Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases

[EN] In this paper we present the development of photonic integrated circuit (PIC) biosensors for the label-free detection of six emerging and endemic swine viruses, namely: African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PPRSV), Porcine Parvovirus (PPV), Porcine Circovirus 2 (PCV2), and Swine Influenza Virus A (SIV). The optical biosensors are based on evanescent wave technology and, in particular, on Resonant Rings (RRs) fabricated in silicon nitride. The novel biosensors were packaged in an integrated sensing cartridge that included a microfluidic channel for buffer/sample delivery and an optical fiber array for the optical operation of the PICs. Antibodies were used as molecular recognition elements (MREs) and were selected based on western blotting and ELISA experiments to ensure the high sensitivity and specificity of the novel sensors. MREs were immobilized on RR surfaces to capture viral antigens. Antibody-antigen interactions were transduced via the RRs to a measurable resonant shift. Cell culture supernatants for all of the targeted viruses were used to validate the biosensors. Resonant shift responses were dose-dependent. The results were obtained within the framework of the SWINOSTICS project, contributing to cover the need of the novel diagnostic tools to tackle swine viral diseases.This work was funded by the EU-2020 program under grant agreement Nº 771649-SWINOSTICS project.Gómez-Gómez, MI.; Sánchez, C.; Peransi, S.; Zurita, D.; Bellieres, L.; Recuero, S.; Rodrigo, M.... (2022). Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases. Sensors. 22(3):1-14. https://doi.org/10.3390/s2203070811422

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations

IntroductionRecombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation.MethodsIn this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter.Results and discussionStarting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients

IN AND OUT UNIVERSITY CLASSROOMS: FACEBOOK AS A TOOL OF CONSTRUCTION OF KNOWLEDGE

Nowadays social networks can be considered as psycho-technological environments (De Kerckove, 1999) [1] with a high identity-making value. Pedagogy cannot ignore these new places, with the aim of making formal training increasingly more ‘connected’ to informal training through Social networks, as Facebook, Twitter, Flickr. This paper aims to present an action-research that is based on new and innovative ways to incorporate technology and social media into teaching methodology (Joosten, 2012) [2], as forms of distance and online education programs. While until now most popular researches try to study the social network influence on young people and on their academic performances (Helou & Rahim, 2012) [3], to analyse the content of user profiles, and try to diversify users and nonusers, our action-research explores the pedagogical relevances of Facebook in interactive laboratorial experiences, carried out with graduating students in the academic year 2012/2013 at the University of Naples "Federico II". We tried to implement frontal lessons with social networks technologies creating a closed group on Facebook and adopting a questionnaire (created ad hoc and tested with a pilot study) to evaluate students’ beliefs on educational scopes of Facebook and its usefulness for teaching learning. Through the exploratory experience of the Facebook groups, students have also realized that externalization/construction of meaning and knowledge is not an individual but a social community act. The appearance, therefore, more interesting is the ability for students to turn knowledge from tacit to explicit. Using metaphors, analogies, and especially through reflections on experiences in common, students are able to explain and to share knowledge, which is difficult to express through purely frontal teaching methods. Teachers, as “digital immigrants”, do not always seem to grasp the potentiality of social networks, which are the preferential medium of exchange and self-representation of their students, considered as “native digitals” (Prensky, 2001) [4]. Native digitals require a rethinking of the traditional didactic structure, employing e-learning usability. The surprising results of our research demonstrate that according to students, Facebook should be useful for educational practices, first as a platform and as an open virtual space for instructional materials and sharing narratives, second as a virtual community of research, where it’s possible to coconstruct knowledge and reflexion upon contents and upon themselves (Strollo, 2012) [5]

IN AND OUT UNIVERSITY CLASSROOMS: FACEBOOK AS A TOOL OF CONSTRUCTION OF KNOWLEDGE

Nowadays social networks can be considered as psycho-technological environments (De Kerckove, 1999) [1] with a high identity-making value. Pedagogy cannot ignore these new places, with the aim of making formal training increasingly more ‘connected’ to informal training through Social networks, as Facebook, Twitter, Flickr. This paper aims to present an action-research that is based on new and innovative ways to incorporate technology and social media into teaching methodology (Joosten, 2012) [2], as forms of distance and online education programs. While until now most popular researches try to study the social network influence on young people and on their academic performances (Helou & Rahim, 2012) [3], to analyse the content of user profiles, and try to diversify users and nonusers, our action-research explores the pedagogical relevances of Facebook in interactive laboratorial experiences, carried out with graduating students in the academic year 2012/2013 at the University of Naples "Federico II". We tried to implement frontal lessons with social networks technologies creating a closed group on Facebook and adopting a questionnaire (created ad hoc and tested with a pilot study) to evaluate students’ beliefs on educational scopes of Facebook and its usefulness for teaching learning. Through the exploratory experience of the Facebook groups, students have also realized that externalization/construction of meaning and knowledge is not an individual but a social community act. The appearance, therefore, more interesting is the ability for students to turn knowledge from tacit to explicit. Using metaphors, analogies, and especially through reflections on experiences in common, students are able to explain and to share knowledge, which is difficult to express through purely frontal teaching methods. Teachers, as “digital immigrants”, do not always seem to grasp the potentiality of social networks, which are the preferential medium of exchange and self-representation of their students, considered as “native digitals” (Prensky, 2001) [4]. Native digitals require a rethinking of the traditional didactic structure, employing e-learning usability. The surprising results of our research demonstrate that according to students, Facebook should be useful for educational practices, first as a platform and as an open virtual space for instructional materials and sharing narratives, second as a virtual community of research, where it’s possible to coconstruct knowledge and reflexion upon contents and upon themselves (Strollo, 2012) [5]

Characterization of Two NMN Deamidase Mutants as Possible Probes for an NMN Biosensor

Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor