DEVELOPMENT OF NOVEL HIGH PERFORMANCE PROTEIN MICROARRAYS FOR DIAGNOSTIC APPLICATIONS

Several application of protein microarray technology in diagnostics have been published and a limited number of protein microarrays is currently available on the In Vitro Diagnostics (IVD) market. Albeit several advantages, related to the miniaturization, the multiplexing capability and the possibility of integrating the immunoassays in biosensing devices, microarrays may still lack of specificity or sensitivity. To overcome these limitations and expand the use of protein microarray platform in diagnostics, the present PhD research aimed at developing innovative approaches to increase the assay specificity and sensitivity, reaching very low detection limits, that are compatible with the use of the proposed devices in diagnostics. Furthermore, the use of protein microarrays has been applied to the characterization of emerging biomarkers: exosomes. First of all, surface immobilized hydrogels have been investigated as reagent reservoir for microarray reagents. They have been demonstrated to store reagents in a dry form, stable over days, in a format easy to transport and to preserve. Moreover, they also acted as chambers able to physically separate analytes or reagents which may cross-react with proteins on the printed arrays. In this way the solution was prevented from spreading over the surface and the assays provided sentitive performances, comparable to standard static incubations. In further studies, the complementarity of information provided by fluorescence-based, label-free IRIS and SP-IRIS microarray platforms has been applied to develop immunoassays useful in the diagnostics of Neurodegenerative Disorders. Specifically, two different assay formats have been exploited. The first part of the work focused on the development of a classical sandwich immunoassay able to detect physiological concentrations of Amyloid-beta peptides, biomarkers for Alzheimer\u2019s disease, in both artificial cerebrospinal fluid and real human samples. The second study was aimed at extending the concept of protein microarrays to extracellular vesicles (i.e., exosomes) detection through surface antigen-antibodies recognition. In this innovative application, the nanoparticles were detected with label-free IRIS (total biomass measurements) and SP-IRIS (particle counting and size distribution). In addition, individual particles were incubated with gold-labeled antibodies to identify biomarkers expressed on their surface

Cultural Misconceptions Surrounding Indigenous Populations in the Western Hemisphere

This group has researched common misconceptions that american culture holds regarding Native North Americans and Latinx immigrants. This was a significant topic for the researchers because these misconceptions are the building blocks for cultural prejudice within Americans. Many of the misconceptions that are perpetuated within American culture regarding native peoples and latinx immigrants have severe, and negative repercussions on those populations. Moreover these misconceptions are naturalized within American culture and have distinctively shaped the way in which Americans view and interact with these minority populations. This group’s research revealed that many of the misconceptions held about Native North Americans and Latinx immigrants represent these populations as marginal, simple, and fixed in time. These representations have influenced American culture in many ways; most prominently in the way in which the socially endorsed, representative bodies within American culture have interacted with these populations

Selectively Fluorinated PAMAM-Arginine Conjugates as Gene Delivery Vectors

: Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. However, an "ideal" PAMAM-based gene delivery vector is still missing due to the high manufacturing costs and non-negligible cytotoxicity associated with the use of high-generation dendrimers, whereas low-generation dendrimers are far from displaying efficient gene transfection. In order to cover this gap in the literature, in this study, we propose the functionalization of the outer primary amines of PAMAM G2 and PAMAM G4 with building blocks bearing fluorinated moieties along with a guanidino functional group. We have designed and synthetized two fluorinated arginine (Arg)-based Michael acceptors which were straightforwardly "clicked" to PAMAM dendrimers without the need for coupling reagents and/or catalysts. The obtained conjugates, in particular, derivative 1 formed starting from the low-cost PAMAM G2 and a building block bearing two trifluoromethyl groups, were able to efficiently complex plasmid DNA, had negligible cytotoxicity, and showed improved gene transfection efficiency as compared to undecorated PAMAM dendrimers and a corresponding unfluorinated PAMAM-Arg derivative, with derivative 1 being two orders of magnitude more efficient than the gold standard branched polyethylenimine, bPEI, 25 kDa. These results highlight the importance of the presence of trifluoromethyl moieties for both gene transfection and a possible future application in 19F magnetic resonance imaging

Simplified high-order Volterra series transfer function for optical transmission links

We develop a simplified high-order multi-span Volterra series transfer function (SHMS- VSTF), basing our derivation on the well-known third-order Volterra series transfer function (VSTF). We notice that when applying an approach based on a recursive method and considering the phased-array factor, the order of the expression for the transfer function grows as 3 raised to the number of considered spans. By imposing a frequency-flat approximation to the higher-order terms that are usually neglected in the commonly used VSTF approach, we are able to reduce the overall expression order to the typical third-order plus a complex correction factor. We carry on performance comparisons between the purposed SH-MS-VSTF, the well-known split-step Fourier method (SSFM), and the third-order VSTF. The SH-MS-VSTF exhibits a uniform improvement of about two orders of magnitude in the normalized mean squared deviation with respect to the other methods. This can be translated in a reduction of the overall number of steps required to fully analyze the transmission link up to 99.75% with respect to the SSFM, and 98.75% with respect to the third-order VSTF, respectively, for the same numerical accuracy

Digital detection of exosomes by interferometric imaging

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation

Introduction: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Methods: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: 1) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, 2) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and 3) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, facilitating subsequent characterization and functional analyses. Results: The optimized final procedure incubates 0.5 mg of peptide for 10 min with 10 µL of Long-arm Cobalt agarose before an overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be directly performed on the agarose beads adding lysis or nucleic-acid extraction buffers, or gently eluted with imidazole or EDTA, rendering a fully competent EV preparation. Discussion: This new isolation methodology is based on the recognition of general membrane characteristics independent of surface markers. It is thus unbiased and can be used in any species EV sample, even in samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, less time-consuming, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down, with the possibility of analyzing very low amounts of sample, and it is compatible with any downstream analyses thanks to the gentle elution procedureThis work has been supported by grants PID 2020-119627GB-I00 and DTS21/00134 from Ministerio Español de Ciencia e Innovación (Spain) from to MY-M. BB is supported by predoctoral industrial contract IND2019_BMD-17100 and JM by a INV-CAM-03 Yo Investigo contract, both from Comunidad Autónoma de Madrid. Work was partially funded from the European Union’s Horizon 2020 research and innovation program under grant agreements No. 951768 (project MARVEL

Education as a strategy for managing occupational-related musculoskeletal pain:a scoping review

BackgroundMusculoskeletal (MSK) pain is the primary contributor to disability worldwide. There is a growing consensus that MSK pain is a recurrent multifactorial condition underpinned by health and lifestyle factors. Studies suggest that education on work-related pain and individualised advice could be essential and effective for managing persistent MSK pain.ObjectiveThe objective of this scoping review was to map the existing educational resources for work-related MSK (WRMSK) pain, and the effects of implementing educational strategies in the workplace on managing WRMSK pain.MethodsThis scoping review assessed original studies that implemented and assessed education as a strategy to manage WMSK pain. Literature search strategies were developed using thesaurus headings (ie, MeSH and CINAHL headings) and free-text search including words related to MSK in an occupational setting. The search was carried out in PubMed, CINAHL, Cochrane Library and Web of Science in the period 12–14 February 2019.ResultsA total of 19 peer-reviewed articles were included and the study design, aim and outcomes were summarised. Of the 19 peer-reviewed articles, 10 randomised controlled trial (RCT) studies assessed the influence of education on work-related MSK pain. Many studies provided a limited description of the education material and assessed/used different methods of delivery. A majority of studies concluded education positively influences work-related MSK pain. Further, some studies reported additive effects of physical activity or ergonomic adjustments.ConclusionsThere is a gap in knowledge regarding the best content and delivery of education of material in the workplace. Although beneficial outcomes were reported, more RCT studies are required to determine the effects of education material as compared with other interventions, such as exercise or behavioural therapy

Addressing Heterogeneity in Direct Analysis of Extracellular Vesicles and Their Analogs by Membrane Sensing Peptides as Pan‐Vesicular Affinity Probes

: Extracellular vesicles (EVs), crucial mediators of cell-to-cell communication, hold significant diagnostic potential due to their ability to concentrate protein biomarkers in bodily fluids. However, challenges in isolating EVs from biological specimens hinder their widespread use. The preferred strategy involves direct analysis, integrating isolation and analysis solutions, with immunoaffinity methods currently dominating. Yet, the heterogeneous nature of EVs poses challenges, as proposed markers may not be as universally present as thought, raising concerns about biomarker screening reliability. This issue extends to EV-mimics, where conventional methods may lack applicability. Addressing these challenges, the study reports on Membrane Sensing Peptides (MSP) as pan-vesicular affinity ligands for both EVs and their non-canonical analogs, streamlining capture and phenotyping through Single Molecule Array (SiMoA). MSP ligands enable direct analysis of circulating EVs, eliminating the need for prior isolation. Demonstrating clinical translation, MSP technology detects an EV-associated epitope signature in serum and plasma, distinguishing myocardial infarction from stable angina. Additionally, MSP allow analysis of tetraspanin-lacking Red Blood Cell-derived EVs, overcoming limitations associated with antibody-based methods. Overall, the work underlines the value of MSP as complementary tools to antibodies, advancing EV analysis for clinical diagnostics and beyond, and marking the first-ever peptide-based application in SiMoA technology

A Proposal of Conformance Tests for CDVS

The MPEG-7 standard has specified an image description tool designed to enable efficient and interoperable visual search applications, allowing visual content matching in images. Visual content matching includes matching of views of objects, landmarks, and printed documents, while being robust to partial occlusions as well as changes in viewpoint, camera parameters, and lighting conditions. Since this has led to a novel set of specifications of the normative parts for this type of standard, conformance testing requires a new approach. This input document proposes a methodology that is being considered for such a purpose