Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project. V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft

Conversion of no/low value waste frying oils into biodiesel and polyhydroxyalkanoates

A sustainable bioprocess was developed for the valorization of a no/low value substrate, i.e. waste frying oils (WFOs) with high content of free fatty acids (FFAs), otherwise unsuitable for biodiesel production. The bioprocess was verified using both recombinant (Escherichia coli) and native (Pseudomonas resinovorans) polyhydroxyalkanoates (PHAs) producing cell factories. Microbial fermentation of WFOs provided a 2-fold advantage: i) the reduction of FFAs content resulting into an upgrading of the "exhausted waste oils" and ii) the production of a bio-based microbial polymer. Proper strain designing and process optimization allowed to achieve up to 1.5 g L-1 of medium chain length, mcl-PHAs, together with an efficient conversion (80% yield) of the treated WFO into biodiesel

A time‐course study of the expression level of synaptic plasticity‐associated genes in un‐lesioned spinal cord and brain areas in a rat model of spinal cord injury: A bioinformatic approach

open8noFunding: This research was funded by the POR-FESR 2019-21, project “Mat2Rep”, Emilia Romagna Region (L.C.) and by the Cluster Tecnologici Nazionali, project IRMI, MIUR (L.C.). Marco Sanna is receiving a fellowship from the program “Alte Competenze” by Emilia Romagna Region. The contribution of “Fondazione Montecatone”, Imola (Italy) is also gratefully acknowledged.“Neuroplasticity” is often evoked to explain adaptation and compensation after acute lesions of the Central Nervous System (CNS). In this study, we investigated the modification of 80 genes involved in synaptic plasticity at different times (24 h, 8 and 45 days) from the traumatic spinal cord injury (SCI), adopting a bioinformatic analysis. mRNA expression levels were analyzed in the motor cortex, basal ganglia, cerebellum and in the spinal segments rostral and caudal to the lesion. The main results are: (i) a different gene expression regulation is observed in the Spinal Cord (SC) segments rostral and caudal to the lesion; (ii) long lasting changes in the SC includes the extracellular matrix (ECM) enzymes Timp1, transcription regulators (Egr, Nr4a1), second messenger associated proteins (Gna1, Ywhaq); (iii) long‐lasting changes in the Motor Cortex includes transcription regulators (Cebpd), neurotransmitters/neuromodulators and receptors (Cnr1, Gria1, Nos1), growth factors and related receptors (Igf1, Ntf3, Ntrk2), second messenger associated proteins (Mapk1); long lasting changes in Basal Ganglia and Cerebellum include ECM protein (Reln), growth factors (Ngf, Bdnf), transcription regulators (Egr, Cebpd), neurotransmitter receptors (Grin2c). These data suggest the molecular mapping as a useful tool to investigate the brain and SC reorganization after SCI.openBaldassarro V.A.; Sanna M.; Bighinati A.; Sannia M.; Gusciglio M.; Giardino L.; Lorenzini L.; Calzà LauraBaldassarro V.A.; Sanna M.; Bighinati A.; Sannia M.; Gusciglio M.; Giardino L.; Lorenzini L.; Calzà Laur

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Treatment and recycling of zinc hydrometallurgical wastes by self-propagating reactions

A novel technique for treating and recycling of a highly toxic solid waste from electrolytic zinc plants, i.e. goethite waste, is proposed. It consists of blending this waste with suitable amount of reducing agents (aluminum or aluminum and silicon) and ferric oxide, and igniting the resulting mixture so that a self-propagating reaction in the form of a combustion wave rapidly travels through the mixture without requiring additional energy. Reactants are converted into two solid products (P-1 and P-2) with different mass, composition and structure, and a gas constituted by SO2. Aluminum and silicon should be preferred as reducing agents, since the reaction product P-1 obtained in larger quantity, is constituted by an amorphous glassy structure of alumino-silicates which embodies heavy metals, such as Pb and Cd. Leaching tests of the reaction products P-1 are also performed to verify the possibility of their disposal. The solid product P-2 on the other hand may be recycled in the roasting unit of the industrial zinc production plant. A waste treatment process is also proposed

Nuclear receptors and differentiation of oligodendrocyte precursor cells

Oligodendrocytes are the cells responsible for myelin formation during development and in adulthood, both for normal myelin turnover and myelin repair. These highly specialized cells derive from the oligodendrocyte precursor cells (OPCs), through a complex differentiation process involving genetic and epigenetic regulation mechanisms, which switch the phenotype from a migratory and replicative precursor to a mature post-mitotic cell. The process is regulated by a plethora of molecules, involving neurotransmitters, growth factors, hormones and other small molecules, and is mainly driven by nuclear receptors (NRs). NRs are transcription factors with heterogeneous ligand-dependent and independent actions which differ for the cell target, the responsive gene and the formation of NR homo- or heterodimers. This chapter highlights the role of NRs in regulating OPC differentiation, also in view of drug discovery strategies aimed at targeting pathological conditions which interfere with both developmental myelination and remyelination in adulthood