Losartan improves the therapeutic effect of metronomic cyclophosphamide in triple negative mammary cancer models

Metronomic chemotherapy refers to the minimum biologically effective doses of a chemotherapy agent given as a continuous regimen without extended rest periods. Drug repurposing is defined as the use of an already known drug for a new medical indication, different from the original one. In oncology the combination of these two therapeutic approaches is called “Metronomics”. The aim of this work is to evaluate the therapeutic effect of cyclophosphamide in a metronomic schedule in combination with the repurposed drug losartan in two genetically different mice models of triple negative breast cancer. Our findings showed that adding losartan to metronomic cyclophosphamide significantly improved the therapeutic outcome. In both models the combined treatment increased the mice's survival without sings of toxicity. Moreover, we elucidated some of the mechanisms of action involved, which include a decrease of intratumor hypoxia, stimulation of the immune response and remodeling of the tumor microenvironment. The remarkable therapeutic effect, the lack of toxicity, the low cost of the drugs and its oral administration, strongly suggest its translation to the clinical setting in the near future.Fil: Mainetti, Leandro Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Rico, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Kaufman, Cintia Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Grillo, Monica Carolina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Guercetti, Julian. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Baglioni, María Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: del Giúdice, Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Capitani, María Celeste. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Fusini, Matías. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Rozados, Viviana Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; ArgentinaFil: Scharovsky, Olga Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Medicas. Instituto de Genetica Experimental; Argentina. Metronomics Global Health Initiative; Franci

Microarray Strategies for Multiplexed Immunochemical Analysis

[eng] The current doctoral thesis titled “Microarray Strategies for Multiplexed Immunochemical Analysis” was carried out in the context of two research projects, expecting to develop multiplexed ligand-binding assays under microarray format for universal applications. On one side, framed by the FoodSmartphone project (ITN-Marie Curie Actions Horizon 2020) the PART I of the thesis was dedicated to generate a multiplexed and high-throughput screening platform based on DNA-directed immobilization (DDI) strategies for the detection of antibiotic residues in cow’s milk according to EU regulations. For this, three class selective monoclonal antibodies, in-house produced, targeting fluoroquinolones, sulfonamides and tylosin were utilized in an indirect competitive immunoassay format, achieving limits of detection in the order of ppb’s. The use of oligonucleotide strains conjugated to hapten molecules allowed site encoding the signal of each hapten-antibody pair, using a fluorescently labelled secondary antibodies for detection. After assay characterization a pre-validation study was performed with the idea to evaluate the application of the novel platform in blind samples, allowing the identification of at least 18 veterinary residues according to regulatory limits and with a sample processing capacity of 180 samples per run in less than 90 minutes. As part of the same project, the implementation of the immunoreagents utilized in the DDI microarray was evaluated in a portable and miniaturized imaging surface plasmon resonance (iSPR) sensor combining smartphone readout system as a proof of concept. It was possible to evidence the binding between one of the monoclonal antibodies to the respective hapten molecule in a label free configuration, for a promising transition towards more user-friendly and cost-effective bioanalytical tools coupled to smartphone systems. On the other hand, the PART II of the thesis was carried out in the context of the PoC4CoV project, attempting to produce point-of-care tests for the rapid detection of SARS-CoV-2 at different levels. Through this work the manufacture of a microarray chip based on SARS-CoV-2 peptides and proteins for personalized immunodiagnostic of COVID-19 was conducted. Initially, a rational design of potential immunogenic linear peptides (15-mer) derived from S and N proteins of the virus was proposed. A total of 28 peptide sequences were selected through computational models and immunogenicity studies and then synthesized also including peptides derived from variants of concern. After platform optimization studies, a multiplexed and high- throughput hybrid microarray chip constituted by full length viral proteins and the selected sequences defined the so called Immuno-μSARS2 chip. This microarray allowed simultaneous IgG and IgM personalized profiling against the selected panel of epitopes with minimum sample requirements, offering an affordable and easy to implement immunodiagnostic tool. The Immuno-μSARS2 chip was successfully implemented in more than 750 human serum samples, achieving a 98 % of clinical sensitivity and 91 % of clinical specificity respecting to the RT-PCR. To extend the potential use of the chip, the integration of artificial intelligence was combined with sample analysis to define immunological signatures based on IgG response disease severity prediction using samples classified according to COVID-19 clinical outcomes. Our platform was able to discriminate with an 81 % of accuracy patients progressing towards irreversible disease condition (Exitus), followed by 70 % accuracy those patients admitted to ICU and with a discriminate in a 66 % those requiring hospitalization, from asymptomatic disease course. At the same time, a few peptide sequences were found as promising immunodiagnostic elements that could potentially replace the use of full-length proteins to generate affordable tests in the future. The methodology applied over this experimental section can be easily translated into other viruses or diseases expecting to improve diagnostic and prognostic performance

Microarray Strategies for Multiplexed Immunochemical Analysis

Programa de Doctorat en Biotecnologia / Tesi realitzada a l'Institut de Química Avançada de Catalunya (IQAC-CSIC)[eng] The current doctoral thesis titled “Microarray Strategies for Multiplexed Immunochemical Analysis” was carried out in the context of two research projects, expecting to develop multiplexed ligand-binding assays under microarray format for universal applications. On one side, framed by the FoodSmartphone project (ITN-Marie Curie Actions Horizon 2020) the PART I of the thesis was dedicated to generate a multiplexed and high-throughput screening platform based on DNA-directed immobilization (DDI) strategies for the detection of antibiotic residues in cow’s milk according to EU regulations. For this, three class selective monoclonal antibodies, in-house produced, targeting fluoroquinolones, sulfonamides and tylosin were utilized in an indirect competitive immunoassay format, achieving limits of detection in the order of ppb’s. The use of oligonucleotide strains conjugated to hapten molecules allowed site encoding the signal of each hapten-antibody pair, using a fluorescently labelled secondary antibodies for detection. After assay characterization a pre-validation study was performed with the idea to evaluate the application of the novel platform in blind samples, allowing the identification of at least 18 veterinary residues according to regulatory limits and with a sample processing capacity of 180 samples per run in less than 90 minutes. As part of the same project, the implementation of the immunoreagents utilized in the DDI microarray was evaluated in a portable and miniaturized imaging surface plasmon resonance (iSPR) sensor combining smartphone readout system as a proof of concept. It was possible to evidence the binding between one of the monoclonal antibodies to the respective hapten molecule in a label free configuration, for a promising transition towards more user-friendly and cost-effective bioanalytical tools coupled to smartphone systems. On the other hand, the PART II of the thesis was carried out in the context of the PoC4CoV project, attempting to produce point-of-care tests for the rapid detection of SARS-CoV-2 at different levels. Through this work the manufacture of a microarray chip based on SARS-CoV-2 peptides and proteins for personalized immunodiagnostic of COVID-19 was conducted. Initially, a rational design of potential immunogenic linear peptides (15-mer) derived from S and N proteins of the virus was proposed. A total of 28 peptide sequences were selected through computational models and immunogenicity studies and then synthesized also including peptides derived from variants of concern. After platform optimization studies, a multiplexed and high- throughput hybrid microarray chip constituted by full length viral proteins and the selected sequences defined the so called Immuno-μSARS2 chip. This microarray allowed simultaneous IgG and IgM personalized profiling against the selected panel of epitopes with minimum sample requirements, offering an affordable and easy to implement immunodiagnostic tool. The Immuno-μSARS2 chip was successfully implemented in more than 750 human serum samples, achieving a 98 % of clinical sensitivity and 91 % of clinical specificity respecting to the RT-PCR. To extend the potential use of the chip, the integration of artificial intelligence was combined with sample analysis to define immunological signatures based on IgG response disease severity prediction using samples classified according to COVID-19 clinical outcomes. Our platform was able to discriminate with an 81 % of accuracy patients progressing towards irreversible disease condition (Exitus), followed by 70 % accuracy those patients admitted to ICU and with a discriminate in a 66 % those requiring hospitalization, from asymptomatic disease course. At the same time, a few peptide sequences were found as promising immunodiagnostic elements that could potentially replace the use of full-length proteins to generate affordable tests in the future. The methodology applied over this experimental section can be easily translated into other viruses or diseases expecting to improve diagnostic and prognostic performance

Bioreceptors for smartphone-based food contaminants detection

A bioreceptor is defined as a biological element that specifically recognizes and binds to its ligand. Receptors from natural origin are characterized by their high affinity and selectivity for a target analyte. In the biosensing field, they are generally used to detect molecules in complex matrices such as milk, tissues and vegetable extracts, feed or urine. The classification of the variety of bioreceptors available is based on their chemical and biological nature. Baring this aspect in mind, they are classified in antibodies, enzymes, nucleic acids or emerging biomimetic receptors. Each one presents certain advantages and limitations considering the target analyte, matrix, affinity, stability, cost or even sensing principle. Bioreceptors became relevant in the food safety field due to their capabilities to identify a wide variety of harmful substances in nutritional matrixes in short periods of time. Also, food sources need to undergo strict regulatory controls to ensure quality and safety for consumers. Nowadays, conventional confirmatory techniques (e.g., chromatographic methods) are based on long sample preparation protocols and therefore, are not suitable for fast and high-throughput analysis. Furthermore, they are generally performed by trained personnel and require expensive equipment. On the other hand, screening biosensing platforms have emerged as alternative tools to process hundreds of samples, with reduced costs and analysis time. Gold standard methods employ biological elements for recognition of distinct target analytes including enzyme linked immunosorbent assay (ELISA) for antibodies, enzymatic assays or polymerase chain reaction (PCR) and its variants for nucleic acids. The integration of biorecognition elements with miniaturized platforms, e.g., smartphones, extend the possibilities for the development of reliable biosensing platforms. In this work, we provide an overview of the most widely used bioreceptors mainly in the food safety field but also including medical and environmental applications to discuss their implementation over biosensors development with special emphasis on those that make use of smartphone readout.This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 720325, FoodSmartphone. We also thank the denomination of consolidated group from the Generalitat de Catalunya: 2017 SGR 1441 (M.-P. Marco). This research was supported by CIBER-BBN, ISCIII, Ministerio de Ciencia e Innovación and Unión Europea – European Regional Development Fund. The group also wants to acknowledge the support from Interdisciplinary Platform Global Health, CSIC.Peer reviewe

DNA directed immobilization (DDI) for the development of an antibody fluorescent microarray for the determination of three family antibiotic residues in milk.

Misuse of antibiotics in food producing animals enhances the possibility to develop antibiotic resistance and can also lead to the appearance of these compounds in derivate products. New strategies to overcome this issue are required. In this context, the FoodSmartphone project will provide analytical tools to ensure food quality concerning pesticides, veterinary products or allergens, from farm to fork. The first approach is based on fluorescent microarrays which emerge as potential screening tools to determine the presence of multiple analytes at the same time. In this study, we present a multiplexed analytical platform based on hapten-oligonucleotide arrays to detect three families of antibiotics. The use of hapten-oligonucleotide conjugates allows the directed immobilization (DDI) of the signal for each antibiotic family by immobilizing the complementary oligo-probes over the surface of glass slide. After hybridization, the cocktail of primary antibodies is added in the samples and this binding is elucidated by the addition of secondary antibodies labeled to a fluorophore which provide the fluorescent signal. Using this system a group of penicillin, sulfonamides and tylosin can be quantified on buffer and milk samples. The format of this assay will provide the basis for its implementation on a Smartphone readout system

ASSURED Point-of-Need Food Safety Screening: A Critical Assessment of Portable Food Analyzers

Standard methods for chemical food safety testing in official laboratories rely largely on liquid or gas chromatography coupled with mass spectrometry. Although these methods are considered the gold standard for quantitative confirmatory analysis, they require sampling, transferring the samples to a central laboratory to be tested by highly trained personnel, and the use of expensive equipment. Therefore, there is an increasing demand for portable and handheld devices to provide rapid, efficient, and on-site screening of food contaminants. Recent technological advancements in the field include smartphone-based, microfluidic chip-based, and paper-based devices integrated with electrochemical and optical biosensing platforms. Furthermore, the potential application of portable mass spectrometers in food testing might bring the confirmatory analysis from the laboratory to the field in the future. Although such systems open new promising possibilities for portable food testing, few of these devices are commercially available. To understand why barriers remain, portable food analyzers reported in the literature over the last ten years were reviewed. To this end, the analytical performance of these devices and the extent they match the World Health Organization benchmark for diagnostic tests, i.e., the Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end-users (ASSURED) criteria, was evaluated critically. A five-star scoring system was used to assess their potential to be implemented as food safety testing systems. The main findings highlight the need for concentrated efforts towards combining the best features of different technologies, to bridge technological gaps and meet commercialization requirements