Synthesis of MIL-Modified Fe3O4 Magnetic Nanoparticles for Enhancing Uptake and Efficiency of Temozolomide in Glioblastoma Treatment.

A nanometric hybrid system consisting of a Fe3O4 magnetic nanoparticles modified through the growth of Fe-based Metal-organic frameworks of the MIL (Materials Institute Lavoiser) was developed. The obtained system retains both the nanometer dimensions and the magnetic properties of the Fe3O4 nanoparticles and possesses increased the loading capability due to the highly porous Fe-MIL. It was tested to load, carry and release temozolomide (TMZ) for the treatment of glioblastoma multiforme one of the most aggressive and deadly human cancers. The chemical characterization of the hybrid system was performed through various complementary techniques: X-ray-diffraction, thermogravimetric analysis, FT-IR and X-ray photoelectron spectroscopies. The nanomaterial showed low toxicity and an increased adsorption capacity compared to bare Fe3O4 magnetic nanoparticles (MNPs). It can load about 12 mg/g of TMZ and carry the drug into A172 cells without degradation. Our experimental data confirm that, after 48 h of treatment, the TMZ-loaded hybrid nanoparticles (15 and 20 µg/mL) suppressed human glioblastoma cell viability much more effectively than the free drug. Finally, we found that the internalization of the MIL-modified system is more evident than bare MNPs at all the used concentrations both in the cytoplasm and in the nucleus suggesting that it can be capable of overcoming the blood-brain barrier and targeting brain tumors. In conclusion, these results indicate that this combined nanoparticle represents a highly promising drug delivery system for TMZ targeting into cancer cells.

The Interplay between Fe3O4 Superparamagnetic Nanoparticles, Sodium Butyrate, and Folic Acid for Intracellular Transport.

Combined treatments which use nanoparticles and drugs could be a synergistic strategy for the treatment of a variety of cancers to overcome drug resistance, low efficacy, and high-dose-induced systemic toxicity. In this study, the effects on human colon adenocarcinoma cells of surface modified Fe3O4 magnetic nanoparticles (MNPs) in combination with sodium butyrate (NaBu), added as a free formulation, were examined demonstrating that the co-delivery produced a cytotoxic effect on malignant cells. Two different MNP coatings were investigated: a simple polyethylene glycol (PEG) layer and a mixed folic acid (FA) and PEG layer. Our results demonstrated that MNPs with FA (FA-PEG@MNPs) have a better cellular uptake than the ones without FA (PEG@MNPs), probably due to the presence of folate that acts as an activator of folate receptors (FRs) expression. However, in the presence of NaBu, the difference between the two types of MNPs was reduced. These similar behaviors for both MNPs likely occurred because of the differentiation induced by butyrate that increases the uptake of ferromagnetic nanoparticles. Moreover, we observed a strong decrease of cell viability in a NaBu dose-dependent manner. Taking into account these results, the cooperation of multifunctional MNPs with NaBu, taking into consideration the particular cancer-cell properties, can be a valuable tool for future cancer treatment.

Anti-angiogenic effect of quercetin and its 8-methyl pentamethyl ether derivative in human microvascular endothelial cells.

Angiogenesis is involved in many pathological states such as progression of tumours, retinopathy of prematurity and diabetic retinopathy. The latter is a more complex diabetic complication in which neurodegeneration plays a significant role and a leading cause of blindness. The vascular endothelial growth factor (VEGF) is a powerful pro-angiogenic factor that acts through three tyrosine kinase receptors (VEGFR-1, VEGFR-2 and VEGFR-3). In this work we studied the anti-angiogenic effect of quercetin (Q) and some of its derivates in human microvascular endothelial cells, as a blood retinal barrier model, after stimulation with VEGF-A. We found that a permethylated form of Q, namely 8MQPM, more than the simple Q, is a potent inhibitor of angiogenesis both in vitro and ex vivo. Our results showed that these compounds inhibited cell viability and migration and disrupted the formation of microvessels in rabbit aortic ring. The addition of Q and more significantly 8MQPM caused recoveries or completely re-establish the transendothelial electrical resistance (TEER) to the control values and suppressed the activation of VEGFR2 downstream signalling molecules such as AKT, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. Taken together, these data suggest that 8MQPM might have an important role in the contrast of angiogenesis-related diseases.

Pericytes in Microvessels: From "Mural" Function to Brain and Retina Regeneration.

