Unexpected different chemoselectivity in the aerobic oxidation of methylated planar catechin and bent epicatechin derivatives catalysed by the Trametes villosa laccase/1-hydroxybenzotriazole system

Unreported methylated catechin and epicatechin derivatives 5 and 6 were synthesized by an oxa-Pictet- Spengler reaction. Catechin 5 shows the B and C rings coplanar because of the formation of a trans junction between the C ring and the newly generated six-term cycle D, in turn condensed to ring B. In contrast, epicatechin 6 presents a bent geometry due to the establishment of a cis junction between the C ring and the newly formed cycle D. The oxidation of compounds 5 and 6 in the presence of the Trametes villosa laccase/1-hydroxybenzotriazole (HBT) system was investigated under aerobic conditions in both a biphasic system and a reverse micelle. The unexpected different chemoselective oxidation at the benzylic position of catechin and epicatechin derivatives 5 and 6 has been rationalized using a molecular modelling approach. These results demonstrate that the Trametes villosa laccase/HBT system represents a useful tool to functionalize the C-2 or C-4 position of phenolic compounds depending on the structural features

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high-redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a lignin-related phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction were affected. Thus, quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets

Click-connected 2-(hydroxyimino)aldehydes for the design of UV-responsive functional molecules

Click chemistry is used to functionalize simple lipophilic and water-soluble molecules, a complex PEGylated phospholipid (DSPE-PEG2000), and two benzylic substrates with the 2-(hydroxyimino)aldehyde (HIA) group. To this end, two terminal alkynes bearing the HIA moiety were synthesized and coupled to different azides through copper(I)-catalyzed azide alkyne cycloaddition (CuAAC). Norrish–Yang photoisomerization (λ= 365 nm, LED source) is successfully obtained, with no interference by the triazole linker, except when the forbidden n-π* carbonyl transition is screened by a remote substituent such as salicylaldehyde. UV-Vis spectrometry suggests a specific interaction of HIAs with Cu(II), whereas no such evidence is found with Cu(I). We thereby show that the CuAAC methodology can be used successfully to obtain HIA-based UV-responsive hydrophilic or lipophilic ligands, phospholipidic components for the construction of liposomes, and macrocycle precursors. © 2020 Wiley-VCH Gmb

Capacity building to boost information and communication skills inside an institute of research

To enhance its visibility, the Library of the Istituto Superiore di Sanità (ISS) along with the Scientific Communication Unit of the same institution delivered a set of informal online training sessions, or webinars, on their fields of expertise: information retrieval, publication, effective communication, and research evaluation, specifically addressed to internal users. The collaboration was extremely useful in terms of improved knowledge on skills available among the personnel of these two services. It increased trust in the competencies of internal staff and at the same time it contributed to develop awareness of the value of the services rendered. Skills to use available online resources for training were improved as well as ISS staff cohesion favouring the development of new collaborations

Per un curricolo della lingua italiana nella scuola di base

The curriculum of the Italian language needs, nowadays, to face the complexity of the current socio-cultural situation which is deeply characterized by multimodality and multiculturalism, by the technological development and by the effects of globalization. In the contribution the first reflections collected during a qualitative investigation, run by the research group of the department of Education, Cultural Heritage and Tourism of the University of Macerata (Italy), are discussed. The investigation highlights interesting practices, often innovative, but that also show the need to give a new meaning to the concept of curriculum.Teachers show consciousness of the effects on the teaching/learning process of the Italian language caused by the current class context’s complexity. Their reference to class activities and strategies they usually promote to actively involve students are numerous and include the students’ enciclopedia (language-subject) and their multimodal experiences in the interaction with the world (language-world). But, often, such processes are not connected to the curriculum and a step is missing to let teachers acquire the needed fullawareness on the modalities they activate and let them foster triangulation process among the language- subject, the language-world and the languageobject, that is, among processes of immersion in the contexts and ability of distancing and reflection.Il curricolo di Italiano richiede oggi una riflessione profonda. Tale necessità trova origine nei cambiamenti socio-culturali che derivano dalla complessità dell’attuale situazione socio-culturale profondamente connotata dalla multimodalità e dalla multiculturalità, dallo sviluppo tecnologico e dagli effetti della globalizzazione.In questa sede riportiamo alcune riflessioni scaturite da un’indagine di tipo qualitativo sulle strategie messe in atto nelle classi in relazione alla didattica dell’italiano, condotta dal gruppo di ricerca afferente al Dipartimento di Scienze della Formazione, dei Beni Culturali e del Turismo dell’Università degli Studi di Macerata. Dall’indagine emergono pratiche interessanti e spesso innovative, ma fanno emergere anche l’esigenza di attribuire un nuovo significato al concetto di curricolo. La complessità attuale impatta nel processo di insegnamento/ apprendimento della lingua italiana e molti sono gli esempi di percorsi e strategie attuate dal docente in cui lo studente è coinvolto in modo attivo sul piano linguistico, percorsi che includono la sua enciclopedia (lingua-soggetto) e le sue esperienze multimodali nell’interazione con il mondo (lingua-mondo). Manca però quel passaggio che consenta agli insegnanti di acquisire consapevolezza delle modalità attivate e di costruire una coerenza tra tali processi e il curricolo esplicito, favorendo processi di triangolazione tra lingua-soggetto, lingua-mondo e lingua-oggetto, ovvero tra processi di immersione nei contesti e capacità di distanziamento e riflessione

Investigating the Paracrine Role of Perinatal Derivatives: Human Amniotic Fluid Stem Cell-Extracellular Vesicles Show Promising Transient Potential for Cardiomyocyte Renewal

Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid-derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of R26pFUCCI2 mice with cell cycle fluorescent tagging by mutually exclusive nuclear signal. mNVCM were stimulated by fetal versus perinatal hAFSC-CM and hAFSC-EVs to identify the most promising formulation for in vivo assessment in a R26pFUCCI2 neonatal mouse model of myocardial infarction (MI) via intraperitoneal delivery. While the perinatal hAFSC secretome did not provide any significant cardiogenic effect, fetal hAFSC-EVs significantly sustained mNVCM transition from S to M phase by 2-fold, while triggering cytokinesis by 4.5-fold over vehicle-treated cells. Treated mNVCM showed disorganized expression of cardiac alpha-actinin, suggesting cytoskeletal re-arrangements prior to cell renewal, with a 40% significant downregulation of Cofilin-2 and a positive trend of polymerized F-Actin. Fetal hAFSC-EVs increased cardiomyocyte cell cycle progression by 1.8-fold in the 4-day-old neonatal left ventricle myocardium short term after MI; however, such effect was lost at the later stage. Fetal hAFSC-EVs were enriched with a short isoform of Agrin, a mediator of neonatal heart regeneration acting by YAP-related signaling; yet in vitro application of YAP inhibitor verteporfin partially affected EV paracrine stimulation on mNVCM. EVs secreted by developmentally juvenile fetal hAFSC can support cardiomyocyte renewal to some extension, via intercellular conveyance of candidates possibly involving Agrin in combination with other factors. These perinatal derivative promising cardiogenic effects need further investigation to define their specific mechanism of action and enhance their potential translation into therapeutic opportunity

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a ligninrelated phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction was affected. Quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets.This work was funded by INDOX (KBBE-2013-7-613549) European project and NOESIS (BIO2014-56388-R) and CTQ2013- 48287-R Spanish National Projects. I. P. and G. S. acknowledge the Spanish Research Council (CSIC) and MINECO for their respective predoctoral fellowships.Peer ReviewedPostprint (published version

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool