Induction of haem oxygenase-1 causes cortical non-haem iron increase in experimental pneumococcal meningitis: evidence that concomitant ferritin up-regulation prevents iron-induced oxidative damage

Desferrioxamine inhibits cortical necrosis in neonatal rats with experimental pneumococcal meningitis, suggesting that iron-induced oxidative damage might be responsible for neuronal damage. We therefore examined the spatial and temporal profile of changes in cortical iron and iron homeostatic proteins during pneumococcal meningitis. Infection was associated with a steady and global increase of non-haem iron in the cortex, particularly in neuronal cell bodies of layer II and V, and in capillary endothelial cells. The non-haem iron increase was associated with induction of haem oxygenase (HO)-1 in neurones, microglia and capillary endothelial cells, whereas HO-2 levels remained unchanged, suggesting that the non-haem iron increase might be the result of HO-1-mediated haem degradation. Indeed, treatment with the haem oxygenase inhibitor tin protoporphyrin (which completely blocked the accumulation of bilirubin detected in HO-1-positive cells) completely prevented the infection-associated non-haem iron increase. The same cells also displayed markedly increased ferritin staining, the increase of which occurred independently of HO activity. At the same time, no increase in DNA/RNA oxidation was observed in infected animals (as assessed by in situ detection of 8-hydroxy[deoxy]guanosine), strongly suggesting that ferritin up-regulation protected the brain from iron-induced oxidative damage. Thus, although pneumococcal meningitis leads to an increase of cortical non-haem iron, protective mechanisms up-regulated in parallel prevent iron-induced oxidative damage. Cortical damage does not appear to be a direct consequence of increased iron, therefore.

The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin

In failing hearts cardiomyocytes undergo alterations in cytoskeleton structure, contractility and viability. It is not known presently, how stress-induced changes of myofibrils correlate with markers for cell death and contractile function in cardiomyocytes. Therefore, we have studied the progression of contractile dysfunction, myofibrillar damage and cell death in cultured adult cardiomyocytes exposed to the cancer therapy doxorubicin. We demonstrate, that long-term cultured adult cardiomyocytes, a well-established model for the study of myofibrillar structure and effects of growth factors, can also be used to assess contractility and calcium handling. Adult rat ventricular myocytes (ARVM) were isolated and cultured for a total of 14 days in serum containing medium. The organization of calcium-handling proteins and myofibrillar structure in freshly isolated and in long-term cultured adult cardiomyocytes was studied by immunofluorescence and electron microscopy. Excitation contraction-coupling was analyzed by fura 2 and video edge detection in electrically paced cardiomyocytes forming a monolayer, and cell death and viability was measured by TUNEL assay, LDH release, MTT assay, and Western blot for LC3. Adult cardiomyocytes treated with Doxo showed apoptosis and necrosis only at supraclinical concentrations. Treated cells displayed merely alterations in cytoskeleton organization and integrity concomitant with contractile dysfunction and up-regulation of autophagosome formation, but no change in total sarcomeric protein content. We propose, that myofibrillar damage contributes to contractile dysfunction prior to cell death in adult cardiomyocytes exposed to clinically relevant concentrations of anthracyclines.

Traditional lesion detection aids

Lesion detection aids ideally aim at increasing the sensitivity of visual caries detection without trading off too much in terms of specificity. The use of a dental probe (explorer), bitewing radiography and fibre-optic transillumination (FOTI) have long been recommended for this purpose. Today, probing of suspected lesions in the sense of checking the 'stickiness' is regarded as obsolete, since it achieves no gain of sensitivity and might cause irreversible tooth damage. Bitewing radiography helps to detect lesions that are otherwise hidden from visual examination, and it should therefore be applied to a new patient. The diagnostic performance of radiography at approximal and occlusal sites is different, as this relates to the 3-dimensional anatomy of the tooth at these sites. However, treatment decisions have to take more into account than just lesion extension. Bitewing radiography provides additional information for the decision-making process that mainly relies on the visual and clinical findings. FOTI is a quick and inexpensive method which can enhance visual examination of all tooth surfaces. Both radiography and FOTI can improve the sensitivity of caries detection, but require sufficient training and experience to interpret information correctly. Radiography also carries the burden of the risks and legislation associated with using ionizing radiation in a health setting and should be repeated at intervals guided by the individual patient's caries risk. Lesion detection aids can assist in the longitudinal monitoring of the behaviour of initial lesions.

A technique for continuous detection of drill liquid in ice cores

When drilling ice cores deeper than ∼100 m, drill liquid is required to maintain ice-core quality and to limit borehole closure. Due to high-pressure air bubbles in the ice, the ice core can crack during drilling and core retrieval, typically at 600–1200 m depth in Greenland. Ice from this 'brittle zone' can be contaminated by drill liquid as it seeps through cracks into the core. Continuous flow analysis (CFA) systems are routinely used to analyse ice for chemical impurities, so the detection of drill liquid is important for validating accurate measurements and avoiding potential instrument damage. An optical detector was constructed to identify drill liquid in CFA tubing by ultraviolet absorption spectroscopy at a wavelength of 290 nm. The set-up was successfully field-tested in the frame of the NEEM ice-core drilling project in Greenland. A total of 27 cases of drill liquid contamination were identified during the analysis of 175 m of brittle zone ice. The analyses most strongly affected by drill liquid contamination include insoluble dust particles, electrolytic conductivity, ammonium, hydrogen peroxide and sulphate. This method may also be applied to other types of drill liquid used at other drill sites.

Vision-based Proximity Detection in Retinal Surgery

In retinal surgery, surgeons face difficulties such as indirect visualization of surgical targets, physiological tremor, and lack of tactile feedback, which increase the risk of retinal damage caused by incorrect surgical gestures. In this context, intraocular proximity sensing has the potential to overcome current technical limitations and increase surgical safety. In this paper, we present a system for detecting unintentional collisions between surgical tools and the retina using the visual feedback provided by the opthalmic stereo microscope. Using stereo images, proximity between surgical tools and the retinal surface can be detected when their relative stereo disparity is small. For this purpose, we developed a system comprised of two modules. The first is a module for tracking the surgical tool position on both stereo images. The second is a disparity tracking module for estimating a stereo disparity map of the retinal surface. Both modules were specially tailored for coping with the challenging visualization conditions in retinal surgery. The potential clinical value of the proposed method is demonstrated by extensive testing using a silicon phantom eye and recorded rabbit in vivo data.

Evaluation offreeze-thaw damage in concrete by ultrasonic imaging

This work studies the use of ultrasonic imaging as an evaluation tool in concrete subjected to freeze–thaw (F–T) cycles. To evaluate the damage in this deterioration process, ultrasonic velocity and attenuation images have been generated from concrete specimens with and without air-entraining agents. Two parameters have been proposed from these ultrasonic images according to our experimental setup: the non-assessable area proportion (NAAP) and a weighted average velocity in terms of the NAAP. The proposed parameters have been compared with the recommended failure criteria of the ASTM and Rilem standards, which employ ultrasonic contact measurements. The principal advantage of the use of ultrasonic images and the proposed methodology in comparison with the ultrasonic velocity measurements by contact is the possibility of detection of incipient damage caused by accelerated freeze–thaw cycles.

Anomaly detection on cup anemometers

The performances of two rotor-damaged commercial anemometers (Vector Instruments A100 LK) were studied. The calibration results (i.e. the transfer function) were very linear, the aerodynamic behavior being more efficient than the one shown by both anemometers equipped with undamaged rotors. No detection of the anomaly (the rotors'damage) was possible based on the calibration results. However, the Fourier analysis clearly revealed this anomaly.

Development and improvement of protection-system algorithms for ground-fault detection and location in synchronous machines

Uno de los defectos más frecuentes en los generadores síncronos son los defectos a tierra tanto en el devanado estatórico, como de excitación. Se produce un defecto cuando el aislamiento eléctrico entre las partes activas de cualquiera de estos devanados y tierra se reduce considerablemente o desaparece. La detección de los defectos a tierra en ambos devanados es un tema ampliamente estudiado a nivel industrial. Tras la detección y confirmación de la existencia del defecto, dicha falta debe ser localizada a lo largo del devanado para su reparación, para lo que habitualmente el rotor debe ser extraído del estator. Esta operación resulta especialmente compleja y cara. Además, el hecho de limitar la corriente de defecto en ambos devanados provoca que el defecto no sea localizable visualmente, pues apenas existe daño en el generador. Por ello, se deben aplicar técnicas muy laboriosas para localizar exactamente el defecto y poder así reparar el devanado. De cara a reducir el tiempo de reparación, y con ello el tiempo en que el generador esta fuera de servicio, cualquier información por parte del relé de protección acerca de la localización del defecto resultaría de gran utilidad. El principal objetivo de esta tesis doctoral ha sido el desarrollo de nuevos algoritmos que permitan la estimación de la localización de los defectos a tierra tanto en el devanado rotórico como estatórico de máquinas síncronas. Respecto al devanado de excitación, se ha presentado un nuevo método de localización de defectos a tierra para generadores con excitación estática. Este método permite incluso distinguir si el defecto se ha producido en el devanado de excitación, o en cualquiera de los componentes del sistema de excitación, esto es, transformador de excitación, conductores de alimentación del rectificador controlado, etc. En caso de defecto a tierra en del devanado rotórico, este método proporciona una estimación de su localización. Sin embargo, para poder obtener la localización del defecto, se precisa conocer el valor de resistencia de defecto. Por ello, en este trabajo se presenta además un nuevo método para la estimación de este parámetro de forma precisa. Finalmente, se presenta un nuevo método de detección de defectos a tierra, basado en el criterio direccional, que complementa el método de localización, permitiendo tener en cuenta la influencia de las capacidades a tierra del sistema. Estas capacidades resultan determinantes a la hora de localizar el defecto de forma adecuada. En relación con el devanado estatórico, en esta tesis doctoral se presenta un nuevo algoritmo de localización de defectos a tierra para generadores que dispongan de la protección de faltas a tierra basada en la inyección de baja frecuencia. Se ha propuesto un método general, que tiene en cuenta todos los parámetros del sistema, así como una versión simplificada del método para generadores con capacidades a tierra muy reducida, que podría resultar de fácil implementación en relés de protección comercial. Los algoritmos y métodos presentados se han validado mediante ensayos experimentales en un generador de laboratorio de 5 kVA, así como en un generador comercial de 106 MVA con resultados satisfactorios y prometedores. ABSTRACT One of the most common faults in synchronous generators is the ground fault in both the stator winding and the excitation winding. In case of fault, the insulation level between the active part of any of these windings and ground lowers considerably, or even disappears. The detection of ground faults in both windings is a very researched topic. The fault current is typically limited intentionally to a reduced level. This allows to detect easily the ground faults, and therefore to avoid damage in the generator. After the detection and confirmation of the existence of a ground fault, it should be located along the winding in order to repair of the machine. Then, the rotor has to be extracted, which is a very complex and expensive operation. Moreover, the fact of limiting the fault current makes that the insulation failure is not visually detectable, because there is no visible damage in the generator. Therefore, some laborious techniques have to apply to locate accurately the fault. In order to reduce the repair time, and therefore the time that the generator is out of service, any information about the approximate location of the fault would be very useful. The main objective of this doctoral thesis has been the development of new algorithms and methods to estimate the location of ground faults in the stator and in the rotor winding of synchronous generators. Regarding the excitation winding, a new location method of ground faults in excitation winding of synchronous machines with static excitation has been presented. This method allows even to detect if the fault is at the excitation winding, or in any other component of the excitation system: controlled rectifier, excitation transformer, etc. In case of ground fault in the rotor winding, this method provides an estimation of the fault location. However, in order to calculate the location, the value of fault resistance is necessary. Therefore, a new fault-resistance estimation algorithm is presented in this text. Finally, a new fault detection algorithm based on directional criterion is described to complement the fault location method. This algorithm takes into account the influence of the capacitance-to-ground of the system, which has a remarkable impact in the accuracy of the fault location. Regarding the stator winding, a new fault-location algorithm has been presented for stator winding of synchronous generators. This algorithm is applicable to generators with ground-fault protection based in low-frequency injection. A general algorithm, which takes every parameter of the system into account, has been presented. Moreover, a simplified version of the algorithm has been proposed for generators with especially low value of capacitance to ground. This simplified algorithm might be easily implementable in protective relays. The proposed methods and algorithms have been tested in a 5 kVA laboratory generator, as well as in a 106 MVA synchronous generator with satisfactory and promising results.

Induction of microsatellite instability by oxidative DNA damage

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the β-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of β-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the β-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 106 wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency ≈300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N′-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This β-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.

Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts

Transcription-coupled repair (TCR) plays an important role in removing DNA damage from actively transcribed genes. It has been speculated that TCR is the most important mechanism for repairing DNA damage in non-dividing cells such as neurons. Therefore, abnormal TCR may contribute to the development of many age-related and neurodegenerative diseases. However, the molecular mechanism of TCR is not well understood. Oligonucleotide DNA triplex formation provides an ideal system to dissect the molecular mechanism of TCR since triplexes can be formed in a sequence-specific manner to inhibit transcription of target genes. We have recently studied the molecular mechanism of triplex-forming oligonucleotide (TFO)-mediated TCR in HeLa nuclear extracts. Using plasmid constructs we demonstrate that the level of TFO-mediated DNA repair activity is directly correlated with the level of transcription of the plasmid in HeLa nuclear extracts. TFO-mediated DNA repair activity was further linked with transcription since the presence of rNTPs in the reaction was essential for AG30-mediated DNA repair activity in HeLa nuclear extracts. The involvement of individual components, including TFIID, TFIIH, RNA polymerase II and xeroderma pigmentosum group A (XPA), in the triplex-mediated TCR process was demonstrated in HeLa nuclear extracts using immunodepletion assays. Importantly, our studies also demonstrated that XPC, a component involved in global genome DNA repair, is involved in the AG30-mediated DNA repair process. The results obtained in this study provide an important new understanding of the molecular mechanisms involved in the TCR process in mammalian cells.

Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease.

There is growing evidence that oxidative stress and mitochondrial respiratory failure with attendant decrease in energy output are implicated in nigral neuronal death in Parkinson disease (PD). It is not known, however, which cellular elements (neurons or glial cells) are major targets of oxygen-mediated damage. 4-Hydroxy-2-nonenal (HNE) was shown earlier to react with proteins to form stable adducts that can be used as markers of oxidative stress-induced cellular damage. We report here results of immunochemical studies using polyclonal antibodies directed against HNE-protein conjugates to label the site of oxidative damage in control subjects (ages 18-99 years) and seven patients that died of PD (ages 57-78 years). All the nigral melanized neurons in one of the midbrain sections were counted and classified into three groups according to the intensity of immunostaining for HNE-modified proteins--i.e., no staining, weak staining, and intensely positive staining. On average, 58% of nigral neurons were positively stained for HNE-modified proteins in PD; in contrast only 9% of nigral neurons were positive in the control subjects; the difference was statistically significant (Mann-Whitney U test; P < 0.01). In contrast to the substantia nigra, the oculomotor neurons in the same midbrain sections showed no or only weak staining for HNE-modified proteins in both PD and control subjects; young control subjects did not show any immunostaining; however, aged control subjects showed weak staining in the oculomotor nucleus, suggesting age-related accumulation of HNE-modified proteins in the neuron. Our results indicate the presence of oxidative stress within nigral neurons in PD, and this oxidative stress may contribute to nigral cell death.

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Damage analysis of bridge structures using vibrational techniques

Much research is currently centred on the detection of damage in structures using vibrational data. The work presented here examined several areas of interest in support of a practical technique for identifying and locating damage within bridge structures using apparent changes in their vibrational response to known excitation. The proposed goals of such a technique included the need for the measurement system to be operated on site by a minimum number of staff and that the procedure should be as non-invasive to the bridge traffic-flow as possible. Initially the research investigated changes in the vibrational bending characteristics of two series of large-scale model bridge-beams in the laboratory and these included ordinary-reinforced and post-tensioned, prestressed designs. Each beam was progressively damaged at predetermined positions and its vibrational response to impact excitation was analysed. For the load-regime utilised the results suggested that the infuced damage manifested itself as a function of the span of a beam rather than a localised area. A power-law relating apparent damage with the applied loading and prestress levels was then proposed, together with a qualitative vibrational measure of structural damage. In parallel with the laboratory experiments a series of tests were undertaken at the sites of a number of highway bridges. The bridges selected had differing types of construction and geometric design including composite-concrete, concrete slab-and-beam, concrete-slab with supporting steel-troughing constructions together with regular-rectangular, skewed and heavily-skewed geometries. Initial investigations were made of the feasibility and reliability of various methods of structure excitation including traffic and impulse methods. It was found that localised impact using a sledge-hammer was ideal for the purposes of this work and that a cartridge `bolt-gun' could be used in some specific cases.

Protein oxidative modifications:new mass spectrometry approaches to detection in biological samples

In inflammatory diseases, release of oxidants leads to oxidative damage to proteins. The precise nature of oxidative damage to individual proteins depends on the oxidant involved. Chlorination and nitration are markers of modification by the myeloperoxidase-H2O2-Cl- system and nitric oxide-derived oxidants, respectively. Although these modifications can be detected by western blotting, currently no reliable method exists to identify the specific sites damage to individual proteins in complex mixtures such as clinical samples. We are developing novel LCMS2 and precursor ion scanning methods to address this. LC-MS2 allows separation of peptides and detection of mass changes in oxidized residues on fragmentation of the peptides. We have identified indicative fragment ions for chlorotyrosine, nitrotyrosine, hydroxytyrosine and hydroxytryptophan. A nano-LC/MS3 method involving the dissociation of immonium ions to give specific fragments for the oxidized residues has been developed to overcome the problem of false positives from ions isobaric to these immonium ions that exist in unmodified peptides. The approach has proved able to identify precise protein modifications in individual proteins and mixtures of proteins. An alternative methodology involves multiple reaction monitoring for precursors and fragment ions are specific to oxidized and chlorinated proteins, and this has been tested with human serum albumin. Our ultimate aim is to apply this methodology to the detection of oxidative post-translational modifications in clinical samples for disease diagnosis, monitoring the outcomes of therapy, and improved understanding of disease biochemistry.

Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray MS

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system. The facility of detection of the oxidized species in complex mixtures was greatly improved compared with direct-injection MS analysis, as they eluted earlier than the native lipids, owing to the decrease in hydrophobicity. In U937 and HL60 cells treated in vitro with t-butylhydroperoxide plus Fe2+, lipid oxidation could not be observed by direct injection, but LC-MS allowed the detection of monohydroperoxides of palmitoyl-linoleoyl and stearoyl-linoleoyl phosphatidylcholines. The levels of hydroperoxides observed in U937 cells were found to depend on the duration and severity of the oxidative stress. In cells treated with HOCl, chlorohydrins of palmitoyloleoyl phosphatidylcholine were observed by LC-MS. The method was able to detect very small amounts of oxidized lipids compared with the levels of native lipids present. The membrane-lipid profiles of these cells were found to be quite resistant to damage until high concentrations of oxidants were used. This is the first report of direct detection by LC-MS of intact oxidized phospholipids induced in cultured cells subjected to oxidative stress.