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The glycosylation status of proteins is increasingly used as biomarker to improve the reliability in the diagnosis and prognosis of diseases as relevant as cancer. This feeds the need for tools that allow its simple and reliable analysis and are compatible with applicability in the clinic. With this objective in mind, this work reports the first bioelectronic immunoplatforms described to date for the determination of glycosylated haptoglobin (Hp) and the simultaneous determination of total and glycosylated Hp. The bioelectronic immunoplatform is based on the implementation of non-competitive bioassays using two different antibodies or an antibody and a lectin on the surface of commercial magnetic microcarriers. The resulting bioconjugates are labeled with the horseradish peroxidase (HRP) enzyme, and after their magnetic capture on disposable electroplatforms, the amperometric transduction using the H2O2/hydroquinone (HQ) system allows the single or multiple detection. The developed immunoplatform achieves limits of detection (LODs) of 0.07 and 0.46 ng mL-1 for total and glycosylated Hp in buffer solution, respectively. The immunoplatform allows accurate determination using simple and relatively short protocols (approx. 75 min) of total and glycosylated Hp in the secretomes of in vitro-cultured colorectal cancer (CRC) cells with different metastatic potentials, which is not feasible, due to lack of sensitivity, by means of some commercial ELISA kits and Western blot methodology. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Spanish Ministerio de Ciencia e Innovación (PID2019-103899RB-I00 and RTI2018-095756-B-I00), AES-ISCIII Program co-founded by FEDER funds (PI17CIII/00045 and PI20CIII/00019 grants), TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349). Sí

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide. Alterations in proteins of the p53-family are a common event in CRC. ΔNp73, a p53-family member, shows oncogenic properties and its effectors are largely unknown. We performed an in-depth proteomics characterization of transcriptional control by ∆Np73 of the secretome of human colon cancer cells and validated its clinical potential. The secretome was analyzed using high-density antibody microarrays and stable isotopic metabolic labeling. Validation was performed by semiquantitative PCR, ELISA, dot-blot and western blot analysis. Evaluation of selected effectors was carried out using 60 plasma samples from CRC patients, individuals carrying premalignant colorectal lesions and colonoscopy-negative controls. In total, 51 dysregulated proteins were observed showing at least 1.5-foldchange in expression. We found an important association between the overexpression of ∆Np73 and effectors related to lymphangiogenesis, vasculogenesis and metastasis, such as brain-derived neurotrophic factor (BDNF) and the putative aminoacyl tRNA synthase complex-interacting multifunctional protein 1 (EMAP-II)-vascular endothelial growth factor C-vascular endothelial growth factor receptor 3 axis. We further demonstrated the usefulness of BDNF as a potential CRC biomarker able to discriminate between CRC patients and premalignant individuals from controls with high sensitivity and specificity. This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project “PI18/00473” and co-funded by the European Union (FEDER funds) and Cátedra UAM-Roche en Medicina de Innovación to GD, and the Ramón y Cajal Programme of the MINECO, PI17CIII/00045 and PI20CIII/00019 research projects from AES-ISCIII to RB. MG-A and JR-C were supported by contracts of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. AM-C FPU predoctoral contract is supported by the MECD. GS-F is a recipient of a predoctoral contract (grant num-ber 1193818N) supported by The Flanders Research Foundation (FWO). Sí

Colorectal cancer (CRC) and diabetes are two of the most prevalent chronic diseases worldwide with dysregulated receptor tyrosine kinase signaling and strong co-occurrence correlation. Plasma autoantibodies represent a promising early diagnostic marker for both diseases before symptoms appear. In this study, we explore the value of autoantibodies against receptor-type tyrosine-protein phosphatase-like N (PTPRN; full-length or selected domains) as diagnostic markers using a cohort of individuals with type 2 diabetes (T2D), CRC, or both diseases or healthy individuals. We show that PTPRN autoantibody levels in plasma discriminated between patients with T2D with and without CRC. Consistently, high PTPRN expression correlated with decreased survival of patients with CRC. Mechanistically, PTPRN depletion significantly reduced invasiveness of CRC cells in vitro and liver homing and metastasis in vivo by means of a dysregulation of the epithelial-mesenchymal transition and a decrease of the insulin receptor signaling pathway. Therefore, PTPRN autoantibodies may represent a particularly helpful marker for the stratification of patients with T2D at high risk of developing CRC. Consistent with the critical role played by tyrosine kinases in diabetes and tumor biology, we provide evidence that tyrosine phosphatases such as PTPRN may hold potential as therapeutic targets in patients with CRC. ispartof: Diabetes vol:71 issue:3 pages:497-510 ispartof: location:United States status: published

13 p.-7 fig., Glioblastomas (GBMs) are the most frequent and highly aggressive brain tumors, being resistant to all cytotoxic and molecularly targeted agents tested so far. There is, therefore, an urgent need to find novel therapeutic approaches and/or alternative targets to bring treatment options to patients. Here, we first show that GBMs express high levels of N-MYC protein, a transcription factor involved in normal brain development. A novel stapled peptide designed to specifically target N-MYC protein monomer, IDP-410, is able to impair the formation of N-MYC/MAX complex and reduce the stability of N-MYC itself. As a result, the viability of GBM cells is compromised. Moreover, the efficacy is found dependent on the levels of expression of N-MYC. Finally, we demonstrate that IDP-410 reduces GBM growth in vivo when administered systemically, both in subcutaneous and intracranial xenografts, reducing the vascularization of the tumors, highlighting a potential relationship between the function of N-MYC and the expression of mesenchymal/angiogenic genes. Overall, our results strengthen the view of N-MYC as a therapeutic target in GBM and strongly suggest that IDP-410 could be further developed to become a first-in-class inhibitor of N-MYC protein, affecting not only tumor cell proliferation and survival, but also the interplay between GBM cells and their microenvironment., This work was supported by Ministerio de Economía y Competitividad: (Acción Estratégica en Salud) and FEDER funds: PI16/01550 to JMS, by “Asociación Española contra el Cancer” grants: INVES192GARG to RG, GCTRA16015SEDA to JMS, and IDEAS20095SÁNC to PSG, and by Ministerio de Ciencia, Innovación y Universidades and FEDER funds: RTI2018-093596 to PSG. MGA was supported by the Young Employment Initiative (Comunidad de Madrid).

Chronic diseases are the leading cause of disability and responsible for about 63% of deaths worldwide. Among the noninfectious chronic diseases with the highest incidence are cancer and neurodegenerative diseases. Although they have been extensively studied in the last years, there is still an urgent need to find and elucidate the molecular mechanisms underlying their formation and progression to get an early diagnosis and find new therapeutic targets of intervention. Beyond other microarray-based proteomic techniques more extensively used because of their commercial availability, such as protein and antibody microarrays, phage microarrays are another kind of protein microarrays useful for the identification and characterization of disease-specific humoral immune responses and to get further insights into these devastating diseases. Here, we describe the integration and utilization of phage microarrays, which offer such a combination of sensitivity and cost-effective multiplexing capabilities that makes them an affordable strategy for the characterization of humoral immune responses in multiple diseases.

The eye is a multifaceted organ organized in several compartments with particular properties that reflect their diverse functions. The prevalence of ocular diseases is increasing, mainly because of its relationship with aging and of generalized lifestyle changes. However, the pathogenic molecular mechanisms of many common eye pathologies remain poorly understood. Considering the unquestionable importance of proteins in cellular processes and disease progression, proteomic techniques, such as protein microarrays, represent a valuable approach to analyze pathophysiological protein changes in the ocular environment. This technology enables to perform multiplex high-throughput protein expression profiling with minimal sample volume requirements broadening our knowledge of ocular proteome network in eye diseases.In this review, we present a brief summary of the main types of protein microarrays (antibody microarrays, reverse-phase protein microarrays, and protein microarrays) and their application for protein change detection in chronic ocular diseases such as dry eye, age-related macular degeneration, diabetic retinopathy, and glaucoma. The validation of these specific protein changes in eye pathologies may lead to the identification of new biomarkers, depiction of ocular disease pathways, and assistance in the diagnosis, prognosis, and development of new therapeutic options for eye pathologies.

The extraordinary plasticity of glioma cells allows them to contribute to different cellular compartments in tumor vessels, reinforcing the vascular architecture. It was recently revealed that targeting glioma-derived pericytes, which represent a big percentage of the mural cell population in aggressive tumors, increases the permeability of the vessels and improves the efficiency of chemotherapy. However, the molecular determinants of this transdifferentiation process have not been elucidated. Here we show that mutations in EGFR stimulate the capacity of glioma cells to function as pericytes in a BMX- (bone marrow and X-linked) and SOX9-dependent manner. Subsequent activation of platelet-derived growth factor receptor beta in the vessel walls of EGFR-mutant gliomas stabilized the vasculature and facilitated the recruitment of immune cells. These changes in the tumor microenvironment conferred a growth advantage to the tumors but also rendered them sensitive to pericyte-targeting molecules such as ibrutinib or sunitinib. In the absence of EGFR mutations, high-grade gliomas were enriched in blood vessels, but showed a highly disrupted blood–brain barrier due to the decreased BMX/SOX9 activation and pericyte coverage, which led to poor oxygenation, necrosis, and hypoxia. Overall, these findings identify EGFR mutations as key regulators of the glioma-to-pericyte transdifferentiation, highlighting the intricate relationship between the tumor cells and their vascular and immune milieu. Our results lay the foundations for a vascular-dependent stratification of gliomas and suggest different therapeutic vulnerabilities determined by the genetic status of EGFR., This work was supported by FONDECYT grant (1140697 to V. Palma), CONICYT Fellowship (to B.S. Casas), by Ministerio de Economía y Competitividad and FEDER funds (PI13/01258 to A. Hernandez-Laín; PI17/01621 to J.M. Sepulveda-S anchez; and PI16/01580 and DTS18/00181 to A. Matheu), by Young Employment Initiative (Comunidad de Madrid) to M. Garranzo-Asensio, by “Asociacion Espanola contra ~ el Cancer” (AECC) grants (INVES192GARG to R. Gargini; GCTRA16015SEDA to J.M. Sepulveda-S anchez); and by Ministerio de Ciencia, Innovacion y Universidades and FEDER funds (RTI2018-093596 to P. Sanchez-Gomez).

This work describes the first electrochemical immunosensor reported for the determination of IL-13 receptor Rα2 (IL-13Rα2), an emerging relevant biomarker in metastatic colon cancer. The approach involves the formation of sandwich immunocomplexes using specific capture (CAb) and biotinylated detector antibodies (BDAb) further labeled with an streptavidin-horseradish peroxidase (Strep-HRP) polymer, onto carboxylic acid-modified magnetic microbeads (HOOC-MBs). Amperometric detection at disposable carbon screen-printed electrodes (SPCEs) using the (H2O2)/hydroquinone (HQ) system was employed to monitor the affinity reactions. The developed immunosensor exhibits a linear calibration plot over the 3.9–100 ng mL−1 concentration range, a LOD of 1.2 ng mL−1 and excellent selectivity against other non-target proteins. The amperometric immunosensor was applied successfully to quantify for the first time the IL-13Rα2 expression in raw lysates of colon cancer cells and to discriminate the metastatic potential of intact cells through recognition of this target extracellular receptor. In comparison with the commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit involving the same immunoreagents, the immunosensor provides a similar LOD in a half-time for the assay.

[Background and Purpose]: The humoral immune response in cancer patients can be used for early detection of the disease. Autoantibodies raised against tumor-associated antigens (TAAs) are promising clinical biomarkers for reliable cancer diagnosis, prognosis, and therapy monitoring. In this study, an electrochemical disposable multiplexed immunosensing platform able to integrate difficult- and easy-to-express colorectal cancer (CRC) TAAs is reported for the sensitive determination of eight CRC-specific autoantibodies., [Methods]: The electrochemical immunosensing approach involves the use of magnetic microcarriers (MBs) as solid supports modified with covalently immobilized HaloTag fusion proteins for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric responses measured using the hydroquinone (HQ)/H2O2 system were related to the levels of autoantibodies in plasma., [Results]: The biosensing platform was applied to the analysis of autoantibodies against 8 TAAs described for the first time in this work in plasma samples from healthy asymptomatic individuals (n=3), and patients with high-risk of developing CRC (n=3), and from patients already diagnosed with colorectal (n=3), lung (n=2) or breast (n=2) cancer. The developed bioplatform demonstrated an improved discrimination between CRC patients and controls (asymptomatic healthy individuals and breast and lung cancer patients) compared to an ELISA-like luminescence test., [Conclusions]: The proposed methodology uses a just-in-time produced protein in a simpler protocol, with low sample volume, and involves cost-effective instrumentation, which could be used in a high-throughput manner for reliable population screening to facilitate the detection of early CRC patients at affordable cost., This work was supported by the financial support of the PI17CIII/00045 grant from the AES-ISCIII program to R.B., cofounded by FEDER funds. The financial support of the CTQ2015-64402-C2-1-R (Spanish Ministerio de Economía y Competitividad) and RTI2018-096135-B-I00 (Ministerio de Ciencia, Innovación y Universidades) Research Projects and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) to P.Y-S., S.C. and JM.P are gratefully acknowledged. G.D. acknowledges the financial support of PI15/00246 grant of the FIS and Cátedra UAM-Roche en Medicina de Innovación. M.G-A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. A.M-C. is a recipient of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte. G.S-F. is recipient of a predoctoral contract (grant number 1193818N) supported by The Flanders Research Foundation (FWO). Predoctoral contracts from the Spanish Ministerio de Economía y Competitividad (E.P.) and Universidad Complutense de Madrid (V.R.V-M.) are also gratefully acknowledged.

© The Author(s) 2019., The p53-family is tightly regulated at transcriptional level. Due to alternative splicing, up to 40 different theoretical proteoforms have been described for p73 and at least 20 and 10 for p53 and p63, respectively. However, only the canonical proteins have been evaluated as autoantibody targets in cancer patients for diagnosis. In this study, we have cloned and expressed in vitro the most upregulated proteoforms of p73, ΔNp73α and ΔNp73β, for the analysis of their seroreactivity by a developed luminescence based immunoassay test using 145 individual plasma from colorectal cancer, premalignant individuals and healthy controls. ∆Np73α seroreactivity showed the highest diagnostic ability to discriminate between groups. The combination of ∆Np73α, ∆Np73β and p73 proteoforms seroreactivity were able to improve their individual diagnostic ability. Competitive inhibition experiments further demonstrated the presence of unique specific epitopes in ΔNp73 isoforms not present in p73, with several colorectal patients showing unique and specific seroreactivity to the ΔNp73 proteoforms. Overall, we have increased the complexity of the humoral immune response to the p53-family in cancer patients, showing that the proteoforms derived from the alternative splicing of p73 possess a higher diagnostic ability than the canonical protein, which might be extensive for p53 and p63 proteins., This work was supported by the Ramon y Cajal programme of the MINECO and the financial support of the PI17CIII/00045 grant from the AES-ISCIII program to R.B., cofounded by FEDER funds. G.D. acknowledges the financial support of PI15/00246 grant of the FIS and Cátedra UAM-Roche en Medicina de Innovación. M.G-A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. We thank the excellent technical support of Maricruz Sánchez. A.M-C. is a recipient of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte.

Alzheimer's disease (AD) is the most common cause of dementia worldwide with 10-30% prevalence in aging population and a high socioeconomic impact. Because AD definitive diagnostic requires post-mortem verification, new approaches to study the disease are necessary. Here, we analyze the humoral immune response in AD to survey whether APP+1 or UBB+1 frameshift proteins, produced as a consequence of the "molecular misreading" alteration in AD occurring in the APP (amyloid precursor protein) and UBB (ubiquitin-B protein) proteins' mRNA, elicit the production of autoantibodies specific of AD. To this end, APP+1 and UBB+1 peptides were expressed in bacteria as 6xHisHalo fusion proteins and after purification to homogeneity their seroreactivity was analyzed using 81 individual sera from AD patients and 43 individual sera from healthy individuals by luminescence beads immunoassay. We found that as a result of the molecular misreading, APP+1 and UBB+1 frameshift peptides produced a humoral immune response in AD patients, whose autoantibody levels are significantly higher in comparison with healthy controls. Their combination with a previously reported panel of four autoantigens specific of AD (ANTXR1, OR8J1, PYGB, and NUPR1) increased their diagnostic ability assessed by receiver operating characteristic (ROC) curves up to an area under the curve (AUC) of 73.5%. Collectively, our results demonstrate that APP+1 and UBB+1 frameshift proteins, non-previously described as AD-specific autoantigens, elicit the production of autoantibodies which might be useful as blood-based biomarkers to aid in the detection of the disease.

This paper describes a dual electrochemical immunoassay for the simultaneous determination of IL-13Rα2 and CDH-17, two biomarkers of emerging relevance in metastatic processes. The sandwich assay uses a screen-printed dual carbon electrode that was electrochemically grafted with p-aminobenzoic acid to allow the covalent immobilization of capture antibodies. A hybrid composed of graphene quantum dots (GQDs) and multiwalled carbon nanotubes (MWCNTs) act as nanocarriers for the detection antibodies and horseradish peroxidase. The use of this hybrid material considerably improves the assay (in comparison to the use of MWCNTs) due to the peroxidase mimicking activity of the GQDs. The method works at a low working potential (0.20 V vs. Ag pseudo-reference electrode) and thus is not readily interfered by unknown electroactive species. The dual immunoassay allows for the selective determination of both biomarkers with LOD values of 1.4 (IL-13sRα2) and 0.03 ng mL−1 (CDH-17). The simultaneous determination of IL-13Rα2 and CDH-17 was accomplished in lysates from breast and colorectal cancer cells with different metastatic potential, and in paraffin-embedded tumor tissues extracts from patients diagnosed with colorectal cancer at different stages. The applicability to discriminate the metastatic potential even in intact cells through the detection of both extracellular receptors has been demonstrated also. The assay can be performed within 3 h, requires small sample amounts (0.5 μg), and has a simple protocol.

The eye is a complex organ comprised of several compartments with exclusive and specialized properties that reflect their diverse functions. Although the prevalence of eye pathologies is increasing, mainly because of its correlation with aging and of generalized lifestyle changes, the pathogenic molecular mechanisms of many common ocular diseases remain poorly understood. Therefore, there is an unmet need to delve into the pathogenesis, diagnosis, and treatment of eye diseases to preserve ocular health and reduce the incidence of visual impairment or blindness. Proteomics analysis stands as a valuable tool for deciphering protein profiles related to specific ocular conditions. In turn, such profiles can lead to real breakthroughs in the fields of ocular science and ophthalmology. Among proteomics techniques, protein microarray technology stands out by providing expanded information using very small volumes of samples. : In this review, we present a brief summary of the main types of protein microarrays and their application for the identification of protein changes in chronic ocular diseases such as dry eye, glaucoma, age-related macular degeneration, or diabetic retinopathy. The validation of these specific protein alterations could provide new biomarkers, disclose eye diseases pathways, and help in the diagnosis and development of novel therapies for eye pathologies.

This paper reports the development of an amperometric immunosensing platform for the determination of cadherin-17 (CDH-17), an atypical adhesion protein involved in the progression, metastatic potential, and survival of high prevalence gastric, hepatocellular, and colorectal tumors. The methodology developed relies on the efficient capture and enzymatic labeling of the target protein on the magnetic microparticles (MBs) surface using commercial antibodies and amperometric transduction at screen-printed carbon electrodes (SCPEs) through the HRP/H2O2/HQ system. The developed immunosensing platform allows the selective determination of the target protein at low ng mL-1 level (LOD of 1.43 ng mL-1) in 45 min and using a single incubation step. The electrochemical immunosensor was successfully used for the accurate determination of the target protein in a small amount (0.5 μg) of raw lysates of colon cancer cells with different metastatic potential as well as in extracts from paraffin embedded cancer colon tissues of different metastatic grade., The financial support of the Spanish Ministerio de Economía y Competitividad, Grants CTQ2015-70023-R and CTQ2015-64402-C2-1-R, and the NANOAVANSENS Program from the Comunidad de Madrid (Grant S2013/MT-3029), and predoctoral contracts from the Spanish Ministerio de Economia y Competitividad (E.P.) and Universidad Complutense de Madrid (V.R.-V.M.) are grate fully acknowledged. R.B. acknowledges the financial support of the PI17CIII/00045 Grant from the AES-ISCIII program. M.G.-A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educacion, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo.

Olive pollen and yellow mustard seeds are major allergenic sources with high clinical relevance. To aid with the identification of IgE-reactive components, the development of sensitive methodological approaches is required. Here, we have combined T7 phage display and protein microarrays for the identification of allergenic peptides and mimotopes from olive pollen and mustard seeds. The identification of these allergenic sequences involved the construction and biopanning of T7 phage display libraries of mustard seeds and olive pollen using sera from allergic patients to both biological sources together with the construction of phage microarrays printed with 1536 monoclonal phages from the third/four rounds of biopanning. The screening of the phage microarrays with individual sera from allergic patients enabled the identification of 10 and 9 IgE-reactive unique amino acid sequences from olive pollen and mustard seeds, respectively. Five immunoreactive amino acid sequences displayed on phages were selected for their expression as His6-GST tag fusion proteins and validation. After immunological characterization, we assessed the IgE-reactivity of the constructs. Our results show that protein microarrays printed with T7 phages displaying peptides from allergenic sources might be used to identify allergenic components -peptides, proteins or mimotopes- through their screening with specific IgE antibodies from allergic patients.

Autoantibodies raised against tumor-associated antigens have shown high promise as clinical biomarkers for reliable diagnosis, prognosis, and therapy monitoring of cancer. An electrochemical disposable biosensor for the specific and sensitive determination of p53-specific autoantibodies has been developed for the first time in this work. This biosensor involves the use of magnetic microcarriers (MBs) modified with covalently immobilized HaloTag fusion p53 protein as solid supports for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric signal using the system hydroquinone/H2O2 was related to the levels of p53-autoantibodies in the sample. The biosensor was applied for the analysis of sera from 24 patients with high-risk of developing colorectal cancer and 6 from patients already diagnosed with colorectal (4) and ovarian (2) cancer. The developed biosensor was able to determine p53 autoantibodies with a...

An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation.