Human Protein Microarray Research Articles

Prefoldin 5 and Anti-prefoldin 5 Antibodies as Biomarkers for Uveitis in Ankylosing Spondylitis.

Objective: Uveitis is the most common extra-articular manifestation of ankylosing spondylitis (AS), for which no diagnostic biomarkers have been identified. This study was conducted to identify biomarker for uveitis in AS.Methods: To identify autoantibodies associated with uveitis in AS, we performed human protein microarray analysis using sera derived from various autoimmune diseases and ELISA analysis of sera derived from AS and rheumatoid arthritis patients. In the curdlan-induced SKG mice model, ophthalmic examination was performed at week 8 post-immunization and histologic examination of the ocular lesions performed at week 16 post-immunization. Serum levels of target antibodies were assessed at various time-points. To evaluate the functional role of specific autoantibodies, an in vitro apoptosis assay using ARPE-19 cells was performed.Results: Reactivity against prefoldin subunit 5 (PFDN5) was identified in AS with uveitis. Levels of anti-PFDN5 antibodies and PFDN5 in sera from AS with uveitis patients were significantly higher than those in AS without uveitis. At week 8, half of curdlan-treated SKG mice developed anterior uveitis, while all of them developed histologically confirmed uveitis at week 16. The levels of anti-PFDN5 antibodies increased over time in the sera of curdlan-treated SKG mice along with increased expression of PFDN5 and apoptosis in the ocular lesions. Knockdown of PFDN5 in ARPE19 cells resulted in increased apoptosis, suggesting a protective role of PFDN5 against cell death in uveitis.Conclusion: AS patients with uveitis have increased levels of anti-PFDN5 antibodies, and our findings suggest that anti-PFDN5 antibodies could provide a biomarker for uveitis in AS.

Elevated levels of autoantibodies against EXD2 and PHAX in the sera of patients with chronic thromboembolic pulmonary hypertension

While circulating autoantibodies have been detected in patients with several cardiovascular diseases, such studies have not been performed for chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH). Here we investigated the production of certain auto-antibodies in CTEPH patients. Initial screening was performed in 5 CTEPH patients and 5 healthy donors (HDs) using a ProtoArray Human Protein Microarray v5.1 containing 9,375 human proteins, and we selected 34 antigens recognized by IgG antibodies more strongly in the sera of CTEPH patients than in the sera of HDs. In subsequent second/third analyses, we validated the auto-antibody level using amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in 96 CTEPH patients and 96 HDs as follows: At the second screening, we used 63 crude peptides derived from those selected 34 antigens and found that the serum levels of autoantibodies for 4 peptides seemed higher in CTEPH patients than in HDs. In third analysis, we used the purified peptides of those selected in second screening and found that serum antibodies against peptides derived from exonuclease 3'-5' domain-containing 2 (EXD2) and phosphorylated adaptor for RNA export (PHAX) were significantly higher in CTEPH patients than in HDs. The serum antibody levels to these antigens were also elevated in PAH patients. The titers against EXD2 peptide decreased after surgical treatment in CTEPH patients. These autoantibodies may be useful as biomarkers of CTEPH and PAH, and further investigations may provide novel insight into the etiology.

Identification of Proteins Interacting with PCSK9 Using a Protoarray Human Protein Microarray.

Proprotein convertase subtilisin-kexin 9 (PCSK9) appears to be involved in multiple processes. A ProtoArray Human Protein Microarray was used to identify proteins interacting with biotinylated PCSK9. Fifteen novel proteins interacting with PCSK9 were identified using this technique. Only two of these proteins, sterol carrier protein 2 and hepatoma-derived growth factor, related protein 3, have known functions. The identification of proteins that could affect the expression/function of PCSK9 is of great interest due to potential implications in personalized medicine for hypercholesterolemic patients.

Circ-UBR5: An exonic circular RNA and novel small nuclear RNA involved in RNA splicing

Circular RNAs (circRNAs) are class of non-coding RNAs formed by back-splicing events as loops, and could be found in all types of organisms. They play important and diverse roles in cell development, growth, and tumorigenesis, but functions of the majority of circRNAs remain enigmatic. Particularly functional phenotypes of great majority of circRNAs are not obvious. Here we randomly selected a circRNA circ-UBR5, which has no obvious functional phenotype in non-small cell lung cancer (NSCLC) cells from our previous research findings, to explore its potential function in cells. Differential expression of circ-UBR5 was detected in paired samples of tumorous tissues and adjacent nontumorous tissues from 59 patients with NSCLC by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Results showed circ-UBR5 expression was significantly downregulated in NSCLC tissues (p < 0.001) and was correlated with tumor differentiation (p = 0.00126), suggesting circ-UBR5 might serve as an index of NSCLC differentiation. Our findings indicated circ-UBR5 could bind splicing regulatory factor QKI, KH domain containing RNA binding (QKI) and NOVA alternative splicing regulator 1 (NOVA1) and U1 small nuclear RNA (snRNA) in the nucleus, revealing circ-UBR5 might be a novel snRNA involved in RNA splicing regulatory process. Moreover, we first presented a highly efficient strategy for finding specific circRNA binding proteins using Human Protein Microarray (Huprot™ Protoarray).

S12. THE ‘AUTOANTIBODYOME’ IN PSYCHOSIS: A PILOT STUDY AND BLUEPRINT FOR BIOMARKER DISCOVERY

BackgroundRecent studies seeking to describe the prevalence and significance of autoantibodies in psychotic disorders can be characterized as ‘top-down’ in theoretical approach; that is, autoantibodies to specific (usually CNS) antigens are sought based on a) the known function of the antigen (e.g. NMDAR) and its putative role in psychosis or b) the clinical observation that these autoantibodies can cause psychosis as part of a more complex neuropsychiatric presentation (e.g. autoimmune encephalitis). No candidate autoantibodies with a clear diagnostic or prognostic role have been definitively established.We sought to take an alternative, ‘bottom-up’ approach to the significance of autoantibodies in psychosis that remains agnostic as to the potential significance of any one antigen. Every individual harbours autoantibodies directed against many thousands of self-antigens and the vast majority are not disease-causing. Indeed production of autoantibodies may represent a physiological, antigen-specific ‘debris-clearing’ response to tissue destruction or damage. It follows that the autoantibody profile of any individual reflects any pathological process that is ongoing in that individual and can thus serve as a ‘readout’ of the disease state in question.MethodsSera from 20 patients with a first episode of psychosis (FEP) (males: n=16; mean age: 29.35 s.d.: 7.07) from the Genetics and Psychosis (GAP) study and 20 matched controls were analysed, using Invitrogen’s ProtoArray v5.1 Human Protein Microarrays, for the presence of IgG to 9486 unique human protein antigens which had been expressed as GST fusion proteins in insect cells, purified and spotted onto slides. Following application of a fluorescent secondary IgG, reactivity patterns were automatically read using a fluorescence scanner. Samples were split into testing and training sets, and the top 50 most differentially expressed and differentially depleted antibodies were then chosen as biomarkers.ResultsThe top 50 expressed biomarkers from the training set correctly identified 100% of psychosis subjects from the testing set, and 80% of healthy controls (OOB estimate of error rate 10%). When training and testing sets were swapped, biomarker overlap was 46% and 90% of psychosis subjects and 90% of controls were correctly identified (OOB estimate of error rate 10%). The top 50 depleted biomarkers from the training set correctly identified 90% of psychosis subjects from the testing set, and 90% of healthy controls (OOB estimate of error rate 10%). When training and testing sets were swapped, depletion biomarker overlap was 2% and 70% of psychosis subjects and 40% of controls were correctly identified (OOB estimate of error rate 45%).DiscussionThe autoantibodyome in FEP differs from that of healthy individuals. In this novel pilot study, a panel of 50 differentially expressed autoantibodies allowed confident discrimination between patients and controls, potentially paving the way for development of antibody-based diagnostics for psychosis using a simple blood test and fewer autoantibodies. Depletion biomarkers, thought to represent antibodies selectively depleted from the blood due to target binding in tissues, had less replicability and utility than expression biomarkers, which may offer insights into the active role of the adaptive immune system in psychosis. Further work will attempt to validate this approach in larger samples, using psychiatric disease controls. This autoantibodyomic approach may also show promise for the identification of predictive and prognostic biomarkers in psychotic disorders.

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.

Identification of the Thioredoxin-Like 2 Autoantibody as a Specific Biomarker for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has a higher risk of death within 5 years of being diagnosed than the other forms of breast cancer. It is the second leading cause of death due to cancer among women. Currently, however, no diagnostic blood-based biomarker exists to identify the early stages of TNBC. To address this point, we utilized a human protein microarray system to identify serum autoantibodies that showed different expression patterns between TNBC and normal serum samples, and identified five autoantibodies showing TNBC-specific expression. Among them, we selected the thioredoxin-like 2 (TXNL2) autoantibody and evaluated its diagnostic relevance by dot blot analysis with the recombinant TXNL2 protein. We demonstrated that the TXNL2 autoantibody showed 2- to 6-fold higher expression in TNBC samples than in normal samples suggesting that serum TXNL2 autoantibodies are potential biomarkers for TNBC.

18 - Myristoylated Methionine Sulfoxide Reductase A Interacts with the Lipid-Binding Protein

Reactive oxygen species (ROS) are produced by a variety of cellular processes including metabolic activity, mitochondria, cellular signaling, and oncogene activity. Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme that can reduce methionine sulfoxide (MetO) in proteins back to methionine (Met). This enzyme plays a protective role in cells against oxidative damage. Although many proteins with MetO are in vitro substrates of MsrA, no in vivo substrates have been confidently identified. In vitro, MsrA can also function as an oxidase that converts methionine to methionine sulfoxide, but it is also not known if it does so in vivo. The cytosolic form of MsrA is myristoylated, and overexpression of the myristoylated form protects the heart from ischemia-reperfusion injury while overexpression of the non-myristoylated form does not. Here we report a novel binding partner of MsrA, STARD3, identified by use of a human protein microarray. ProtoArray Human Protein Microarray v5.0 contains 9,483 human proteins from multiple protein classes along with 2,016 controls. STARD3 was scored as an interacting protein with high probability. STARD3 contains the star-related lipid transfer (START) domain, and proteins with that domain are implicated in lipid and sterol metabolism. STARD3 is known to bind cholesterol and transport it from the endoplasmic reticulum to the late endosome. We overexpressed both myristoylated MsrA and STARD3 in HEK293 cells and immunoprecipitated cell homogenates with anti-STARD3 antibody. We demonstrated that myristoylated MsrA was co-immunoprecipitated with STARD3, but non-myristoylated form wasn’t. This indicates that myristoylation is required for the interaction. Met307 of STARD3 is known to be essential for lipid binding, and we found that it is also required for the interaction of STARD3 and myristoylated MsrA. Finally, we showed by confocal microscopy that myristoylated MsrA was colocalized with STARD3 at late endosomes/lysosomes. We conclude that myristoylated MsrA binds to STARD3 on the cytoplasmic surface of late endosomes.

UTILITY OF DISEASE-LINKED DEPLETION OF AUTOANTIBODIES AS BIOMARKERS FOR EARLY, BLOOD-BASED DETECTION AND MONITORING OF ALZHEIMER’S AND OTHER NEURODEGENERATIVE DISEASES

