Contributing to the management of viral infections through simple immunosensing of the arachidonic acid serum level.

A trendsetting direct competitive-based biosensing tool has been developed and implemented for the determination of the polyunsaturated fatty acid arachidonic acid (ARA), a highly significant biological regulator with decisive roles in viral infections. The designed methodology involves a competitive reaction between the target endogenous ARA and a biotin-ARA competitor for the recognition sites of anti-ARA antibodies covalently attached to the surface of carboxylic acid-coated magnetic microbeads (HOOC-MµBs), followed by the enzymatic label of the biotin-ARA residues with streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The resulting bioconjugates were magnetically trapped onto the sensing surface of disposable screen-printed carbon transducers (SPCEs) to monitor the extent of the biorecognition reaction through amperometry. The operational functioning of the exhaustively optimized and characterized immunosensing bioplatform was highly convenient for the quantitative determination of ARA in serum samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-) and respiratory syncytial virus (RSV)-infected individuals in a rapid, affordable, trustful, and sensitive manner.

ΔNp73 and its effector targets promote colorectal peritoneal carcinosis and predict survival.

Peritoneal metastasis of colorectal origin appears in ~10-15% of patients at the time of diagnosis and in 30-40% of cases with disease progression. Locoregional spread through the peritoneum is considered stage IVc and is associated with a poor prognosis. The development of a regional therapeutic strategy based on cytoreductive surgery, and hyperthermic intra-abdominal chemotherapy has significantly altered the course of the disease. Although recent evidence supports the benefits of cytoreductive surgery, the benefits of hyperthermic intra-abdominal chemotherapy are, however, still a matter of debate. Understanding the molecular alterations underlying the disease is crucial for developing new therapeutic strategies. Here, we evaluated the involvement in peritoneal dissemination of the oncogenic isoform of TP73, ΔNp73, and its effector targets in in vitro and mouse models, and in 30 patients diagnosed with colorectal peritoneal metastasis. In an orthotopic mouse model, we observed that tumor cells overexpressing ΔNp73 present a higher avidity for the peritoneum and that extracellular vesicles secreted by ΔNp73-upregulating tumor cells enhance their dissemination. In addition, we identified that tumor cells overexpressing ΔNp73 present with dysregulation of genes associated with an epithelial/mesothelial-to-mesenchymal transition (MMT) and that mesothelial cells exposed to the conditioned medium of tumor cells with upregulated ΔNp73 present a mesenchymal phenotype. Lastly, ΔNp73 and its effector target RNAs were dysregulated in our patient series, there were positive correlations between ΔNp73 and its effector targets, and MSN and ITGB4 (ΔNp73 effectors) predicted patient survival. In conclusion, ΔNp73 and its effector targets are involved in the peritoneal dissemination of colorectal cancer and predict patient survival. The promotion of the EMT/MMT and modulation of the adhesion capacity in colorectal cancer cells might be the mechanisms triggered by ΔNp73. Remarkably, ΔNp73 protein is a druggable protein and should be the focus of future studies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Breaking barriers in electrochemical biosensing using bioinspired peptide and phage probes.

Electrochemical biosensing continues to advance tirelessly, overcoming barriers that have kept it from leaving research laboratories for many years. Among them, its compromised performance in complex biological matrices due to fouling or receptor stability issues, the limitations in determining toxic and small analytes, and its use, conditioned to the commercial availability of commercial receptors and the exploration of natural molecular interactions, deserved to be highlighted. To address these challenges, in addition to the intrinsic properties of electrochemical biosensing, its coupling with biomimetic materials has played a fundamental role, among which bioinspired phage and peptide probes stand out. The versatility in design and employment of these probes has opened an unimaginable plethora of possibilities for electrochemical biosensing, improving their performance far beyond the development of highly sensitive and selective devices. The state of the art offers robust electroanalytical biotools, capable of operating in complex samples and with exciting opportunities to discover and determine targets regardless of their toxicity and size, the commercial availability of bioreceptors, and prior knowledge of molecular interactions. With all this in mind, this review offers a panoramic, novel, and updated vision of both the tremendous advances and opportunities offered by the combination of electrochemical biosensors with bioinspired phage and peptide probes and the challenges and research efforts that are envisioned in the immediate future.