Pericytes are branched cells located in the wall of capillary blood vessels that are found throughout the body, embedded within the microvascular basement membrane and wrapping endothelial cells, with which they establish a strong physical contact. Pericytes regulate angiogenesis, vessel stabilization, and contribute to the formation of both the blood-brain and blood-retina barriers by Angiopoietin-1/Tie-2, platelet derived growth factor (PDGF) and transforming growth factor (TGF) signaling pathways, regulating pericyte-endothelial cell communication. Human pericytes that have been cultured for a long period give rise to multilineage progenitor cells and exhibit mesenchymal stem cell (MSC) features. We focused our attention on the roles of pericytes in brain and ocular diseases. In particular, pericyte involvement in brain ischemia, brain tumors, diabetic retinopathy, and uveal melanoma is described. Several molecules, such as adenosine and nitric oxide, are responsible for pericyte shrinkage during ischemia-reperfusion. Anti-inflammatory molecules, such as IL-10, TGFß, and MHC-II, which are increased in glioblastoma-activated pericytes, are responsible for tumor growth. As regards the eye, pericytes play a role not only in ocular vessel stabilization, but also as a stem cell niche that contributes to regenerative processes in diabetic retinopathy. Moreover, pericytes participate in melanoma cell extravasation and the genetic ablation of the PDGF receptor reduces the number of pericytes and aberrant tumor microvessel formation with important implications for therapy efficacy. Thanks to their MSC features, pericytes could be considered excellent candidates to promote nervous tissue repair and for regenerative medicine.

Neuroinflammation and neurohormesis in the pathogenesis of Alzheimer's disease and Alzheimer-linked pathologies: modulation by nutritional mushrooms.

Human life develops and expands not only in time and space, but also in the retrograde permanent recollection and interweaving of memories. Therefore, individual human identity depends fully on a proper access to the autobiographical memory. Such access is hindered or lost under pathological conditions such as Alzheimer's disease, including recently associated oxidant pathologies, such as ocular neural degeneration occurring in glaucoma or neurosensorial degeneration occurring in Menière's disease. Oxidative stress and altered antioxidant systems have been suggested to play a role in the aetiology of major neurodegenerative disorders, and altered expression of genes sensing oxidative stress, as well as decreased cellular stress response mechanisms could synergistically contribute to the course of these oxidant disorders. Thus, the theory that low levels of stress can produce protective responses against the pathogenic processes is a frontier area of neurobiological research focal to understanding and developing therapeutic approaches to neurodegenerative disorders. Herein, we discuss cellular mechanisms underlying AD neuroinflammatory pathogenesis that are contributory to Alzheimer's disease. We describe endogenous cellular defence mechanism modulation and neurohormesis as a potentially innovative approach to therapeutics for AD and other neurodegenerative conditions that are associated with mitochondrial dysfunction and neuroinflammation. Particularly, we consider the emerging role of the inflammasome as an important component of the neuroprotective network, as well as the importance of Coriolus and Hericium nutritional mushrooms in redox stress responsive mechanisms and neuroprotection.

Recombinant laccase: I. Enzyme cloning and characterization.

We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white-rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET-28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl-ß-d-thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II.

Cellular stress response, sirtuins and UCP proteins in Alzheimer disease: role of vitagenes.

Alzheimer's Disease (AD) is a neurodegenerative disorder affecting up to one third of individuals reaching the age of 80. Different integrated responses exist in the brain to detect oxidative stress which is controlled by several genes termed Vitagenes. Vitagenes encode for cytoprotective heat shock proteins (Hsp), as well as thioredoxin, sirtuins and uncouple proteins (UCPs). In the present study we evaluate stress response mechanisms in plasma and lymphocytes of AD patients, as compared to controls, in order to provide evidence of an imbalance of oxidant/antioxidant mechanisms and oxidative damage in AD patients and the possible protective role of vitagenes.We found that the levels of Sirt-1 and Sirt-2 in AD lymphocytes were significantly higher than in control subjects. Interestingly, analysis of plasma showed in AD patients increased expression of Trx, a finding associated with reduced expression of UCP1, as compared to control group.This finding can open up new neuroprotective strategies, as molecules inducing this defense mechanisms can represent a therapeutic target to minimize the deleterious consequences associated to oxidative stress, such as in brain aging and neurodegenerative disorders.

Hyaluronan-Cyclodextrin Conjugates as Doxorubicin Delivery Systems.

In the last years, nanoparticles based on cyclodextrins have been widely investigated for the delivery of anticancer drugs. In this work, we synthesized nanoparticles with a hyaluronic acid backbone functionalized with cyclodextrins under green conditions. We functionalized hyaluronic acid with two different molecular weights (about 11 kDa and 45 kDa) to compare their behavior as doxorubicin delivery systems. We found that the new hyaluronan-cyclodextrin conjugates increased the water solubility of doxorubicin. Moreover, we tested the antiproliferative activity of doxorubicin in the presence of the new cyclodextrin polymers in SK-N-SH and SK-N-SH-PMA (over-expressing CD44 receptor) cancer cells. We found that hyaluronan-cyclodextrin conjugates improved the uptake and antiproliferative activity of doxorubicin in the SK-N-SH-PMA compared to the SK-N-SH cell line at the ratio 8/1 doxorubicin/polymer. Notably, the system based on hyaluronan (45 kDa) was more effective as a drug carrier and significantly reduced the IC50 value of doxorubicin by about 56%. We also found that hyaluronic acid polymers determined an improved antiproliferative activity of doxorubicin (IC50 values are on average reduced by about 70% of free DOXO) in both cell lines at the ratio 16/1 doxorubicin/polymer.

Self-assembled BiFeO3@MIL-101 nanocomposite for antimicrobial applications under natural sunlight.

In this paper, we report on the synthesis of a new hybrid photocatalytic material activated by natural sunlight irradiation. The material consists of multiferroic nanoparticles of bismuth ferrite (BFO) modified through the growth of the Fe-based MIL-101 framework. Material characterization, conducted using various techniques (X-ray diffraction, transmission electron microscopy, FTIR, and X-ray photoelectron spectroscopies), confirmed the growth of the MIL-101 metal-organic framework on the BFO surface. The obtained system possesses the intrinsic photo-degradative properties of BFO nanoparticles significantly enhanced by the presence of MIL-101. The photocatalytic activity of this material was tested in antibacterial experiments conducted under natural sunlight exposure within the nanocomposite concentration range of 100-0.20 µg/ml. The MIL-modified BFO showed a significant decrease in both Minimum Inhibiting Concentration and Minimum Bactericide Concentration values compared to bare nanoparticles. This confirms the photo-activating effect of the MIL-101 modification. In particular, they show an increased antimicrobial activity against the tested Gram-positive species and the ability to begin to inhibit the growth of the four Escherichia coli strains, although at the maximum concentration tested. These results suggest that the new nanocomposite BiFeO3@MOF has been successfully developed and has proven to be an effective antibacterial agent against a wide range of microorganisms and a potential candidate in disinfection processes.

Cytostatic Effects of Polyethyleneimine Surfaces on the Mesenchymal Stromal Cell Cycle.

Polyelectrolytes assembled layer-by-layer (PEMs) are commonly used as functional coatings to build-up biological interfaces, particularly suitable as compatible layers for the interaction with a biological medium, providing suitable conditions to promote or prevent cell seeding while maintaining the phenotype. The proper assessment of the biocompatibility of PEMs and the elucidation of the related mechanisms are therefore of paramount importance. In this study, we report in detail the effect of two different PEM endings, polystyrene sulfonate (PSS) and polyethylenimine (PEI), respectively, on the cell adhesion, growth, and viability of human bone mesenchymal stromal cells (MSCs). The results have shown that PSS-ended substrates appear to be the most suitable to drive the cell adhesion and phenotype maintenance of MSCs, showing good biocompatibility. On the contrary, while the cells seem to adhere more quickly and strongly on the PEI-ended surfaces, the interaction with PEI significantly affects the growth and viability, reducing the cell spreading capability, by sequestering the adhesion molecules already in the very early steps of cell-substrate contact. These results point to the promotion of a cytostatic effect of PEI, rather than the often-claimed cytotoxicity.

Aerobic Exercise and Metabolic Syndrome: The Role of Sympathetic Activity and the Redox System.

BACKGROUND: Aerobic exercise can greatly assist in reducing collateral effects of metabolic syndrome (MetS). Moreover, aerobic exercise is associated with sympathetic activation and adaptive responses to sustain muscle engagement, changes in the release of Orexin A, a pleiotropic neuropeptide. AIM: The aim of this study was to analyze the beneficial effects of aerobic exercise without dietary changes, in a cohort of MetS subjects, focusing on the role of sympathetic and orexinergic activity. Several blood parameters linked to MetS ROS production, heart rate, galvanic skin response, d-ROM test, and Orexin A serum levels were evaluated in ten males with MetS (BMI 30-34.9) before and after a period of 6 months of aerobic exercise compared to ten healthy subjects. METHODS: Ten male subjects (aged 54 ± 4.16) with MetS (MetS group) and ten healthy males (aged 49.7 ± 2.79, Healthy group) were told about the study protocol and possible risks, signed the informed consent, and voluntarily participated in the study. Several blood parameters were evaluated in the two tested groups and were re-evaluated in the MetS group after 6 months of training (MetS6M group). The training protocol consisted of more than 30 min/day of walking (average speed of 4.5 km/h) and 3 days/week of aerobic activities (jogging under heart rate control - 120-140 bpm for 45 min). RESULTS: The results showed that exercise induced a significant increase in GSR and plasma Orexin A but no significant increase in d-ROM values. Significant decreases in the serum ALT enzyme, triglycerides, and total cholesterol were found, while the HDL levels were significantly higher. Finally, a significant reduction of BMI and resting HR were reported. CONCLUSION: The results of this study confirm that physical activity is associated with sympathetic activation, having a pivotal role against adverse effects linked to MetS. Moreover, this study demonstrates that, in patients with MetS, Orexin A is involved in hormonal adaptations to exercise.

The double effect of walnut septum extract (Juglans regia L.) counteracts A172 glioblastoma cell survival and bacterial growth.

Walnut (Juglans regia L.) is considered to be a 'superfood' for its multiple protective actions on human health. Walnut extracts have proven antitumor activity in different cancer cell lines. However, the efficacy of septum extract against glioblastoma has still not been investigated. Glioblastoma is the most difficult type of brain cancer to treat. The standard therapy, based on temozolomide, causes several side effects, including neutropenia and lymphocytopenia, which often favor the onset of opportunistic infections. In the present study, the chemical profile of the Sicilian walnut septum ethanolic extract was analyzed using high­performance liquid chromatography (HPLC)­diode array detection and HPLC­electrospray ionization tandem mass spectrometry. The potential cytostatic activity of the extract against the human A172 glioblastoma cell line was investigated and the results showed that the extract could decrease cancer cell proliferation and migration. Using cytofluorimetric analyses and caspase­3 assays, the pro­apoptotic action of walnut extract was demonstrated. Furthermore, the evaluation of the antibacterial activity highlighted the efficacy of the extract in reducing Gram­positive and Gram­negative bacterial growth, most of which were resistant to the antibiotic, ciprofloxacin. Finally, Prediction of Activity Spectra for Substances analysis showed the predicted antitumor and antibacterial activity of HPLC detected compounds. The promising results could provide novel perspective in the field of chemotherapeutic co­adjuvants.

Classical VEGF, Notch and Ang signalling in cancer angiogenesis, alternative approaches and future directions (Review).

Angiogenesis is the formation of new vessels starting from pre-existing vasculature. Tumour environment is characterized by 'aberrant angiogenesis', whose main features are tortuous and permeable blood vessels, heterogeneous both in their structure and in efficiency of perfusion and very different from normal vessels. Therapeutic strategies targeting the three pathways chiefly involved in tumour angiogenesis, VEGF, Notch and Ang signalling, have been identified to block the vascular supply to the tumour. However, phenomena of toxicity, development of primary and secondary resistance and hypoxia significantly blunted the effects of anti-angiogenic drugs in several tumour types. Thus, different strategies aimed to overcome these problems are imperative. The focus of the present review was some principal 'alternative' approaches to classic antiangiogenic therapies, including the cyclooxygenase-2 (COX-2) blockade, the use of oligonucleotide complementary to the miRNA to compete with the mRNA target (antimiRs) and the inhibition of matrix metalloproteinases (MMPs). The role of blood soluble VEGFA as a predictive biomarker during antiangiogenic therapy in gastric, ovarian and colorectal cancer was also examined.

Effect of Some Growth Factors on Tissue Transglutaminase Overexpression Induced by ß-Amyloid in Olfactory Ensheathing Cells.

Herein, we assessed in a particular glial cell type, called olfactory ensheathing cells (OECs), the effect of some growth factors (GFs) on tissue transglutaminase (TG2) overexpression induced by amyloid-beta (Aß) with native full-length peptide 1-42 or by fragments, 25-35 or 35-25, as control. Previously, we demonstrated that TG2 overexpression induced by some stressors was down-regulated by GFs exposure in OECs. To monitor cell viability, an MTT test was used, while TG2 expression was examined using immunocytochemical and Western blot analysis. We also considered the involvement of the TG2-mediated apoptotic pathway. Vimentin expression was evaluated as well. Reactive oxygen species and reduced glutathione levels were utilized to test the oxidative intracellular status. Lactate dehydrogenase released into the medium, as a marker of necrotic cell death, was evaluated. We found that in OECs exposed to Aß(1-42) or Aß(25-35) for 24 h, TG2 expression increased, and we observed that the protein appeared prevalently localized in the cytosol. The pre-treatment with GFs, basic fibroblast growth factor (bFGF) or glial-derived neurotrophic factor (GDNF), down-regulated the TG2 level, which was prevalently limited to the nuclear compartment. Vimentin expression and caspase cleavage showed a significant enhancement in Aß(1-42) and Aß(25-35) exposed cells. The pre-treatment with bFGF or GDNF was able to restore the levels of the proteins to control values, and the intracellular oxidative status modified by the exposure to Aß(1-42) or Aß(25-35). Our data suggest that both bFGF or GDNF could be an innovative mechanism to contrast TG2 expression, which plays a key role in Alzheimer's disease.

A high sensitivity amperometric biosensor using laccase as biorecognition element.

An amperometric flow biosensor, using laccase from Rigidoporus lignosus as bioelement was developed. The laccase was kinetically characterized towards various phenolics both in solution and immobilized to a hydrophilic matrix by carbodiimide chemistry. A bioreactor connected to an amperometric flow cell by a FIA system was filled with the immobilized enzyme and the operational conditions of this biosensor were optimized as regards pH. Under the adopted experimental conditions, the immobilized enzyme oxidizes all the substrate molecules avoiding the need of cumbersome calibration procedures. The biosensor sensitivity, which was found to be 100 nA/microM for some of the tested substrates, resulted to be constant for more than 100 working days. This biosensor permits the detection of phenolics in aqueous solutions at concentrations in the nanomolar range and was successfully used to detect phenolics in wastewaters from olive oil mill without sample preparation.

Structure-activity relationship on fungal laccase from Rigidoporus lignosus: a Fourier-transform infrared spectroscopic study.

The structure and thermal stability of a laccase from Rigidoporus lignosus (Rl) was analysed by Fourier-transform infrared (FT-IR) spectroscopy. The enzyme was depleted of copper atoms, then part of the apoenzyme was re-metalled and these two forms of the protein were analysed as well. The enzymatic activity, lost by the removal of copper atoms, was restored in the re-metalled apoenzyme and resulted similar to that of native protein. The infrared data indicated that the enzyme contains a large amount of beta-sheets and a small content of alpha-helices, and it displayed a marked thermostability showing the T(m) at 92.5 degrees C. The apoenzyme and the re-metalled apoenzyme did not show remarkable differences in the secondary structure with respect to the native protein, but the thermal stability of the apoenzyme was dramatically reduced showing a T(m) close to 72 degrees C, while the re-metalled protein displayed the T(m) at 90 degrees C. These data indicate that copper atoms, beside their role in catalytic activity, play also an important role on the stabilisation of the structure of Rl laccase. About 35% of the polypeptide chain is buried and/or constitutes a particular compact structure, which, beside copper atoms, is probably involved in the high thermal stability of the protein. Another small part of the structure is particularly sensitive to high temperatures and it could be the cause of the loss of enzymatic activity when the temperature is raised above 45-50 degrees C.

The structure of Rigidoporus lignosus Laccase containing a full complement of copper ions, reveals an asymmetrical arrangement for the T3 copper pair.

Laccase is a multicopper blue oxidase that couples the four-electron reduction of oxygen with the oxidation of a broad range of organic substrates, including phenols and arylamines. The enzyme is the object of intense biotechnological research, due to its employment in bioremediation of soils and water as well as in other biotechnological applications. We report here the cDNA and protein sequences, the post-translational modifications, the crystallization and X-ray structure determination of a laccase from the white-rot fungus Rigidoporus lignosus. The amino acid residues sequence deduced from cDNA clearly identified a pre-sequence of 21 residues representing the signal for extra-cellular localization. Mass spectrometry analysis performed on the salvage enzyme, confirmed the deduced sequence and precisely mapped two glycosylation sites at Asn337 and Asn435, determining the nature of the bound glycosidic moieties. The crystal structure was determined at 1.7A resolution from perfectly hemihedrally twinned crystals, by molecular replacement technique. While the overall structure closely resembled those reported for other fungal laccases, the analysis of the T2/T3 trinuclear cluster revealed an unprecedented coordination sphere for the T3 copper pair. No bridging oxygen ligand was present between the two T3 copper ions, which were no longer symmetrically coordinated. The observed structure could represent an intermediate along the process of four-electron reduction of oxygen to water taking place at the trinuclear copper cluster.

Effect of acidic phospholipids on the structural properties of recombinant cytosolic human glyoxalase II.

A peculiar characteristic of highly concentrated cytosolic recombinant human glyoxalase II (GII) solutions is to undergo partial precipitation. Previous work indicated that anionic phospholipids (PLs) exert a noncompetitive inhibition on the enzymatic activity of the soluble enzyme. In this study, FTIR spectroscopy was used to analyze the structural properties and the thermal stability of the soluble protein in the absence and in the presence of liposomes made of different phospholipids (PLs). The structural analysis was performed on the precipitate as well. The interaction of acidic PLs with GII lowered the thermal stability of the enzyme and inhibited protein intermolecular interactions (aggregation) brought about by thermal denaturation. Infrared data indicated that ionic and hydrophobic interactions occur between GII and acidic PLs causing small changes in the secondary structure of the enzyme. No interactions of the protein with egg phosphatidylcholine liposomes were detected. The results are consistent with the destabilization of the protein tertiary structure, and indicate that GII possesses hydrophobic part(s) that interact with the acyl chains of PLs. Data on precipitated GII did not show remarkable modification of secondary structure, suggesting that hydrophobic stretches of the enzyme may also be involved in the protein-protein association (precipitation) at high GII concentration. The alterations in the GII structure and the noncompetitive inhibition exerted by acidic PLs are strictly related.

A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element.

Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold surface. A flow biosensor, using the monolayer of laccase as bioelement and a glassy carbon electrode as amperometric transduction system, was developed. Although the amount of the immobilised enzyme (about 140 ng/cm2 effective surface area) was tiny, the biosensor showed a sensitivity of 3 nA/microM when 1,4-hydroquinone was used as substrate, and a half-life of 35 days. The proposed device permits detection of phenols in aqueous solutions at concentrations in the low micromolar range, i.e. below European Community limits. The biosensor was successfully used to detect phenols in wastewaters from an olive oil mill after minimal sample preparation (incubation of the aqueous sample with sodium borohydride for a few minutes) to suppress the current due to oxidised compounds present in the wastewaters.

In silico study of structural determinants modulating the redox potential of Rigidoporus lignosus and other fungal laccases.

Laccases are multicopper oxidases in which substrate oxidation takes place at the type-1 (T1) copper site. The redox potential (E (0)) significantly varies amongst members of the family and is a key parameter for substrate specificity. Despite sharing highly conserved features at the T1 copper site, laccases span a large range of E (0), suggesting that the influence of the metal secondary coordination sphere is important. In silico analysis of structural determinants modulating the E (0) of Rigidoporus lignosus and other fungal laccases indicated that different factors can be considered. First, the length of the T1 copper coordinating histidine bond is observed to be longer in high E (0) laccases than in low E (0) ones. The hydrophobic environment around the T1 copper site appeared as another important structural determinant in modulating the E (0), with a stronger hydrophobic environment correlating with higher E (0). The analysis of hydrogen bonding network (HBN) around the T1-binding pocket revealed that the amino acids building up the metal binding site strongly interact with neighbouring residues and contribute to the stabilization of the protein folds. Changes in these HBNs that modified the Cu1 preferred coordination geometry lead to an increase of E (0). The presence of axial ligands modulates the E (0) of T1 to different extent. Stacking interactions between aromatic residues located in the second coordination shell and the metal ion coordination histidine imidazole ring have also been identified as a factor that modulates the E (0). The electrostatic interactions between the T1 copper site and backbone carbonyl oxygen indicate that Cu1-CO=NH distance is longer in the high E (0) laccases. In short, the in silico study reported herein identifies several structural factors that may influence the E (0) of the examined laccases. Some of these are dependent on the nature of the coordination ligands at the T1 site, but others can be ascribed to the hydrophobic effects, HBNs, axial ligations, stacking and electrostatic interactions, not necessary directly interacting with the copper metal.