We have shown that autoantibodies are ubiquitous in human blood and that specific changes in autoantibody profiles occur that reflect the presence of disease. Autoantibody production is presumably increased in response to the appearance of disease-specific tissue debris in the blood. For AD, our previous studies have exploited this phenomenon and developed panels of autoantibodies that can be used to distinguish healthy control subjects from those with early-stage AD pathology at mild cognitive impairment (MCI), and at more advanced mild-moderate AD with high overall accuracy. Here, we show that early AD pathology can also be linked to the selective depletion of specific brain-reactive autoantibodies, which also has utility for AD detection at both the MCI and mild-moderate AD stages, potentially opening the door to preclinical detection. Sera from over 300 subjects, including 50 MCI (with low CSF Aβ42) and 50 mild-moderate AD subjects, as well as those with Parkinson's disease, and multiple sclerosis were screened with human protein microarrays containing 9,486 potential antigen targets to identify autoantibodies that are selectively depleted due to the presence of disease. For each disease, 50 autoantibodies with depleted titers relative to controls were identified and evaluated using Random Forest and Receiver Operating Characteristic (ROC) curves for their ability to distinguish disease subjects from age- and sex-matched controls, and from individuals with other neurodegenerative diseases. Results demonstrate that detection of depleted autoantibodies in serum can readily differentiate disease subjects from age- and sex-matched controls with high overall accuracy, sensitivity, and specificity. Depletion biomarkers were able to distinguish subjects with early AD pathology at MCI from controls with 90.0% overall accuracy in the Testing Set (sensitivity=88.0%; specificity=92.0%). Comparable accuracies were obtained for identification of subjects with mild-moderate AD, early-stage Parkinson's disease and multiple sclerosis. Results demonstrate that monitoring the disease-linked depletion of brain-reactive autoantibodies in blood has utility for early detection and staging of AD and other neurodegenerative diseases. Since autoantibody depletion may reflect neuropathological events prior to significant neurodegeneration, we hypothesize that depleted autoantibody biomarkers may be particularly useful for preclinical disease detection, perhaps years before the onset of symptoms.

Autoantibodies as diagnostic biomarkers for the detection and subtyping of multiple sclerosis

The goal of this preliminary proof-of-concept study was to use human protein microarrays to identify blood-based autoantibody biomarkers capable of diagnosing multiple sclerosis (MS). Using sera from 112 subjects, including 51 MS subjects, autoantibody biomarkers effectively differentiated MS subjects from age- and gender-matched normal and breast cancer controls with 95.0% and 100% overall accuracy, but not from subjects with Parkinson's disease. Autoantibody biomarkers were also useful in distinguishing subjects with the relapsing-remitting form of MS from those with the secondary progressive subtype. These results demonstrate that autoantibodies can be used as noninvasive blood-based biomarkers for the detection and subtyping of MS.

221 - STARD3 Interacts with Myristoylated Methionine Sulfoxide Reductase A

Methionine sulfoxide reductase A (MsrA) is a component of the oxidative defense system that reduces methionine sulfoxide (MetO) in proteins back to methionine. Although many proteins with MetO are in vitro substrates of MsrA, no in vivo substrates have been confidently identified. In vitro, MsrA can also function as an oxidase that converts methionine to methionine sulfoxide, but it is also not known if it does so in vivo. The cytosolic form of MsrA is myristoylated, and overexpression of the myristoylated form protects the heart from ischemia-reperfusion injury while overexpression of the non-myristoylated form does not. Previous efforts in our laboratory to identify proteins that interact in vivo with MsrA had not been successful. We now report use of a human protein microarray to identify a protein that binds to MsrA. The ProtoArray Human Protein Microarray v5.0 contains 9,483 human proteins from multiple protein classes along with 2,016 controls. Each protein was expressed as an N-terminal GST- tagged protein and printed in duplicate on the nitrocellulose-coated slide glass. We incubated the slide with biotinylated human MsrA and interrogated the array with streptavidin tagged with Alexa Fluor 647. STARD3 emerged as an interacting protein with a high probability score. We co-expressed MsrA and STARD3 in HEK-293 cells, immunoprecipitated from a homogenate with anti-STARD3, and confirmed that MsrA was co-immunoprecipitated. When non-myristoylated MsrA was co-expressed, no interaction with STARD3 was detected. STARD3 contains the star-related lipid transfer (START) domain, and proteins with that domain have been implicated in lipid and steroid metabolism. STARD3 is known to bind cholesterol and transport it to the late endosome. We conclude that it also binds to myristoylated MsrA, and we are now investigating the physiological significance of the interaction.

Selectivity Determination of a Small Molecule Chemical Probe Using Protein Microarray and Affinity Capture Techniques.

Small molecule selectivity is an essential component of candidate drug selection and target validation. New technologies are required to better understand off-target effects, with particular emphasis needed on broad protein profiling. Here, we describe the use of a tritiated chemical probe and a 9000 human protein microarray to discern the binding selectivity of an inhibitor of the mRNA decapping scavenger enzyme DcpS. An immobilized m7GTP resin was also used to assess the selectivity of a DcpS inhibitor against mRNA cap-associated proteins in whole cell extracts. These studies confirm the exquisite selectivity of diaminoquinazoline DcpS inhibitors, and highlight the utility of relatively simple protein microarray and affinity enrichment technologies in drug discovery and chemical biology.

FGFR3 promotes angiogenesis-dependent metastasis of hepatocellular carcinoma via facilitating MCP-1-mediated vascular formation.

The biological role of fibroblast growth factor receptor 3 (FGFR3) in tumor angiogenesis of hepatocellular carcinoma (HCC) has not been discussed before. Our previous work had indicated FGFR3 was overexpressed in HCC, and silencing FGFR3 in Hu7 cells could regulate tumorigenesis via down-regulating the phosphorylation level of key members of classic signaling pathways including ERK and AKT. In the present work, we explored the role of FGFR3 in angiogenesis-dependent metastasis by using SMMC-7721 and QGY-7703 stable cell lines. Our results indicated FGFR3 could regulate in vitro cell migration ability and in vivo lung metastasis ability of HCC, which was in accordance with increased angiogenesis ability in vitro and in vivo. Using the supernatant from SMMC-7721/FGFR3 cells, we conducted a human angiogenesis protein microarray including 43 angiogenesis factors and found that FGFR3 modulated angiogenesis and metastasis of HCC mainly by promoting the protein level of monocyte chemotactic protein 1 (MCP-1). Silencing FGFR3 by short hairpin RNA (shRNA) could reduce MCP-1 level in lysates and supernatant of QGY-7703 cells and SMMC-7721 cells. Silencing MCP-1 in QGY-7703 or SMMC-7721 cells could induce similar phenotypes compared with silencing FGFR3. Our results suggested FGFR3 promoted metastasis potential of HCC, at least partially if not all, via facilitating MCP-1-mediated angiogenesis, in addition to previously found cell growth and metastasis. MCP-1, a key medium between HCC cells and HUVECs, might be a novel anti-vascular target in HCC.

Antitumor effects of calgranulin B internalized in human colon cancer cells.

Calgranulin B is a small, calcium-binding protein expressed in neutrophils that is secreted into the tumor microenvironment in cancer cases. We previously showed that calgranulin B levels are increased in the stools of colorectal cancer patients. In patient tumor tissues, calgranulin B protein levels correlated with the presence of stromal inflammatory cells surrounding tumor cells, and calgranulin B promoter methylation was observed in both paired human tissues and colon cancer cell lines. Cell lines did not express calgranulin B, but in vitro studies showed that colon cancer cells internalized extracellular calgranulin B, while other types of cancer cells did not. Calgranulin B internalization led to reduced cell proliferation and increased apoptotic cell death. AKT and ERK signals were also increased after calgranulin B treatment, as were p53, β-catenin, E-cadherin and cleaved caspase-3 levels. Additionally, a human protein microarray identified aurora A kinase as a calgranulin B binding partner, and binding inhibited aurora A kinase activity in a dose-dependent manner. Our findings demonstrate the antitumor effects of calgranulin B in the inflammatory microenvironment and suggest that calgranulin B could be potentially efficacious in the treatment of colon cancer.

Detection of Alzheimer's disease at mild cognitive impairment and disease progression using autoantibodies as blood-based biomarkers

IntroductionThere is an urgent need to identify biomarkers that can accurately detect and diagnose Alzheimer's disease (AD). Autoantibodies are abundant and ubiquitous in human sera and have been previously demonstrated as disease-specific biomarkers capable of accurately diagnosing mild-moderate stages of AD and Parkinson's disease. MethodsSera from 236 subjects, including 50 mild cognitive impairment (MCI) subjects with confirmed low CSF Aβ42 levels, were screened with human protein microarrays to identify potential biomarkers for MCI. Autoantibody biomarker performance was evaluated using Random Forest and Receiver Operating Characteristic curves. ResultsAutoantibody biomarkers can differentiate MCI patients from age-matched and gender-matched controls with an overall accuracy, sensitivity, and specificity of 100.0%. They were also capable of differentiating MCI patients from those with mild-moderate AD and other neurologic and non-neurologic controls with high accuracy. DiscussionAutoantibodies can be used as noninvasive and effective blood-based biomarkers for early diagnosis and staging of AD.

Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity.

The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer.

Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification. To explore their role in cancer, we compared 5,473 tumor-normal genome pairs to identify snoRNAs with frequent copy number loss. The SNORD50A-SNORD50B snoRNA locus was deleted in 10-40% of 12 common cancers, where its loss was associated with reduced survival. A human protein microarray screen identified direct SNORD50A and SNORD50B RNA binding to K-Ras. Loss of SNORD50A and SNORD50B increased the amount of GTP-bound, active K-Ras and hyperactivated Ras-ERK1/ERK2 signaling. Loss of these snoRNAs also increased binding by farnesyltransferase to K-Ras and increased K-Ras prenylation, suggesting that KRAS mutation might synergize with SNORD50A and SNORD50B loss in cancer. In agreement with this hypothesis, CRISPR-mediated deletion of SNORD50A and SNORD50B in KRAS-mutant tumor cells enhanced tumorigenesis, and SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types. SNORD50A and SNORD50B snoRNAs thus directly bind and inhibit K-Ras and are recurrently deleted in human cancer.

Potential utility of autoantibodies as blood-based biomarkers for early detection and diagnosis of Parkinson’s disease

IntroductionThere is a great need to identify readily accessible, blood-based biomarkers for Parkinson’s disease (PD) that are useful for accurate early detection and diagnosis. This advancement would allow early patient treatment and enrollment into clinical trials, both of which would greatly facilitate the development of new therapies for PD. MethodsSera from a total of 398 subjects, including 103 early-stage PD subjects derived from the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) study, were screened with human protein microarrays containing 9,486 potential antigen targets to identify autoantibodies potentially useful as biomarkers for PD. A panel of selected autoantibodies with a higher prevalence in early-stage PD was identified and tested using Random Forest for its ability to distinguish early-stage PD subjects from controls and from individuals with other neurodegenerative and non-neurodegenerative diseases. ResultsResults demonstrate that a panel of selected, blood-borne autoantibody biomarkers can distinguish early-stage PD subjects (90% confidence in diagnosis) from age- and sex-matched controls with an overall accuracy of 87.9%, a sensitivity of 94.1% and specificity of 85.5%. These biomarkers were also capable of differentiating patients with early-stage PD from those with more advanced (mild-moderate) PD with an overall accuracy of 97.5%, and could distinguish subjects with early-stage PD from those with other neurological (e.g., Alzheimer’s disease and multiple sclerosis) and non-neurological (e.g., breast cancer) diseases. ConclusionThese results demonstrate, for the first time, that a panel of selected autoantibodies may prove to be useful as effective blood-based biomarkers for the diagnosis of early-stage PD.

NDRG1 promotes growth of hepatocellular carcinoma cells by directly interacting with GSK-3β and Nur77 to prevent β-catenin degradation.

The N-myc downstream regulated gene 1 (NDRG1) is significantly associated with advanced tumor stages and poor survival of hepatocellular carcinoma (HCC), thereby implicating it as a potential target for HCC treatment. We aim to further understand its biological roles in hepatocarcinogenesis, as a means to exploit it for therapeutic purposes. By screening using the ProtoArray® Human Protein Microarrays, we identified glycogen synthase kinase 3β (GSK-3β) and the orphan nuclear receptor (Nur77) as potential interaction partners of NDRG1. These interactions were confirmed in HCC cell lines in vitro by co-immunoprecipitation; and co-localizations of NDRG1 with GSK-3β and Nur77 were observed by immunofluorescence staining. Additionally, high levels of NDRG1 competitively bind to GSK-3β and Nur77 to allow β-catenin to escape degradation, with consequent elevated levels of downstream oncogenic genes. In vivo, we consistently observed that NDRG1 suppression in HCC xenografts decreased β-catenin levels and its downstream target Cyclin D1, with concomitant tumor growth inhibition. Clinically, the over-expression of NDRG1 in HCC patient samples is positively correlated with GSK-3β-9ser (|”‚ R | = 0.28, p = 0.01), Nur77 (|”‚ R | = 0.42, p < 0.001), and β-catenin (| R |= 0.32, p = 0.003) expressions. In conclusion, we identified GSK-3β and Nur77 as novel interaction partners of NDRG1. These protein-protein interactions regulate the turnover of β-catenin and subsequent downstream signaling mediated by β-catenin in HCC cells, and provides potential targets for future therapeutic interventions.

Relationship between plasma TRAIL before 20 weeks' gestation and pregnancy induced hypertension

Objective To assess the relationship between maternal plasma tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) before 20 weeks' gestation and hypertensive disorder complicating pregnancy (HDCP); and to evaluate the predictive value of plasma TRAIL for HDCP. Methods A 2-phase screening/validation study was designed.In the screening phase, a nested, case-controlled study was performed, the plasma samples collected before 20 weeks' gestation from 20 women who later developed HDCP and 20 age-and gestation week-matched controls were tested in prospective screening test for protein expression profiling during pregnancy and HDCP. Plasma samples were analyzed by a human protein microarray technology designed to detect 507 proteins simultaneously. Differently expressed proteins' functional annotation and clustering were performed by using of Database for Annotation, Visualization and Integrated Discovery (DAVID) and Gene Ontology (GO) database. The TRAIL level of plasma samples obtained before 20 weeks' gestation from 53 women who later developed HDCP and 106 similarly matched controls were further validated by ELISA and 62 clinical risk factors were investigated. Logistic regression and ROC analysis were used to evaluate the relationship between TRAIL and HDCP and its predictive value for HDCP. Results In protein microarray analysis, 23 proteins expressed differently before 20 weeks' gestation between the two groups. Further validation results showed that TRAIL levels in HDCP patients were lower significantly (45.7±13.1) pg/ml than those in healthy pregnant controls(51.2±14.7)pg/ml, P=0.021. Multiple factor logistic regression analysis of 159 pregnancies showed that three features were finally entering the logistic model, they were: anemia (OR=4.87, 95% CI 1.05-24.26), pre-pregnancy BMI (OR=1.72, 95% CI 1.35-2.19) and TRAIL (OR=0.96, 95% CI 0.92-0.99). The predictive accuracy of logistic model was 81.8%. The model significantly increases the predictive value (AUC=0.81, 95% CI 0.73-0.87) compared to TRAIL as independent predictor (AUC=0.59, 95% CI 0.51-0.67). Conclusions Totally 23 proteins were expressed differentially before 20 weeks' gestation in plasma of women who later developed HDCP, confirming that HDCP is a heterogeneous disease with different biological changes. The data suggests that plasma TRAIL levels relate with the development of HDCP and its combination with pre-pregnancy BMI and anemia have a high predictive value for HDCP before 20 weeks' gestation.(Chin J Lab Med, 2015, 38: 522-527) Key words: Hypertension, pregnancy-induced; Receptors, TNF-related apoptosis-inducing ligand; Protein array analysis