Protein Microarray Technique Research Articles

Naked-Eye Readable Microarray for Rapid Profiling of Antibodies against Multiple SARS-CoV-2 Variants.

Novel high-throughput protein detection technologies are critically needed for population-based large-scale SARS-CoV-2 antibody detection as well as for monitoring quality and duration of immunity against virus variants. Current protein microarray techniques rely heavily on labeled transduction methods that require sophisticated instruments and complex operations, limiting their clinical potential, particularly for point-of-care (POC) applications. Here, we developed a label-free and naked-eye readable microarray (NRM) based on a thickness-sensing plasmon ruler, enabling antibody profiling within 30 min. The NRM chips provide 100% accuracy for neutralizing antibody detection by efficiently screening antigen types and experimental conditions and allow for the profiling of antibodies against multiple SARS-CoV-2 variants in clinical samples. We further established a flexible "barcode" NRM assay with a simple tape-based operation, enabling an effective smartphone-based readout and analysis. These results demonstrate new strategies for high-throughput protein detection and highlight the potential of novel protein microarray techniques for realistic clinical applications.

Bearing the brunt: Mongolian khulan (Equus hemionus hemionus) are exposed to multiple influenza A strains

The majority of influenza A virus strains are hosted in nature by avian species in the orders of Anseriformes and Charadriformes. A minority of strains have been able to cross species boundaries and establish themselves in novel non-avian hosts. Influenza viruses of horses, donkeys, and mules represent such successful events of avian to mammal influenza virus adaptation. Mongolia has over 3 million domestic horses and is home to two wild equids, the Asiatic wild ass or khulan (Equus hemionus hemionus), and Przewalski’s horse (Equus ferus przewalskii). Domestic and wild equids are sympatric across most of their range in Mongolia. Epizootic influenza A virus outbreaks among Mongolian domestic horses have been frequently recorded. However, the exposure, circulation and relation to domestic horse influenza A virus outbreaks among wild equids is unknown. We evaluated serum samples of Asiatic wild asses in Mongolia for antibodies against influenza A viruses, using modified protein microarray technique. We detected antibodies against hemagglutinin (H) H1, H3, H5, H7, H8 and H10 influenza A viruses. Asiatic wild asses may represent a previously unidentified influenza A virus reservoir in an ecosystem shared with populations of domestic horses in which influenza strains circulate.

Herb-partitioned moxibustion alleviates colon injuries in ulcerative colitis rats.

AIMTo observe the effect of herb-partitioned moxibustion (HPM) on expression of colonic cytokines in ulcerative colitis (UC) rats.METHODSA UC rat model was established by protein immunization in combination with topical chemical stimulation. Rats in the HPM group (n = 8) received HPM at bilateral Tianshu (ST25) points. The gross injury and pathological scores of the colon were recorded. The expression profile of colonic cytokines was assayed using the protein microarray technique. Specific differential cytokines were selected and verified by ELISA. The corresponding UniProt Accessions of the differentially expressed cytokines were retrieved in the UniProt database. The pathways involved were analyzed with the help of the KEGG PATHWAY database. The DAVID database was used for functional cluster and pathway analysis.RESULTSHPM improved colon injuries in UC rats, manifested by accelerated repair of ulcers and alleviation of inflammation, and the gross injury and pathological scores both significantly decreased (P < 0.01). Fold change > 1.3 or < 0.77 was taken as the screening standard. There were 77 down-regulated and 9 up-regulated differentially expressed colonic cytokines in the HPM group compared with the model group, and expression of 20 differed significantly (P < 0.05). Twelve of the 20 significantly differentially expressed cytokines [β-catenin, interleukin-1 receptor 6 (IL-1R6), IL-1β, B7-1, nerve growth factor receptor, AMP-activated protein kinase-α1, neuropilin-2, orexin A, adipocyte differentiation-related protein, IL-2, Fas and FasL] were up-regulated in the model group (n = 3, compared with the normal group) but down-regulated in the HPM group (n = 3, compared with the model group). Functional cluster analysis showed that the differentially expressed colonic cytokines in the HPM group regulated apoptosis and protein phosphorylation. KEGG pathway analysis showed that 52 down-regulated and 7 up-regulated differentially expressed colonic cytokines in the HPM group had pathways. The pathways that interacted between the cytokines and their receptors accounted for the largest proportion (28 of the down-regulated and 5 of the up-regulated cytokines).CONCLUSIONHPM promotes the repair of colon injuries in UC rats, which is related to the regulation of several abnormally expressed cytokines.

Effects of acupuncture on the number of associated protein phosphorylation in brain tissues of MCAO rats based on protein microarray technique

To investigate the effects of acupuncture on the number of associated phosphorylated proteins in brain tissues of middle cerebral artery occlusion (MCAO) rats, based on the protein microarray technique. The MCAO model was prepared according to the modified occlusion method using occlusion lines. Forty healthy Sprague-Dawley (SD) rats were randomly divided into 4 groups using the lottery method: a sham operation group, a model group, a control point group and an acupoint group, with 10 rats in each group. Rats in the sham operation group and the model group only received binding without acupuncture. Rats in the acupoint group received acupuncture at Dazhui (GV 14), Baihui (GV 20) and Shuigou (GV 25); rats in the control point group received acupuncture at non-acupoint control points. The needle was twisted once for 1 min after insertion and another time in the middle of the 30 min needle retaining. Acupuncture was conducted once every 12 h for 6 consecutive times. At the end of the experiment, the neurological impairment score was collected, and cells of the ischemic brain tissues were extracted. The protein phosphorylation of the related signaling was detected using the 720 phosphorylated antibody microarray technique, and the differentially expressed proteins between groups were screened. The neurological impairment scores after 72 h of treatment: compared with the sham operation group, the scores of the model group, the control point group and the acupoint group were significantly increased (P<0.01); compared with the model group, the scores of the acupoint group and the control point group were significantly decreased (P<0.01, P<0.05); the score of the acupoint group was better than that of the control point group (P<0.05). The results of the protein microarray: compared with the sham operation group, 48 proteins showed up-regulated phosphorylation (≥1.5 times) in the model group and the down-regulated was 28; compared with the model group, 35 proteins showed up-regulated phosphorylation in the control point group, and the down-regulated was 24. There were 29 proteins showing up-regulated phosphorylation in the acupoint group and the down-regulated was 51. The numbers of proteins involved in the function and signal transduction pathways were also different. Acupuncture at Dazhui (GV 14), Baihui (GV 20) and Shuigou (GV 25) can effectively repair brain injury. The ischemic injury of brain tissue may be caused by imbalance of a variety of proteins, and acupuncture can promote brain tissue repair by multi-functional and multi-channel regulation of the protein disorders.

Nuclear vasohibin-2 promotes cell proliferation by inducing G0/G1 to S phase progression.

As a member of the vasohibin (VASH2) family, VASH2 is localized intracellularly as a nuclear and cytoplasmic type. Cytoplasmic VASH2 is associated with carcinoma angiogenesis and malignant transformation and promotes cancer growth. However, the function of nuclear VASH2 has yet to be investigated. The aim of the present study was to detect the nuclear VASH2 expression profile in human organs and tissues by protein microarray technique. To examine the function of nuclear VASH2, we analyzed the relationship between nuclear VASH2 and Ki-67, and stably constructed VASH2 overexpression and knockdown in LO2 and HepG2 cell lines, based on a previous study in hepatic cells. The study was conducted using bromodeoxyuridine, immunofluorescent staining, western blot analysis and flow cytometry. Nuclear VASH2 was highly expressed in actively dividing cells in normal and cancer tissues. There was a significant positive correlation between nuclear VASH2 and Ki-67, indicating that nuclear VASH2 positively correlated with cell proliferation in normal and cancer tissues. The bromodeoxyuridine (BrdU) proliferation test showed that nuclear VASH2 increased the S-phase population and promoted cell proliferation, while VASH2 knockdown reduced BrdU absorbance. Cell cycle analysis revealed that nuclear VASH2 overexpression increased the S-phase population in LO2 and HepG2 cells, while nuclear VASH2 knockdown reduced the S-phase population and increased the G0/G1 population. The findings of this study challenge the classic view of VASH2, which was previously reported as an angiogenesis factor. Furthermore, to the best of our knowledge, these results are the first clinical data indicating that nuclear VASH2, but not cytoplasmic VASH2, promotes cell proliferation by driving the cell cycle from the G0/G1 to Sphase.

