Carbon Nanotubes as Activating Tyrosinase Supports for the Selective Synthesis of Catechols

A series of redox catalysts based on the immobilization of tyrosinase on multiwalled carbon nanotubes has been prepared by applying the layer-by-layer principle. The oxidized nanotubes (ox-MWCNTs) were treated with poly­(diallyl dimethylammonium chloride) (PDDA) and tyrosinase to yield ox-MWCNTs/PDDA/tyrosinase <b>I</b>. Catalysts <b>II</b> and <b>III</b> have been prepared by increasing the number of layers of PDDA and enzyme, while <b>IV</b> was obtained by co-immobilization of tyrosinase with bovine serum albumin (ox-MWCNTs/PDDA/BSA-tyrosinase). Attempts to covalently bind tyrosinase provided weakly active systems. The coating of the enzyme based on the simple layer-by-layer principle has afforded catalysts <b>I–III</b>, with a range of activity from 21 units/mg (multilayer, <b>II</b>) to 66 units/mg (monolayer, <b>I</b>), the best system being catalyst <b>IV</b> (80 units/mg). The novel catalysts were fully characterized by scanning electron microscopy and atomic force microscopy, showing increased activity with respect to that of the native enzyme. These catalysts were used in the selective synthesis of catechols by oxidation of <i>meta</i>- and <i>para</i>-substituted phenols in an organic solvent (CH₂Cl₂) as the reaction medium. It is worth noting that immobilized tyrosinase was able to catalyze the oxidation of very hindered phenol derivatives that are slightly reactive with the native enzyme. The increased reactivity can be ascribed to a stabilization of the immobilized tyrosinase. The novel catalysts <b>I</b> and <b>IV</b> retained their activity for five subsequent reactions, showing a higher stability in organic solvent than under traditional buffer conditions

Molecular and Functional Characterization of Three Different Postzygotic Mutations in <i>PIK3CA</i>-Related Overgrowth Spectrum (PROS) Patients: Effects on PI3K/AKT/mTOR Signaling and Sensitivity to PIK3 Inhibitors

<div><p>Background</p><p><i>PIK3CA</i>-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic <i>PIK3CA</i> mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed.</p><p>Methods</p><p>We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 <i>in vitro</i>.</p><p>Results and Conclusion</p><p>Our data indicate that patients’ cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients.</p></div

Clinical and mutational spectrum of the three index cases.

<p><b>a</b> Patient 1, clinically diagnosed with MCAP, showing diffuse capillary malformation at the age of 2 months and cutaneous syndactyly between the 2^nd and 3^rd toes. The <i>PIK3CA</i> c.241 G>A [p.E81K] mutation detected by Sanger sequencing in affected cells and tissues of patient 1 showed varying levels of the mutant allele depending on the tissue tested. The mutation was absent in the patient's blood and in her parents. <b>b</b> Macrodactyly of the right 4^th finger in patient 2, diagnosed with FAO, at the age of 17 years. Sequence of <i>PIK3CA</i> exon 20 in blood and cultured fibroblasts obtained from patient 2 showing that the mutation is undetectable in these samples. <b>c</b> Patient 3, at the age of 15 months before surgical intervention; note the disproportion of the left 2^nd and 3^rd fingers and the subcutaneous mass at the left deltoid region. Sanger sequencing validation of the c.3140 A>T [p.H1047L] mutation detected with targeted deep sequencing in the biopsy from the 2^nd finger of patient 3. <b>d</b> List of samples and mutations detected with targeted deep sequencing. Coverage indicates the mean average of reads on target in the regions of interest (ROI) while frequency denotes the percentage of reads with the mutation.</p

Overactivation of the PI3K/Akt pathway is abrogated by pharmacological inhibition of PI3K in all patients tested.

<p><b>a</b> The indicated values are the result of the densitometric analysis of the phosphorylated forms of Akt and p70S6K normalized against total Akt and the loading control, respectively. The presented results are representative of at least three independent sets of experiments (bars represent standard deviation of the mean). <b>b</b> Immunoblot analysis of phospho-Akt (Ser473), phospho-Akt (Thr308), total Akt and phospho-p70S6K (Ser371) in mutant cells (fibroblasts from biopsies of FAO patient 2, and from left [L] and right [R] leg biopsies of MCAP patient 1) compared to IMR90 primary human normal fibroblasts (Ctrl). β-Actin was used as a loading control. Cells were treated with the PI3K inhibitor wortmannin (10μM) for 24 hours in the absence of growth factor stimulation. The presented results are representative of at least three independent sets of experiments. <b>c</b> Patients' affected cells are dependent on PI3K activity for proliferation. Primary fibroblasts obtained from biopsies were cultured in the presence or absence of wortmannin (10μM) and LY294002 (25μM). At the indicated time points, the proliferation index was determined using the WST-1 assay. The results were also confirmed by cell counting with trypan blue staining (data not shown). Each assay was performed in 6 replicates and the experiment was repeated six times. Statistical analysis was performed using Student’s t-tail test; *<i>P</i><0.05, which was considered statistically significant (bars represent standard deviation of the mean).</p