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.

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.

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.

Metabolic Reprogramming Helps to Define Different Metastatic Tropisms in Colorectal Cancer.

Approximately 25% of colorectal cancer (CRC) patients experience systemic metastases, with the most frequent target organs being the liver and lung. Metabolic reprogramming has been recognized as one of the hallmarks of cancer. Here, metabolic and functional differences between two CRC cells with different metastatic organotropisms (metastatic KM12SM CRC cells to the liver and KM12L4a to the lung when injected in the spleen and in the tail vein of mice) were analysed in comparison to their parental non-metastatic isogenic KM12C cells, for a subsequent investigation of identified metabolic targets in CRC patients. Meta-analysis from proteomic and transcriptomic data deposited in databases, qPCR, WB, in vitro cell-based assays, and in vivo experiments were used to survey for metabolic alterations contributing to their different organotropism and for the subsequent analysis of identified metabolic markers in CRC patients. Although no changes in cell proliferation were observed between metastatic cells, KM12SM cells were highly dependent on oxidative phosphorylation at mitochondria, whereas KM12L4a cells were characterized by being more energetically efficient with lower basal respiration levels and a better redox management. Lipid metabolism-related targets were found altered in both cell lines, including LDLR, CD36, FABP4, SCD, AGPAT1, and FASN, which were also associated with the prognosis of CRC patients. Moreover, CD36 association with lung metastatic tropism of CRC cells was validated in vivo. Altogether, our results suggest that LDLR, CD36, FABP4, SCD, FASN, LPL, and APOA1 metabolic targets are associated with CRC metastatic tropism to the liver or lung. These features exemplify specific metabolic adaptations for invasive cancer cells which stem at the primary tumour.

In-depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer.

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.

Aryl-hydrocarbon receptor-interacting protein regulates tumorigenic and metastatic properties of colorectal cancer cells driving liver metastasis.

BACKGROUND: Liver metastasis is the primary cause of colorectal cancer (CRC)-associated death. Aryl-hydrocarbon receptor-interacting protein (AIP), a putative positive intermediary in aryl-hydrocarbon receptor-mediated signalling, is overexpressed in highly metastatic human KM12SM CRC cells and other highly metastatic CRC cells. METHODS: Meta-analysis and immunohistochemistry were used to assess the relevance of AIP. Cellular functions and signalling mechanisms mediated by AIP were assessed by gain-of-function experiments and in vitro and in vivo experiments. RESULTS: A significant association of high AIP expression with poor CRC patients' survival was observed. Gain-of-function and quantitative proteomics experiments demonstrated that AIP increased tumorigenic and metastatic properties of isogenic KM12C (poorly metastatic) and KM12SM (highly metastatic to the liver) CRC cells. AIP overexpression dysregulated epithelial-to-mesenchymal (EMT) markers and induced several transcription factors and Cadherin-17 activation. The former induced the signalling activation of AKT, SRC and JNK kinases to increase adhesion, migration and invasion of CRC cells. In vivo, AIP expressing KM12 cells induced tumour growth and liver metastasis. Furthermore, KM12C (poorly metastatic) cells ectopically expressing AIP became metastatic to the liver. CONCLUSIONS: Our data reveal new roles for AIP in regulating proteins associated with cancer and metastasis to induce tumorigenic and metastatic properties in colon cancer cells driving liver metastasis.

Binary MoS2 nanostructures as nanocarriers for amplification in multiplexed electrochemical immunosensing: simultaneous determination of B cell activation factor and proliferation-induced signal immunity-related cytokines.

A dual immunosensor is reported for the simultaneous determination of two important immunity-related cytokines: BAFF (B cell activation factor) and APRIL (a proliferation-induced signal). Sandwich-type immunoassays with specific antibodies (cAbs) and a strategy for signal amplification based on labelling the detection antibodies (dAbs) with binary MoS2/MWCNTs nanostructures and using horseradish peroxidase (HRP) were implemented. Amperometric detection was carried out at screen-printed dual carbon electrodes (SPdCEs) through the hydroquinone HQ/H2O2 system. The developed dual immunosensor provided limit of detection (LOD) of 0.08 and 0.06 ng mL-1 for BAFF and APRIL, respectively, and proved to be useful for the determination of both cytokines in cancer cell lysates and serum samples from patients diagnosed with autoimmune diseases and cancer. The obtained results agreed with those found using ELISA methodologies.

Spatial Proteomic Analysis of Isogenic Metastatic Colorectal Cancer Cells Reveals Key Dysregulated Proteins Associated with Lymph Node, Liver, and Lung Metastasis.

Metastasis is the primary cause of colorectal cancer (CRC) death. The liver and lung, besides adjacent lymph nodes, are the most common sites of metastasis. Here, we aimed to study the lymph nodes, liver, and lung CRC metastasis by quantitative spatial proteomics analysis using CRC cell-based models that recapitulate these metastases. The isogenic KM12 cell system composed of the non-metastatic KM12C cells, liver metastatic KM12SM cells, and liver and lung metastatic KM12L4a cells, and the isogenic non-metastatic SW480 and lymph nodes metastatic SW620 cells, were used. Cells were fractionated to study by proteomics five subcellular fractions corresponding to cytoplasm, membrane, nucleus, chromatin-bound proteins, and cytoskeletal proteins, and the secretome. Trypsin digested extracts were labeled with TMT 11-plex and fractionated prior to proteomics analysis on a Q Exactive. We provide data on protein abundance and localization of 4710 proteins in their different subcellular fractions, depicting dysregulation of proteins in abundance and/or localization in the most common sites of CRC metastasis. After bioinformatics, alterations in abundance and localization for selected proteins from diverse subcellular localizations were validated via WB, IF, IHC, and ELISA using CRC cells, patient tissues, and plasma samples. Results supported the relevance of the proteomics results in an actual CRC scenario. It was particularly relevant that the measurement of GLG1 in plasma showed diagnostic ability of advanced stages of the disease, and that the mislocalization of MUC5AC and BAIAP2 in the nucleus and membrane, respectively, was significantly associated with poor prognosis of CRC patients. Our results demonstrate that the analysis of cell extracts dilutes protein alterations in abundance in specific localizations that might only be observed studying specific subcellular fractions, as here observed for BAIAP2, GLG1, PHYHIPL, TNFRSF10A, or CDKN2AIP, which are interesting proteins that should be further analyzed in CRC metastasis.

Seroreactivity Against Tyrosine Phosphatase PTPRN Links Type 2 Diabetes and Colorectal Cancer and Identifies a Potential Diagnostic and Therapeutic Target.

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.

Multiomics Profiling of Alzheimer's Disease Serum for the Identification of Autoantibody Biomarkers.

New biomarkers of Alzheimer's disease (AD) with a diagnostic value in preclinical and prodromal stages are urgently needed. AD-related serum autoantibodies are potential candidate biomarkers. Here, we aimed at identifying AD-related serum autoantibodies using protein microarrays and mass spectrometry-based methods. To this end, an untargeted complementary screening using high-density (42,100 antigens) and low-density (384 antigens) planar protein-epitope signature tag (PrEST) arrays and an immunoprecipitation protocol coupled to mass spectrometry analysis were used for serum autoantibody profiling. From the untargeted screening phase, 377 antigens corresponding to 338 proteins were selected for validation. Out of them, IVD, CYFIP1, and ADD2 seroreactivity was validated using 128 sera from AD patients and controls by PrEST-suspension bead arrays, and ELISA or luminescence Halotag-based bead immunoassay using full-length recombinant proteins. Importantly, IVD, CYFIP1, and ADD2 showed in combination a noticeable AD diagnostic ability. Moreover, IVD protein abundance in the prefrontal cortex was significantly two-fold higher in AD patients than in controls by western blot and immunohistochemistry, whereas CYFIP1 and ADD2 were significantly down-regulated in AD patients. The panel of AD-related autoantigens identified by a comprehensive multiomics approach may provide new insights of the disease and should help in the blood-based diagnosis of Alzheimer's disease. Mass spectrometry raw data are available in the ProteomeXchange database with the access number PXD028392.

Multiplexed magnetic beads-assisted amperometric bioplatforms for global detection of methylations in nucleic acids.

This work reports the first electrochemical bioplatform developed for the multidetection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in DNA, DNA N6-methyladenine (6mA) and RNA N6-methyladenosine (m6A) methylations at global level. Direct competitive immunoassays were implemented on the surface of magnetic beads (MBs) and optimized for the single amperometric determination of different targets varying in length, sequence and number of methylations on screen-printed carbon electrodes. After evaluating the sensitivity and selectivity of such determinations and the confirmation of no cross-reactivity, a multiplexed disposable platform allowing the simultaneous determination of the mentioned four methylation events in only 45 min has been prepared. The multiplexed bioplatform was successfully applied to the determination of m6A in cellular total RNA and of 5-mC, 5-hmC and 6mA in genomic DNA extracted from tissues. The developed bioplatform showed its usefulness to discriminate the aggressiveness of cancerous cells and between healthy and tumor tissues of colorectal cancer patients.

Phage Microarrays for Screening of Humoral Immune Responses.

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.

Protein Microarrays for Ocular 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.

Magnetic microbeads-based amperometric immunoplatform for the rapid and sensitive detection of N6-methyladenosine to assist in metastatic cancer cells discrimination.

