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

Intensification of bioconversion processes: design and development of reactors with high biocatalyst loading

The aim of the research program was to address some open issues related to immobilization of biological systems (enzymes, microrganisms) for bioprocess applications. The research pathway is ultimately targetted at the development of novel and effective tools for bioprocess intensification based on the use of immobilizer biocatalysts. The attention has been specifically focused on two processes: an enzymatic and a microbial process. Conversion of the synthetic dye Remazol Brilliant Blue R (RBBR) by means of crude laccase mixtures from Pleurotus ostreatus. The study aimed at the optimization of the enzymes immobilization protocol and at the characterization of the RBBR conversion by means of free and immobilizer enzymes. The immobilization process was optimized with reference to the covalent binding on granular supports - EUPERGIT C 250L©. In particular, operating conditions were selected so as to maximize the immobilization yield. A reactor for the activity assay of the immobilised enzymes was designed and set-up. The kinetics of RBBR conversion by means of immobilized laccase mixtures was characterised using a purposely designed fixed bed reactor. A kinetic model has been best-fitted against experimental data relative to conversion degree and reactor space-time to assess kinetic parameters. The role of mass transport phenomena between the phases and of adsorption during heterogeneous enzymatic conversion of the dye were investigated. Based on data collected in the experimental campaign and on kinetic models developed, two alternative processes for remediation of dye-bearing wastewaters have been assessed: i) a batch Stirred Tank Reactor (STR) operated with a homogeneous liquid phase containing both the crude laccase mixture and the dye; ii) a Continuous Fixed Bed Reactor (CFBR) loaded with laccases immobilised on EUPERGIT© and continuously fed with the dye-bearing liquid stream. The results highlighted the role played by biocatalyst stability and immobilisation yield on the selection of the best option in terms of the volume of wastewater cumulatively treated in either option. Conversion of the phenol by means of Pseudomonas sp. OX1 biofilm in threephase bioreactors. The study has addressed both experimental and theoretical aspects of the problem. The experimental study has been directed to the assessment of the growth of P. sp OX1 biofilm on silica-based granular supports. Two bench-scale reactor typologies were investigated: a three Phase Circulating (3PC) reactor and an Internal Loop Airlift (ILA) reactor. Tests with the 3PC reactor were directed to optimize the conditions for biofilm growth. Tests with the ILA reactor were aimed at investigating the effect of dilution rate on the performance of the system operated with a mature biofilm that had been previously partly grown up on the solid carrier. The theoretical work has addressed the analysis of the bifurcational and dynamic patterns of an ILA reactor operated with biofilm of P. sp. OX1 attached on granular solids. The role of the growth rate of suspended and immobilised cells using phenol as carbon source, the adhesion rate of suspended cells on carrier surface and the detachment rate of biofilm from the solids carrier were investigated. The dependence of bifurcational patterns on detachment coefficient and dilution rate has been analysed. Results from the theoretical study and from tests carried out with the ILA reactor were compared: the model properly reproduces the functional dependence of the steady state behaviour of the biofilm on the dilution rate observed during the tests

Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae

Heterologous expression of Pleurotus ostreatus POXC and POXA1b laccases in two yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae, was performed. Both transformed hosts secreted recombinant active laccases, although K. lactis was much more effective than S. cerevisiae. rPOXA1b transformants always had higher secreted activity than rPOXC transformants did. The lower tendency of K. lactis with respect to S. cerevisiae to hyperglycosylate recombinant proteins was confirmed. Recombinant laccases from K. lactis were purified and characterised. Specific activities of native and recombinant POXA1b are similar. On the other hand, rPOXC specific activity is much lower than that of the native protein, perhaps due to incomplete or incorrect folding. Both recombinant laccase signal peptides were correctly cleaved, with rPOXA1b protein having two C-terminal amino acids removed. The availability of the established recombinant expression system provides better understanding of laccase structure-function relationships and allows the development of new oxidative catalysts through molecular evolution techniques

Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae

Heterologous expression of Pleurotus ostreatus POXC and POXA1b laccases in two yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae, was performed. Both transformed hosts secreted recombinant active laccases, although K. lactis was much more effective than S. cerevisiae. rPOXA1b transformants always had higher secreted activity than rPOXC transformants did. The lower tendency of K. lactis with respect to S. cerevisiae to hyperglycosylate recombinant proteins was confirmed. Recombinant laccases from K. lactis were purified and characterised. Specific activities of native and recombinant POXA1b are similar. On the other hand, rPOXC specific activity is much lower than that of the native protein, perhaps due to incomplete or incorrect folding. Both recombinant laccase signal peptides were correctly cleaved, with rPOXA1b protein having two C-terminal amino acids removed. The availability of the established recombinant expression system provides better understanding of laccase structure-function relationships and allows the development of new oxidative catalysts through molecular evolution techniques

Reduction of phenol content and toxicity in olive oil mill waste waters with the ligninolytic fungus Pleurotus ostratus

Olive oil mill waste waters (OMW) constitute a major environmental problem because of the large amount produced and the toxicity of the phenolic compounds present. Several of these aromatic compounds can be assimilated to many of the components of lignin. Only few microorganisms, mainly “white-rot” basidiomycete, are able to degrade lignin by means of oxidative reactions catalysed by phenol oxidases and peroxidases. Both the low degree of specificity which characterizes these enzymes, and the structural relationships of many aromatic pollutants with the natural substrates of the enzymes, have suggested the use of ligninolytic organisms and of their enzymes for the treatment of these kinds of substrates. This paper investigates the ability of the “white-rot” basidiomycete Pleurotus ostreatus and particularly of the phenol oxidases it produces in the detoxification of OMW phenol compounds. Treatment of OMW with purified phenol oxidase showed a significant reduction of phenolic content, but no decrease of its toxicity was observed when tested on Bacillus cereus. Otherwise, the effect of processing OMW with the entire microorganism resulted in a noticeable detoxification of the waste with concomitant abatement of the phenol content