STUDIO DEL PROCESSO DI MATURAZIONE ED ATTIVAZIONE DI FOSFOLIPASI A2 FUNGINE

TbSP1 (Tuber borchii secreted protein 1) è una fosfolipasi che si accumula nello strato interno della parete cellulare e nello spazio extracellulare in miceli, corpi fruttiferi e micorrize del fungo simbiotico T. borchii. Un’analisi dei livelli di mRNA di TbSP1 in funghi cresciuti in vivo in differenti condizioni nutrizionali ha evidenziato una regolazione in risposta alla disponibilità di nutrienti. In particolare, terreni carenti di carbonio o di azoto, inducono un significativo aumento nell’espressione di TbSP1. E’ stato precedentemente dimostrato che la proteina è in grado di attivarsi mediante auto rimozione della porzione N-terminale sia in vitro che in vivo. Il meccanismo del processo di digestione di TbSP1 è di tipo intermolecolare ed endoproteasico. Il sito proteasico è localizzato sul frammento di 14.5 kDa ed è distinto dal sito fosfolipasico. Nel presente lavoro di tesi viene descritta la caratterizzazione biochimica di TbSP1 con la determinazione dei parametri cinetici della reazione fosfolipasica così come delle costanti di affinità per il calcio di TbSP1. E’ stata inoltre analizzata l’importanza della presenza dello ione calcio in relazione alle due attività di TbSP1 e sono stati condotti esperimenti al fine di identificare i residui responsabili dell’attività proteasica. Inoltre, TbSP1 viene confrontata con altre fosfolipasi A2 fungine. In particolare è stata indagata la possibilità che due proteine da T. melanosporum e N. crassa, omologhe a TbSP1, abbiano lo stesso meccanismo di maturazione ed attivazione. Infine, viene analizzata l’attività fosfolipasica e l’eventuale capacità di digestione in trans di una forma stabile di TbSP1 da parte di alcune tra le principali PLA2 descritte in letteratura

the allergen mus m 1 0102 cysteine residues and molecular allergology

Abstract Mus m 1.0102 is a member of the mouse Major Urinary Protein family, belonging to the Lipocalins superfamily. Major Urinary Proteins (MUPs) are characterized by highly conserved structural motifs. These include a disulphide bond, involved in protein oxidative folding and protein structure stabilization, and a free cysteine residue, substituted by serine only in the pheromonal protein Darcin (MUP20). The free cysteine is recognized as responsible for the onset of inter- or intramolecular thiol/disulphide exchange, an event that favours protein aggregation. Here we show that the substitution of selected cysteine residues modulates Mus m 1.0102 protein folding, fold stability and unfolding reversibility, while maintaining its allergenic potency. Recombinant allergens used for immunotherapy or employed in allergy diagnostic kits require, as essential features, conformational stability, sample homogeneity and proper immunogenicity. In this perspective, recombinant Mus m 1.0102 might appear reasonably adequate as lead molecule because of its allergenic potential and thermal stability. However, its modest resistance to aggregation renders the protein unsuitable for pharmacological preparations. Point mutation is considered a winning strategy. We report that, among the tested mutants, C138A mutant acquires a structure more resistant to thermal stress and less prone to aggregation, two events that act positively on the protein shelf life. Those features make that MUP variant an attractive lead molecule for the development of a diagnostic kit and/or a vaccine

Early versus delayed antiretroviral therapy based on genotypic resistance test: Results from a large retrospective cohort study

Rapid start of antiretroviral therapy (ART) pending genotypic resistance test (GRT) has been recently proposed, but the effectiveness of this strategy is still debated. The rate of virological success (VS), defined as HIV-RNA\u2009<\u200950 copies/ml, with and without GRT was compared in drug-na\uefve individuals enrolled in the Italian ARCA cohort who started ART between 2015 and 2018. 521 individuals started ART: 397 without GRT (pre-GRT group) and 124 following GRT (post-GRT group). Overall, 398 (76%) were males and 30 (6%) were diagnosed with AIDS. In the pre-GRT group, baseline CD4+\u2009cell counts were lower (p\u2009<\u20090.001), and viral load was higher (p\u2009<\u20090.001) than in the post-GRT group. The estimated probability of VS in pre-GRT versus post-GRT group was 72.54% (CI95 : 67.78-76.60) versus 66.94% (CI95 : 57.53-74.26) at Week 24 and 92.40% (CI95 : 89.26-94.62) versus 92.92% (CI95 : 86.35-96.33) at Week 48, respectively (p\u2009=\u20090.434). At Week 48, VS was less frequent among individuals with baseline CD4+\u2009cell counts <200 versus >500 (90.33% vs. 97.33%), log viral load <5.00 versus >5.70 log10 cps/ml (97.17% vs 78.16%; p\u2009<\u20090.001), and those treated with protease inhibitors or non-nucleoside reverse transcriptase inhibitors versus those treated with integrase strand transfer inhibitors (p\u2009<\u20090.001). The rate of VS does not seem to be affected by an early ART initiation pending GRT results, but it could be influenced by the composition of the ART regimen, as well as immuno-virological parameters

Real-life data on potential drug-druginteractions in patients with chronic hepatitis C viral infection undergoing antiviral therapy with interferon-free DAAs in the PITER Cohort Study

Background There are few real-life data on the potential drug-drug interactions (DDIs) between anti-HCV direct-acting antivirals (DAAs) and the comedications used. Aim To assess the potential DDIs of DAAs in HCV-infected outpatients, according to the severity of liver disease and comedication used in a prospective multicentric study. Methods Data from patients in 15 clinical centers who had started a DAA regimen and were receiving comedications during March 2015 to March 2016 were prospectively evaluated. The DDIs for each regimen and comedication were assigned according to HepC Drug Interactions (www.hep-druginteractions.org) Results Of the 449 patients evaluated, 86 had mild liver disease and 363 had moderate-to-severe disease. The use of a single comedication was more frequent among patients with mild liver disease (p = 0.03), whereas utilization of more than three drugs among those with moderate- to-severe disease (p = 0.05). Of the 142 comedications used in 86 patients with mild disease, 27 (20%) may require dose adjustment/closer monitoring, none was contraindicated. Of the 322 comedications used in 363 patients with moderate-to-severe liver disease, 82 (25%) were classified with potential DDIs that required only monitoring and dose adjustments; 10 (3%) were contraindicated in severe liver disease. In patients with mild liver disease 30% (26/86) used at least one drug with a potential DDI whereas of the 363 patients with moderate-to-severe liver disease, 161 (44%) were at risk for one or more DDI. Conclusions Based on these results, we can estimate that 30 - 44% of patients undergoing DAA and taking comedications are at risk of a clinically significant DDI. This data indicates the need for increased awareness of potential DDI during DAA therapy, especially in patients with moderate- to-severe liver disease. For several drugs, the recommendation related to the DDI changes from "dose adjustment/closer monitoring", in mild to moderate liver disease, to "the use is contraindicated" in severe liver disease

Impact of COVID-19 pandemic on outpatient visit volume in cancer patients: Results of COMETA multicenter retrospective observational study

ObjectiveTo evaluate the impact of the COVID-19 pandemic on first and follow-up visits for cancer outpatients.MethodsThis is a multicenter retrospective observational study involving three Comprehensive Cancer Care Centers (CCCCs): IFO, including IRE and ISG in Rome, AUSL-IRCCS of Reggio Emilia, and IRCCS Giovanni Paolo II in Bari) and one oncology department in a Community Hospital (Saint'Andrea Hospital, Rome). From 1 January 2020 and 31 December 2021, we evaluated the volume of outpatient consultations (first visits and follow-up), comparing them with the pre-pandemic year (2019). Results were analyzed by quarter according to the Rt (real-time indicator used to assess the evolution of the pandemic). IFO and IRCCS Giovanni Paolo II were “COVID-free” while AUSL-IRCCS RE was a “COVID-mixed” Institute. Depending on the Rt, Sain't Andrea Hospital experienced a “swinging” organizational pathway (COVID-free/ COVID-mixed).ResultsRegarding the “first appointments”, in 2020 the healthcare facilities operating in the North and Center of Italy showed a downward trend. In 2021, only AUSL-IRCCS RE showed an upward trend. Regarding the “follow-up”, only AUSL IRCCS RE showed a slight up-trend in 2020. In 2021, IFO showed an increasing trend, while S. Andrea Hospital showed a negative plateau. Surprisingly, IRCCS Giovanni Paolo II in Bari showed an uptrend for both first appointment and follow-ups during pandemic and late pandemic except for the fourth quarter of 2021.ConclusionsDuring the first pandemic wave, no significant difference was observed amongst COVID-free and COVID-mixed Institutes and between CCCCs and a Community Hospital. In 2021 (“late pandemic year”), it has been more convenient to organize COVID-mixed pathway in the CCCCs rather than to keep the Institutions COVID-free. A swinging modality in the Community Hospital did not offer positive results in term of visit volumes. Our study about the impact of COVID-19 pandemic on visit volume in cancer outpatients may help health systems to optimize the post-pandemic use of resources and improve healthcare policies

Intravenous methylprednisolone pulses in hospitalised patients with severe COVID-19 pneumonia, A double-blind, randomised, placebo-controlled trial

