Protein Microarray Technology Research Articles

Host response during Yersinia pestis infection of human bronchial epithelial cells involves negative regulation of autophagy and suggests a modulation of survival-related and cellular growth pathways.

Yersinia pestis (Yp) causes the re-emerging disease plague, and is classified by the CDC and NIAID as a highest priority (Category A) pathogen. Currently, there is no approved human vaccine available and advances in early diagnostics and effective therapeutics are urgently needed. A deep understanding of the mechanisms of host response to Yp infection can significantly advance these three areas. We employed the Reverse Phase Protein Microarray (RPMA) technology to reveal the dynamic states of either protein level changes or phosphorylation changes associated with kinase-driven signaling pathways during host cell response to Yp infection. RPMA allowed quantitative profiling of changes in the intracellular communication network of human lung epithelial cells at different times post infection and in response to different treatment conditions, which included infection with the virulent Yp strain CO92, infection with a derivative avirulent strain CO92 (Pgm-, Pst-), treatment with heat inactivated CO92, and treatment with LPS. Responses to a total of 111 validated antibodies were profiled, leading to discovery of 12 novel protein hits. The RPMA analysis also identified several protein hits previously reported in the context of Yp infection. Furthermore, the results validated several proteins previously reported in the context of infection with other Yersinia species or implicated for potential relevance through recombinant protein and cell transfection studies. The RPMA results point to strong modulation of survival/apoptosis and cell growth pathways during early host response and also suggest a model of negative regulation of the autophagy pathway. We find significant cytoplasmic localization of p53 and reduced LC3-I to LC3-II conversion in response to Yp infection, consistent with negative regulation of autophagy. These studies allow for a deeper understanding of the pathogenesis mechanisms and the discovery of innovative approaches for prevention, early diagnosis, and treatment of plague.

Protein Microarrays in Proteome-wide Applications

Proteomics aims at understanding of protein status under certain physiological or diseased conditions in a large scale. Protein microarray technology has emerged as a promising approach for a wide variety of applications for scientific and clinical research on a whole-proteome scale. Herein, the aim of this review is to summarize the most recent developments in the applications of protein microarrays for biomarker profiling, enzyme substrate profiling, small molecule profiling, protein-protein interaction profiling, and antibody specificity profiling.

Developing Prospects of Microarray Proteomics

“Proteomics” can be defined as a field of research studying proteins in terms of their function, expression, structure, modification and their interaction in physiological and in pathological states. It is not only a powerful research tools for the proteomics, but also a new means of early diagnosis, prognosis and treatment evaluation in the clinical application. Protein microarray technology possesses some of the greatest potential for providing direct information on protein function and potential drug targets. In the near future, protein chips may allow construction of complete relational databases for metabolic and signal transduction pathways. For example, functional protein microarrays are ideal tools suited for the mapping of biological pathways. The principle, classification, preparation, application, advantages, disadvantages and development prospects of the Microarray Proteomics are introduced. Protein microarray technology possesses some of the greatest potential for providing direct information on protein function and potential drug targets. We strongly believe that functional protein microarrays will soon become an indispensible and invaluable tool in proteomics research and systems biology.

579: Senescence associated secretory phenotype: a signal for parturition?

Senescence of placenta and fetal membranes have been documented in term labor tissues in contrast to term not in labor. This is associated with a unique set of biomarkers called Senescence Associated Secretory Phenotype (SASP). This study documented histologic characteristics of senescence at term and the differences in amniotic fluid SASP markers at term (labor vs. non labor) to further confirm senescence associate changes associated with labor. Amniochorionic membranes (n=7) and amniotic fluids (AF) were collected from normal term deliveries; Group 1: Term labor (TL) and vaginal delivery Group 2: Term not in labor (TNIL). Term labor AF (n=29) samples were collected by transvaginal amniocentesis and TNIL (n=33) by transabdominal amniocentesis. Membranes were subjected to transmission electron microscopy (TEM) to document senescence associated changes. A Fast protein microarray technology was used to analyze 14 SASP biomarkers. Associations between SASP markers transformed to log base2 and Term L were examined using generalized linear models. Spearman correlation was used to determine among the entire cohort if any of the SASP markers were correlated. TEM showed signs of senescence in TL membranes characterized by enlarged cells and organelles, (heterochromatic nuclei, endoplasmic reticulum and mitochondria) than TNIL (Fig 1). Of the 14 markers studied, 7 markers showed significant differences. There were significant associations between GM-CSF, IL-6 and IL-8 (All p<0.01) and increased log odds of having term L. In contrast, there were significant associations between anti-cell death markers, eotaxin (p=0.056), Fas Ligand, osteoprotegerin (both p<0.0001), and adhesion molecule ICAM-1 (P<0.0001) with a decreased log odds of having term L. Senescence is associated with TL and AF SASP markers at TL are uterotonic agents.Domination of anti-cell death markers at TNIL validate that senescence generate noninfectious inflammatory markers that may signal parturition by the fetus.

Optimisation of protein microarray techniques for analysis of the plasma proteome: Minimisation of non-specific binding interactions

Components of the plasma proteome, particularly serum albumin, have been shown to compromise the accuracy of protein microarray technologies through non-specific binding interactions. Optimisation of array conditions is imperative to help address these problems. Here we demonstrate how modifications to array printing conditions and processing methodology can influence the reliability of data output. In particular, we demonstrate that whilst some glycerol is necessary to maintain specific binding signals, it also increases non-specific binding of albumin. Concentrations of 20% glycerol in the printing buffers are therefore recommended. The findings presented here provide opportunities for increased accuracy in plasma protein detection for possible future diagnostic applications.

Evolution of the strategies for screening and identifying human tumor antigens.

The prevailing view is that not only can some of the tumor antigens be used as biosensors for cancers, but also they may indeed be used as targets for immunotherapy. The identification of tumor antigens becomes a vital step in oncology research. Both the humoral immune system and the cellular immune system are activated in response to a tumor antigen in vivo of patients with tumor. Immune effector molecules and cells can be used to screen and identify tumor antigens. Specific T cells, including CD8(+) and CD4(+) T cells, can identify T cell epitopes, and specific antibodies in sera can identify B cell epitopes. The researchers have studied this area for decades. Initially, they explored tumor antigens with the use of 1-D SDS-PAGE and sandwich ELISAs. Since 1990s, CTL screening approach and peptide elution approach had been established. After that, SEREX, SERPA and protein microarray technology have become the mainstream highthroughput strategies for identifying tumor antigens. There are some other approaches, such as combinatorial peptide libraries, representational difference analysis of cDNA and bioinformatics methods. This review's aim is to describe the generation, the theory, the key protocols and the application of some main techniques and provide their benefits and drawbacks.

Site-selective protein immobilization by covalent modification of GST fusion proteins.

The immobilization of functional proteins onto solid supports using affinity tags is an attractive approach in recent development of protein microarray technologies. Among the commonly used fusion protein tags, glutathione S-transferase (GST) proteins have been indispensable tools for protein-protein interaction studies and have extensive applications in recombinant protein purification and reversible protein immobilization. Here, by utilizing pyrimidine-based small-molecule probes with a sulfonyl fluoride reactive group, we report a novel and general approach for site-selective immobilization of Schistosoma japonicum GST (sjGST) fusion proteins through irreversible and specific covalent modification of the tyrosine-111 residue of the sjGST tag. As demonstrated by sjGST-tagged eGFP and sjGST-tagged kinase activity assays, this immobilization approach offers the advantages of high immobilization efficiency and excellent retention of protein structure and activity.

Abstract 869: Evaluate breast cancer diagnostic antibody specificity by using high density protein microarray technology

Abstract Detection sensitivity and target specificity are the two key features for a good diagnostic monoclonal antibody. There are a number of well-established technology platforms to quantitatively measure antibody affinity. However, there is no complete technology solution to evaluate antibody target specificity. Antibody cross-reactivity could lead to false diagnostic reports. Currently, high density protein microarray technology is the best technology platform available for antibody specificity evaluation. Here we will showcase how we utilized protein microarray technology to evaluate target specificity for a couple of commonly used breast cancer IHC diagnostic antibodies. Our data suggested that cross-reactivity is one of the reasons to create off-target staining for breast cancer diagnostics. We will also show how we can utilize OriGene protein microarray chip technology to develop UltraMABs, the Ultra-specific monoclonal antibodies for anatomic pathology application. Citation Format: Donghui Ma, Jian Chen, Wei Fu, Julie McDowell, Kehu Yuan. Evaluate breast cancer diagnostic antibody specificity by using high density protein microarray technology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 869. doi:10.1158/1538-7445.AM2014-869

MERS-CoV: Bridging the Knowledge Gaps.

MERS-CoV: Bridging the Knowledge Gaps.

Microarray Discovery of New OGT Substrates: The Medulloblastoma Oncogene OTX2 Is O-GlcNAcylated

O-GlcNAc transferase (OGT) is a serine/threonine glycosyltransferase that is essential for development and continues to be critically important throughout life. Understanding OGT’s complex biology requires identifying its substrates. Here we demonstrate the utility of a microarray approach for discovering novel OGT substrates. We also report a rapid method to validate OGT substrates that combines in vitro transcription-translation with O-GlcNAc mass tagging. Among the validated new OGT targets is Orthodenticle homeobox 2 (OTX2), a transcription factor critical for brain development, which is primarily expressed only during early embryogenesis and in medulloblastomas, where it functions as an oncogene. We show that endogenous OTX2 from a medulloblastoma cell line is O-GlcNAcylated at several sites. Our results demonstrate that protein microarray technology, combined with the target validation strategy we report, is useful for identifying biologically important OGT substrates, including substrates not present in most tissue types or cell lines.

Protein Microarrays for Studies of Drug Mechanisms and Biomarker Discovery in the Era of Systems Biology

Protein microarray technology is one of the most powerful tools presently available for proteomic studies. Numerous types of protein microarrays have been widely and successfully applied for both basic biological studies and clinical researches, including those designed to characterize protein-protein, protein-nucleic acid, protein-drug/small molecule and antibody-antigen interactions. In the past decade, a variety of protein microarrays have been developed, including those spotted with whole proteomes, smaller peptides, antibodies, and lectins. Featured as high-throughput, miniaturized, and capable of parallel analysis, the power of protein microarrays has already been demonstrated many times in both basic research and clinical applications. In this review, we have summarized the latest developments in the production and application of protein microarrays. We discuss several of the most important applications of protein microarray, ranging from proteome microarrays for large scale identification of protein-protein interactions to lectin microarrays for live cell surface glycan profiling, with special emphasis on their use in studies of drug mechanisms and biomarker discovery. Already with tremendous success, we envision protein microarrays will become an indispensible tool for any systems-wide studies, fostering the integration of basic research observations to clinically useful applications.

Biomarker Analysis with Grating Coupled Surface Plasmon Coupled Fluorescence

One of the greatest hurdles in protein microarray technology has been the limited sensitivity. However, the strategy of amplifying the SPR signal with fluorescence improves sensitivity. Herein, we report a simple and efficient method, Grating Coupled Surface Plasmon Coupled Fluorescence (GCSPCF) with a peptide microarray to screen sera of human subjects and a mouse strain with autistic-like behavior for quantification of the epitope specificities of their circulating antibodies. The GCSPCF peptide microarray is a novel assay that facilitates high-throughput screening of antibody epitope specificities or binding of interacting analytes with a small quantity (<50 μl) of sample. This study utilized over 600 peptides with the eventual goal of being able to identify antibodies that could be used as biomarkers of particular disorders. The GCSPCF technology was able to detect antibodies to the tripeptide glutathione; it was also capable of sequential screening antibodies of different immunoglobulin isotypes (IgG1, IgG4 and IgE) to the same peptide. The GCSPCF broad-based screening approach was able to begin the identification of Original Research Article Mendoza et al.; JABB, Article no. JABB.2014.001 2 epitope-specific antibody binding patterns in an effort to develop a predictive signature that could be further developed for clinical trials. In addition to evaluation of antibody specificities with hundreds of spotted peptides at multiple regions of interest (ROI), the addition of spotted antibodies could provide an even more complete biomarker profile with capture of analytes, such as stress proteins and cytokines. Differences between a mouse strain with normal behaviors (C57BL/6J) and a strain with autistic-like behaviors (BTBR T + tf/J) as well as healthy and autistic participants are used for comparative analyses.

Protein microarray technology: how far off is routine diagnostics?

The nature of protein microarray platforms is favorable for multiplexing, leading to the development of tools for personalised medicine and highly precise diagnostics. However, to date, only a limited number of protein microarrays are available in the in vitro diagnostics (IVD) market. This review article will focus on the following operational challenges that are crucial for the use of microarrays in clinical settings: (1) probe printing and quality control; (2) procurement of bio-reagents and antibody cross-reactivity; (3) mass transport limitations and assay automation; (4) calibration and quantification. A selection of microarray assays applicable to IVD and a summary of the diagnostic products currently available on the market are provided.

Development and validation of protein microarray technology for simultaneous inflammatory mediator detection in human sera.

Biomarkers, including cytokines, can help in the diagnosis, prognosis, and prediction of treatment response across a wide range of disease settings. Consequently, the recent emergence of protein microarray technology, which is able to quantify a range of inflammatory mediators in a large number of samples simultaneously, has become highly desirable. However, the cost of commercial systems remains somewhat prohibitive. Here we show the development, validation, and implementation of an in-house microarray platform which enables the simultaneous quantitative analysis of multiple protein biomarkers. The accuracy and precision of the in-house microarray system were investigated according to the Food and Drug Administration (FDA) guidelines for pharmacokinetic assay validation. The assay fell within these limits for all but the very low-abundant cytokines, such as interleukin- (IL-) 10. Additionally, there were no significant differences between cytokine detection using our microarray system and the “gold standard” ELISA format. Crucially, future biomarker detection need not be limited to the 16 cytokines shown here but could be expanded as required. In conclusion, we detail a bespoke protein microarray system, utilizing well-validated ELISA reagents, that allows accurate, precise, and reproducible multiplexed biomarker quantification, comparable with commercial ELISA, and allowing customization beyond that of similar commercial microarrays.

Abstract P6-04-01: Protein pathway activation mapping of LCM tumor epithelium from I-SPY 1 TRIAL surgical specimens reveals activation of receptor tyrosine kinase drug target signaling networks in the non-responding patient cohort