Identification of proteomic biomarkers of preeclampsia using protein microarray and tandem mass spectrometry

Preeclampsia (PE) is the leading cause of death of the fetus and the mother. The exact pathomechanism has not so far been clarified. PE coexists with many other diseases, but it is often difficult to explain the association between them and find a clear reason for their occurrence. There are many predictive factors, but none are highly specific in preeclampsia. The diagnosis of preeclampsia seems to be very complex, which is another argument for the exploration of knowledge on this subject. Although many of the discoveries have hitherto been made in the field of proteomics, still no single specific biomarker of preeclampsia has been discovered. Research at the genome level is important because it can help us understand the genetic predisposition of patients affected by this disease. Nevertheless, researchers have recently become more interested in the pathophysiology of PE, and they are trying to answer the question: what is the real, direct cause of preeclampsia? Thus, the discovery of a protein that is a good predictor of preeclampsia development would significantly accelerate the medical care of pregnant women, and consequently reduce the risk of occurrence of HELLP syndrome and fetal death. Apart from the predictive and diagnostic function, such a discovery would help us to better understand the pathogenesis of preeclampsia and to find in the future a medical drug to suppress this disease. In order to make a breakthrough in this field, scientists need to use the most modern methods of proteomics, which allow for the analysis of small amounts of biological material in the shortest possible time, thereby giving a lot of information about existing proteins in the sample. Such optimization allows two methods, most commonly used by researchers: tandem mass spectrometry and protein microarray technique.

The higher frequency of IgA deficiency among Swedish twins is not explained by HLA haplotypes.

Serum immunoglobulin A (IgA) concentrations were determined in 12 600 adult Swedish twins, applying a high-throughput reverse-phase protein microarray technique. The prevalence of IgA deficiency (IgAD) was found to be 1:241 in monozygotic (MZ) twins and 1:198 in dizygotic (DZ) twins. Hence, the prevalence in twins is markedly elevated as compared with the normal Swedish adult population (1:600). The twins did not show a difference in the frequency of HLA haplotypes in comparison with almost 40 000 healthy Swedish controls. As expected, the risk-conveying HLA alleles A*01, B*08 and DRB1*01 were overrepresented among the IgAD twins and were also associated with significantly lower mean serum IgA concentrations in the twin cohort. In contrast, significantly higher mean IgA concentrations were found among individuals carrying the protective HLA alleles B*07 and DRB1*15. Exome sequencing data from two MZ twin pairs discordant for the deficiency showed no differences between the siblings. Model fitting analyses derived a heritability of 35% and indicate that genetic influences are modestly important for IgAD. The probandwise concordance rates for IgAD were found to be 31% for MZ and 13% for DZ twins.

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.

Protein-Protein Interaction Analysis in Blood Plasma of Patients with Myelodysplastic Syndromes By Surface Plasmon Resonance Imaging and Mass Spectrometry

