Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

Background: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53- activating agents, for the treatment of tumors that retain wild-type p53.

The Thermal Effects of Therapeutic Lasers with 810 and 904 nm Wavelengths on Human Skin

Objective: To investigate the effect of therapeutic infrared class 3B laser irradiation on skin temperature in healthy participants of differing skin color, age, and gender. Background: Little is known about the potential thermal effects of Low Level Laser Therapy (LLLT) irradiation on human skin. Methods: Skin temperature was measured in 40 healthy volunteers with a thermographic camera at laser irradiated and control (non-irradiated) areas on the skin. Six irradiation doses (2-12 J) were delivered from a 200mW, 810nm laser and a 60mW, 904nm laser, respectively. Results: Thermal effects of therapeutic LLLT using doses recommended in the World Association for Laser Therapy (WALT) guidelines were insignificant; below 1.5 degrees C in light, medium, and dark skin. When higher irradiation doses were used, the 60mW, 904 nm laser produced significantly (p < 0.01) higher temperatures in dark skin (5.7, SD +/- 1.8 degrees C at 12 J) than in light skin, although no participants requested termination of LLLT. However, irradiation with a 200mW, 810nm laser induced three to six times more heat in dark skin than in the other skin color groups. Eight of 13 participants with dark skin asked for LLLT to be stopped because of uncomfortable heating. The maximal increase in skin temperature was 22.3 degrees C. Conclusions: The thermal effects of LLLT at doses recommended by WALT-guidelines for musculoskeletal and inflammatory conditions are negligible (< 1.5 degrees C) in light, medium, and dark skin. However, higher LLLT doses delivered with a strong 3B laser (200mW) are capable of increasing skin temperature significantly and these photothermal effects may exceed the thermal pain threshold for humans with dark skin color.

Development of a DNA-dosimeter system for monitoring the effects of solar-ultraviolet radiation

Solar radiation sustains and affects all life forms on Earth. In recent years, the increase in environmental levels of solar-UV radiation due to depletion of the stratospheric ozone layer, as a result of anthropogenic emission of destructive chemicals, has highlighted serious issues of social concern. This becomes still more dramatic in tropical and subtropical regions, where the intensity of solar radiation is higher. To better understand the impact of the harmful effects of solar-UV radiation on the DNA molecule, we developed a reliable biological monitoring system based on the exposure of plasmid DNA to artificial UV lamps and sunlight. The determination and quanti. cation of different types of UV photoproducts were performed through the use of specific DNA repair enzymes and antibodies. As expected, a significant number of CPDs and 6-4PPs was observed when the DNA-dosimeter system was exposed to increasing doses of UVB radiation. Moreover, CPDs could also be clearly detected in plasmid DNA when this system was exposed to either UVA or directly to sunlight. Interestingly, although less abundant, 6-4PPs and oxidative DNA damage were also generated after exposure to both UVA and sunlight. These results confirm the genotoxic potential of sunlight, reveal that UVA may also produce CPDs and 6-4PPs directly in naked DNA and demonstrate the applicability of a DNA-dosimeter system for monitoring the biological effects of solar-UV radiation.

Increasing the symmetry of drug crystals: a monoclinic conformational polymorph of the platelet antiaggregating agent ticlopidine hydrochloride

Ticlopidine hydrochloride (TICLID (R)) is a platelet antiaggregating agent whose use as a potent antithrombotic pharmaceutical ingredient is widespread, even though this drug has not been well characterized in the solid state. Only the crystal phase used for drug product manufacturing is known. Here, a new polymorph of ticlopidine hydrochloride was discovered and its structure was determined. While the antecedent polymorph crystallizes in the triclinic space group P (1) over bar, the new crystal phase was solved in the monoclinic space group P2(1)/c. Both polymorphs crystallize as racemic mixtures of enantiomeric (ticlopidine)(+) cations. Detailed geometrical and packing comparisons between the crystal structures of the two polymorphs have allowed us to understand how different supramolecular architectures are assembled. It was feasible to conclude that the main difference between the two polymorphs is a rotation of about 120 degrees on the bridging bond between the thienopyridine and o-chlorobenzyl moieties. The differential o-chlorobenzyl conformation is related to changeable patterns of weak intermolecular contacts involving this moiety, such as edge-to-face Cl center dot center dot center dot pi and C-H center dot center dot center dot pi interactions in the new polymorph and face-to-face pi center dot center dot center dot pi contacts in the triclinic crystal phase, leading to a symmetry increase in the ticlopidine hydrochloride solid state form described for the first time in this study. Other conformational features are slightly different between the two polymorphs, such as the thienopyridine puckerings and the o-chlorophenyl orientations. These conformational characteristics were also correlated to the crystal packing patterns.

Interference with Hemozoin Formation Represents an Important Mechanism of Schistosomicidal Action of Antimalarial Quinoline Methanols

Background: The parasitic trematode Schistosoma mansoni is one of the major causative agents of human schistosomiasis, which afflicts 200 million people worldwide. Praziquantel remains the main drug used for schistosomiasis treatment, and reliance on the single therapy has been prompting the search for new therapeutic compounds against this disease. Our group has demonstrated that heme crystallization into hemozoin (Hz) within the S. mansoni gut is a major heme detoxification route with lipid droplets involved in this process and acting as a potential chemotherapeutical target. In the present work, we investigated the effects of three antimalarial compounds, quinine (QN), quinidine (QND) and quinacrine (QCR) in a murine schistosomiasis model by using a combination of biochemical, cell biology and molecular biology approaches. Methodology/Principal Findings: Treatment of S. mansoni-infected female Swiss mice with daily intraperitoneal injections of QN, and QND (75 mg/kg/day) from the 11(th) to 17(th) day after infection caused significant decreases in worm burden (39%-61%) and egg production (42%-98%). Hz formation was significantly inhibited (40%-65%) in female worms recovered from QN- and QND-treated mice and correlated with reduction in the female worm burden. We also observed that QN treatment promoted remarkable ultrastructural changes in male and female worms, particularly in the gut epithelium and reduced the granulomatous reaction to parasite eggs trapped in the liver. Microarray gene expression analysis indicated that QN treatment increased the expression of transcripts related to musculature, protein synthesis and repair mechanisms. Conclusions: The overall significant reduction in several disease burden parameters by the antimalarial quinoline methanols indicates that interference with Hz formation in S. mansoni represents an important mechanism of schistosomicidal action of these compounds and points out the heme crystallization process as a valid chemotherapeutic target to treat schistosomiasis.

Continuous monitoring of ascorbate transport through neuroblastoma cells with a ruthenium oxide hexacyanoferrate modified microelectrode

The uptake of ascorbate by neuroblastoma cells using a ruthenium oxide hexacyanoferrate (RuOHCF)-modified carbon fiber disc (CFD) microelectrode (r = 14.5 mu m) was investigated. By use of the proposed electrochemical sensor the amperometric determination of ascorbate was performed at 0.0 V in minimum essential medium (MEM, pH = 7.2) with a limit of detection of 25 mu mol L(-1). Under the optimum experimental conditions, no interference from MEM constituents and reduced glutathione (used to prevent the oxidation of ascorbate during the experiments) was noticed. The stability of the RuOHCF-modified electrode response was studied by measuring the sensitivity over an extended period of time (120 h), a decrease of around 10% being noticed at the end of the experiment. The rate of ascorbate uptake by control human neuroblastoma SH-SY5Y cells, and cells transfected with wild-type Cu,Zn-superoxide dismutase (SOD WT) or with a mutant typical of familial amyotrophic lateral sclerosis (SOD G93A), was in agreement with the level of oxidative stress in these cells. The usefulness of the RuOHCF-modified microelectrode for in vivo monitoring of ascorbate inside neuroblastoma cells was also demonstrated.

Single reaction interface flow system for chemiluminescent monitoring of mannitol based on its hydroxyl radical scavenger activity

A single reaction interface flow analysis (SIFA) system for the monitoring of mannitol in pharmaceutical formulations and human urine is presented. The developed approach takes advantage of the mannitol scavenger aptitude to inhibit the chemiluminescent reaction between luminol and myoglobin in the absence of H(2)O(2). The SIFA system facilitated the fully automation of the developed methodology, allowing the in-line reproducible handling of chemical species with a very short lifetime as is the case of the hydroxyl radical generated in the abovementioned luminol/myoglobin reaction. The proposed methodology allowed the determination of mannitol concentrations between 25 mmol L(-1) and 1 mol L(-1), with good precision (R.S.D. < 4.7%, n = 3) and a sampling frequency of about 60 h(-1). The procedure was applied to the determination of mannitol in pharmaceuticals and in human urine samples Without any pretreatment process. The results obtained for pharmaceutical formulations were statistically comparable to those provided by the reference method (R.D. < 4.6%); recoveries values obtained in the analysis of spiked urine samples (between 94.9 and 105.3% of the added amount) were also satisfactory. (C) 2008 Elsevier B.V. All rights reserved.

A new monitor for routine thermal and epithermal neutron fluence rate monitoring in k(0) INAA

The Zr-Au set for monitoring the thermal and epithermal neutron fluence rate and the epithermal spectrum parameter a is not always practicable for routine application of INAA in well-thermalized facilities. An alternative set consisting of Cr, Au and Mo provides values for the thermal neutron fluence rate, f and alpha that are not significantly different from those found via the Zr-Au method and the Cd-covered Zr-method. The IRMM standard SMELS-II was analyzed using the (Au-Cr-Mo) monitor and a good agreement was obtained. (C) 2008 Elsevier Ltd. All rights reserved.

Validity and reliability of the Portuguese version of the Epilepsy Medication Treatment Complexity Index for Brazil

We evaluated the reliability and validity of a Brazilian-Portuguese version of the Epilepsy Medication Treatment Complexity Index (EMTCI). Interrater reliability was evaluated with the intraclass correlation coefficient (ICC), and validity was evaluated by correlation of mean EMTCI scores with the following variables: number of antiepileptic drugs (AEDs), seizure control, patients` perception of seizure control, and adherence to the therapeutic regimen as measured with the Morisky scale. We studied patients with epilepsy followed in a tertiary university-based hospital outpatient clinic setting, aged 18 years or older, independent in daily living activities, and without cognitive impairment or active psychiatric disease. ICCs ranged from 0.721 to 0.999. Mean EMTCI scores were significantly correlated with the variables assessed. Higher EMTCI scores were associated with an increasing number of AEDs, uncontrolled seizures, patients` perception of lack of seizure control, and poorer adherence to the therapeutic regimen. The results indicate that the Brazilian-Portuguese EMTCI is reliable and valid to be applied clinically in the country. The Brazilian-Portuguese EMTCI version may be a useful tool in developing strategies to minimize treatment complexity, possibly improving seizure control and quality of life in people with epilepsy in our milieu. (C) 2011 Elsevier Inc. All rights reserved.

Monitoring stress tolerance and occurrences of upper respiratory illness in basketball players by means of psychometric tools and salivary biomarkers

The aim of the study was to evaluate the possible relationships between stress tolerance, training load, banal infections and salivary parameters during 4 weeks of regular training in fifteen basketball players. The Daily Analysis of Life Demands for Athletes` questionnaire (sources and symptoms of stress) and the Wisconsin Upper Respiratory Symptom Survey were used on a weekly basis. Salivary cortisol and salivary immunoglobulin A (SIgA) were collected at the beginning (before) and after the study, and measured by enzyme-linked immunosorbent assay (ELISA). Ratings of perceived exertion (training load) were also obtained. The results from ANOVA with repeated measures showed greater training loads, number of upper respiratory tract infection episodes and negative sensation to both symptoms and sources of stress, at week 2 (p < 0.05). Significant increases in cortisol levels and decreases in SIgA secretion rate were noted (before to after). Negative sensations to symptoms of stress at week 4 were inversely and significantly correlated with SIgA secretion rate. A positive and significant relationship between sources and symptoms of stress at week 4 and cortisol levels were verified. In summary, an approach incorporating in conjunction psychometric tools and salivary biomarkers could be an efficient means of monitoring reaction to stress in sport. Copyright (C) 2010 John Wiley & Sons, Ltd.

The process of drug dispensing and distribution at four Brazilian hospitals: A multicenter descriptive study

A multicenter descriptive study was carried out in two steps: an interview with providers involved in the medication processes, and then non-participating observation of their environment and practices. Only one hospital was found to have a bar-coding, dispensing system connected to a computerized prescription system. fit all participating hospitals at least 90% of the drugs were dispensed and distributed as unit doses, but in none of them did pharmacists assess prescriptions. The study findings showed that the processes of drug dispensing and distribution in Brazilian hospitals encounter several problems, mostly associated to work environment conditions and inadequacy in drug ordering and requests.

Evaluation of three brands of drug interaction software for use in intensive care units

Objective To evaluate drug interaction software programs and determine their accuracy in identifying drug-drug interactions that may occur in intensive care units. Setting The study was developed in Brazil. Method Drug interaction software programs were identified through a bibliographic search in PUBMED and in LILACS (database related to the health sciences published in Latin American and Caribbean countries). The programs` sensitivity, specificity, and positive and negative predictive values were determined to assess their accuracy in detecting drug-drug interactions. The accuracy of the software programs identified was determined using 100 clinically important interactions and 100 clinically unimportant ones. Stockley`s Drug Interactions 8th edition was employed as the gold standard in the identification of drug-drug interaction. Main outcome Sensitivity, specificity, positive and negative predictive values. Results The programs studied were: Drug Interaction Checker (DIC), Drug-Reax (DR), and Lexi-Interact (LI). DR displayed the highest sensitivity (0.88) and DIC showed the lowest (0.69). A close similarity was observed among the programs regarding specificity (0.88-0.92) and positive predictive values (0.88-0.89). The DIC had the lowest negative predictive value (0.75) and DR the highest (0.91). Conclusion The DR and LI programs displayed appropriate sensitivity and specificity for identifying drug-drug interactions of interest in intensive care units. Drug interaction software programs help pharmacists and health care teams in the prevention and recognition of drug-drug interactions and optimize safety and quality of care delivered in intensive care units.

Sensorless synchronization method for thin-film direct optical broadband monitoring system

Optical monitoring systems are necessary to manufacture multilayer thin-film optical filters with low tolerance on spectrum specification. Furthermore, to have better accuracy on the measurement of film thickness, direct monitoring is a must. Direct monitoring implies acquiring spectrum data from the optical component undergoing the film deposition itself, in real time. In making film depositions on surfaces of optical components, the high vacuum evaporator chamber is the most popular equipment. Inside the evaporator, at the top of the chamber, there is a metallic support with several holes where the optical components are assembled. This metallic support has rotary motion to promote film homogenization. To acquire a measurement of the spectrum of the film in deposition, it is necessary to pass a light beam through a glass witness undergoing the film deposition process, and collect a sample of the light beam using a spectrometer. As both the light beam and the light collector are stationary, a synchronization system is required to identify the moment at which the optical component passes through the light beam.

Connection Admission Control for a WDM Optical Network Based on Traffic Monitoring and Bandwidth Release

We proposed a connection admission control (CAC) to monitor the traffic in a multi-rate WDM optical network. The CAC searches for the shortest path connecting source and destination nodes, assigns wavelengths with enough bandwidth to serve the requests, supervises the traffic in the most required nodes, and if needed activates a reserved wavelength to release bandwidth according to traffic demand. We used a scale-free network topology, which includes highly connected nodes ( hubs), to enhance the monitoring procedure. Numerical results obtained from computational simulations show improved network performance evaluated in terms of blocking probability.

Monitoring Services for Industrial Applications

Sao Paulo Research Foundation (FAPESP) in Brazil