Abstract Background: Identification of alternative therapeutic strategies is critically needed for patients who do not achieve complete clinical response in the neoadjuvant setting, and in particular, from that cohort with the most aggressive disease (shortest relapse-free survival (RFS)). The I-SPY 1 TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides longitudinal biopsy specimens and extensive molecular and clinical/pathological characterization of non-responding patients. Methods: Tumor epithelium was procured by Laser Capture Microdissection (LCM) from frozen surgical specimens of 27 patients who did not achieve complete pathological response in the I-SPY1 TRIAL. Reverse Phase Protein Microarray (RPPA) technology was used to measure the levels of 120 key signaling proteins including drug targets for Phase I-III and FDA-approved therapeutics. Cox proportional hazard univariate and multivariate analyses were used to evaluate RFS correlates. Significance threshold was p = &amp;lt;0.05. Results: Univariate analysis of pathway activation revealed a number of actionable/druggable targets, particularly receptor tyrosine kinase (RTK) activation, where increased phosphorylation of ALK (Y1586), EGFR (Y1173), ERBB3 (Y1289), cABL (T735), and FAK (Y576/Y577) was significantly associated with aggressive disease (shorter RFS). Higher levels of SRC (Y527) inhibitory phosphorylation were also significantly associated with early recurrence. Coordinate activation of downstream targets of these RTKs, such as RAS-GRF S916, SHC (Y317), MEK (S217/221) and the Aurora kinases were also associated with early relapse in this study set. In multivariate analyses controlling for receptor subtype (HR+/HER2-, triple-negative (TN), HER2+), the associations between shorter RFS and RTK pathway activation including EGFR, ERBB3, cABL and FAK activation and SRC inhibition, as well as RAS-GRF and SHC phosphorylation remained significant. Conclusions: This unique functional protein pathway/proteomic analysis of LCM-isolated tumor epithelium by RPPA in surgical specimens taken from patients who did not achieve complete pathological response provides evidence for a significant number of druggable RTK pathway targets in patients with residual disease who are most at risk to die from breast cancer. Activation of ALK, EGFR, ERBB3, ABL, FAK, along with downstream RAS-ERK and Aurora kinase signaling activation in this cohort provide new insights into tumor progression along with distinct, actionable information on new therapeutic modalities to test in patients with progressing disease. Identification of new therapeutic strategies for patients who progress or recur early after neoadjuvant therapy is critical and, if validated in larger independent cohorts, could lead to rationalized personalized therapy-based trials. This strategy is being utilized in the I SPY 2 TRIAL as we introduce pathway-directed therapeutics in the neoadjuvant setting and are employing the RPPA platform to determine whether these agents are impacting their intended pathway. This work was partially supported by NIH grants CA31946 and CA33601. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-01.

Optimization of the measurement of outdoor airborne allergens using a protein microarrays platform

Increased knowledge on allergenic molecules in the environmental air helps in the information on environmental air quality and in the prevention and treatment of allergies. The aim of this study is to develop and validate a new methodology for the simultaneous detection and quantification of several airborne allergens using protein microarray technology, which has been created for the clinical detection of allergens. The immunological method was performed with Immuno Solid-phase Allergen Chip (ISAC) inhibition assay. Reagents for the validation studies include the following: (1) three sera from patients allergic to grass pollen each with different IgE levels as the detection reagents, (2) recombinant Phl p 1 major allergen as the inhibitor for the inhibition assays, (3) “natural” Phl p 1 released by Phleum pratense (timothy grass) pollen grains as the “biologically” relevant aeroallergen and (4) samples of airborne pollens collected by a Multi-vial Cyclone Sampler for comparison of levels of pollen detection versus the protein allergen detection by the microarray assay. The results obtained showed that ISAC inhibition is a sensitive technique able to detect 2.1 pg/mL of Phl p 1 and the allergens released from 1 grain of natural pollen. Also, the airborne allergen samples analyzed showed a good correlation with the concentration of grass pollen in the air. The use of ISAC inhibition will greatly improve future airborne simultaneous allergen quantification, becoming a valuable option in air quality control.

Alloimmunization To HLA Class I Antigens But Not H-Y Antigens Is Associated With Transfusion In Children With Sickle Cell Disease

IntroductionPatients with sickle cell disease (SCD) are at high risk for graft rejection after hematopoietic stem cell transplant (HSCT) especially when using reduced intensity conditioning (RIC). Alloimmunization from red blood cell (RBC) transfusions, critical to the management of SCD, may predispose these patients to HSCT rejection. Alloimmunization to major or minor histocompatibility antigens (mHA) may occur after RBC transfusion due to residual white blood cells or platelets present in RBC units even after leukoreduction. In mice, despite extensive leukoreduction, RBC transfusions caused alloimmunization and subsequent HSCT rejection after RIC.We conducted a cross-sectional study to determine if RBC transfusions are associated with alloantibody formation to human leukocyte antigens (HLA) and mHA, the target of the host versus graft rejection in HLA-identical transplantation. For mHA, we assessed immunity to H-Y antigens, mHA encoded on the Y chromosome. We have previously demonstrated the importance of H-Y antibodies in a variety of settings including chronic graft versus host disease (in male recipients of female grafts) and rejection of renal transplants (in female recipients of male grafts). MethodsWe enrolled 114 nulliparous pediatric SCD (SS or Sβ0) patients: 58 females on chronic transfusion, 32 males on chronic transfusion, and 24 females who had never been transfused. Serum was evaluated for HLA antibodies using the FlowPRA® (panel reactive antibody) screening test which detects IgG antibodies to the majority of HLA class I and II antigens. Serum was also tested for IgG antibodies against 5 H-Y antigens (DDX3Y [DBY], EIF1AY, RPS4Y1, UTY, ZFY) using protein microarray technology with each H-Y-seropositive threshold determined as a microarray mean fluorescence intensity (MFI) > the median MFI + 2.5 quartiles measured in 60 adult healthy males. ResultsChronically transfused patients had a higher prevalence of HLA class I antibodies than never transfused patients (33.3% vs. 12.5%, p=0.074). This increased prevalence of HLA class I antibodies in the chronic transfusion group was significant when examining the number of patients who tested positive to more than 25% of the panel (17.8% vs. 0%, p=0.022). Few patients had detectable HLA class II antibodies with no significant differences between chronically transfused and non-transfused patients.Few SCD patients had detectable H-Y alloantibodies with no significant differences in chronically transfused females, chronically transfused males, and non-transfused females.Chronic Transfusion (n=90)Never Transfused (n=24)PHLA class I PRA >3%30 (33.3%)3 (12.5%)0.074HLA class I PRA >25%16 (17.8%)0 (0%)0.022*HLA class II PRA >3%6 (6.7%)3 (12.5%)0.39HLA class II PRA >25%3 (3.3%)1 (4.2%)1.00HLA class I or II PRA >3%33 (36.7%)6 (25.0%)0.34H-Y antigenChronic Transfusion Female (n=58)Chronic Transfusion Male (n=32)Never Transfused Female (n=24)DBY1 (1.7%)0 (0%)0 (0%)EIF1AY0 (0%)0 (0%)0 (0%)RPS4Y12 (3.4%)0 (0%)0 (0%)UTY2 (3.4%)2 (6.3%)1 (4.2%)ZFY1 (1.7%)0 (0%)0 (0%)Any H-Y4 (6.9%) [p= 0.90]2 (6.3%)1 (4.2%) ConclusionsLeukocyte-reduced RBC transfusions appear to engender immunization to class I HLA but not class II HLA or H-Y antigens in pediatric SCD patients. Since hematopoietic progenitor cells express class I HLA, transfusion-related immunity to these antigens could pose a barrier to engraftment in HLA-disparate transplants. Since these antigens are also expressed on platelets, class I HLA antibodies in SCD could have important implications for platelet transfusion support during HSCT. While RBC transfusion does not appear to be an adequate stimulus for H-Y alloantibody formation, it is possible that exposure to mHA during transfusion could cause a cellular rather than a humoral immune response that may contribute to future HSCT graft rejection. Our result that few (6%) nulliparous female children with SCD had detectable H-Y alloantibodies is also a significant finding considering that 19-41% of healthy adult females had antibodies to at least one H-Y antigen in previous studies. Finally our finding that 25% of never transfused children with SCD had a positive HLA PRA is unexpected for an “unsensitized” group; its significance is being further investigated. Disclosures:No relevant conflicts of interest to declare.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV): a perpetual challenge.

Ann Saudi Med 2013 September-October www.annsaudimed.net 427 The emergence of a novel human coronavirus recently renamed the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the Arabian Peninsula has created global alarm because it is the causative agent of a severe and frequently fatal acute respiratory illness (SARI) resembling the illness caused by severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV).1-4 The case fatality rate (CFR) in patients infected with MERS-CoV is high—estimated at 43% in 147 patients reported so far by World Health Organization (WHO).3 This rate is higher than that of SARS—estimated at 15%, and is strongly ageand sex-dependent.4 Although the source of virus in patients with sporadic infection remains unknown, it appears likely to be some species of animal.4,5 Clear evidence of limited human-to-human transmission of MERS-CoV has now been documented in several case clusters, including particularly family members and patients in health care facilities,6-8 but all such clusters have, at least thus far, been limited in extent. However, a real concern persists that the virus will adapt to interhuman transmission and switch from an aborted epidemic to a pandemic similar to the SARS-CoV epidemic in 2003–2004. MERS-CoV is transmitted through droplets and contact. In the case of invasive respiratory procedures, MERS-CoV is transmitted through airborne route.2 Early diagnosis and strict implementation of the core components for infection prevention and control programs are crucial for preventing epidemic amplification.2 In the absence of an effective vaccine and a specific antiviral treatment, there is an urgent need to rapidly identify potential therapeutics.

A Microliter-Scale High-throughput Screening System with Quantum-Dot Nanoprobes for Amyloid-β Aggregation Inhibitors

The aggregation of amyloid β protein (Aβ) is a key step in the pathogenesis of Alzheimer’s disease (AD), and therefore inhibitory substances for Aβ aggregation may have preventive and/or therapeutic potential for AD. Here we report a novel microliter-scale high-throughput screening system for Aβ aggregation inhibitors based on fluorescence microscopy-imaging technology with quantum-dot Nanoprobes. This screening system could be analyzed with a 5-µl sample volume when a 1536-well plate was used, and the inhibitory activity could be estimated as half-maximal effective concentrations (EC50). We attempted to comprehensively screen Aβ aggregation inhibitors from 52 spices using this system to assess whether this novel screening system is actually useful for screening inhibitors. Screening results indicate that approximately 90% of the ethanolic extracts from the spices showed inhibitory activity for Aβ aggregation. Interestingly, spices belonging to the Lamiaceae, the mint family, showed significantly higher activity than the average of tested spices. Furthermore, we tried to isolate the main inhibitory compound from Satureja hortensis , summer savory, a member of the Lamiaceae, using this system, and revealed that the main active compound was rosmarinic acid. These results demonstrate that this novel microliter-scale high-throughput screening system could be applied to the actual screening of Aβ aggregation inhibitors. Since this system can analyze at a microscopic scale, it is likely that further minimization of the system would easily be possible such as protein microarray technology.

Psychiatric patient stratification using biosignatures based on cerebrospinal fluid protein expression clusters

Psychiatric disorders are caused by perturbed molecular pathways that affect brain circuitries. The identification of specific biosignatures that are the result of altered pathway activities in major depression, bipolar disorder and schizophrenia can contribute to a better understanding of disease etiology and aid in the implementation of diagnostic assays.In the present study we identified disease-specific protein biosignatures in cerebrospinal fluid of depressed (n: 36), bipolar (n: 27) and schizophrenic (n: 35) patients using the Reverse Phase Protein Microarray technology. These biosignatures were able to stratify patient groups in an objective manner according to cerebrospinal fluid protein expression patterns. Correct classification rates were over 90%. At the same time several protein sets that play a role in neuronal growth, proliferation and differentiation (NEGR1, NPDC1), neurotransmission (SEZ6) and protection from oxidative damage (GPX3) were able to distinguish diseased from healthy individuals (n: 35) indicating a molecular signature overlap for the different psychiatric phenotypes. Our study is a first step toward implementing a psychiatric patient stratification system based on molecular biosignatures. Protein signatures may eventually be of use as specific and sensitive biomarkers in clinical trials not only for patient diagnostic and subgroup stratification but also to follow treatment response.