All-Diamond Microelectrodes as Solid State Probes for Localized Electrochemical Sensing.

The fabrication of an all-diamond microprobe is demonstrated for the first time. This ME (microelectrode) assembly consists of an inner boron doped diamond (BDD) layer and an outer undoped diamond layer. Both layers were grown on a sharp tungsten tip by chemical vapor deposition (CVD) in a stepwise manner within a single deposition run. BDD is a material with proven potential as an electrochemical sensor. Undoped CVD diamond is an insulating material with superior chemical stability in comparison to conventional insulators. Focused ion beam (FIB) cutting of the apex of the ME was used to expose an electroactive BDD disk. By cyclic voltammetry, the redox reaction of ferrocenemethanol was shown to take place at the BDD microdisk surface. In order to ensure that the outer layer was nonelectrically conductive, a diffusion barrier for boron atoms was established seeking the formation of boron-hydrogen complexes at the interface between the doped and the undoped diamond layers. The applicability of the microelectrodes in localized corrosion was demonstrated by scanning amperometric measurements of oxygen distribution above an Al-Cu-CFRP (Carbon Fiber Reinforced Polymer) galvanic corrosion cell.

Pervasive sign epistasis between conjugative plasmids and drug-resistance chromosomal mutations.

Multidrug-resistant bacteria arise mostly by the accumulation of plasmids and chromosomal mutations. Typically, these resistant determinants are costly to the bacterial cell. Yet, recently, it has been found that, in Escherichia coli bacterial cells, a mutation conferring resistance to an antibiotic can be advantageous to the bacterial cell if another antibiotic-resistance mutation is already present, a phenomenon called sign epistasis. Here we study the interaction between antibiotic-resistance chromosomal mutations and conjugative (i.e., self-transmissible) plasmids and find many cases of sign epistasis (40%)--including one of reciprocal sign epistasis where the strain carrying both resistance determinants is fitter than the two strains carrying only one of the determinants. This implies that the acquisition of an additional resistance plasmid or of a resistance mutation often increases the fitness of a bacterial strain already resistant to antibiotics. We further show that there is an overall antagonistic interaction between mutations and plasmids (52%). These results further complicate expectations of resistance reversal by interdiction of antibiotic use.

PERK inhibition in zebrafish mimics human Wolcott-Rallison syndrome phenotypes.

Wolcott-Rallison Syndrome (WRS) is the most common cause of permanent neonatal diabetes mellitus among consanguineous families. The diabetes associated with WRS is non-autoimmune, insulin-requiring and associated with skeletal dysplasia and growth retardation. The therapeutic options for WRS patients rely on permanent insulin pumping or on invasive transplants of liver and pancreas. WRS has a well identified genetic cause: loss-of-function mutations in the gene coding for an endoplasmic reticulum kinase named PERK (protein kinase R-like ER kinase). Currently, WRS research is facilitated by cellular and rodent models with PERK ablation. While these models have unique strengths, cellular models incompletely replicate the organ/system-level complexity of WRS, and rodents have limited scalability for efficiently screening potential therapeutics. To address these challenges, we developed a new in vivo model of WRS by pharmacologically inhibiting PERK in zebrafish. This small vertebrate displays high fecundity, rapid development of organ systems and is amenable to highly efficient in vivo drug testing. PERK inhibition in zebrafish produced typical WRS phenotypes such as glucose dysregulation, skeletal defects, and impaired development. PERK inhibition in zebrafish also produced broad-spectrum WRS phenotypes such as impaired neuromuscular function, compromised cardiac function and muscular integrity. These results show that zebrafish holds potential as a versatile model to study WRS mechanisms and contribute to the identification of promising therapeutic options for WRS.

Cross-talk between neurons and astrocytes in response to bilirubin: early beneficial effects.

Hyperbilirubinemia remains one of the most frequent clinical diagnoses in the neonatal period. This condition may lead to the deposition of unconjugated bilirubin (UCB) in the central nervous system, causing nerve cell damage by molecular and cellular mechanisms that are still being clarified. To date, all the studies regarding bilirubin-induced neurological dysfunction were performed in monotypic nerve cell cultures. The use of co-cultures, where astrocyte-containing culture inserts are placed on the top of neuron cultures, provides the means to directly evaluate the cross-talk between these two different cell types. Therefore, this study was designed to evaluate whether protective or detrimental effects are produced by astrocytes over UCB-induced neurodegeneration. Our experimental model used an indirect co-culture system where neuron-to-astrocyte signaling was established concomitantly with the 24 h exposure to UCB. In this model astrocytes abrogated the well-known UCB-induced neurotoxic effects by preventing the loss of cell viability, dysfunction and death by apoptosis, as well as the impairment of neuritic outgrowth. To this protection it may have accounted the induced expression of the multidrug resistance-associated protein 1 and the 3.5-fold increase in the values of S100B, when communication between both cells was established independently of UCB presence. In addition, the presence of astrocytes in the neuronal environment preserved the UCB-induced increase in glutamate levels, but raised the basal concentrations of nitric oxide and TNF-α although no UCB effects were noticed. Our data suggest that bidirectional signalling during astrocyte-neuron recognition exerts pro-survival effects, stimulates neuritogenesis and sustains neuronal homeostasis, thus protecting cells from the immediate UCB injury. These findings may help explain why irreversible brain damage usually develops only after the first day of post-natal life.

Artifact level produced by different femoral head prostheses in CT imaging: diamond coated silicon nitride as total hip replacement material.

Commercial femoral head prostheses (cobalt-chromium alloy, yttria partially stabilized zirconia (Y-PSZ) and alumina) and new silicon nitride ceramic ones (nanocrystalline diamond coated and uncoated) were compared in terms of artifact level production by computed tomography (CT). Pelvis examination by CT allows the correct diagnosis of some pathologies (e.g. prostate and colon cancer) and the evaluation of the prosthesis-bone interface in post-operative joint surgery. Artifact quantification is rarely seen in literature despite having a great potential to grade biomaterials according to their imaging properties. Materials' characteristics (density and effective atomic number), size and geometry of the prostheses can cause more or less artifact. A quantification procedure based on the calculation of four statistical parameters for the Hounsfield pixel values (mean, standard deviation, mean squared error and worst case error) is presented. CT sequential and helical scanning modes were performed. Results prove the artifact reproducibility and indicate that the cobalt-chromium and Y-PSZ are the most artifact-inducing materials, while alumina and silicon nitride (diamond coated and uncoated) ceramic ones present a low level of artifact. Considering the excellent biocompatibility and biotribological behaviour reported in earlier works, combined with the high medical imaging quality here assessed, diamond coated silicon nitride ceramics are arising as new materials for joint replacement.

Carbon Nanostructures-Silica Aerogel Composites for Adsorption of Organic Pollutants.

Silica aerogels are a class of materials that can be tailored in terms of their final properties and surface chemistry. They can be synthesized with specific features to be used as adsorbents, resulting in improved performance for wastewater pollutants' removal. The purpose of this research was to investigate the effect of amino functionalization and the addition of carbon nanostructures to silica aerogels made from methyltrimethoxysilane (MTMS) on their removal capacities for various contaminants in aqueous solutions. The MTMS-based aerogels successfully removed various organic compounds and drugs, achieving adsorption capacities of 170 mg⋅g-1 for toluene and 200 mg⋅g-1 for xylene. For initial concentrations up to 50 mg⋅L-1, removals greater than 71% were obtained for amoxicillin, and superior to 96% for naproxen. The addition of a co-precursor containing amine groups and/or carbon nanomaterials was proven to be a valuable tool in the development of new adsorbents by altering the aerogels' properties and enhancing their adsorption capacities. Therefore, this work demonstrates the potential of these materials as an alternative to industrial sorbents due to their high and fast removal efficiency, less than 60 min for the organic compounds, towards different types of pollutants.

Customizing 3D Structures of Vertically Aligned Carbon Nanotubes to Direct Neural Stem Cell Differentiation.