Gold Nanoparticle-Decorated Catalytic Micromotor-Based Aptassay for Rapid Electrochemical Label-Free Amyloid-ß42 Oligomer Determination in Clinical Samples from Alzheimer's Patients.

Micromotor (MM) technology offers a valuable and smart on-the-move biosensing microscale approach in clinical settings where sample availability is scarce in the case of Alzheimer's disease (AD). Soluble amyloid-ß protein oligomers (AßO) (mainly AßO42) that circulate in biological fluids have been recognized as a molecular biomarker and therapeutic target of AD due to their high toxicity, and they are correlated much more strongly with AD compared to the insoluble Aß monomers. A graphene oxide (GO)-gold nanoparticles (AuNPs)/nickel (Ni)/platinum nanoparticles (PtNPs) micromotors (MMGO-AuNPs)-based electrochemical label-free aptassay is proposed for sensitive, accurate, and rapid determination of AßO42 in complex clinical samples such as brain tissue, cerebrospinal fluid (CSF), and plasma from AD patients. An approach that implies the in situ formation of AuNPs on the GO external layer of tubular MM in only one step during MM electrosynthesis was performed (MMGO-AuNPs). The AßO42 specific thiolated-aptamer (AptAßO42) was immobilized in the MMGO-AuNPs via Au-S interaction, allowing for the selective recognition of the AßO42 (MMGO-AuNPs-AptAßO42-AßO42). AuNPs were smartly used not only to covalently bind a specific thiolated-aptamer for the design of a label-free electrochemical aptassay but also to improve the final MM propulsion performance due to their catalytic activity (approximately 2.0× speed). This on-the-move bioplatform provided a fast (5 min), selective, precise (RSD < 8%), and accurate quantification of AßO42 (recoveries 94-102%) with excellent sensitivity (LOD = 0.10 pg mL-1) and wide linear range (0.5-500 pg mL-1) in ultralow volumes of the clinical sample of AD patients (5 µL), without any dilution. Remarkably, our MM-based bioplatform demonstrated the competitiveness for the determination of AßO42 in the target samples against the dot blot analysis, which requires more than 14 h to provide qualitative results only. It is also important to highlight its applicability to the potential analysis of liquid biopsies as plasma and CSF samples, improving the reliability of the diagnosis given the heterogeneity and temporal complexity of neurodegenerative diseases. The excellent results obtained demonstrate the analytical potency of our approach as a future tool for clinical/POCT (Point-of-care testing) routine scenarios.

Bringing to Light the Importance of the miRNA Methylome in Colorectal Cancer Prognosis Through Electrochemical Bioplatforms.

This work reports the first electrochemical bioplatforms developed for the determination of the total contents of either target miRNA or methylated target miRNA. The bioplatforms are based on the hybridization of the target miRNA with a synthetic biotinylated DNA probe, the capture of the formed DNA/miRNA heterohybrids on the surface of magnetic microcarriers, and their recognition with an antibody selective to these heterohybrids or to the N6-methyladenosine (m6A) epimark. The determination of the total or methylated target miRNA was accomplished by labeling such secondary antibodies with the horseradish peroxidase (HRP) enzyme. In both cases, amperometric transduction was performed on the surface of disposable electrodes after capturing the resulting HRP-tagged magnetic bioconjugates. Because of their increasing relevance in colorectal cancer (CRC) diagnosis and prognosis, miRNA let-7a and m6A methylation were selected. The proposed electrochemical bioplatforms showed attractive analytical and operational characteristics for the determination of the total and m6A-methylated target miRNA in less than 75 min. These bioplatforms, innovative in design and application, were applied to the analysis of total RNA samples extracted from cultured cancer cells with different metastatic profiles and from paired healthy and tumor tissues of patients diagnosed with CRC at different stages. The obtained results demonstrated, for the first time using electrochemical platforms, the potential of interrogating the target miRNA methylation level to discriminate the metastatic capacities of cancer cells and to identify tumor tissues and, in a pioneering way, the potential of the m6A methylation in miRNA let-7a to serve as a prognostic biomarker for CRC.

Micromotor-based electrochemical immunoassays for reliable determination of amyloid-ß (1-42) in Alzheimer's diagnosed clinical samples.

Alzheimer's disease (AD), in addition to being the most common cause of dementia, is very difficult to diagnose, with the 42-amino acid form of Aß (Aß-42) being one of the main biomarkers used for this purpose. Despite the enormous efforts made in recent years, the technologies available to determine Aß-42 in human samples require sophisticated instrumentation, present high complexity, are sample and time-consuming, and are costly, highlighting the urgent need not only to develop new tools to overcome these limitations but to provide an early detection and treatment window for AD, which is a top-challenge. In recent years, micromotor (MM) technology has proven to add a new dimension to clinical biosensing, enabling ultrasensitive detections in short times and microscale environments. To this end, here an electrochemical immunoassay based on polypyrrole (PPy)/nickel (Ni)/platinum nanoparticles (PtNPs) MM is proposed in a pioneering manner for the determination of Aß-42 in left prefrontal cortex brain tissue, cerebrospinal fluid, and plasma samples from patients with AD. MM combines the high binding capacity of their immunorecognition external layer with self-propulsion through the catalytic generation of oxygen bubbles in the internal layer due to decomposition of hydrogen peroxide as fuel, allowing rapid bio-detection (15 min) of Aß-42 with excellent selectivity and sensitivity (LOD = 0.06 ng/mL). The application of this disruptive technology to the analysis of just 25 µL of the three types of clinical samples provides values concordant with the clinical values reported, thus confirming the potential of the MM approach to assist in the reliable, simple, fast, and affordable diagnosis of AD by determining Aß-42.

Pursuing precision in medicine and nutrition: the rise of electrochemical biosensing at the molecular level.

In the era that we seek personalization in material things, it is becoming increasingly clear that the individualized management of medicine and nutrition plays a key role in life expectancy and quality of life, allowing participation to some extent in our welfare and the use of societal resources in a rationale and equitable way. The implementation of precision medicine and nutrition are highly complex challenges which depend on the development of new technologies able to meet important requirements in terms of cost, simplicity, and versatility, and to determine both individually and simultaneously, almost in real time and with the required sensitivity and reliability, molecular markers of different omics levels in biofluids extracted, secreted (either naturally or stimulated), or circulating in the body. Relying on representative and pioneering examples, this review article critically discusses recent advances driving the position of electrochemical bioplatforms as one of the winning horses for the implementation of suitable tools for advanced diagnostics, therapy, and precision nutrition. In addition to a critical overview of the state of the art, including groundbreaking applications and challenges ahead, the article concludes with a personal vision of the imminent roadmap.

Functional Proteomics Characterization of the Role of SPRYD7 in Colorectal Cancer Progression and Metastasis.

SPRY domain-containing protein 7 (SPRYD7) is a barely known protein identified via spatial proteomics as being upregulated in highly metastatic-to-liver KM12SM colorectal cancer (CRC) cells in comparison to its isogenic poorly metastatic KM12C CRC cells. Here, we aimed to analyze SPRYD7's role in CRC via functional proteomics. Through immunohistochemistry, the overexpression of SPRYD7 was observed to be associated with the poor survival of CRC patients and with an aggressive and metastatic phenotype. Stable SPRYD7 overexpression was performed in KM12C and SW480 poorly metastatic CRC cells and in their isogenic highly metastatic-to-liver-KM12SM-and-to-lymph-nodes SW620 CRC cells, respectively. Upon upregulation of SPRYD7, in vitro and in vivo functional assays confirmed a key role of SPRYD7 in the invasion and migration of CRC cells and in liver homing and tumor growth. Additionally, transient siRNA SPRYD7 silencing allowed us to confirm in vitro functional results. Furthermore, SPRYD7 was observed as an inductor of angiogenesis. In addition, the dysregulated SPRYD7-associated proteome and SPRYD7 interactors were elucidated via 10-plex TMT quantitative proteins, immunoproteomics, and bioinformatics. After WB validation, the biological pathways associated with the stable overexpression of SPRYD7 were visualized. In conclusion, it was demonstrated here that SPRYD7 is a novel protein associated with CRC progression and metastasis. Thus, SPRYD7 and its interactors might be of relevance in identifying novel therapeutic targets for advanced CRC.

Benefits of FAIMS to Improve the Proteome Coverage of Deteriorated and/or Cross-Linked TMT 10-Plex FFPE Tissue and Plasma-Derived Exosomes Samples.

The proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = -45 and CV = -60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with FAIMS in comparison to the LC-MS/MS analysis without FAIMS. Our results highlight the potential of mass spectrometry analyses with FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.

Histopathologic Changes in Congenital Corneal Stromal Dystrophy: Report of 4 Cases in 2 Families.

Corneal dystrophies are hereditary diseases affecting the corneal tissue; they are bilateral, symmetrical and unrelated to environmental or systemic conditions. Congenital corneal stromal dystrophy is a very rare autosomal dominant dystrophy that is caused by a mutation in the DCN gene that encodes decorin (a proteoglycan of the extracellular matrix). We herein report 4 cases of congenital stromal corneal dystrophy in 2 families, highlighting the previously undescribed histopathologic features, the possible differential diagnosis of this entity and the key role played by decorin staining in its diagnosis.

Flat clathrin lattices are linked to metastatic potential in colorectal cancer.

Clathrin assembles at the cells' plasma membrane in a multitude of clathrin-coated structures (CCSs). Among these are flat clathrin lattices (FCLs), alternative clathrin structures that have been found in specific cell types, including cancer cells. Here we show that these structures are also present in different colorectal cancer (CRC) cell lines, and that they are extremely stable with lifetimes longer than 8 h. By combining cell models representative of CRC metastasis with advanced fluorescence imaging and analysis, we discovered that the metastatic potential of CRC is associated with an aberrant membranous clathrin distribution, resulting in a higher prevalence of FCLs in cells with a higher metastatic potential. These findings suggest that clathrin organization might play an important yet unexplored role in cancer metastasis.

Brain and immune system-derived extracellular vesicles mediate regulation of complement system, extracellular matrix remodeling, brain repair and antigen tolerance in Multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated central nervous system disease whose course is unpredictable. Finding biomarkers that help to better comprehend the disease's pathogenesis is crucial for supporting clinical decision-making. Blood extracellular vesicles (EVs) are membrane-bound particles secreted by all cell types that contain information on the disease's pathological processes. PURPOSE: To identify the immune and nervous system-derived EV profile from blood that could have a specific role as biomarker in MS and assess its possible correlation with disease state. RESULTS: Higher levels of T cell-derived EVs and smaller size of neuron-derived EVs were associated with clinical relapse. The smaller size of the oligodendrocyte-derived EVs was related with motor and cognitive impairment. The proteomic analysis identified mannose-binding lectin serine protease 1 and complement factor H from immune system cell-derived EVs as autoimmune disease-associated proteins. We observed hepatocyte growth factor-like protein in EVs from T cells and inter-alpha-trypsin inhibitor heavy chain 2 from neurons as white matter injury-related proteins. In patients with MS, a specific protein profile was found in the EVs, higher levels of alpha-1-microglobulin and fibrinogen ß chain, lower levels of C1S and gelsolin in the immune system-released vesicles, and Talin-1 overexpression in oligodendrocyte EVs. These specific MS-associated proteins, as well as myelin basic protein in oligodendrocyte EVs, correlated with disease activity in the patients with MS. CONCLUSION: Neural-derived and immune-derived EVs found in blood appear to be good specific biomarkers in MS for reflecting the disease state.

Electrochemical biotool for the dual determination of epithelial mucins associated to prognosis and minimal residual disease in colorectal cancer.

This work reports a dual immunoplatform for the simultaneous detection of two epithelial glycoproteins of the mucin family, mucin 1 (MUC1) and mucin 16 (MUC16), whose expression is related to adverse prognosis and minimal residual disease (MRD) in colorectal cancer (CRC). The developed immunoplatform involves functionalised magnetic microparticles (MBs), a set of specific antibody pairs (a capture antibody, cAb, and a biotinylated detector antibody b-dAb labelled with a streptavidin-horseradish peroxidase, Strep-HRP, polymer) for each target protein and amperometric detection at dual screen-printed carbon electrodes (SPdCEs) using the hydroquinone (HQ)/horseradish peroxidase (HRP)/H2O2 system. This dual immunoplatform allows, under the optimised experimental conditions, to achieve LOD values of 50 and 1.81 pg mL-1 (or mU mL-1) for MUC1 and MUC16, respectively, and adequate selectivity for the determination of the two targets in the clinic. The developed immunoplatform was employed to analyse CRC cell protein extracts (1.0 µg/determination) with different metastatic potential providing results in agreement with those obtained by blotting technologies but using affordable and applicable point-of-care instruments. This new biotool also emerges competitive in state-of-the-art electrochemical immunoplatforms seeking a compromise among simplicity, reduction of test time and analytical characteristics.

Proteomics analysis of prefrontal cortex of Alzheimer's disease patients revealed dysregulated proteins in the disease and novel proteins associated with amyloid-ß pathology.

BACKGROUND: Alzheimer's disease (AD) is a progressive, chronic, and neurodegenerative disease, and the most common cause of dementia worldwide. Currently, the mechanisms underlying the disease are far from being elucidated. Thus, the study of proteins involved in its pathogenesis would allow getting further insights into the disease and identifying new markers for AD diagnosis. METHODS: We aimed here to analyze protein dysregulation in AD brain by quantitative proteomics to identify novel proteins associated with the disease. 10-plex TMT (tandem mass tags)-based quantitative proteomics experiments were performed using frozen tissue samples from the left prefrontal cortex of AD patients and healthy individuals and vascular dementia (VD) and frontotemporal dementia (FTD) patients as controls (CT). LC-MS/MS analyses were performed using a Q Exactive mass spectrometer. RESULTS: In total, 3281 proteins were identified and quantified using MaxQuant. Among them, after statistical analysis with Perseus (p value < 0.05), 16 and 155 proteins were defined as upregulated and downregulated, respectively, in AD compared to CT (Healthy, FTD and VD) with an expression ratio ≥ 1.5 (upregulated) or ≤ 0.67 (downregulated). After bioinformatics analysis, ten dysregulated proteins were selected as more prone to be associated with AD, and their dysregulation in the disease was verified by qPCR, WB, immunohistochemistry (IHC), immunofluorescence (IF), pull-down, and/or ELISA, using tissue and plasma samples of AD patients, patients with other dementias, and healthy individuals. CONCLUSIONS: We identified and validated novel AD-associated proteins in brain tissue that should be of further interest for the study of the disease. Remarkably, PMP2 and SCRN3 were found to bind to amyloid-ß (Aß) fibers in vitro, and PMP2 to associate with Aß plaques by IF, whereas HECTD1 and SLC12A5 were identified as new potential blood-based biomarkers of the disease.

Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer.

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 µg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability.

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

p53 and p63 Proteoforms Derived from Alternative Splicing Possess Differential Seroreactivity in Colorectal Cancer with Distinct Diagnostic Ability from the Canonical Proteins.

Colorectal cancer (CRC) is the third most common cancer and the second most frequent cause of cancer-related death worldwide. The detection in plasma samples of autoantibodies against specific tumor-associated antigens has been demonstrated to be useful for the early diagnosis of CRC by liquid biopsy. However, new studies related to the humoral immune response in cancer are needed to enable blood-based diagnosis of the disease. Here, our aim was to characterize the humoral immune response associated with the different p53 and p63 proteoforms derived from alternative splicing and previously described as aberrantly expressed in CRC. Thus, here we investigated the diagnostic ability of the twelve p53 proteoforms and the eight p63 proteoforms described to date, and their specific N-terminal and C-terminal end peptides, by means of luminescence HaloTag beads immunoassays. Full-length proteoforms or specific peptides were cloned as HaloTag fusion proteins and their seroreactivity analyzed using plasma from CRC patients at stages I-IV (n = 31), individuals with premalignant lesions (n = 31), and healthy individuals (n = 48). p53γ, Δ40p53ß, Δ40p53γ, Δ133p53γ, Δ160p53γ, TAp63α, TAp63δ, ΔNp63α, and ΔNp63δ, together with the specific C-terminal end α and δ p63 peptides, were found to be more seroreactive against plasma from CRC patients and/or individuals with premalignant lesions than from healthy individuals. In addition, ROC (receiver operating characteristic) curves revealed a high diagnostic ability of those p53 and p63 proteoforms to detect CRC and premalignant individuals (AUC higher than 85%). Finally, electrochemical biosensing platforms were employed in POC-like devices to investigate their usefulness for CRC detection using selected p53 and p63 proteoforms. Our results demonstrate not only the potential of these biosensors for the simultaneous analysis of proteoforms' seroreactivity, but also their convenience and versatility for the clinical detection of CRC by liquid biopsy. In conclusion, we here show that p53 and p63 proteoforms possess differential seroreactivity in CRC patients in comparison to controls, distinctive from canonical proteins, which should improve the diagnostic panels for obtaining a blood-based biomarker signature for CRC detection.

Electrochemical bioplatform for the determination of the most common and carcinogenic human papillomavirus DNA.

Nucleic acid-based analytical bioplatforms have gained importance as diagnostic tests for genomics and as early detection tools for diseases such as cancer. In this context, we report the development of an amperometric bioplatform for the determination of a specific human papillomavirus type 16 (HPV16) sequence. The bioplatform utilizes an immune-nucleic acid hybrid-sandwich assay. A biotinylated RNA capture probe (RNAbCp), complementary to the selected HPV16 target DNA sequence, was immobilised on the surface of streptavidin coated magnetic microbeads (Strep-MBs). The RNA/DNA heteroduplex resulting from the hybridization of the RNAbCP and the HPV16 target sequence was recognised by a commercial antibody that specifically bound to the heteroduplex (AbDNA-RNA). A horseradish-peroxide labeled secondary antibody (antiIgG-HRP) was used for the detection of AbDNA-RNA. Relying on amperometric detection of the resulting HRP-labeled magnetic bioconjugates captured on screen-printed electrodes (SPCEs) in the presence of H2O2 and hydroquinone (HQ), the biotool achieved a low limit of detection (0.5 pM) for the synthetic HPV16 target DNA. In addition, the developed bioplatform was able to discriminate between HPV16 positive and negative human cancer cells using only 25 ng of amplified DNA in a test time of 45 min.

Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases.

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (-0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories.

In-depth quantitative proteomics analysis revealed C1GALT1 depletion in ECC-1 cells mimics an aggressive endometrial cancer phenotype observed in cancer patients with low C1GALT1 expression.

BACKGROUND: Endometrial cancer (EC) is the most common cancer of the female reproductive organs. Despite the good overall prognosis of most low-grade ECs, FIGO I and FIGO II patients might experience tumor recurrence and worse prognosis. The study of alterations related to EC pathogenesis might help to get insights into underlying mechanisms involved in EC development and progression. METHODS: Core tumoral samples were used to investigate the role of C1GALT1 in EC by immunohistochemistry (IHC). ECC-1 cells were used as endometrioid EC model to investigate the effect of C1GALT1 depletion using C1GALT1 specific shRNAs. SILAC quantitative proteomics analyses and cell-based assays, PCR, qPCR, WB, dot-blot and IHC analyses were used to identify, quantify and validate dysregulation of proteins. RESULTS: Low C1GALT1 protein expression levels associate to a more aggressive phenotype of EC. Out of 5208 proteins identified and quantified by LC-MS/MS, 100 proteins showed dysregulation (log2fold-change ≥ 0.58 or ≤-0.58) in the cell protein extracts and 144 in the secretome of C1GALT1 depleted ECC-1 cells. Nine dysregulated proteins were validated. Bioinformatics analyses pointed out to an increase in pathways associated with an aggressive phenotype. This finding was corroborated by loss-of-function cell-based assays demonstrating higher proliferation, invasion, migration, colony formation and angiogenesis capacity in C1GALT1 depleted cells. These effects were associated to the overexpression of ANXA1, as demonstrated by ANXA1 transient silencing cell-based assays, and thus, correlating C1GALT and ANXA1 protein expression and biological effects. Finally, the negative protein expression correlation found by proteomics between C1GALT1 and LGALS3 was confirmed by IHC. CONCLUSION: C1GALT1 stably depleted ECC-1 cells mimic an EC aggressive phenotype observed in patients and might be useful for the identification and validation of EC markers of progression.