This work describes the preparation of an immunoplatform for the sensitive and selective determination of N6-methyladenosine (m6A). The simple and fast protocol involves for the first time the use of micromagnetic immunoconjugates to establish a direct competitive assay between the m6A target and a biotinylated RNA oligomer bearing a single m6A enzymatically labelled with a commercial conjugate of streptavidin-peroxidase (Strep-HRP) as tracer. The cathodic current change measured in the presence of H2O2/hydroquinone (HQ) at screen-printed carbon electrodes (SPCEs) upon surface capturing the magnetic bioconjugates is inversely proportional to the m6A target concentration. After evaluating the effect of key variables, the analytical characteristics were established for the determination of three different targets: the N6-methyladenosine-5'-triphosphate (m6ATP) ribonucleotide, a short synthetic RNA oligomer bearing a single m6A and the positive control provided in a commercial colorimetric kit for m6A-RNA quantification. The obtained results show that this immunoplatform is competitive with other methods reported to date, achieving an improved sensitivity (limit of detection of 0.9 pM for the short synthetic oligomer) using a much simpler and faster protocol (~1 h) and disposable electrodes for the transduction. Furthermore, the applicability for discriminating the metastatic potential of cancer cells by directly analyzing a small amount of raw total RNA without enriching or fragmenting was also preliminary assessed.

Prognostic Role of Aryl Hydrocarbon Receptor Interacting Protein (AIP) Immunohistochemical Expression in Patients with Resected Gastric Carcinomas.

Aryl hydrocarbon receptor (AHR) interacting protein (AIP) is a chaperone which binds to inactive AHR in the cell cytoplasm. AHR is best known for mediating the toxicity of halogenated aromatics, but it has also been linked to carcinogenesis and tumor progression in several tumor types. Our aims are to assess the features of AIP immunohistochemical (IHC) staining and to evaluate its possible role as a prognostic marker in gastric cancer (GC). Retrospective study of 147 cases of resected GC. Clinicopathological features were collected, tissue microarrays were constructed for AIP IHC and statistical analysis were performed. AIP staining was observed in 50.3% of tumors. All AIP-positive cases exhibited cytoplasmic or membranous staining, variably associated with nuclear co-staining. 93.2% of AIP-positive tumors showed AIP immunoreactivity in 100% of cells. Staining intensity was mild, moderate and intense in 33.8%, 13.5% and 52.7% of cases. Tumors were stratified according to AIP staining intensity into low expression (no or mild AIP immunoreactivity) and high expression (moderate or intense AIP immunoreactivity). 36.6% of our cases showed high AIP expression. High AIP expression was significantly and independently correlated to tumor progression and cancer death. Tumors with high AIP expression showed lower survival and higher progression rates. AIP expression might be useful for determining GC prognosis. More studies are needed to clarify the role of AHR pathway in GC, AIP expression and its potential use as a surrogate marker for selecting patients for AHR modulation therapy.

Multiplexed monitoring of a novel autoantibody diagnostic signature of colorectal cancer using HaloTag technology-based electrochemical immunosensing platform.

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.

Identification of tumor-associated antigens with diagnostic ability of colorectal cancer by in-depth immunomic and seroproteomic analysis.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer related death worldwide. Its diagnosis at early stages would significantly improve the survival of CRC patients. The humoral immune response has been demonstrated useful for cancer diagnosis, predating clinical symptoms up to 3 years. Here, we employed an in-depth seroproteomic approach to identify proteins that elicit a humoral immune response in CRC patients. The seroproteomic approach relied on the immunoprecipitation with patient-derived autoantibodies of proteins from CRC cell lines with different metastatic properties followed by LC-MS/MS. After bioinformatics, we focused on 31 targets of CRC autoantibodies. After WB and IHC validation, ERP44 and TALDO1 showed potential to discriminate disease-free and metastatic CRC patients, and time to recurrence of CRC patients in stage II. Using plasma samples of 30 healthy individuals, 28 premalignant individuals, and 32 CRC patients, nine out of 13 selected targets for seroreactive analysis showed significant diagnostic ability to discriminate either CRC patients or premalignant subjects from controls. Our results suggest that the here defined panel of CRC autoantibodies and their target proteins should be included in CRC blood-based biomarker panels to get a clinically useful blood-based diagnostic signature for CRC detection. SIGNIFICANCE: Colorectal cancer is one of the deadliest cancer types mainly due to its late diagnosis. Its early diagnosis, therefore, is of great importance since it would significantly improve the survival of CRC patients. In our work, the in-depth seroproteomic analysis of colorectal cancer using isolated IgGs from colorectal cancer patients and controls and protein extract of colorectal cancer cells provide the identification of valuable biomarkers with diagnostic and prognostic ability of the disease.

Protein Microarrays: Valuable Tools for Ocular Diseases Research.

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.