IntroductionThe protein microarrays are becoming the leading technology in proteomic research area. They enable to implement both features of proteins that can be altered in disease as quantitative proteomics (levels in biological samples) as well as functional proteomics (determination of their selective interactions with other biomolecules). Protein microarray techniques such as sandwich immunoassays, antigen capture immunoassay and direct immunoassays use labeling and antibodies. However, these labels can interfere with the analyte binding site and thus affect protein activity. Using antibodies requires a prior knowledge of the studied proteins which is in the case of a heterogeneous disease with little knowledge in its proteomic field a huge disadvantage. Surface plasmon resonance (SPR) is a label-free and direct method allowing quantification as well as monitoring of protein-protein interactions simultaneously and in real time.Myelodysplastic syndrome (MDS) is a heterogeneous group of hematological malignancies. It affects pluripotent hematopoietic stem cell and is manifested by variety of clinical symptoms according to predominant involvement of development lineage. The high risk of MDS to transform into acute myeloid leukemia makes it a suitable model for study of biological processes leading to leukemia development.In this work, we use SPR imaging for simultaneous screening of blood plasma of MDS patients followed by mass spectrometry (MS) for identification of interacting partners and analysis of protein network properties. Proteins, whose levels are either elevated during MDS disease or interaction with their receptors/ligands is a part of signaling pathway, were employed.MethodSPR imaging system with polarization contrast and internal referencing was combined with dispersionless microfluidics for parallel screening of blood plasma samples. Proteins involved in pathogenesis of MDS and their physiological counterparts were immobilized under flow to create 6x6 sensing spots. Specifically, these include integrin αMβ2 (LFA1), intercellular adhesion molecule 1 (ICAM1), integrin α4β1 (VLA4), vascular cell adhesion protein 1 (VCAM1) and cytotoxic T-lymphocyte protein 4 (CTLA4). The sensor surface functionalization was optimized with respect to its ability to provide a low-fouling sensing surface with biologically active receptors. Plasma samples of controls and MDS patients were flowed along the functionalized surface and differences in individual interactions were evaluated. Selected interacting partners were further identified using 2D-HPLC/ESI-MS/MS. Identified proteins were analyzed by String Networks and Power Graph Analysis.Results and ConclusionSignificant differences in the protein profiles among different MDS groups of patients as well as relative to control healthy donors were observed using SPR imaging; tens of interacting proteins were identified by mass spectrometry. Protein interaction networks were explored through clustering of proteins into groups that share the same biological function, are similarly localized in the cell, or are known to be a part of a complex. Identified proteins are involved in several processes; regulation of immune system, ubiquitinylation and protein degradation, cell signaling, hemostasis, protein synthesis, cell adhesion, metastasis, and inhibition of blood coagulation. Using of Power Graphs, a novel representation of (protein) networks, provided valuable insight into the existence of protein complexes, their internal organization, and their relationships. Interaction networks also indicated possible pathways involved in MDS pathogenesis (especially Src tyrosine kinases). The results showed that SPR biosensors are a promising tool for the diagnosis and follow-up efficiency treatment in complex heterogeneous malignancies. DisclosuresNo relevant conflicts of interest to declare.

AB0012 Functional Characterization of P.E383k Blau Syndrome-Related Mutation

BackgroundThe Blau syndrome (BS) is a rare autosomal dominant autoinflammatory disease clinically characterized by symmetrical arthritis, granulomatous dermatitis and recurrent uveitis. The disease is caused by single mutations in CARD15/NOD2,...

P.2.003 Sub-anaesthetic ketamine modulates intrinsic blood oxygen level-dependent (BOLD) connectivity between the hippocampus and the prefrontal cortex in the rat

so may potentially reverse isolation-induced cognitive impairments. The aims of this study were to utilise protein microarray, immunohistochemical and glutamate microsensor techniques to further characterise isolationinduced hippocampal changes, and examine the ability of a 5-HT6 receptor antagonist, SB-399885, to reverse the neurochemical and behavioural alterations in this model. Male Lister hooded rats (University of Nottingham or Charles River UK) were weaned on post-natal day 21−24 and housed individually or in groups (3−4). Rats received minimal handling until assessment (starting 5−6 weeks later) of locomotor activity, NOD, and contextual and cue-mediated CER. Rats received i.p. vehicle (1% Tween 80, 1ml/kg) or 10mg/kg SB-399885 on six occasions (n = 11/housing-treatment combination) either 30min prior to testing or immediately after CER acquisition to preclude potential nociceptive/affective confounds. Rats were killed 24 h after the final injection and hippocampi collected for quantification of VGLUT1−3 expression using Western blots, cell proliferation using Ki67 immunohistochemistry and expression of intracellular signalling molecules using protein microarray. Glutamate signalling in hippocampal slices from separate groups of drug-free rats (n = 7/housing condition) was measured using enzyme-coated microsensors. Isolation-rearing prevented discrimination of the novel object during NOD (P> 0.05 versus familiar, whereas group-housed P 0.05 versus isolate and group vehicle controls in each case). Isolation also elevated Rac/CDC42 (Rho GTPases which regulate microtubule stabilisation and dendrite morphogenesis, and have been implicated in schizophrenia), while SB-399885 decreased TAK1 and pSTAT3 (members of the SAPK/JNK signalling cascade, whose stimulation via Rac/CDC42 reduces pSTAT3) in isolates only. Isolation tended to lower basal extracellular glutamate in hippocampal slices, from 4.32±1.40mM to 1.99±0.41mM (P= 0.0708), but the combination of 3mM SB-399885 and 120mM KCl stimulated glutamate release irrespective of housing. The 5-HT6 receptor antagonist reversed isolationinduced cognitive impairments and partially reversed deficits in hippocampal cell proliferation in this neurodevelopmental model of schizophrenia. These findings further support the use of 5-HT6 antagonists to treat cognitive dysfunction, and the value of isolation-reared rats to investigate the underlying neurobiology of schizophrenia and evaluate novel treatments for the cognitive symptoms.

TOMM34 expression in early invasive breast cancer: a biomarker associated with poor outcome

Appropriate mitochondrial functioning in normal cells depends on proper functioning of mitochondrial translocation machinery, of which translocase of the outer membrane of mitochondria (TOMM) plays important role. The aim of this study was to explore the expression of TOMM34 in invasive breast cancer (BC) with relevance to BC molecular subtypes and patients' outcome. Gene expression data of 128 BC were analysed using artificial neuronal network (ANN) analysis to identify differentially expressed genes between BC with distant metastases and that without distant metastases. TOMM34 expression was assessed in a large series of BC (n = 1,061) with long-term follow-up using tissue microarray and immunohistochemistry. TOMM34 protein expression was quantitatively measured using the novel reverse phase protein microarray (RPPA) technique. ANN analysis revealed TOMM34 gene transcript as one of the top differentially expressed gene correlated with BC distant metastasis. Protein expression of TOMM34 was associated with features of aggressive behaviour including higher tumour grade, advanced nodal stage, larger tumour size and lymphovascular invasion. TOMM34 over-expression was significantly associated with shorter BC-specific survival and metastasis-free survival independent of standard prognostic parameters. TOMM34 protein expression was quantified by RPPA which showed that the mean expression values of TOMM34 were higher in samples demonstrating features of poor outcome. This study demonstrates at translational protein expression level that TOMM34 is a marker of poor prognosis in BC. Our findings underscore the role played by mitochondrial machinery in BC progression and warrant their validation on a prospective basis.

Facing current quantification challenges in protein microarrays.

The proteome is highly variable and differs from cell to cell. The reasons are posttranslational modifications, splice variants, and polymorphisms. Techniques like next-generation sequencing can only give an inadequate picture of the protein status of a cell. Protein microarrays are able to track these changes on the level they occur: the proteomic level. Therefore, protein microarrays are powerful tools for relative protein quantification, to unveil new interaction partners and to track posttranslational modifications. This papers gives an overview on current protein microarray techniques and discusses recent advances in relative protein quantification.

The Role of Proteomics in the Diagnosis and Treatment of Women's Cancers: Current Trends in Technology and Future Opportunities

Technological and scientific innovations over the last decade have greatly contributed to improved diagnostics, predictive models, and prognosis among cancers affecting women. In fact, an explosion of information in these areas has almost assured future generations that outcomes in cancer will continue to improve. Herein we discuss the current status of breast, cervical, and ovarian cancers as it relates to screening, disease diagnosis, and treatment options. Among the differences in these cancers, it is striking that breast cancer has multiple predictive tests based upon tumor biomarkers and sophisticated, individualized options for prescription therapeutics while ovarian cancer lacks these tools. In addition, cervical cancer leads the way in innovative, cancer-preventative vaccines and multiple screening options to prevent disease progression. For each of these malignancies, emerging proteomic technologies based upon mass spectrometry, stable isotope labeling with amino acids, high-throughput ELISA, tissue or protein microarray techniques, and click chemistry in the pursuit of activity-based profiling can pioneer the next generation of discovery. We will discuss six of the latest techniques to understand proteomics in cancer and highlight research utilizing these techniques with the goal of improvement in the management of women's cancers.

Protein microarrays and novel detection platforms

The field of proteomics has undergone rapid advancements over the last decade and protein microarrays have emerged as a promising technological platform for the challenging task of studying complex proteomes. This gel-free approach has found an increasing number of applications due to its ability to rapidly and efficiently study thousands of proteins simultaneously. Different protein microarrays, including capture arrays, reverse-phase arrays, tissue microarrays, lectin microarrays and cell-free expression microarrays, have emerged, which have demonstrated numerous applications for proteomics studies including biomarker discovery, protein interaction studies, enzyme–substrate profiling, immunological profiling and vaccine development, among many others. The need to detect extremely low-abundance proteins in complex mixtures has provided motivation for the development of sensitive, real-time and multiplexed detection platforms. Conventional label-based approaches like fluorescence, chemiluminescence and use of radioactive isotopes have witnessed substantial advancements, with techniques like quantum dots, gold nanoparticles, dye-doped nanoparticles and several bead-based methods now being employed for protein microarray studies. In order to overcome the limitations posed by label-based technologies, several label-free approaches like surface plasmon resonance, carbon nanotubes and nanowires, and microcantilevers, among others, have also advanced in recent years, and these methods detect the query molecule itself. The scope of this article is to outline the protein microarray techniques that are currently being used for analytical and function-based proteomics and to provide a detailed analysis of the key technological advances and applications of various detection systems that are commonly used with microarrays.

Proteomic Studies Identify Citron Rho Interacting Kinase (CRIK), a Novel Protein That Regulates Proliferation and Survival In Multiple Myeloma Cells

AbstractAbstract 2958 PURPOSE:Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. Using an antibody-based protein microarray technique, we identified Citron Rho Interacting Kinase (CRIK) as a protein that was highly expressed in primary multiple myeloma (MM) cells compared to normal plasma cell. We therefore sought to investigate the functional role of CRIK in MM cells. Methods:Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM and 4 healthy control using high-throughput proteomic analysis with antibody-based protein microarray (Clontech, CA). MM.1S, OPM2, RPMI8226, and INA6 were used to perform functional validation. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform and qPCR. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, OPM2, and INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by BrdU, MTT, PI and Apo2.7 staining, and flow cytometry analysis, respectively. Results:We first showed that CRIK was overexpressed in 12 patients with MM compared to normal CD138+ cells obtained from healthy controls using high-throughput protein microarray. We further confirmed CRIK expression using immunohistochemistry in the same samples of MM patients. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. Knockdown of CRIK in MM cell lines led to an anti-proliferative and pro-apoptotic effect in all MM cell lines tested. Indeed, CRIK-knockdown MM cells were characterized by a reduction of 60–80% in the proliferation rate, supported by reduction of DNA synthesis and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity of caspase-3, caspase-8, caspase-9, and parp cleavage was also demonstrated in CRIK knockdown cells compared to scramble probe transfected cells. We also showed that CRIK knockdown led to cytokinesis in MM cell lines, indicating a possible mechanism of inhibition of proliferation of these cells. Conclusion:In this study, we show that MM cells express a high level of a novel protein CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of MM cells. CRIK could be a critical therapeutic target in MM. Disclosures:Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Rapid and simultaneous detection of Ureaplasma parvum and Chlamydia trachomatis antibodies based on visual protein microarray using gold nanoparticles and silver enhancement

Based on gold-labeled silver stain (GLSS) method, we developed the visual protein microarray for simultaneous, sensitive, and specific detection of Ureaplasma parvum and Chlamydia trachomatis using N-terminus multiple-banded antigen (NMBA) of U. parvum and major outer membrane protein of C. trachomatis. The specific antigens were immobilized on glass surface that was treated with 3-glycidoxypropyltrimethoxysilane, and they were used as the capturing probes to recognize the complementary target antibodies binding to the detecting probes of Nano-gold-Staphylococcal protein A (SPA). In the “sandwich” format, Nano-gold-SPA probe was used as an indicator and GLSS was applied to amplify the detection signals and produce black image on array spots, which were visible with naked eyes. In our model arrays, the detection limit of protein microarray was as low as 2 ng/mL, and the lowest titer of detectable antibody was 1:128; thus, this sensitivity was comparable to the fluorescent detection method. The visual simultaneous protein microarrays were used to detect total 186 clinical samples, which had been determined by enzyme-linked immunosorbent assay (ELISA) and fluorescence quantitative real-time polymerase chain reaction; the results were identical and no distinct difference ( P > 0.05) existed between them. Our results demonstrate that we have developed the visual protein microarray technique, which is of high sensitivity and high specificity, and it may have potential in clinical applications.

Citron Rho Interacting Kinase (CRIK) Regulates Survival in IL-6 Dependent Multiple Myeloma Cells.

Abstract 1825Poster Board I-851 PurposeRecent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. We have previously demonstrated that Citron Rho Interacting Kinase (CRIK) is overexpressed in primary multiple myeloma (MM) cells, as compared to the normal plasma cell counterpart, using an antibody-based protein microarray technique. We therefore sought to investigate the functional role of CRIK in MM cells. MethodsWe determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM.1S, RPMI8226, and INA6 MM cell lines were used in this study. Protein expression has been studied by immunoblotting. Gene expression analysis has been assessed using the Affymetrix U133A platform. Lentivirus was used to knockdown CRIK in MM cell lines (MM.1S, RPMI8226, INA6). DNA synthesis, cell survival, cell cycle profiling and apoptosis were assessed by thymidine uptake, MTT, PI and Annexin/PI staining and flow cytometric analysis, respectively. ResultsOverexpression of CRIK has been confirmed in primary CD138+ tumor cells isolated from bone marrow of 12 patients with MM, as compared to normal plasma cells obtained from healthy donors. We found that CRIK-knockdown exerted an anti-proliferative and pro-apoptotic effect only in IL-6-dependent MM cell line INA6; in contrast, no effect on proliferation and survival was observed in MM1.S and RPMI8226. Indeed, INA6 CRIK-knockdown cells were characterized by a reduction in the proliferation rate, associated with decreased S-phase and G2/M phase cell cycle arrest. Moreover, induction of cytotoxicity was also demonstrated in CRIK knockdown cells compared to scramble probe transfected or non-transfected cells. We also showed that CRIK knockdown led to cytokinesis in INA6, indicating a possible mechanism for inhibition of proliferation of these cells. We next correlated CRIK gene expression level (CIT) with prognosis using previously published gene expression datasets and found that CRIK correlated with poor prognosis. ConclusionIn this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of proliferation and survival of IL-6 dependent MM cells. Moreover, CRIK protein expression correlated with poor survival in patients with MM. DisclosuresAnderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Proteomic Analysis of Multiple Myeloma Identifies Upregulation of CRIK Protein, a Novel Protein Regulating Migration and Adhesion

PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers.METHODS: We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform.RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients.CONCLUSION: In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.

Proteomics analysis in post‐transplant lymphoproliferative disorders

Little is known about the biology of post-transplant lymphoproliferative disorders (PTLDs). The objective of this study was to determine the molecular alterations that occur at the protein level in patients with PTLDs. Six tumor samples from adult patients with PTLD and four benign lymph nodes were studied using protein microarray technique. Proteins that were dysregulated included proteins in the PI3K/mTOR, NFkB and HSP90 pathways. Inhibitors of these proteins induced cytotoxicity and apoptosis in EBV+ve and -ve cell lines. These results provide insight into pathways that are dysregulated in PTLD and can be targeted in future clinical trials.