Neural tissue-related illnesses have a high incidence and prevalence in society. Despite intensive research efforts to enhance the regeneration of neural cells into functional tissue, effective treatments are still unavailable. Here, a novel therapeutic approach based on vertically aligned carbon nanotube forests (VA-CNT forests) and periodic VA-CNT micropillars produced by thermal chemical vapor deposition is explored. In addition, honeycomb-like and flower-like morphologies are created. Initial viability testing reveals that NE-4C neural stem cells seeded on all morphologies survive and proliferate. In addition, free-standing VA-CNT forests and capillary-driven VA-CNT forests are created, with the latter demonstrating enhanced capacity to stimulate neuritogenesis and network formation under minimal differentiation medium conditions. This is attributed to the interaction between surface roughness and 3D-like morphology that mimics the native extracellular matrix, thus enhancing cellular attachment and communication. These findings provide a new avenue for the construction of electroresponsive scaffolds based on CNTs for neural tissue engineering.

Disruptions of circadian rhythms, sleep, and stress responses in zebrafish: New infrared-based activity monitoring assays for toxicity assessment.

Behavioural disruptions are sensitive indicators of alterations to normal animal physiology and can be used for toxicity assessment. The small vertebrate zebrafish is a leading model organism for toxicological studies. The ability to continuously monitor the toxicity of drugs, pollutants, or environmental changes over several days in zebrafish can have high practical application. Although video-recordings can be used to monitor short-term zebrafish behaviour, it is challenging to videorecord prolonged experiments (e.g. circadian behaviour over several days) because of the darkness periods (nights) and the heavy data storage and image processing requirements. Alternatively, infrared-based activity monitors, widely used in invertebrate models such as drosophila, generate simple and low-storage data and could optimize large-scale prolonged behavioural experiments in zebrafish, thus favouring the implementation of high-throughput testing strategies. Here, we validate the use of a Locomotor Activity Monitor (LAM) to study the behaviour of zebrafish larvae, and we characterize the behavioural phenotypes induced by abnormal light conditions and by the Parkinsonian toxin MPP+. When zebrafish were deprived from daily light-cycle synchronization, the LAM detected various circadian disruptions, such as increased activity period, phase shifts, and decreased inter-daily stability. Zebrafish exposed to MPP+ (10, 100, 500 µM) showed a concentration-dependent decrease in activity, sleep disruptions, impaired habituation to repetitive startles (visual-motor responses), and a slower recovery to normal activity after the startle-associated stress. These phenotypes evidence the feasibility of using infrared-based LAM to assess multi-parameter behavioural disruptions in zebrafish. The procedures in this study have wide applicability and may yield standard methods for toxicity testing.

Impact of atomic layer deposited TiO2 on the photocatalytic efficiency of TiO2/w-VA-CNT nanocomposite materials.

Titanium oxide (TiO2) has been widely investigated as a photocatalytic material, and the fact that its performance depends on its crystalline structure motivates further research on the relationship between preparation methods and material properties. In this work, TiO2 thin films were grown on non-functionalized wave-like patterned vertically aligned carbon nanotubes (w-VA-CNTs) via the atomic layer deposition (ALD) technique. Grazing incidence X-ray diffraction (GIXRD) analysis revealed that the structure of the TiO2/VA-CNT nanocomposites varied from amorphous to a crystalline phase with increasing deposition temperature, suggesting a "critical deposition temperature" for the anatase crystalline phase formation. On the other hand, scanning transmission electron microscopy (STEM) studies revealed that the non-functionalized carbon nanotubes were conformally and homogeneously coated with TiO2, forming a nanocomposite while preserving the morphology of the nanotubes. X-ray photoelectron spectroscopy (XPS) provided information about the surface chemistry and stoichiometry of TiO2. The photodegradation experiments under ultraviolet (UV) light on a model pollutant (Rhodamine B, RhB) revealed that the nanocomposite comprised of anatase crystalline TiO2 grown at 200 °C (11.2 nm thickness) presented the highest degradation efficiency viz 55% with an illumination time of 240 min. Furthermore, its recyclability was also demonstrated for multiple cycles, showing good recovery and potential for practical applications.

Automated analysis of activity, sleep, and rhythmic behaviour in various animal species with the Rtivity software.

Behavioural studies provide insights into normal and disrupted biological mechanisms. In many research areas, a growing spectrum of animal models-particularly small organisms-is used for high-throughput studies with infrared-based activity monitors, generating counts per time data. The freely available software to analyse such data, however, are primarily optimized for drosophila and circadian analysis. Researchers investigating other species or non-circadian behaviour would thus benefit from a more versatile software. Here we report the development of a free and open-source software-Rtivity-allowing customisation of species-specific parameters, and offering a versatile analysis of behavioural patterns, biological rhythms, stimulus responses, and survival. Rtivity is based on the R language and uses Shiny and the recently developed Rethomics package for a user-friendly graphical interface without requiring coding skills. Rtivity automatically assesses survival, computes various activity, sleep, and rhythmicity parameters, and performs fractal analysis of activity fluctuations. Rtivity generates multiple informative graphs, and exports structured data for efficient interoperability with common statistical software. In summary, Rtivity facilitates and enhances the versatility of the behavioural analysis of diverse animal species (e.g. drosophila, zebrafish, daphnia, ants). It is thus suitable for a broad range of researchers from multidisciplinary fields such as ecology, neurobiology, toxicology, and pharmacology.

Advances in RF Glow Discharge Optical Emission Spectrometry Characterization of Intrinsic and Boron-Doped Diamond Coatings.

Accurate determination of the effective doping range within diamond thin films is important for fine-tuning of electrical conductivity. Nevertheless, it is not easily attainable by the commonly adopted techniques. In this work, pulsed RF glow discharge optical emission spectrometry (GD-OES) combined with ultrafast sputtering (UFS) is applied for the first time to acquire elemental depth profiles of intrinsic diamond coatings and boron content bulk distribution in films. The GD-OES practical advances presented here enabled quick elemental profiling with noteworthy depth resolution and determination of the film interfaces. The erosion rates and layer thicknesses were measured using differential interferometric profiling (DIP), demonstrating a close correlation between the coating thickness and the carbon/hydrogen gas ratio. Moreover, DIP and the adopted semiquantification methodology revealed a nonhomogeneous bulk distribution of boron within the diamond crystalline structure, i.e., boron doping is both substitutional and interstitial within the diamond framework. DIP measurements also showed that effective boron doping is not linearly correlated to the increasing content introduced into the diamond coating. This is a finding well supported by X-ray diffraction (XRD) Rietveld refinement and X-ray photoelectron spectroscopy (XPS). This work demonstrates the advantage of applying advanced GD-OES operation modes due to its ease of use, affordability, accuracy, and high-speed depth profile analysis capability.

Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin.

Microglia are the main players of the brain immune response. They act as active sensors that rapidly respond to injurious insults by shifting into different activated states. Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation and oxidative stress in both neurons and astrocytes. We recently reported that microglial phagocytic phenotype precedes the release of pro-inflammatory cytokines upon UCB exposure. We investigated whether and how microglia microenvironment influences the response to UCB. Our findings revealed that conditioned media derived from UCB-treated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an attempt to curtail microglial over activation. Conditioned medium from UCB-challenged neurons, although down-regulating tumor necrosis factor-α and interleukin-1ß promoted the release of interleukin-6 and nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared with UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated neurons intensified the phagocytic properties manifested by microglia under direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with UCB evidenced that sensitized microglia were able to prevent neurite outgrowth impairment and cell death. In conclusion, our data indicate that stressed neurons signal microglial clearance functions, but also overstimulate its inflammatory potential ultimately leading to microglia demise.

Neuroprotective Properties of Food-Borne Polyphenols in Neurodegenerative Diseases.

Fruits and vegetables are the richest source of polyphenols in the regular human diet [...].

Multilayer Diamond Coatings Applied to Micro-End-Milling of Cemented Carbide.

Cobalt-cemented carbide micro-end mills were coated with diamond grown by chemical vapor deposition (CVD), with the purpose of micro-machining cemented carbides. The diamond coatings were designed with a multilayer architecture, alternating between sub-microcrystalline and nanocrystalline diamond layers. The structure of the coatings was studied by transmission electron microscopy. High adhesion to the chemically pre-treated WC-7Co tool substrates was observed by Rockwell C indentation, with the diamond coatings withstanding a critical load of 1250 N. The coated tools were tested for micro-end-milling of WC-15Co under air-cooling conditions, being able to cut more than 6500 m over a period of 120 min, after which a flank wear of 47.8 µm was attained. The machining performance and wear behavior of the micro-cutters was studied by scanning electron microscopy and energy-dispersive X-ray spectroscopy. Crystallographic analysis through cross-sectional selected area electron diffraction patterns, along with characterization in dark-field and HRTEM modes, provided a possible correlation between interfacial stress relaxation and wear properties of the coatings. Overall, this work demonstrates that high adhesion of diamond coatings can be achieved by proper combination of chemical attack and coating architecture. By preventing catastrophic delamination, multilayer CVD diamond coatings are central towards the enhancement of the wear properties and mechanical robustness of carbide tools used for micro-machining of ultra-hard materials.

Boron Doped Diamond for Real-Time Wireless Cutting Temperature Monitoring of Diamond Coated Carbide Tools.

Among the unique opportunities and developments that are currently being triggered by the fourth industrial revolution, developments in cutting tools have been following the trend of an ever more holistic control of manufacturing processes. Sustainable manufacturing is at the forefront of tools development, encompassing environmental, economic, and technological goals. The integrated use of sensors, data processing, and smart algorithms for fast optimization or real time adjustment of cutting processes can lead to a significant impact on productivity and energy uptake, as well as less usage of cutting fluids. Diamond is the material of choice for machining of non-ferrous alloys, composites, and ultrahard materials. While the extreme hardness, thermal conductivity, and wear resistance of CVD diamond coatings are well-known, these also exhibit highly auspicious sensing properties through doping with boron and other elements. The present study focuses on the thermal response of boron-doped diamond (BDD) coatings. BDD coatings have been shown to have a negative temperature coefficient (NTC). Several approaches have been adopted for monitoring cutting temperature, including thin film thermocouples and infrared thermography. Although these are good solutions, they can be costly and become impractical for certain finishing cutting operations, tool geometries such as rotary tools, as well as during material removal in intricate spaces. In the scope of this study, diamond/WC-Co substrates were coated with BDD by hot filament chemical vapor deposition (HFCVD). Scanning electron microscopy, Raman spectroscopy, and the van der Pauw method were used for morphological, structural, and electrical characterization, respectively. The thermal response of the thin diamond thermistors was characterized in the temperature interval of 20-400 °C. Compared to state-of-the-art temperature monitoring solutions, this is a one-step approach that improves the wear properties and heat dissipation of carbide tools while providing real-time and in-situ temperature monitoring.

Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation.

Microglia constitute the brain's immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, upregulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target.

N-methyl-aspartate receptor and neuronal nitric oxide synthase activation mediate bilirubin-induced neurotoxicity.

Hyperbilirubinemia may lead to neurotoxicity and neuronal death. Although the mechanisms of nerve cell damage by unconjugated bilirubin (UCB) appear to involve a disruption of the redox status and excitotoxicity, the contribution of nitric oxide (NO·) and of N-methyl-D-aspartate (NMDA) glutamate receptors is unclear. We investigated the role of NO· and NMDA glutamate receptors in the pathways of nerve cell demise by UCB. Neurons were incubated with 100 micromol/L UCB, in the presence of 100 micromol/L human serum albumin for 4 h at 37ºC, alone or in combination with N-ω-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of neuronal nitric oxide synthase [nNOS]), hemoglobin (an NO· scavenger) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (an NMDA-receptor antagonist). Exposure to UCB led to increased expression of nNOS and production of both NO· and cyclic guanosine 3',5'-monophosphate (cGMP), along with protein oxidation and depletion of glutathione. These events concurred for cell dysfunction and death and were counteracted by L-NAME. Moreover, the UCB-induced loss of neuronal viability was abolished by hemoglobin, whereas the activation of nNOS and production of both NO· and cGMP were counteracted by MK-801, resulting in significant protection from cell dysfunction and death. These results reinforce the involvement of oxidative stress by showing that nerve cell damage by UCB is mediated by NO· and therefore is counteracted by NO· inhibitors or scavengers. Our findings strongly suggest that the activation of nNOS and neurotoxicity occur through the engagement of NMDA receptors. These data reveal a role for overstimulation of glutamate receptors in mediating oxidative damage by UCB.

Polyphenols from Food and Natural Products: Neuroprotection and Safety.

Polyphenols are naturally occurring micronutrients that are present in many food sources. Besides being potent antioxidants, these molecules may also possess anti-inflammatory properties. Many studies have highlighted their potential role in the prevention and treatment of various pathological conditions connected to oxidative stress and inflammation (e.g., cancer, and cardiovascular and neurodegenerative disorders). Neurodegenerative diseases are globally one of the main causes of death and represent an enormous burden in terms of human suffering, social distress, and economic costs. Recent data expanded on the initial antioxidant-based mechanism of polyphenols' action by showing that they are also able to modulate several cell-signaling pathways and mediators. The proposed benefits of polyphenols, either as protective/prophylactic substances or as therapeutic molecules, may be achieved by the consumption of a natural polyphenol-enriched diet, by their use as food supplements, or with formulations as pharmaceutical drugs/nutraceuticals. It has also been proved that the health effects of polyphenols depend on the consumed amount and their bioavailability. However, their overconsumption may raise safety concerns due to the accumulation of high levels of these molecules in the organism, particularly if we consider the loose regulatory legislation regarding the commercialization and use of food supplements. This review addresses the main beneficial effects of food polyphenols, and focuses on neuroprotection and the safety issues related to overconsumption.

Physical Structure and Electrochemical Response of Diamond-Graphite Nanoplatelets: From CVD Synthesis to Label-Free Biosensors.

Hybrid diamond-graphite nanoplatelet (DGNP) thin films are produced and applied to label-free impedimetric biosensors for the first time, using avidin detection as a proof of concept. The DGNPs are synthesized by microwave plasma chemical vapor deposition through H2/CH4/N2 gas mixtures in a reproducible and rapid single-step process. The material building unit consists of an inner two-dimensional-like nanodiamond with preferential vertical alignment covered by and covalently bound to nanocrystalline graphite grains, exhibiting {111}diamond||{0002}graphite epitaxy. The DGNP films' morphostructural aspects are of interest for electrochemical transduction, in general, and for Faradaic impedimetric biosensors, in particular, combining enhanced surface area for biorecognition element loading and facile Faradaic charge transfer. Charge transfer rate constants in phosphate buffer saline/[Fe(CN)6]4- solution are shown to increase up to 5.6 × 10-3 cm s-1 upon N2 addition to DGNP synthesis. For the impedimetric detection of avidin, biotin molecules are covalently bound as avidin specific recognition elements on (3-aminopropyl)triethoxysilane-functionalized DGNP surfaces. Avidin quantification is attained within the 10-1000 µg mL-1 range following a logarithmic dependency. The limits of detection and of quantitation are 1.3 and 6.4 µg mL-1 (19 and 93 nM), respectively, and 2.3 and 13.8 µg mL-1 (33 and 200 nM) when considering the nonspecific response of the sensors.

Bilirubin injury to neurons: contribution of oxidative stress and rescue by glycoursodeoxycholic acid.

It is well established that high levels of unconjugated bilirubin (UCB) can be toxic to the central nervous system, and oxidative stress is emerging as a relevant event in the mechanisms of UCB encephalopathy. In contrast, the hydrophilic bile acid, ursodeoxycholic acid (UDCA), has been reported as a cytoprotective and antioxidant molecule. In this study, we investigated if exposure of rat neurons in primary culture to clinically relevant concentrations of UCB leads to oxidative injury. The contribution of oxidative stress in UCB neurotoxicity was further investigated by examining whether the reduction of NO production by NAME, an inhibitor of nitric oxide synthase, prevents the disruption of the redox status and neuronal damage. Moreover, we evaluated the ability of glycoursodeoxycholic acid (GUDCA), the most relevant conjugated derivative in the serum of patients treated with UDCA, to abrogate the UCB-induced oxidative damage. Cultured rat neurons were incubated with 50 or 100microM UCB in the presence of 100microM human serum albumin, alone or in combination with 100microM NAME or with 50microM GUDCA, for 4h at 37 degrees C. Protein carbonyls, 4-hydroxy-2-nonenal-protein adducts, intracellular glutathione content and cell death were determined. The results obtained showed that UCB induces protein oxidation and lipid peroxidation, while diminishes the thiol antioxidant defences, events that were correlated with the extent of cell death. Moreover, these events were counteracted by NAME and abrogated in the presence of GUDCA. Collectively, this study shows that oxidative stress is one of the pathways associated with neuronal viability impairment by UCB, and that GUDCA significantly prevents such effects from occurring. These findings corroborate the antioxidant properties of the bile acid and point to a new therapeutic approach for UCB-induced neurotoxicity due to oxidative stress.