Rationale: Pulse glucocorticoid therapy is used in hyperinflammation related to coronavirus 2019 (COVID-19). We evaluated the efficacy and safety of pulse intravenous methylprednisolone in addition to standard treatment in COVID-19 pneumonia. Methods: In this multicenter, randomised, double-blind, placebo-controlled trial, 304 hospitalised patients with Covid-19 pneumonia were randomised to receive 1 g of methylprednisolone intravenously for 3 consecutive days or placebo in addition to standard dexamethasone. The primary outcome was the duration of the patient hospitalisation, calculated as the time interval between randomisation and hospital discharge without the need of supplementary oxygen. The key secondary outcomes were survival free from invasive ventilation with orotracheal intubation and overall survival. Results: Overall, 112 of 151 (75.4%) patients in the pulse methylprednisolone arm and 111 of 150 (75.2%) in the placebo arm were discharged from hospital without oxygen within 30 days from randomisation. Median time to discharge was similar in both groups [15 days (95% confidence interval (CI), 13.0 to 17.0) and 16 days (95%CI, 13.8 to 18.2); hazard ratio (HR), 0.92; 95% CI 0.71-1.20; p=0.528]. No significant differences between pulse methylprednisolone and placebo arms were observed in terms of admission to Intensive Care Unit with orotracheal intubation or death (20.0% versus 16.1%; HR, 1.26; 95%CI, 0.74-2.16; p=0.176), or overall mortality (10.0% versus 12.2%; HR, 0.83; 95%CI, 0.42-1.64; p=0.584). Serious adverse events occurred with similar frequency in the two groups. Conclusions: Methylprenisolone pulse therapy added to dexamethasone was not of benefit in patients with COVID-19 pneumonia. Message of the study: Pulse glucocorticoid therapy is used for severe and/or life threatening immuno-inflammatory diseases. The addition of pulse glucocorticoid therapy to the standard low dose of dexamethasone scheme was not of benefit in patients with COVID-19 pneumonia

Safety assessment of titanium dioxide (E171) as a food additive

Acknowledgements: The Panel wishes to thank the following for the support provided to this scientific output: Ana Campos Fernandes, Laura Ciccolallo, Esraa Elewa, Galvin Eyong, Christina Kyrkou, Irene Munoz, Giorgia Vianello, the members of the SCER Cross-cutting WG nanotechnologies: Jacqueline Castenmiller, Mohammad Chaudhry, Roland Franz, David Gott, Stefan Weigel and the former member of the SCER Cross-cutting WG Genotoxicity Maciej Stepnik. The FAF Panel wishes to acknowledge all European competent institutions, Member State bodies and other organisations that provided data for this scientific output.Peer reviewedPublisher PD

Conserved cysteines mutagenesis in Major Urinary Protein: a new insight in folding stability and reversibility

INTRODUCTION: The mouse Major Urinary Proteins constitute a family of pheromone binding proteins, while representing the major mouse allergen for humans. They belong to the Lipocalins superfamily sharing an eight-stranded, antiparallel β-barrel fold stabilized by a highly conserved disulfide bridge. Here, we have investigated the role of the disulfide bridge (C64-C157) and of the free C138 residue on the protein folding stability and ligand-binding property. METHODS: The three recombinant proteins bearing the substitutions C138A, C157A or C138-157A were studied by means of Circular Dichroism and intrinsic/extrinsic Fluorescence spectroscopies. RESULTS: Preliminary structural characterization confirmed that the mutants retain a native-like fold. The mutant C138A was able to bind the fluorescent probe N-phenyl-naphthylamine with the same affinity of the wt protein, while the mutants bearing the substitution of C157 revealed a weaker binding of the ligand. Thermal unfolding experiments attributed a significant loss of thermal stability only to the mutants C157A and C138-157A. Similarly, GndHCl unfolding studies revealed that the substitution of C157 residue determined an increased sensitivity to the denaturant. Complete reversibility of the denaturation process was obtained only with C138A which, after gradual sample cooling or progressive denaturant subtraction, recovered the native conformational properties. CONCLUSIONS: We ascribe the decrease of the binding affinity, of the thermal stability and of the resistance to GndHCl observed for C157A to the lack of the disulfide bridge. It is worth noting that the mutation C138A does not perturb the protein functionality, and in addition it suppresses the free and reactive thiol group that contributes to incorrect disulphide bond formation and irreversible unfolding. Further studies will take advantage of this stable and refolding protein to investigate its potential role both as carrier protein and human allergen

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin. METHODS: We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. RESULTS: We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites. CONCLUSIONS: Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia