major challenge. We present a novel miRNA-based signature for detecting AD from blood samples. We apply next-generation sequencing to miRNAs from blood samples of 48 AD patients and 22 unaffected controls, yielding a total of 140 unique mature miRNAs with significantly changed expression levels. Of these, 82 have higher and 58 have lower abundance in AD patient samples. We selected a panel of 12 miRNAs for an RT-qPCR analysis on a larger cohort of 202 samples, comprising not only AD patients and healthy controls but also patients with other CNS illnesses. These included mild cognitive impairment, which is assumed to represent a transitional period before the development of AD, as well as multiple sclerosis, Parkinson disease, major depression, bipolar disorder and schizophrenia. miRNA target enrichment analysis of the selected 12 miRNAs indicates an involvement of miRNAs in nervous system development, neuron projection, neuron projection development and neuron projection morphogenesis. Using this 12-miRNA signature, we differentiate between AD and controls with an accuracy of 93%, a specificity of 95% and a sensitivity of 92%. The differentiation of AD from other neurological diseases is possible with accuracies between 74% and 78%. The differentiation of the other CNS disorders from controls yields even higher accuracies. The data indicate that deregulated miRNAs in blood might be used as biomarkers in the diagnosis of AD or other neurological diseases.

Title
Microarray-based transcriptomic signatures to help improving the success rate of ad clinical trials

Journal
Conference

Overview
Additional samples (40 AD et 37 CT) were issued from a CRO (Precision Med, San Diego, CA). Therefore it is critical to expand the identification and use of clinically relevant biomarkers which can contribute to the renewal of AD drug development.

Year
2011

Authors
Pando, MP;Carriere, J;Desire, L;Beurdeley, P;Barber, I;

Link
https://www.researchgate.net/profile/Laurent_Desire/publication/304483730_MICROARRAY-BASED_TRANSCIPTOMIC_SIGNATURES_TO_HELP_IMPROVING_THE_SUCCESS_RATE_OF_AD_CLINICAL_TRIALS/links/5770dac808ae6219474884e4.pdf

CSF Cell Pellet

Title
MSPrecise: A molecular diagnostic test for multiple sclerosis using next generation sequencing

Journal
Gene

Overview
By lumbar puncture in accordance with IRB-approved protocols at UT Southwestern Medical Center, the University of Massachusetts Memorial Medical Center (UMass), John Hopkins University (JHU), or purchased from a commercial biorepository (PrecisionMed, Solana Beach).

Year
2015

Authors
Rounds, WH;Salinas, EA;Wilks, TB;Levin, MK;Ligocki, AJ;Ionete, C;Pardo, CA;Vernino, S;Greenberg, BM;Bigwood, DW;Eastman, EM;Cowell, LG;Monson, NL;;

Link
https://www.sciencedirect.com/science/article/pii/S0378111915008215

Abstract
We have previously demonstrated that cerebrospinal fluid-derived B cells from early relapsing-remitting multiple sclerosis (RRMS) patients that express a VH4 gene accumulate specific replacement mutations. These mutations can be quantified as a score that identifies such patients as having or likely to convert to RRMS. Furthermore, we showed that next generation sequencing is an efficient method for obtaining the sequencing information required by this mutation scoring tool, originally developed using the less clinically viable single-cell Sanger sequencing. To determine the accuracy of MSPrecise, the diagnostic test that identifies the presence of the RRMS-enriched mutation pattern from patient cerebrospinal fluid B cells. Cerebrospinal fluid cell pellets were obtained from RRMS and other neurological disease (OND) patient cohorts. VH4 gene segments were amplified, sequenced by next generation sequencing and analyzed for mutation score. The diagnostic test showed a sensitivity of 75% on the RRMS cohort and a specificity of 88% on the OND cohort. The accuracy of the test in identifying RRMS patients or patients that will develop RRMS is 84%. MSPrecise exhibits good performance in identifying patients with RRMS irrespective of time with RRMS. Copyright © 2015. Published by Elsevier B.V.

BLOOD

Title
Intracellular clusterin interacts with brain isoforms of the bridging integrator 1 and with the microtubule-associated protein Tau in Alzheimer’s disease

Journal
PLoS ONE

Overview
… were obtained from commercial sources and analyzed anonymously. Human blood and cerebrospinal fluid (CSF) from clinically diagnosed Alzheimer’s disease patients and aged controls were purchased from PrecisionMed, Inc (www.precisionmed.com). CSF samples were aliquoted and stored at −80°C until analysis. Genomic DNA was extracted from blood cells using the QIAamp DNA blood midi prep (Qiagen). DN

Year
2014

Authors
Zhou, Y;Hayashi, I;Wong, J;Tugusheva, K;Renger, JJ;Zerbinatti, C;

Link
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103187

Abstract
Sporadic or late-onset Alzheimer’s disease (AD) is expected to affect 50% of individuals reaching 85 years of age. The most significant genetic risk factor for late-onset AD is the e4 allele of APOE gene encoding apolipoprotein E, a lipid carrier shown to modulate brain amyloid burden. Recent genome-wide association studies have uncovered additional single nucleotide polymorphisms (SNPs) linked to AD susceptibility, including those in the CLU and BIN1 genes encoding for clusterin (CLU) and the bridging integrator 1 (BIN1) proteins, respectively. Because CLU has been implicated in brain amyloid-β (Aβ) clearance in mouse models of amyloid deposition, we sought to investigate whether an AD-linked SNP in the CLU gene altered Aβ42 biomarker levels in the cerebrospinal fluid (CSF). Instead, we found that the CLU rs11136000 SNP modified CSF levels of the microtubule-associated protein Tau in AD patients. We also found that an intracellular form of CLU (iCLU) was upregulated in the brain of Tau overexpressing Tg4510 mice, but not in Tg2576 amyloid mouse model. By overexpressing iCLU and Tau in cell culture systems we discovered that iCLU was a Tau-interacting protein and that iCLU associated with brain-specific isoforms of BIN1, also recently identified as a Tau-binding protein. Through expression analysis of CLU and BIN1 variants, we found that CLU and BIN1 interacted via their coiled-coil motifs. In co-immunoprecipitation studies using human brain tissue, we showed that iCLU and the major BIN1 isoform expressed in neurons were associated with modified Tau species found in AD. Finally, we showed that expression of certain coding CLU variants linked to AD risk led to increased levels of iCLU. Together, our findings suggest that iCLU and BIN1 interaction might impact Tau function in neurons and uncover potential new mechanisms underlying the etiology of Tau pathology in AD.

Title
Genomic Dissection of Bipolar Disorder and Schizophrenia, Including 28 Subphenotypes

Journal
Cell

Overview
Purchased blood samples were obtained from PrecisionMed International by Pharmacia and Upjohn Corporation, and were collected from diagnosed subjects with schizophrenia and schizoaffective disorder. All studies were reviewed by both central and local institutional review boards, depending on the study site, before recruitment of subjects started. Protocol amendments were approved while the study was in progress and before the data were unblinded.

Year
2018

Authors
Bipolar Disorder and Schizophrenia Working Group of the Psychiatric Genomics Consortium

Link
https://www.cell.com/cell/abstract/S0092-8674(18)30658-5

Abstract
Schizophrenia and bipolar disorder are two distinct diagnoses that share symptomology. Understanding the genetic factors contributing to the shared and disorder-specific symptoms will be crucial for improving diagnosis and treatment. In genetic data consisting of 53,555 cases (20,129 bipolar disorder [BD], 33,426 schizophrenia [SCZ]) and 54,065 controls, we identified 114 genome-wide significant loci implicating synaptic and neuronal pathways shared between disorders. Comparing SCZ to BD (23,585 SCZ, 15,270 BD) identified four genomic regions including one with disorder-independent causal variants and potassium ion response genes as contributing to differences in biology between the disorders. Polygenic risk score (PRS) analyses identified several significant correlations within case-only phenotypes including SCZ PRS with psychotic features and age of onset in BD. For the first time, we discover specific loci that distinguish between BD and SCZ and identify polygenic components underlying multiple symptom dimensions. These results point to the utility of genetics to inform symptomology and potential treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

Title
Simultaneous Measurement of 3-Chlorotyrosine and 3,5-Dichlorotyrosine in Whole Blood, Serum and Plasma by Isotope Dilution HPLC-MS-MS

Journal
J Anal Toxicol

Overview
A convenience set of 100 serum and 100 blood samples was purchased from Tennessee Blood Services to assess background levels of each analyte in volunteers with no reported inflammatory disease (healthy). A total of 175 serum and blood samples collected from patients with declared disease states were purchased from BioreclamationIVT (Baltimore, MD), Bioserve (Beltsville, MD), Precision-Med (Solana Beach, CA), and Discovery Life Sciences (Los Osos, CA). Thirty-five samples for each of the following disease states were purchased: rheumatoid arthritis (RA), atherosclerosis (ATH), cystic fibrosis (CF), cardiovascular disease (CVD), and inflammatory bowel disease (IBD). This study used de-identified serum, plasma, and blood acquired from commercial sources, and thus, the work was determined not human subjects research as specified in 45 CFR 46.102(f).

Year
2016

Authors
Crow, BS;Quiñones-González, J;Pantazides, BG;Perez, JW;Winkeljohn, WR;Garton, JW;Thomas, JD;Blake, TA;Johnson, RC;

Link
https://academic.oup.com/jat/article-abstract/40/4/264/1750454

Abstract
Chlorine is a public health concern and potential threat due to its high reactivity, ease and scale of production, widespread industrial use, bulk transportation, massive stockpiles and history as a chemical weapon. This work describes a new, sensitive and rapid stable isotope dilution method for the retrospective detection and quantitation of two chlorine adducts. The biomarkers 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr) were isolated from the pronase digest of chlorine exposed whole blood, serum or plasma by solid-phase extraction (SPE), separated by reversed-phase HPLC and detected by tandem mass spectrometry (MS-MS). The calibration range is 2.50-1,000 ng/mL (R2 ≥ 0.998) with a lowest reportable limit (LRL) of 2.50 ng/mL for both analytes, an accuracy of ≥93% and an LOD of 0.443 ng/mL for Cl-Tyr and 0.396 ng/mL for Cl2-Tyr. Inter- and intra-day precision of quality control samples had coefficients of variation of ≤10% and ≤7.0%, respectively. Blood and serum samples from 200 healthy individuals and 175 individuals with chronic inflammatory disease were analyzed using this method to assess background levels of chlorinated tyrosine adducts. Results from patients with no known inflammatory disease history (healthy) showed baseline levels of <LRL-4.26 ng/mL Cl-Tyr and <LRL Cl2-Tyr. Patients with inflammatory disease had baseline levels of <LRL-15.4 ng/mL Cl-Tyr and <LRL-5.22 ng/mL Cl2-Tyr. Blood exposed to 2.02 ppm chlorine gas for 15 min produced 941 ng/mL Cl-Tyr and 223 ng/mL Cl2-Tyr. This high-throughput method has been developed and analytically validated for the diagnosis of human exposure to chlorine. Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Title
Evidence for HTR1A and LHPP as interacting genetic risk factors in major depression

Journal
Mol. Psychiatry

Overview
…of Ashkenazi descent. These samples were collected by PrecisionMed (Solana Beach, CA, USA). Utah and Ashkenazi individuals ascertained without respect to depression status were used as controls. Initial logistic regression…

Year
2009

Authors
Neff, CD;Abkevich, V;Packer, JC;Chen, Y;Potter, J;Riley, R;Davenport, C;DeGrado Warren, J;Jammulapati, S;Bhathena, A;Choi, WS;Kroeger, PE;Metzger, RE;Gutin, A;Skolnick, MH;Shattuck, D;Katz, DA;

Link
https://www.nature.com/articles/mp20088

Abstract
The HTR1A -1019C>G genotype was associated with major depression in the Utah population. Linkage analysis on Utah pedigrees with strong family histories of major depression including only cases with the HTR1A -1019G allele revealed a linkage peak on chromosome 10 (maximum HLOD=4.4). Sequencing of all known genes in the linkage region revealed disease-segregating single-nucleotide polymorphisms (SNPs) in LHPP. LHPP SNPs were also associated with major depression in both Utah and Ashkenazi populations. Consistent with the linkage evidence, LHPP associations depended on HTR1A genotype. Lhpp or a product of a collinear brain-specific transcript, therefore, may interact with Htr1a in the pathogenesis of major depression.

Title
Drug treatment of Alzheimer’s disease patients leads to expression changes in peripheral blood cells

Journal
Alzheimers Dement

Overview
…because of unclear drug treatment. Blood samples from each patient were provided through a Clinical Research Organization (PrecisionMed; San Diego, CA) after complying with full patient consent regulations. Patients who had a…

Year
2010

Authors
Calciano, MA;Zhou, W;Snyder, PJ;Einstein, R;

Link
https://www.sciencedirect.com/science/article/pii/S1552526010000038

Abstract
Increasing cholinergic activity has been the primary mechanism for treating dementia due to Alzheimer’s disease. However, the effectiveness of cholinesterase inhibitors (ChEIs) is still widely debated. The identification of specific biomarkers capable of identifying patients more likely to respond to these treatments could potentially provide specific evidence to clearly address this controversy through patient stratification. The goal of this study was to determine the feasibility of discovering biomarkers specific for the treatment of Alzheimer’s disease. Peripheral blood was collected from a cohort of patients treated with different ChEIs. Total RNA was isolated and profiled on the human Genome-Wide SpliceArray (GWSA) to test the feasibility of discriminating the different treatment subgroups of subjects based on the expression patterns generated from the Genome-Wide SpliceArray. Specific expression differences were identified for the various treatment groups that lead to a clear separation between patients treated with ChEIs versus naïve patients when Principal Component Analysis was performed on probe sets selected for differential expression. In addition, specific probe sets were identified to be dependent on the inhibitor used among the treated patients. Distinct separation between non-treated, galantamine, donepezil, and rivastigmine-treated patients was clearly identified based on small sets of expression probes. The ability to identify drug-specific treatment expression differences strengthens the potential for using peripheral gene signatures for the identification of individuals responding to drug treatment. Copyright 2010 The Alzheimer

Title
Validation of AclarusDx™, a blood-based transcriptomic signature for the diagnosis of Alzheimer’s disease

Journal
J. Alzheimers Dis.

Overview
Additional blood samples, which were only used in the training set, were pur- chased from PrecisionMed (San Diego, CA, USA) Samples from PrecisionMed were recruited according to US legisla- tion and after IRB approval.

Year
2012

Authors
Fehlbaum-Beurdeley, P;Sol, O;Désiré, L;Touchon, J;Dantoine, T;Vercelletto, M;Gabelle, A;Jarrige, AC;Haddad, R;Lemarié, JC;Zhou, W;Hampel, H;Einstein, R;Vellas, B;

Link
https://content.iospress.com/articles/journal-of-alzheimers-disease/jad120637

Abstract
Biomarkers have gained an increased importance in the past years in helping physicians to diagnose Alzheimer’s disease (AD). This study was designed to identify a blood-based, transcriptomic signature that can differentiate AD patients from control subjects. The performance of the signature was then evaluated for robustness in an independent blinded sample population. RNA was extracted from 177 blood samples (90 AD patients and 87 controls) and gene expression profiles were generated using the human Genome-Wide Splice Array™. These profiles were used to establish a signature to differentiate AD patients from controls. Subsequently, prediction results were optimized by establishing grey zone boundaries that discount prediction scores near the disease status threshold. Signature validation was then performed on a blinded independent cohort of 209 individuals (111 AD and 98 controls). The AclarusDx™ signature consists of 170 probesets which map to 136 annotated genes, a significant number of which are associated with inflammatory, gene expression, and cell death pathways. Additional signature genes are known to interact with pathways involved in amyloid and tau metabolism. The validation sample set, after removal of 45 individuals with prediction profile scores within the grey zone, consisted of 164 subjects. The AclarusDx™ performance on this validation cohort had a sensitivity of 81.3% (95% CI: [73.3%; 89.3%]); and a specificity of 67.1% (95% CI: [56.3%; 77.9%]). AclarusDx™ is a non-invasive blood-based transcriptomic test that, in combination with standard assessments, can provide physicians with objective information to support the diagnosis of AD.

Urine

Title
BRAF V600E mutations in urine and plasma cell-free DNA from patients with Erdheim-Chester disease

Journal
Oncotarget

Overview
Previous to this study, accuracy of the urine-based ddPCR BRAF V600E assay was verified in 89 urine specimens from 50 healthy control samples (Precision Med; Solana Beach, CA) and 39 samples from 20 patients with known positive BRAF V600E mutation tissue biopsies.

Year
2014

Authors
Janku, F;Vibat, CRT;Kosco, K;Holley, VR;Cabrilo, G;

Link
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4116506/

Abstract
Urine and plasma cfDNA were quantified by a droplet digital PCR (ddPCR; QX-100, BioRad; Hercules, CA) assay to a 44bp amplicon of _RNase_ P, a single-copy gene. Quantified DNA (12.4ng to 60ng) was used for a two-step PCR assay for rare mutant allele detection of a 31bp region containing _BRAF_ V600E (Figure ​(Figure1).1 [/pmc/articles/PMC4116506/figure/F1/]). The first step involved pre-amplification with two primers flanking the _BRAF_ V600E locus, where both primers contain non-complementary 5′ tags which hybridize to second round primers. A complementary blocking oligonucleotide suppressed wt _BRAF_ amplification, achieving enrichment of the mutant _BRAF_ V600E sequence within the pre-amplification step. The second step entailed a duplex ddPCR reaction using FAM (V600E _BRAF_) and VIC (wt _BRAF_) TaqMan probes to enable differentiation of mutant versus wild-type quantification, respectively. The RainDrop ddPCR instrument (RainDance; Billerica, MA) was used for PCR droplet separation, fluorescent reading, and counting droplets containing mutant sequence, wt sequence, or unreacted probe. For a given patient sample, the assay reported _BRAF_ V600E mutation fragments detected as a percentage of detected wt _BRAF_. Previous to this study, accuracy of the urine-based ddPCR _BRAF_ V600E assay was verified in 89 urine specimens from 50 healthy control samples (Precision Med; Solana Beach, CA) and 39 samples from 20 patients with known positive _BRAF_ V600E mutation tissue biopsies as determined in a CLIA laboratory.[17] Thresholds for mutation detection in urine were determined by assessing these data using a classification tree. Minimizing the percentage of false negatives was given a higher importance than minimizing false positives. Thresholds were defined as no detection – wt (0.107%). For plasma detection, plasma from 13 patients with wt _BRAF_ metastatic cancer was used to determine a threshold for detection of _BRAF_ V600E mutations. For plasma, >0.094% mutant, equivalent to three standard deviations (0.021%) above the mean of wt _BRAF_ controls (0.031%), was considered positive for _BRAF_ V600E mutation.

Genotyping

Title
Meta-analysis for genome-wide association study identifies multiple variants at the BIN1 locus associated with late-onset Alzheimer’s disease

Journal
PLoS ONE

Overview
… Effect in Alzheimer’s Dementia (LEADe) trial [39]–[40] , 180 MCI subjects from the Vitamin E trial who have converted to AD during the course of the study [18], 216 probable AD subjects enrolled by PrecisionMed for case/control study and 149 subjects from clinical trial A3041005 which is a phase II trial investigating CP-457920 (a selective alpha5 GABAA receptor inverse agonist) in Alzheimer’s …

Year
2011

Authors
Hu, X;Pickering, E;Liu, YC;Hall, S;Fournier, H;Katz, E;Dechairo, B;John, S;Van Eerdewegh, P;Soares, H;, ;

Link
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016616

Abstract
Recent GWAS studies focused on uncovering novel genetic loci related to AD have revealed associations with variants near CLU, CR1, PICALM and BIN1. In this study, we conducted a genome-wide association study in an independent set of 1034 cases and 1186 controls using the Illumina genotyping platforms. By coupling our data with available GWAS datasets from the ADNI and GenADA, we replicated the original associations in both PICALM (rs3851179) and CR1 (rs3818361). The PICALM variant seems to be non-significant after we adjusted for APOE e4 status. We further tested our top markers in 751 independent cases and 751 matched controls. Besides the markers close to the APOE locus, a marker (rs12989701) upstream of BIN1 locus was replicated and the combined analysis reached genome-wide significance level (p = 5E-08). We combined our data with the published Harold et al. study and meta-analysis with all available 6521 cases and 10360 controls at the BIN1 locus revealed two significant variants (rs12989701, p = 1.32E-10 and rs744373, p = 3.16E-10) in limited linkage disequilibrium (r²  =  0.05) with each other. The independent contribution of both SNPs was supported by haplotype conditional analysis. We also conducted multivariate analysis in canonical pathways and identified a consistent signal in the downstream pathways targeted by Gleevec (P = 0.004 in Pfizer; P = 0.028 in ADNI and P = 0.04 in GenADA). We further tested variants in CLU, PICALM, BIN1 and CR1 for association with disease progression in 597 AD patients where longitudinal cognitive measures are sufficient. Both the PICALM and CLU variants showed nominal significant association with cognitive decline as measured by change in Clinical Dementia Rating-sum of boxes (CDR-SB) score from the baseline but did not pass multiple-test correction. Future experiments will help us better understand potential roles of these genetic loci in AD pathology.

Title
Three-cohort targeted gene screening reveals a non-synonymous TRKA polymorphism associated with schizophrenia

Journal
J Psychiatr Res

Overview
2. Materials and methods. 2.1. Cohorts. 2.1.1. USA. The USA cohort consisted of 491 Caucasian schizophrenic patients and 298 unrelated controls. Samples were derived from the PrecisionMed Inc. Human Biological Sample Bank collection (San Diego, California, USA).

Year
2009

Authors
van Schijndel, JE;van Loo, KM;van Zweeden, M;Djurovic, S;Andreassen, OA;Hansen, T;Werge, T;Kallunki, P;Pedersen, JT;Martens, GJ;

Link
https://www.sciencedirect.com/science/article/pii/S0022395609000867

Abstract
Schizophrenia is a complex neurodevelopmental disorder that is thought to be induced by an interaction between predisposing genes and environmental stressors. To identify predisposing genetic factors, we performed a targeted (mostly neurodevelopmental) gene approach involving the screening of 396 selected non-synonymous single-nucleotide polymorphisms (SNPs) in three independent Caucasian schizophrenia case-control cohorts (USA, Denmark and Norway). A meta-analysis revealed ten non-synonymous SNPs that were nominally associated with schizophrenia, nine of which have not been previously linked to the disorder. Risk alleles are in TRKA (rs6336), BARD1 (rs28997576), LAMA5 (rs3810548), DKK2 (rs7037102), NOD2 (rs2066844) and RELN (rs2229860), whereas protective alleles are in NOD2 (rs2066845), NRG1 (rs10503929), ADAM7 (rs13259668) and TNR (rs859427). Following correction for multiple testing, the most attractive candidate for further study concerns SNP rs6336 (q=0.12) that causes the substitution of an evolutionarily highly conserved amino acid residue in the kinase domain of the neurodevelopmentally important receptor TRKA. Thus, TRKA signaling may represent a novel susceptibility pathway for schizophrenia.

Title
A γ-secretase polymorphism and complex disorders Chapter B4

Journal
Book

Overview
…patients; undifferentiated type (295.9): 126 patients). USA. A total of 793 Caucasian individuals were derived from the PrecisionMed Sample Bank collection (San Diego, California, USA). Controls (n= 300) had no (family) history.

Year
2016

Authors
van Zweeden, M;van Schijndel, JE;Djurovic, S;

Link
https://core.ac.uk/download/pdf/16151311.pdf#page=170

EDTA Plasma

Title
Exosome-Derived miR-25-3p and miR-92a-3p Stimulate Liposarcoma Progression

Journal
Cancer Res.

Overview
Healthy donor blood used in the discovery and in the validation sets was purchased respectively from PrecisionMed and ZenBio. For plas- ma collection protocol, PrecisionMed centrifuged tubes at 2,440 rpm or 1,200 A g …

Year
2017

Authors
Casadei, L;Calore, F;Creighton, CJ;Guescini, M;Batte, K;Iwenofu, OH;Zewdu, A;Braggio, DA;Bill, KL;Fadda, P;Lovat, F;Lopez, G;Gasparini, P;Chen, JL;Kladney, RD;Leone, G;Lev, D;Croce, CM;Pollock, RE;

Link
http://cancerres.aacrjournals.org/content/early/2017/06/14/0008-5472.CAN-16-2984.short

Abstract
Despite the development of combined modality treatments against liposarcoma in recent years, a significant proportion of patients respond only modestly to such approaches, possibly contributing to local or distant recurrence. Early detection of recurrent or metastatic disease could improve patient prognosis by triggering earlier clinical intervention. However, useful biomarkers for such purposes are lacking. Using both patient plasma samples and cell lines, we demonstrate here that miR-25-3p and miR-92a-3p are secreted by liposarcoma cells through extracellular vesicles and may be useful as potential biomarkers of disease. Both miR-25-3p and miR-92a-3p stimulated secretion of proinflammatory cytokine IL6 from tumor-associated macrophages in a TLR7/8-dependent manner, which in turn promoted liposarcoma cell proliferation, invasion, and metastasis via this interaction with the surrounding microenvironment. Our findings provide novel and previously unreported insight into liposarcoma progression, identifying communication between liposarcoma cells and their microenvironment as a process critically involved in liposarcoma progression. This study establishes the possibility that the pattern of circulating miRNAs may identify recurrence prior to radiological detectability while providing insight into disease outcome and as a possible approach to monitor treatment efficacy. Cancer Res; 77(14); 3846-56. ©2017 AACR. ©2017 American Association for Cancer Research.

Title
Plasma microRNA biomarkers for detection of mild cognitive impairment: biomarker validation study

Journal
Aging (Albany NY)

Overview
…and CSF analysis. MATERIALS AND METHODS. Plasma samples. K2EDTA Plasma samples from 50 MCI patients and 50 AMC were obtained from a commercial vendor PrecisionMed (Solana Beach, California). The samples…

Year
2013

Authors
Sheinerman, KS;Tsivinsky, VG;Abdullah, L;Crawford, F;Umansky, SR;

Link
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3883708/

Abstract
A minimally invasive test for early detection and monitoring of Alzheimer’s and other neurodegenerative diseases is a highly unmet need for drug development and planning of patient care. Mild Cognitive Impairment (MCI) is a syndrome characteristic of early stages of many neurodegenerative diseases. Recently, we have identified two sets of circulating brain-enriched miRNAs, the miR-132 family (miR-128, miR-132, miR-874) normalized per miR-491-5p and the miR-134 family (miR-134, miR-323-3p, miR-382) normalized per miR-370, capable of differentiating MCI from age-matched control (AMC) with high accuracy. Here we report a biomarker validation study of the identified miRNA pairs using larger independent sets of age- and gender- matched plasma samples. The biomarker pairs detected MCI with sensitivity, specificity and overall accuracy similar to those obtained in the first study. The miR-132 family biomarkers differentiated MCI from AMC with 84%-94% sensitivity and 96%-98% specificity, and the miR-134 family biomarkers demonstrated 74%-88% sensitivity and 80-92% specificity. When miRNAs of the same family were combined, miR-132 and miR-134 family biomarkers demonstrated 96% and 87% overall accuracy, respectively. No statistically significant differences in the biomarker concentrations in samples obtained from male and female subjects were observed for either MCI or AMC. The present study also demonstrated that the highest sensitivity and specificity are achieved with pairs of miRNAs whose concentrations in plasma are highly correlated.

Title
Methylation patterns of cell-free plasma DNA in relapsing-remitting multiple sclerosis

Journal
J. Neurol. Sci.

Overview
The project was supported by the NS060311 grant from NINDS (VL) and philanthropy to the Rush University MS Center (RB, DS). Authors are grateful to Melinda Kopec, R.N., Cynthia Dendrinos, R.N., Carmen Petrizzo, R.N. and Raquel Brillante, N.P., for their invaluable help with the collection of blood samples and clinical data. VL is grateful to Dr. John Flax (PrecisionMed, Inc.) for a subset of plasma samples, and to Dr. Joel Peek (Microarrays, Inc.) for printing microarrays and technical support with microarray experiments.

Year
2010

Authors
Liggett, T;Melnikov, A;Tilwalli, S;Yi, Q;Chen, H;Replogle, C;Feng, X;Reder, A;Stefoski, D;Balabanov, R;Levenson, V;

Link
https://www.sciencedirect.com/science/article/pii/S0022510X09010211

Abstract
There is growing interest for identification of new targets for biomarker development in multiple sclerosis (MS). The goal of this study was to compare the concentration and the methylation patterns of cell-free plasma DNA (cfpDNA) in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy individuals. Three 30-patient cohorts were examined: patients with RRMS, in either remission or exacerbation, and healthy individuals as controls. Concentration of cfpDNA was determined using a standard fluorometric assay. Patterns of methylation in 56 gene promoters were determined by a microarray-based assay (MethDet-56). The data were analyzed to identify statistically relevant differences among the study groups. The concentration of cfpDNA in patients with RRMS was four to eight-fold higher compared to healthy controls. Significant differences in cfpDNA methylation patterns were detected in all three comparisons: RRMS patients in remission versus healthy controls were recognized with 79.2% sensitivity and 92.9% specificity; RRMS patients in exacerbation versus healthy controls were recognized with 75.9% sensitivity and 91.5% specificity; and RRMS patients in exacerbation versus those in remission were recognized with 70.8% sensitivity and 71.2% specificity. Based on our findings, we conclude that patients with RRMS display unique disease- and state-specific changes of cfpDNA. Our findings are of clinical significance as they could be used in the development of potentially new biomarkers for MS. This is the first report in our knowledge describing such changes of cfpDNA in patients with MS.

Prospective Collection

Title
Effectiveness of the selective D4 antagonist sonepiprazole in schizophrenia: a placebo-controlled trial

Journal
Biol. Psychiatry

Overview
Douglas Hospital Centre, Borough of Verdun, Montreal, Quebec, Canada; Rosalinda Sepulveda Garcia, M.D., Hospital Psiquiatrico No. 22 International Medical Support Services, Monterrey NL; Mexicol Hernan Silva, M.D., Clínica Psiquiatrica Hospital Clinico Univ. de Chile, Santiago, Chile; Amarendra Singh, M.D., Hotel Dieu Hospital, Kingston, Ontario, Canada; Seymour Solodar, M.D., PrecisionMed Inc., Duluth, Georgia; Omar Kawas Valle, M.D., Hospital Universitario de Nuevo Leon, Monterrey, Mexico; Walter Vieweg, M.D., Veterans Adminstration Medical Center, Richmond, Virginia; Sasanto Wibisono, M.D., Psychiatric Department, Cipto Mangunkusumo General Hospital (RSCM), Jakarta, Indonesia; Ching-Kuan Wu, M.D., Chang Gung Memorial Hospital KaoShiung, KaoShiung County, Taiwan.

Year
2004

Authors
Corrigan, MH;Gallen, CC;Bonura, ML;Merchant, KM;

Link
https://www.sciencedirect.com/science/article/pii/S000632230301076X

Abstract
Selective localization of dopamine D(4) receptors in the prefrontal cortex and preferential affinity of clozapine for the dopamine D(4) receptor over the D(2) receptor led to the hypothesis that the superior efficacy of clozapine may be mediated via blockade of the D(4) receptor. This hypothesis was tested by evaluating sonepiprazole, a selective D(4) dopamine antagonist, in schizophrenia patients. We treated 467 hospitalized schizophrenia patients with scores of > or = 60 on the Positive and Negative Syndrome Scale (PANSS) with sonepiprazole, olanzapine, or placebo once daily for 6 weeks. The primary efficacy end point was the mean change from baseline in the PANSS total score at 6 weeks. Secondary efficacy end points were the mean change from baseline in the PANSS factor scores, the Brief Psychiatric Rating Scale score, the Clinical Global Impressions Severity of Illness score, and the Calgary Depression Scale score. No statistically significant differences were observed between placebo and any sonepiprazole dose on the primary or any secondary end point after 6 weeks of treatment. Statistically significant differences, favoring olanzapine over placebo, were observed on all efficacy end points but the Calgary Depression Scale. Sonepiprazole was ineffective for the treatment of patients with schizophrenia.

Title
Multiplexed Quantification of Proglucagon-Derived Peptides by Immunoaffinity Enrichment and Tandem Mass Spectrometry after a Meal Tolerance Test

Journal
Clin. Chem.

Overview
… criteria (interassay CV and extrapolated concentration ≤20% from nominal). Sample stability was tested through 3 freeze–thaw cycles. STUDY PROTOCOL We sponsored a study written and executed at PrecisionMed (Solano Beach, CA). All protocols underwent Ethics Committee review and approval, and all participants provided informed consent before undergoing study procedures and activities.

Year
2016

Authors
Lee, AY;Chappell, DL;Bak, MJ;Judo, M;Liang, L;Churakova, T;Ayanoglu, G;Castro-Perez, J;Zhou, H;Previs, S;Souza, SC;Lassman, ME;Laterza, OF;

Link
http://clinchem.aaccjnls.org/content/62/1/227.short

Abstract
Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity. We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy. Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay. IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal. © 2015 American Association for Clinical Chemistry.

TAU Data

Title
Divergent CSF τ alterations in two common tauopathies: Alzheimer’s disease and progressive supranuclear palsy

Journal
J. Neurol. Neurosurg. Psychiatry

Overview
Institute of Neurological Disorders and Stroke-Society for Progressive Supranuclear Palsy (NINDS-SPSP) criteria.16 A second set of patients with AD (n=13) that were matched in age, education and gender, which were purchased from Precision Med (Solana Beach, California).

Year
2015

Authors
Wagshal, D;Sankaranarayanan, S;Guss, V;Hall, T;Berisha, F;Lobach, I;Karydas, A;Voltarelli, L;Scherling, C;Heuer, H;Tartaglia, MC;Miller, Z;Coppola, G;Ahlijanian, M;Soares, H;Kramer, JH;Rabinovici, GD;Rosen, HJ;Miller, BL;Meredith, J;Boxer, AL;

Link
https://jnnp.bmj.com/content/86/3/244.short

Abstract
Elevated CSF τ is considered a biomarker of neuronal injury in newly developed Alzheimer’s disease (AD) and mild cognitive impairment (MCI) criteria. However, previous studies have failed to detect alterations of τ species in other primary tauopathies. We assessed CSF τ protein abnormalities in AD, a tauopathy with prominent Aβ pathology, and progressive supranuclear palsy (PSP), a primary tauopathy characterised by deposition of four microtubule-binding repeat (4R) τ with minimal Aβ pathology. 26 normal control (NC), 37 AD, and 24 patients with PSP participated in the study. AD and PSP were matched for severity using the clinical dementia rating sum of boxes (CDR-sb) scores. The INNO BIA AlzBio3 multiplex immunoassay was used to measure CSF Aβ, total τ, and ptau181. Additional, novel ELISAs targeting different N-terminal and central τ epitopes were developed to examine CSF τ components and to investigate interactions between diagnostic group, demographics and genetic variables. PSP had lower CSF N-terminal and C-terminal τ concentrations than NC and AD measured with the novel τ ELISAs and the standard AlzBio3 τ and ptau assays. AD had higher total τ and ptau levels than NC and PSP. There was a gender by diagnosis interaction in AD and PSP for most τ species, with lower concentrations for male compared to female patients. CSF τ fragment concentrations are different in PSP compared with AD despite the presence of severe τ pathology and neuronal injury in both disorders. CSF τ concentration likely reflects multiple factors in addition to the degree of neuronal injury. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Title
Quantification of tau in cerebrospinal fluid by immunoaffinity enrichment and tandem mass spectrometry

Journal
Clin. Chem.

Overview
An interassay CV 20%. HEALTHY AND AD SAMPLES A panel of age-matched CSF from donors with normal cognition (n 50) and donors with a diagnosis of AD (n 50) were obtained from PrecisionMed. Tau concentrations…

Year
2014

Authors
McAvoy, T;Lassman, ME;Spellman, DS;Ke, Z;Howell, BJ;Wong, O;Zhu, L;Tanen, M;Struyk, A;Laterza, OF;

Link
http://clinchem.aaccjnls.org/content/60/4/683.short

Abstract
Cerebrospinal fluid (CSF) tau is a common biomarker for Alzheimer disease (AD). Measurements of tau have historically been performed using immunoassays. Given the molecular diversity of tau in CSF, the selectivity of these immunoassays has often been questioned. Therefore, we aimed to develop an analytically sensitive and selective immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) (IA-MS) assay. IA-MS sample analysis involved the addition of an internal standard, immunoaffinity purification of tau using a tau monoclonal antibody coupled to magnetic beads, trypsin digestion, and quantification of a surrogate tau peptide by LC-MS/MS using a Waters Trizaic nanoTile ultraperformance LC microfluidic device. Further characterization of tau peptides was performed by full-scan MS using a Thermo Orbitrap LC-MS. CSF samples from a cohort of age-matched controls and patients with AD were analyzed by the IA-MS method as well as a commercially available immunoassay. The IA-MS assay had intra- and interassay imprecision values of 3.2% to 8.1% CV and 7.8% to 18.9% C, respectively, a mean recovery of 106%, and a limit of quantification of 0.25 pmol/L and was able to quantify tau concentrations in all human specimens tested. The IA-MS assay showed a correlation of R(2) = 0.950 against a total-tau immunoassay. In patients with AD, tau was increased approximately 2-fold. Combining immunoaffinity enrichment with microflow LC-MS/MS analysis is an effective approach for the development of a highly selective assay to measure total tau and, potentially, other posttranslationally modified forms of tau in CSF.

OTHER

Title
An ultra-sensitive clinical biomarker assay: quantitation of thymus and activation-regulated chemokine in human plasma

Journal
Bioanalysis

Overview
ProteoGenex, Inc. (CA, USA). EDTA plasma samples of healthy control subjects, as well as patients with mild, moderate, severe and very severe AD were purchased from PrecisionMed, Inc. (CA, USA). ▪ Sample collection. Human plasma…

Year
2014

Authors
Zhao, X;Delgado, L;Weiner, R;Laterza, OF;

Link
https://www.future-science.com/doi/abs/10.4155/bio.14.72

Abstract
Thymus and activation-regulated chemokine (TARC) is a Th2 type, pro-allergic secreted chemokine. TARC in plasma/serum has been proposed as a marker for disease activity of atopic dermatitis (AD) and as a pharmacodynamic readout in the clinical development of novel agents for the treatment of AD. An ultra-sensitive electrochemiluminescence assay for TARC in human plasma was developed and analytically validated. The assay demonstrated excellent performance characteristics, including precision, sensitivity, dilution linearity, accuracy and specificity. Stability and biological variability of TARC in plasma were also assessed for clinical sample analysis and data interpretation. The improved sensitivity allowed the measurement of approximately 90% TARC inhibition from baseline levels of healthy subjects and >90% TARC inhibition from baseline levels of AD patients after drug treatment. A validated TARC electrochemiluminescence assay enables pharmacodynamic assessment in the development of AD therapeutics.

Title
Carbon monoxide and iron modulate plasmatic coagulation in Alzheimer’s disease

Journal
Curr Neurovasc Res

Overview
All subjects were confirmed to be disease-free, nonsmokers, and not pregnant. With regard to AD patient samples, they were obtained from a commercial vendor (PrecisionMed, Inc., Solana Beach, CA, USA). This vendor…

Year
2015

Authors
Nielsen, VG;Pretorius, E;Bester, J;Jacobsen, WK;Boyle, PK;Reinhard, JP;

Link
https://pdfs.semanticscholar.org/5161/43b8cd7b996f07360ffaeb519701a598dbab.pdf

Abstract
Alzheimer’s disease (AD) is a significant source of morbidity and mortality for millions of people worldwide, and multiple potential etiologies have been postulated to contribute to AD. Among these, spontaneous cerebral emboli and increased cerebral and circulating heme oxygenase (Hmox) activity in AD patients are of particular interest, as two of the products of Hmox activity, carbon monoxide (CO) and iron enhance plasmatic coagulation and modify the ultrastructure of thrombi. We hypothesized that patients afflicted with AD would have coagulation kinetics modulated by CO and iron. Using viscoelastic assessments of coagulation, it was determined with a small cohort (n=11) of AD patients that all had enhancement of coagulation by CO, iron, or both. In a complementary fashion, it was determined that a separate cohort (n=12) of AD patients had thrombi with ultrastructural features consistent with iron and CO exposure as assessed with scanning electron microscopy. Further, when stratified by normal or abnormally increased serum ferritin concentrations (which can be increased by Hmox), the AD patients with abnormal ferritin concentrations had significantly thinner fibrin fiber diameters, not unlike that noted when normal plasma is mixed with iron or CO. In sum, AD patients were noted to have plasmatic coagulation kinetic and thrombus ultrastructural changes consistent with exposure to CO and iron. Future investigation of CO and iron in the pathogenesis of Alzheimer’s disease is warranted.

Title
Incidence of tardive dyskinesia in older adult patients treated with olanzapine or conventional antipsychotics

Journal
J Geriatr Psychiatry Neurol

Overview
J. Steven Lamberti, MD, Psychiatry, Rochester, NY; Ronald P. Landbloom, MD, Regions Hospital, Behavior Health Research, St. Paul, MN; Gunnar Larson, MD, Zablocki VA Medical Center, Milwaukee, WI; Michael McLarnon, MD, PrecisionMed, Inc., Duluth, GA; Gregory F. Oxenkrug, MD, PhD, St. Elizabeth’s Medical Center, Boston, MA; William M. Petrie, MD, Psychiatry Consultants, PC, Nashville, TN; Nunzio Pomara, MD, Nathan Kline Institute for Psychiatric Research, Orangeburg, NY; Stephen Rappaport, MD, Agewell LTD, Indianapolis, IN; Stephen Scheinthal, DO, University of Medicine & Dentistry of New Jersey.

Year
2015

Authors
Kinon, BJ;Kollack-Walker, S;Jeste, D;Gupta, S;Chen, L;Case, M;Chen, J;Stauffer, V;

Link
http://journals.sagepub.com/doi/abs/10.1177/0891988714541867

Abstract
The risk of persistent tardive dyskinesia (TD) was compared in patients with acute psychosis or agitation aged 55 years or older who were treated with olanzapine (OLZ) or conventional antipsychotic (CNV) drug therapy. Patients without TD were randomized to treatment with OLZ (2.5-20 mg/d; n = 150) or CNV (dosed per label; n = 143). Following a 6-week drug tapering/initiation period, patients without TD were treated with OLZ or CNV for up to 1 year. The a priori defined primary outcome end point was persistent TD defined as Abnormal Involuntary Movement Scale (AIMS) scores = 2 on at least 2 items or ≥3 on at least 1 item (items 1-7) lasting at least for 1 month (Criterion A). Post hoc analyses assessed persistent TD meeting the criterion of moderate severity defined as AIMS score ≥3 on at least 1 item persisting for 1 month (Criterion B) and probable TD defined as elevated AIMS scores (Criterion A or B) not persisting for 1 month. Treatment groups were compared using Kaplan-Meier curve with log-rank exact test. On average, patients were 78 years of age; the predominant diagnosis was dementia (76.7% in the OLZ group and 82.5% in the CNV group). Approximately, 40.6% of patients in the CNV group received haloperidol. No significant difference in time to developing persistent TD was observed during treatment with OLZ or CNV (cumulative incidence: OLZ, 2.5% [95% confidence interval [95% CI]: 0.5-7.0]; CNV, 5.5% [95% CI: 2.1-11.6], P = .193). The exposure-adjusted event rates per 100 person-years were not significantly different between treatment groups: OLZ (2.7) and CNV (6.3; ratio: 0.420; 95% CI: 0.068-1.969). Post hoc analyses revealed a significantly lower risk of at least moderately severe persistent TD persisting for 1 month (P = .012) and probable TD not persisting for 1 month (Criterion A, P = .030; Criterion B, P = .048) in OLZ-treated patients. For those patients without significant extrapyramidal symptoms at baseline, significantly more patients in the CNV treatment group developed treatment-emergent parkinsonism than for patients in the OLZ treatment group (CNV: 70%, 35 of 50 patients; OLZ 44%, 25 of 57 patients; P = .011). No significant difference between the groups was observed for treatment-emergent akathisia (CNV: 6%, 7 of 117 patients; OLZ: 10%, 13 of 130 patients; P = .351). The cumulative incidence of persistent TD was low and the risk of persistent TD did not differ significantly among predominantly older adult patients having dementia with acute psychosis or agitation treated with OLZ or CNV. © The Author(s) 2014.

Title
Dickkopf-related Protein 3 as a Sensitive and Specific Marker for Cerebrospinal Fluid Leaks

Journal
Otology & Neurotology

Overview
Biweekly injections of recombinant protein were made into rabbits and a goat (Cocalico Biologicals, Inc, Reamstown, PA, U.S.A.). The initial injections were made in complete Freund’s adjuvant and the remaining injections in incomplete Freund’s adjuvant. After eight injections sera were extracted and tested for activity. Antibodies were affinity purified over a column in which the immunogen was immobilized on Sepharose beads. Reagents Human cerebrospinal fluid and human sera from normal health subjects were obtained from PrecisionMed, Inc. (Solana Beach, CA, U.S.A.).

Year
2016

Authors
Michaelides, EM;Kuang, H;Pieribone, VA;

Link
https://journals.lww.com/otology-neurotology/Abstract/2016/03000/Dickkopf_related_Protein_3_as_a_Sensitive_and.17.aspx

Abstract
Hypothesis: Cerebrospinal fluid (CSF) can be identified by using an enzyme-linked immunosorbent assay (ELISA) for Dickkopf-related protein 3 (DKK3). Background: Cerebrospinal fluid leakage from the subarachnoid space is a potentially alarming condition that, left unrepaired, may result in increased risk of meningitis and encephalitis. Current biochemical methods of CSF leak detection involve using beta-2-transferrin-based or beta trace protein-based assays, both of which, at present, have limitations that hinder practical clinical application. This study presents the immunological detection of the CSF-enriched protein DKK3 as a method for detection of a CSF leak. Methods: Antibodies against DKK3 were generated in rabbits and goats immunized with recombinant human DKK3. Varying dilutions and combinations of human CSF and serum were tested on immunoblots and sandwich ELISA using antibodies to DKK3. Results: ELISA data show that there is a negligible amount of detectable DKK3 in serum samples compared with CSF samples. Inclusion of sera (up to 30%) in a sample containing CSF failed to produce a positive signal, whereas concentrations of CSF as low as 1% produced a positive signal. The minimum concentration required for reliable CSF detection in a sandwich ELISA was determined to be 0.5 μl. Conclusion: ELISA sandwich assays for DKK3 can reliably detect the presence of as little as 0.5 μl of human CSF, even in the presence of excessive serum. This study provides quantitative evidence of the utility of DKK3 immunoreactivity as an assay for the presence of CSF in samples that contain contaminating sera. The robustness of this assay has allowed for the development of a rapid, point of care test for the detection of CSF in clinical and surgical setting.

Title
Die Vaskuläre Biobank – hands on

Journal
Gefässchirurgie

Overview
World Medical Association https://www.wma.net/what-we-do/medical-ethics/ German Biobank Node http://bbmri.de German Biobank Alliance http://bbmri.de/ueber-gbn/german-biobank- alliance/ PrecisionMed https://www.precisionmed.com BioServe https://www.bioserve.com

Year
2018

Authors
Reutersberg, B;Peters, A;Hakimi, M;Pelisek, J;Eckstein, H;Jahns, R;Busch, A;

Link
https://link.springer.com/content/pdf/10.1007/s00772-018-0369-9.pdf

Title
Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

Journal
J Extracell Vesicles

Overview
… 842 9 22057275 210 Teunissen CE Tumani H Bennett JL Berven FS Brundin L Comabella M Consensus guidelines for CSF and blood biobanking for CNS biomarker studies Mult Scler Int 2011 246412 22096631 211 PrecisionMed PrecisionMed 2013 [cited 2013 April 5]. Available from: http://www.precisionmed.com/cerebrospinal-fluid. 212 Irani DN Anderson C Gundry R Cotter R Moore S Kerr DA Cleavage of cystatin C …
Year
2013

Authors
Witwer, KW;Buzás, EI;Bemis, LT;Bora, A;Lässer, C;Lötvall, J;Nolte-‘t Hoen, EN;Piper, MG;Sivaraman, S;Skog, J;Théry, C;Wauben, MH;Hochberg, F;

Link
https://www.tandfonline.com/doi/abs/10.3402/jev.v2i0.20360

Abstract
The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.

Title
Analysis of flavin-containing monooxygenase 3 genotype data in populations administered the anti-schizophrenia agent olanzapine

Journal
Drug Metab Lett

Overview
Briefly, European American male (840) and female (444) cases had a median age of 48 and 45, respectively. The affected subjects were from a large multi-center study of patients with schizophrenia recruited by PrecisionMed Inc., (PMI) (Solana Beach, CA).

Year
2008

Authors
Cashman, JR;Zhang, J;Nelson, MR;Braun, A;

Link
https://www.ingentaconnect.com/content/ben/dml/2008/00000002/00000002/art00005

Abstract
Flavin-containing monooxygenase 3 (FMO3) genotype data for European-, Latin-, African- and Asian-American schizophrenia patients administered olanzapine were compared to age-, gender-, and race/ethnicity-matched controls. Single nucleotide polymorphisms and haplotypes associated with case-control status was undertaken to determine the potential role of FMO3 in olanzapine therapeutic response. The relationship between side effects and FMO3 genotype and allele frequencies was also studied. For European Americans, significant differences in individual cases versus controls were observed between FMO3 158 and 257 alleles and genotype frequencies and schizophrenia delusions, hallucinations, and weight gain/increased appetite but this was not observed in a replicated population. For Latin Americans, a significant difference in individual cases versus controls was observed for FMO3 158 and 257 for schizophrenia delusions as well as hallucinations and delusions. Sleepiness and weight gain was associated with allele 308. In African Americans, a comparison of allele frequency and diagnosis showed a significant dependence on allele 158 in individual cases versus controls. FMO3 genotype and allele frequency was not significantly associated with auditory hallucinations or delusions. For Asian Americans, no significant difference in allele or genotype frequency and auditory hallucination and delusions was observed in individual cases versus controls. In female Asian American, allele frequency for FMO3 257 was significantly associated with diagnosis and in males, genotype frequency for FMO3 257 and diagnosis was significantly associated.

Title
Lessons from the Case Studies

Journal
Book

Overview
Methodology used to derive and train the computational model is not available, Yes (PrecisionMed International housed specimens, Quest Diagnostics performed biomarker measurements, Applied Clinical Intelligence performed data analysis). Yes, Unknown.

Year
2012

Authors
Micheel, CM;Nass, SJ;Omenn, GS;

Link
https://www.ncbi.nlm.nih.gov/books/NBK202164/

Abstract
In Chapter 5 [/books/n/nap13297/ch5/], the committee also recommends that FDA communicate the investigational device exemption (IDE) requirements for omics-based tests used in clinical trials. Commercial developers, such as those examined in the case studies, may be more familiar with IDE requirements than academic institutions. In several of the case studies, companies and FDA held a pre-IDE meeting to determine whether an IDE would be required for the company’s test development process. FDA determined that an IDE was not needed for both the AlloMap and Tissue of Origin tests because the test was not directing patient therapy in the studies proposed to assess the test.3 Physicians can now use the test for that purpose, however. Agendia reported that it received an IDE for MammaPrint that helped clarify the process and requirements for the de novo 510(k),4 and Vermillion reported that it received an IDE for OVA1.5 Two ongoing prospective studies (the TAI-LORx and RxPONDER trials) direct patient management on the basis of the Onco_type_ DX Recurrence Score. For both trials, information required for approval of investigational use of Onco_type_ DX in the trial was submitted as part of an investigational new drug application to FDA.6 In the Duke case study, the investigators did consult FDA regarding the need for an IDE (FDA, 2011a [/books/n/nap13297/ch4/#ref_000115]). In 2009, FDA sent a letter to Duke stating that the omics-based tests being studied in the three clinical trials named in the IOM statement of task needed to go through the IDE process (Chan, 2009). In response, the investigators made some changes to the protocol of the studies, and Duke contacted FDA for further clarification about whether an IDE was still required (FDA, 2011a [/books/n/nap13297/ch4/#ref_000115]; Potti, 2009). The Duke Institutional Review Board (IRB) determined that an IDE was not needed when it did not receive a reply from FDA7 (FDA, 2011a [/books/n/nap13297/ch4/#ref_000115]). However, in retrospect, the Duke IRB recognized that an IDE should have been obtained for the omics-based tests because the tests were used to direct patient management in the clinical trials (FDA, 2011a [/books/n/nap13297/ch4/#ref_000115]).

Title
The Relationship of Asthma to Suicidal Thoughts and Behavior in United States Youth

Journal
Thesis

Overview
They enrolled 63 patients into the study after admission to Lund University Hospital after a suicide attempt, and they enrolled 47 healthy control subjects from the Neuropsychiatric and Psychiatric Clinics at the University Hospitals in Malmö and Linköping, Sweden, and Precision Med, San Diego, California. They compared the suicide attempters and healthy control subjects on depressive symptoms evaluated

Year
2018

Authors
Barber, B;;

Link
http://search.proquest.com/openview/a72ec6295e5d13ba66630c61c5583d33/1?pq-origsite=gscholar&cbl=18750&diss=y


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