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Oxidative stress, an imbalance between reactive oxygen species (ROS) and endogenous antioxidants, plays an important role in the development of neurodegenerative diseases, including Parkinson’s. The human brain is vulnerable to oxidative stress because of the high rate of oxygen that it needs and the high levels of polyunsaturated fatty acids, which are substrates of lipid peroxidation. Natural antioxidants inhibit oxidation and reduce oxidative stress, preventing cancer, inflammation, and neurodegenerative disorders. Furthermore, in the literature, it is reported that antioxidants, due to their possible neuroprotective activity, may offer an interesting option for better symptom management, even Parkinson’s disease (PD). Natural antioxidants are usually found in several foods, such as fruits, vegetables, meat, fish, and oil, and in food wastes, such as seeds, peels, leaves, and skin. They can help the system of endogenous antioxidants, protect or repair cellular components from oxidative stress, and even halt lipid, protein, and DNA damage to neurons. This review will examine the extent of knowledge from the last ten years, about the neuroprotective potential effect of natural antioxidants present in food and food by-products, in in vivo and in vitro PD models. Additionally, this study will demonstrate that the pool of dietary antioxidants may be an important tool in the prevention of PD and an opportunity for cost savings in the public health area.

Background: Experimental evidence indicates that Naloxone (NLX) holds antioxidant properties. The present study aims at verifying the hypothesis that NLX could prevent oxidative stress induced by hydrogen peroxide (H2O2) in PC12 cells. Methods: To investigate the antioxidant effect of NLX, initially, we performed electrochemical experiments by means of platinum-based sensors in a cell-free system. Subsequently, NLX was tested in PC12 cells on H2O2-induced overproduction of intracellular levels of reactive-oxygen-species (ROS), apoptosis, modification of cells' cycle distribution and damage of cells’ plasma membrane. Results: This study reveals that NLX counteracts intracellular ROS production, reduces H2O2-induced apoptosis levels, and prevents the oxidative damage-dependent increases of the percentage of cells in G2/M phase. Likewise, NLX protects PC12 cells from H2O2- induced oxidative damage, by preventing the lactate dehydrogenase (LDH) release. Moreover, electrochemical experiments confirmed the antioxidant properties of NLX. Conclusion: Overall, these findings provide a starting point for studying further the protective effects of NLX on oxidative stress.

Until now, research has been performed mainly in men, with a low recruitment of women; consequentially, biological, physiological, and physio-pathological mechanisms are less understood in women. Obviously, without data obtained on women, it is impossible to apply the results of research appropriately to women. This issue also applies to medical devices (MDs), and numerous problems linked to scarce pre-market research and clinical trials on MDs were evidenced after their introduction to the market. Globally, some MDs are less efficient in women than in men and sometimes MDs are less safe for women than men, although recently there has been a small but significant decrease in the sex and gender gap. As an example, cardiac resynchronization defibrillators seem to produce more beneficial effects in women than in men. It is also important to remember that MDs can impact the health of healthcare providers and this could occur in a sex- and gender-dependent manner. Recently, MDs’ complexity is rising, and to ensure their appropriate use they must have a sex–gender-sensitive approach. Unfortunately, the majority of physicians, healthcare providers, and developers of MDs still believe that the human population is only constituted by men. Therefore, to overcome the gender gap, a real collaboration between the inventors of MDs, health researchers, and health providers should be established to test MDs in female and male tissues, animals, and women.

Abstract Tissue engineering is rapidly progressing toward clinical application. In the musculoskeletal field, there has been an increasing necessity for bone and cartilage replacement. Despite the promising translational potential of tissue engineering approaches, careful attention should be given to the quality of developed constructs to increase the real applicability to patients. After a general introduction to musculoskeletal tissue engineering, this narrative review aims to offer an overview of methods, starting from classical techniques, such as gene expression analysis and histology, to less common methods, such as Raman spectroscopy, microcomputed tomography, and biosensors, that can be employed to assess the quality of constructs in terms of viability, morphology, or matrix deposition. A particular emphasis is given to standards and good practices (GXP), which can be applicable in different sectors. Moreover, a classification of the methods into destructive, noninvasive, or conservative based on the possible further development of a preimplant quality monitoring system is proposed. Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages, including minimal invasiveness, making them suitable for the development of preimplant quality control systems.

Polymeric permselective films are frequently used for amperometric biosensors to prevent electroactive interference present in the target matrix. Phenylenediamines are the most commonly used for the deposition of shielding polymeric films against interfering species; however, even phenolic monomers have been utilized in the creation of these films for microsensors and biosensors. The purpose of this paper is to evaluate the performances of electrosynthesized polymers, layered by means of constant potential amperometry (CPA), of naturally occurring compound zingerone (ZING) and its dimer dehydrozingerone (ZING DIM), which was obtained by straight oxidative coupling reaction. The polymers showed interesting shielding characteristics against the main interfering species, such as ascorbic acid (AA): actually, polyZING exhibited an AA shielding aptitude comprised between 77.6 and 99.6%, comparable to that obtained with PPD. Moreover, a marked capability of increased monitoring of hydrogen peroxide (HP), when data were compared with bare metal results, was observed. In particular, polyZING showed increases ranging between 55.6 and 85.6%. In the present work, the molecular structures of the obtained polymers have been theorized and docking analyses were performed to understand their peculiar characteristics better. The structures were docked using the Lamarckian genetic algorithm (LGA). Glutamate biosensors based on those polymers were built, and their performances were compared with biosensors based on PPD, which is the most widespread polymer for the construction of amperometric biosensors.

According to the WHO, the overall age-standardized cancer rate keeps declining, and the number of cases diagnosed each year increases, remaining among the leading causes of death in 91 out of 172 recorded countries. In this context, novel cancer prediction and therapeutic protocols are compulsory. The effect of a Stachys circinata L’Hér dichloromethane extract (ScDME) on cell redox homeostasis and tumor proliferation was investigated. HepG2 cell feedback mechanisms to oxidative stress exposure were evaluated by determining catalase (CAT) and reduced glutathione (GSH), following the supply with ScDME (0.0–5.7 µg/µL). Cytotoxicity of ScDME against the human umbilical vein endothelial cell (HUVEC) and two human cancer cell lines (breast: MCF7; liver: HepG2) was evaluated by the MTT assay. H2O2-stressed HepG2 cells supplied with the S. circinata extracts exhibited significantly increased CAT and GSH activity as compared to unsupplied ones. The anti-inflammatory activity of the extracts was evaluated by real time-qPCR on IL-1, IL-6 and TNF-α expression. As a result, this research points out that S. circinata dichloromethane extract owns anti-inflammatory and anti-proliferative properties against MCF7 and HepG2 cells and activates CAT and GSH of the HepG2 cells’ antioxidant enzyme system.

Electrochemical microsensors and biosensors have been widely used in many fields, in particular neurochemical monitoring, because of their features. Usually, hydrogen peroxide (HP), obtained as a by-product of an enzymatic reaction, is the detected compound on transducers made of precious metals, in particular platinum. The over-time increase in the price of platinum and its alloys requires the use of miniaturizable low-cost supports that can be suitably modified with the deposition of Pt particles; among them, graphite is the most widespread. In the present paper, carbon-composition resistors (CCRs) and pencil leads (PLs) of different diameters (0.3, 0.5 and 2.0 mm), mainly made up of graphite, clay and some other components were used as carbonaceous support for the deposition of platinum. Platinizations were carried out by means of cyclic voltammetry (CV) and constant potential amperometry (CPA) techniques. On the platinized supports, hydrogen peroxide (HP) and ascorbic acid (AA) sensitivity were assessed in order to verify the possibility of using them as transducers of amperometric biosensors. All the used protocols determined the occurrence of HP monitoring, not appreciable on carbonaceous surfaces. We chose 0.3 mm Ø PLs for the construction of glucose biosensors by appropriately modifying the platinum surface layering, a permselective polymer, an enzyme booster, the glucose oxidase (GOx) enzyme and a containing network. The biosensor constructed in this way demonstrated a behavior comparable to that obtained using classic platinum wires.

Background: Oxidative stress plays an important role in neurodegeneration, pain and inflammation. (R)-(-)-linalool (LIN) is endowed with neuroprotective, anti-nociceptive and anti-inflammatory properties. Purpose: The present study aims at investigating the hypothesis that LIN's neuroprotective, antinociceptive and anti-inflammatory properties descend from its ability to act as antioxidant. The study challenges this hypothesis by verifying whether LIN may counteract hydrogen peroxide (H2O2)-induced oxidative stress in PC12 cells. Methods: In H2O2-exposed PC12 cells, LIN was tested on a) cell viability, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), b) damage of plasma membrane, measured by lactate dehydrogenase (LDH) release, c) intracellular levels of reactive-oxygen-species (ROS), d) apoptosis and e) cell cycle distribution. Results: Under H2O2-induced cell viability reduction, LIN protects PC12 cells. Likewise, LIN protects cells from oxidative damage by preventing the H2O2-dependent increase of LDH release, counteracts intracellular ROS overproduction and reduces H2O2-induced apoptosis. Finally, the results of the cell cycle analysis from cells exposed to H2O2 indicate that LIN incubation reduces the number of cells induced into quiescence by H2O2 in the G2/M phase. Conclusions: These findings indicate that LIN protects PC12 cells from H2O2-induced oxidative stress. This mechanism could justify the neuroprotective, anti-nociceptive and anti-inflammatory effects of this compound and suggest LIN as a potential therapeutic agent for the management oxidative stress-mediated pain.

The purpose of the present review is to give an update regarding the classification, the epidemiology, the clinical manifestation, the diagnoses, and the treatment of the Rickettsiae species present in the Mediterranean area. We performed a comprehensive search, through electronic databases (Pubmed – MEDLINE) and search engines (Google Scholar), of peer-reviewed publications (articles, reviews, and books). The availability of new diagnostic tools, including Polymerase Chain Reaction and nucleotide sequencing has significantly modified the classification of intracellular bacteria, including the order Rickettsiales with more and more new Rickettsia species recognized as human pathogens. Furthermore, emerging Rickettsia species have been found in several countries and are often associated with unique clinical pictures that may challenge the physician in the early detection of the diseases. Rickettsial infections include a wide spectrum of clinical presentations ranging from a benign to a potentially life treating disease that requires prompt recognition and proper management. Recently, due to the spread of SARS-CoV-2 infection, the differential diagnosis with COVID-19 is of crucial importance. The correct understanding of the clinical features, diagnostic tools, and proper treatment can assist clinicians in the management of Rickettsioses in the Mediterranean area.

Abstract By identifying endogenous molecules in brain extracellular fluid metabolomics can provide insight into the regulatory mechanisms and functions of sleep. Here we studied how the cortical metabolome changes during sleep, sleep deprivation and spontaneous wakefulness. Mice were implanted with electrodes for chronic sleep/wake recording and with microdialysis probes targeting prefrontal and primary motor cortex. Metabolites were measured using ultra performance liquid chromatography-high resolution mass spectrometry. Sleep/wake changes in metabolites were evaluated using partial least squares discriminant analysis, linear mixed effects model analysis of variance, and machine-learning algorithms. More than 30 known metabolites were reliably detected in most samples. When used by a logistic regression classifier, the profile of these metabolites across sleep, spontaneous wake, and enforced wake was sufficient to assign mice to their correct experimental group (pair-wise) in 80–100% of cases. Eleven of these metabolites showed significantly higher levels in awake than in sleeping mice. Some changes extend previous findings (glutamate, homovanillic acid, lactate, pyruvate, tryptophan, uridine), while others are novel (D-gluconate, N-acetyl-beta-alanine, N-acetylglutamine, orotate, succinate/methylmalonate). The upregulation of the de novo pyrimidine pathway, gluconate shunt and aerobic glycolysis may reflect a wake-dependent need to promote the synthesis of many essential components, from nucleic acids to synaptic membranes.

In this study, carbon screen-printed sensors (C-SPEs) were functionalized with a high reactivity carbonaceous material (HRCM) to measure the ascorbic acid (AA) concentration in fresh-cut fruit (i.e., watermelon and apple) with a low content of vitamin C. HRCM and the functionalized working electrodes (WEs) were characterized by SEM and TEM. The increases in the electroactive area and in the diffusion of AA molecules towards the WE surface were evaluated by cyclic voltammetry (CV) and chronoamperometry. The performance of HRCM-SPEs were evaluated by CV and constant potential amperometry compared with the non-functionalized C-SPEs and MW-SPEs nanostructured with multi-walled carbon nanotubes. The results indicated that SPEs functionalized with 5 mg/mL of HRCM and 10 mg/mL of MWCNTs had the best performances. HRCM and MWCNTs increased the electroactive area by 1.2 and 1.4 times, respectively, whereas, after functionalization, the AA diffusion rate towards the electrode surface increased by an order of 10. The calibration slopes of HRCM and MWCNTs improved from 1.9 to 3.7 times, thus reducing the LOD of C-SPE from 0.55 to 0.15 and 0.28 μM, respectively. Finally, the functionalization of the SPEs proved to be indispensable for determining the AA concentration in the watermelon and apple samples.

This work provides companies in the fresh-cut produce sector with an Ascorbate Bluetooth© Analyzer (ABA), a screen-printed sensor-based device for ascorbic acid (AA) detection, for quality control all along the supply chain. The amperometric detection of AA on fresh and fresh-cut parsley, under correct and incorrect storage temperature, allowed us to investigate the kinetics of AA decay in response to oxidative stress. The role of ascorbate oxidase (AOx) and ascorbate peroxidase (APx) was studied. ABA was used in situ by unskilled personnel. Treatments influenced AA decay kinetics, which were linear in fresh parsley, and non-linear in fresh-cut. Two hours at 28 °C immediately after chopping, the resilience of the fresh-cut parsley was reduced, even though the cold chain was restored. Two hours at −2 °C caused a rapid loss of AA until its complete decay after 72 h. Significant differences between treatments were observed in both the expression and activity of AOx and APx. ABA registered sudden changes of parsley AA following unpredicted variations of temperature during processing or transport. It was useful to remedy the effects of unexpected flaws in the cold chain, which can be proposed for quality preservation of different fresh-cut produce.

A new telemetric system for the electrochemical monitoring of dissolved oxygen is showed. The device, connected with two amperometric sensors, has been successfully applied to the wireless detection of the extracellular oxygen in the central complex of freely-walking Gromphadorhina portentosa. The unit was composed of a potentiostat, a two-channel sensor conditioning circuit, a microprocessor module, and a wireless serial transceiver. The amperometric signals were digitalized and sent to a notebook using a 2.4 GHz transceiver while a serial-to-USB converter was connected to a second transceiver for completing the communication bridge. The software, running on the laptop, allowed to save and graph the oxygen signals. The electronics showed excellent stability and the acquired data was linear in a range comprised between 0 and -165 nA, covering the entire range of oxygen concentrations. A series of experiments were performed to explore the dynamics of dissolved oxygen by exposing the animals to different gases (nitrogen, oxygen and carbon dioxide), to low temperature and anesthetic agents (chloroform and triethylamine). The resulting data are in agreement with previous O2 changes recorded in the brain of awake rats and mice. The proposed system, based on simple and inexpensive components, can constitute a new experimental model for the exploration of central complex neurochemistry and it can also work with oxidizing sensors and amperometric biosensors.

The importance of physiological glutamate has been widely demonstrated in cognitive and memory processes, as well as in neurotransmission. The involvement of physiological glutamate in several pathologies has also been established. Therefore, analytical devices for studying variations in physiological glutamate are of fundamental importance, particularly in preclinical studies. The necessary knowledge to develop and characterize biosensors for glutamate detection is often restricted to only a few research groups. However, many more groups have sought to implant such analytical devices to study the glutamatergic system in vivo. On this basis, a series of studies was undertaken to explore the medium-term storage of biosensors, thereby allowing their usage results to be differentiated from their construction and characterization processes to facilitate the wider diffusion and use of such sensors. Therefore, it has become vital to determine the best storage conditions to extend the life and functionality of these biosensors, especially due to the diachronic instability of the enzyme present on the surface. In the present study, we analyzed the impact of glycols, such as glycerol and triethylene glycol, as enzyme stabilizers coupled with long-term storage at low temperatures (−20 and −80 °C) on biosensor performance. The biosensors were observed for 5 months and evaluated for their enzymatic activity by measuring the VMAX(app) and KM(app). The analytical features were also evaluated in terms of the Linear Region Slope, which is one the most important parameters for indicating the efficiency and the sensitivity of biosensors. Interestingly, both glycols proved to be capable of increasing enzymatic activity and maintaining good biosensor efficiency over time. Moreover, the combination with low-temperature storage highlighted the different behaviors of the two glycols. In particular, glycerol was more effective in stabilizing the enzyme and maintaining analytical performance when the biosensors were stored at −20 °C. Instead, triethylene glycol performed the same function as glycerol but when the biosensors were stored at −80 °C.

Pompia is a Citrus species belonging to Sardinian endemic biodiversity. Health benefits were attributed to its flavedo rind extracts and essential oils while the juice qualities have never been investigated. In this paper, the antioxidant, antimicrobial, and other biological properties of Pompia juice were studied. A combined LCMS/electrochemical/biological approach was used to clarify a still debated phylogeny of this species and to explain the role of its juice phenolic compounds. A closer phylogenetic relationship with lemon and citron, rather than oranges was suggested. Sensors-based electrochemical measures, together with LCMS qualitative and quantitative analyses, revealed a high contribution of ascorbic acid and phenolics with low redox potential, isorhamnetin 3-O-rutinoside, diosmin, and diosmetin 6,8-diglucoside, to antioxidant capacity. The biological assays demonstrated a marked effect of low concentration of Pompia juice against reactive oxygen species (ROS) starting from 50 µg mL−1, and a moderate capacity to reduce ROS damages on cell membrane. Treatments with Pompia juice also resulted in a significant reduction (20%) of the metabolic activity of SW48 colon cancer cells. Lastly, MIC, MBC, and MBIC antimicrobial assays demonstrated that Pompia and lemon juices have inhibitory and antibiofilm effects against the pathogenic bacteria Pseudomonas aeruginosa, Streptococcus aureus, and Enterococcus faecalis.

The impaired activity of tyrosinase and laccase can provoke serious concerns in the life cycles of mammals, insects and microorganisms. Investigation of inhibitors of these two enzymes may lead to the discovery of whitening agents, medicinal products, anti-browning substances and compounds for controlling harmful insects and bacteria. A small collection of novel reversible tyrosinase and laccase inhibitors with a phenylpropanoid and hydroxylated biphenyl core was prepared using naturally occurring compounds and their activity was measured by spectrophotometric and electrochemical assays. Biosensors based on tyrosinase and laccase enzymes were constructed and used to detect the type of protein-ligand interaction and half maximal inhibitory concentration (IC50). Most of the inhibitors showed an IC50 in a range of 20–423 nM for tyrosinase and 23–2619 nM for laccase. Due to the safety concerns of conventional tyrosinase and laccase inhibitors, the viability of the new compounds was assayed on PC12 cells, four of which showed a viability of roughly 80% at 40 µM. In silico studies on the crystal structure of laccase enzyme identified a hydroxylated biphenyl bearing a prenylated chain as the lead structure, which activated strong and effective interactions at the active site of the enzyme. These data were confirmed by in vivo experiments performed on the insect model Tenebrio molitur.

Glutamate is a major excitatory neurotransmitter in the brain. It is involved in many normal physiological brain activities, but also neurological disorders and excitotoxicity. Hence, glutamate measurement is important both in clinical and pre-clinical studies. Pre-clinical studies often use amperometric biosensors due to their low invasiveness and the relatively small size of the devices. These devices also provide fast, real-time measurements because of their high sensitivity. In the present study, diethylene glycol (DEG), neopentyl glycol (NPG), triethylene glycol (TEG), and glycerol (GLY) were used to increase the long-term stability of glutamate biosensors. The evaluation was made by measuring variations of the main enzymatic (VMAX and KM) and analytical (Linear Region Slope (LRS)) parameters. Of the glycols tested, TEG was the most promising stabilizer, showing about twice as high VMAX maintained over a greater duration than with other stabilizers tested. It is also yielded the most stable linear region slope (LRS) values over the study duration. Moreover, we highlighted the ability of glycols to interact with enzyme molecules to form a containment network, able to maintain all the layered components of the biosensor adhering to the transducer.

The hepatocellular carcinoma is one of the most common malignant tumour with high level of mortality rate due to its rapid progression and high resistance to conventional chemotherapies. Thus, the search for novel therapeutic leads is of global interest. Herein, a small set of derivatives of magnolol 1 and honokiol 2, the main components of Magnolia grandiflora and Magnolia obovata, were evaluated in in vitro assay using tumoral hepatocytes. The pro-drug approach was applied as versatile strategy to the improve bioactivity of the compounds by careful transformation of the hydroxyl groups of magnolol 1 and honokiol 2 in suitable ester derivatives. Compounds 10 and 11 resulted to be more potent than the parental honokiol 2 at concentration down to 1 μM with complete viability of treated fibroblast cells up to concentrations of 80 μM. The combination of a butyrate ester and a bare phenol-OH group in the honokiol structure seemed to play a significant role in the antiproliferative activity identifying an interesting pharmacological clue against hepatocellular carcinoma.

The use of amperometric biosensors has attracted particular attention in recent years, both from researchers and from companies, as they have proven to be low-cost, reliable, and very sensitive devices, with a wide range of uses in different matrices. The continuous development of amperometric biosensors, since their use involves an enzyme, is specifically aimed at keeping and increasing the catalytic properties of the loaded protein, so as to be able to use the same device over time. The present study aimed to investigate the impact of glycerol and polysaccharides, in the presence of polycationic substances to constitute a hydrogel, in enhancing the enzymatic and analytic performance of a glucose biosensor. Initially, it was possible to verify how the deposition of the starch-based hydrogel, in addition to allowing the electropolymerization of the poly(p-phenylenediamine) polymer and the maintenance of its ability to shield the ascorbic acid, did not substantially limit the permeability towards hydrogen peroxide. Moreover, different biosensor designs, loading a mixture containing all the components (alone or in combination) and the enzyme, were tested in order to evaluate the changes of the apparent enzyme kinetic parameters, such as VMAX and KM, and analytical response in terms of Linear Region Slope, highlighting how the presence of all components (starch, glycerol, and polyethyleneimine) were able to substantially enhance the performance of the biosensors. The surface analysis of the biosensors was performed by scanning electron microscope (SEM). More, it was shown that the same performances were kept unchanged for seven days, proving the suitability of this biosensor design for short- and mid-term use.

Cholangiocarcinoma (CCA) is a highly-aggressive malignancy arising from the biliary tree, characterized by a steady increase in incidence globally and a high mortality rate. Most CCAs are diagnosed in the advanced and metastatic phases of the disease, due to the paucity of signs and symptoms in the early stages. This fact, along with the poor results of the local and systemic therapies currently employed, is responsible for the poor outcome of CCA patients and strongly supports the need for novel therapeutic agents and strategies. In recent years, the introduction of next-generation sequencing technologies has opened new horizons for a better understanding of the genetic pathophysiology of CCA and, consequently, for the identification and evaluation of new treatments tailored to the molecular features or alterations progressively elucidated. In this review article, we describe the potential targets under investigation and the current molecular therapies employed in biliary tract cancers. In addition, we summarize the main drugs against CCA under evaluation in ongoing trials and describe the preliminary data coming from these pioneering studies.

Molecular biomarkers are very important in biology, biotechnology and even in medicine, but it is quite hard to convert biology-related signals into measurable data. For this purpose, amperometric biosensors have proven to be particularly suitable because of their specificity and sensitivity. The operation and shelf stability of the biosensor are quite important features, and storage procedures therefore play an important role in preserving the performance of the biosensors. In the present study two different designs for both glucose and lactate biosensor, differing only in regards to the containment net, represented by polyurethane or glutharaldehyde, were studied under different storage conditions (+4, −20 and −80 °C) and monitored over a period of 120 days, in order to evaluate the variations of kinetic parameters, as VMAX and KM, and LRS as the analytical parameter. Surprisingly, the storage at −80 °C yielded the best results because of an unexpected and, most of all, long-lasting increase of VMAX and LRS, denoting an interesting improvement in enzyme performances and stability over time. The present study aimed to also evaluate the impact of a short-period storage in dry ice on biosensor performances, in order to simulate a hypothetical preparation-conservation-shipment condition.

Ethyl alcohol may be considered one of the most widespread central nervous system (CNS) depressants in Western countries. Because of its toxicological and neurobiological implications, the detection of ethanol in brain extracellular fluid (ECF) is of great importance. In a previous study, we described the development and characterization of an implantable biosensor successfully used for the real-time detection of ethanol in the brain of freely-moving rats. The implanted biosensor, integrated in a low-cost telemetry system, was demonstrated to be a reliable device for the short-time monitoring of exogenous ethanol in brain ECF. In this paper we describe a further in-vitro characterization of the above-mentioned biosensor in terms of oxygen, pH and temperature dependence in order to complete its validation. With the aim of enhancing ethanol biosensor performance, different enzyme loadings were investigated in terms of apparent ethanol Michaelis-Menten kinetic parameters, viz. IMAX, KM and linear region slope, as well as ascorbic acid interference shielding. The responses of biosensors were studied over a period of 28 days. The overall findings of the present study confirm the original biosensor configuration to be the best of those investigated for in-vivo applications up to one week after implantation.

The present paper aims to miniaturize a graphite-epoxy and synthetic zeolite-modified graphite-epoxy composite macroelectrode as a quasi-microelectrode aiming in vitro and also, envisaging in vivo simultaneous electrochemical detection of dopamine (DA) and ascorbic acid (AA) neurotransmitters, or DA detection in the presence of AA. The electrochemical behavior and the response of the designed materials to the presence of dopamine and ascorbic acid without any protective membranes were studied by cyclic voltammetry and constant-potential amperometry techniques. The catalytic effect towards dopamine detection was proved for the synthetic zeolite-modified graphite-epoxy composite quasi-microelectrode, allowing increasing the sensitivity and selectivity for this analyte detection, besides a possible electrostatic attraction between dopamine cation and the negative surface of the synthetic zeolite and electrostatic repulsion with ascorbic acid anion. Also, the synthetic zeolite-modified graphite-epoxy composite quasi-microelectrode gave the best electroanalytical parameters for dopamine detection using constant-potential amperometry, the most useful technique for practical applications.

L-glutamate is one the most important excitatory neurotransmitter at the central nervous system level and it is implicated in several pathologies. So, it is very important to monitor its variations, in real time in animal models’ brain. The present study aimed to develop and characterize a new amperometric glutamate biosensor design that exploits the selectivity of Glutamate Oxidase (GluOx) for l-glutamate, and the capability of a small molecule as propylene glycol (PG), never used before, to influence and extend the stability and the activity of enzyme. Different designs were evaluated by modifying the main components in their concentrations to find the most suitable design. Moreover, enzyme concentrations from 100 U/mL up to 200 U/mL were verified and different PG concentrations (1%, 0.1% and 0.05%) were tested. The most suitable selected design was Ptc/PPD/PEI(1%)2/GlutOx5/PG(0.1%) and it was compared to the same already described design loading PEDGE, instead of PG, in terms of over-time performances. The PG has proved to be capable of determining an over-time stability of the glutamate biosensor in particular in terms of linear region slope (LRS) up to 21 days.

The Ca2+ sensitizer levosimendan (LEV) improves myocardial contractility by enhancing the sensitivity of the contractile apparatus to Ca2+. In addition, LEV promotes Ca2+ entry through L-type channels in human cardiac myocytes. In this study, which was performed using microdialysis, infusion of LEV at 0.25 μM for 160 min increased dopamine (DA) concentrations (up to fivefold baseline) in dialysates from the striatum of freely moving rats. Ca2+ omission from the perfusion fluid abolished baseline DA release and greatly decreased LEV-induced DA release. Reintroduction of Ca2+ in the perfusion fluid restored LEV-induced DA release. Chelation of intracellular Ca2+ by co-infusing 1,2-bis (o-amino-phenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM, 0.2 mM) did not affect basal DA release and scarcely affected LEV-induced increases in dialysate DA. In addition, co-infusion of the L-type (Cav 1.1 – 1.3) voltage-sensitive Ca2+-channel inhibitor nifedipine failed to inhibit LEV-induced increases in dialysate DA, which, in contrast, was inhibited by co-infusion of the N-type (Cav 2.2) voltage-sensitive Ca2+-channel inhibitor ω-conotoxin GVIA. We conclude that LEV promotes striatal extracellular Ca2+ entry through N-type Ca2+ channels with a consequent increase in DA release. Keywords:: levosimendan, microdialysis, striatal dopamine, calcium entry

This study investigated the mechanisms and kinetics of nitric oxide (NO) generation by derivatives of Piloty’s acid (NO-donors) under physiological conditions. In order to qualitatively and quantitatively measure NO release, electron paramagnetic resonance (EPR) was carried out with NO spin trapping. In addition, voltammetric techniques, including cyclic voltammetry and constant potential amperometry, were used to confirm NO release from Piloty’s acid and its derivatives. The resulting data showed that Piloty’s acid derivatives are able to release NO under physiological conditions. In particular, electron-withdrawing substituents favoured NO generation, while electron-donor groups reduced NO generation. In vitro microdialysis, performed on PC12 cell cultures, was used to evaluate the dynamical secretion of dopamine induced by the Piloty’s acid derivatives. Although all the studied molecules were able to induce DA secretion from PC12, only those with a slow release of NO have not determined an autoxidation of DA itself. These results confirm that the time-course of NO-donors decomposition and the amount of NO released play a key role in dopamine secretion and auto-oxidation. This information could drive the synthesis or the selection of compounds to use as potential drugs for the therapy of Parkinson’s disease (PD).

The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S) pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S) shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented.