Failure modes and effects analysis of total skin electron irradiation technique.

PURPOSE: Total skin electron irradiation (TSEI) is a radiotherapy technique which consists of an homogeneous body surface irradiation by electrons. This treatment requires very strict technical and dosimetric conditions, requiring the implementation of multiple controls. Recently, the Task Group 100 report of the AAPM has recommended adapting the quality assurance program of the facility to the risks of their processes. MATERIALS AND METHODS: A multidisciplinary team evaluated the potential failure modes (FMs) of every process step, regardless of the management tools applied in the installation. For every FM, occurrence (O), severity (S) and detectability (D) by consensus was evaluated, which resulted in the risk priority number (RPN), which permitted the ranking of the FMs. Subsequently, all the management tools used, related to the TSEI process, were examined and the FMs were reevaluated, to analyze the effectiveness of these tools and to propose new management tools to cover the greater risk FMs. RESULTS: 361 FMs were identified, 103 of which had RPN ≥80, initially, and 41 had S ≥ 8. Taking this into account the quality management tools FMs were reevaluated and only 30 FMs had RPN ≥80. The study of these 30 FMs emphasized that the FMs that involved greater risk were related to the diffuser screen placement and the patient's position during treatment. CONCLUSIONS: The quality assurance program of the facility has been adapted to the risk of this treatment process, following the guidelines proposed by the TG-100. However, clinical experience continually reveals new FMs, so the need for periodic risk analysis is required.

Restriction of trophic factors and nutrients induces PARKIN expression.

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder and manifests at old age. While many details of its pathogenesis remain to be elucidated, in particular the protein and mitochondrial quality control during stress responses have been implicated in monogenic PD variants. Especially the mitochondrial kinase PINK1 and the ubiquitin ligase PARKIN are known to cooperate in autophagy after mitochondrial damage. As autophagy is also induced by loss of trophic signaling and PINK1 gene expression is modulated after deprivation of cytokines, we analyzed to what extent trophic signals and starvation stress regulate PINK1 and PARKIN expression. Time course experiments with serum deprivation and nutrient starvation of human SH-SY5Y neuroblastoma cells and primary mouse neurons demonstrated phasic induction of PINK1 transcript up to twofold and PARKIN transcript levels up to sixfold. The corresponding threefold starvation induction of PARKIN protein was limited by its translocation to lysosomes. Analysis of primary mouse cells from PINK1-knockout mice indicated that PARKIN induction and lysosomal translocation occurred independent of PINK1. Suppression of the PI3K-Akt-mTOR signaling by pharmacological agents modulated PARKIN expression accordingly. In conclusion, this expression survey demonstrates that PARKIN and PINK1 are coregulated during starvation and suggest a role of both PD genes in response to trophic signals and starvation stress.

Practical diagnosis of red cell disorders in southern Spain.

BACKGROUND: Hereditary red cell disorders are associated with a protective effect against malaria, which results in an increased prevalence in malaria-endemic areas. Migratory flows from these areas are resulting in a marked increase in such abnormalities in Southern Spain. METHODS: All hemoglobin disorders diagnosed between 1997 and 2010 have been recorded. Since 2008, we have performed systematic screening for hemoglobinopathies on African patients. A high-pressure liquid chromatography system was used as screening method for structural hemoglobinopathies and for separation of hemoglobin (Hb) F and A(2). RESULTS: We detected 666 cases in patients of foreign origin and 308 in native Spanish patients. Thalassemias (thal) are the most frequent disorders amongst the local population: ß-thal minor, 57.1% (176/308); α-thal, 18.2% (56/308), and δß-thal, 7.8% (24/308). In ethnic minorities, there is a huge variety of hemoglobinopathies: heterozygous Hb S, 45% (300/666); heterozygous Hb C, 15% (100/666); ß-thal minor, 13.7% (91/666); α-thal, 10.2% (68/666); Hb SS in 14 patients, and Hb CC in 9 patients. Of the native patients, 14 were found to have Hb AS and 9 Hb AC. CONCLUSION: Given the modern migratory flows, greater knowledge of these disorders is needed by all medical staff, and new practical and cost/time-effective diagnostic approaches have to be devised.

Extraction, purification and characterization of the plant-produced HPV16 subunit vaccine candidate E7 GGG.

Several studies indicated that biopharmaceuticals based on the recombinant protein E7 of human papillomavirus (HPV) can serve as therapeutic vaccines preventing the development of cancer in women infected with high-risk types of HPV such as HPV16. Here, we report effective extraction and purification of a plant-produced E7GGG-lichenase fusion protein, an HPV16 subunit vaccine candidate, from Nicotiana benthamiana plants, to a high yield. The target contains the modified HPV16 E7 protein internally fused to the surface loop of a truncated, hexa-His- and KDEL-tagged variant of bacterial lichenase, and has been previously shown to possess anti-cancer activity in an animal model. We purified the protein using a combination of immobilized metal-ion affinity chromatography and gel filtration. The achieved purity of the final product was 99% as confirmed by Coomassie or SYPRO Ruby staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical size exclusion chromatography coupled with multi-angle laser light scattering. The overall yield was 50% corresponding to 0.1g of protein per 1 kg plant biomass. Only slight changes in these parameters were observed during the process scale-up from 50 g to 1 kg of processed leaf biomass.

NFATc3 is required for chronic hypoxia-induced pulmonary hypertension in adult and neonatal mice.

Pulmonary hypertension occurs with prolonged exposure to chronic hypoxia in both adults and neonates. The Ca(2+)-dependent transcription factor, nuclear factor of activated T cells isoform c3 (NFATc3), has been implicated in chronic hypoxia-induced pulmonary arterial remodeling in adult mice. Therefore, we hypothesized that NFATc3 is required for chronic hypoxia-induced pulmonary hypertension in adult and neonatal mice. The aim of this study was to determine whether 1) NFATc3 mediates chronic hypoxia-induced increases in right ventricular systolic pressure in adult mice; 2) NFATc3 is activated in neonatal mice exposed to chronic hypoxia; and 3) NFATc3 is involved in chronic hypoxia-induced right ventricular hypertrophy and pulmonary vascular remodeling in neonatal mice. Adult mice were exposed to hypobaric hypoxia for 2, 7, and 21 days. Neonatal mouse pups were exposed for 7 days to hypobaric chronic hypoxia within 2 days after delivery. Hypoxia-induced increases in right ventricular systolic pressure were absent in NFATc3 knockout adult mice. In neonatal mice, chronic hypoxia caused NFAT activation in whole lung and nuclear accumulation of NFATc3 in both pulmonary vascular smooth muscle and endothelial cells. In addition, heterozygous NFATc3 neonates showed less right ventricular hypertrophy and pulmonary artery wall thickness in response to chronic hypoxia than did wild-type neonates. Our results suggest that NFATc3 mediates pulmonary hypertension and vascular remodeling in both adult and neonatal mice.

[Hypercoagulable state due to acquired protein C resistance, harbinger of colonic neoplasm?]. / Hipercoagulabilidad debida a resistencia adquirida a la proteína C activada: primer signo de neoplasia de colon?

Hereditary activated protein C resistance (aPCR) has been identified as an important risk factor for the occurrence of thromboembolic events. It is most frequently hereditary, and caused by a point mutation in factor V, named Factor V Leiden (FVL), which renders it resistant to the anticoagulant action of circulating protein C. However, aPCR can also be found in absence of FVL (acquired aPCR), associated to lupus anticoagulant, pregnancy or neoplasms. We report a case of deep venous thrombosis (DVT) in a 54 year-old woman, with no digestive symptoms and negative screening for biochemical tumor markers, who presented with DVT from FVL-negative aPCR, one year before being diagnosed of colonic adenocarcinoma. Once complete remission of the carcinoma was reached, aPCR returned to normal values. In thrombophilia screening studies, the finding of aPCR may be caused by acute-phase reactants or neoplastic processes, and therefore require evolutive evaluation and genetic search for FVL.

Carotenoid photooxidation in photosystem II.

Carotenoids are known to function as light-harvesting pigments and they play important roles in photoprotection in both plant and bacterial photosynthesis. These functions are also important for carotenoids in photosystem II. In addition, beta-carotene recently has been found to function as a redox intermediate in an alternate pathway of electron transfer within photosystem II. This redox role of a carotenoid in photosystem II is unique among photosynthetic reaction centers and stems from the very highly oxidizing intermediates that form in the process of water oxidation. In this minireview, an overview of the electron-transfer reactions in photosystem II is presented, with an emphasis on those involving carotenoids. The carotenoid composition of photosystem II and the physical methods used to study the structure of the redox-active carotenoid are reviewed. Possible roles of carotenoid cations in photoprotection of photosystem II are discussed.

Photochemical behavior of xanthophylls in the recombinant photosystem II antenna complex, CP26.

The steady state absorption and fluorescence spectroscopic properties of the xanthophylls, violaxanthin, zeaxanthin, and lutein, and the efficiencies of singlet energy transfer from the individual xanthophylls to chlorophyll have been investigated in recombinant CP26 protein overexpressed in Escherichia coli and then refolded in vitro with purified pigments. Also, the effect of the different xanthophylls on the extents of static and dynamic quenching of chlorophyll fluorescence has been investigated. Absorption, fluorescence, and fluorescence excitation demonstrate that the efficiency of light harvesting from the xanthophylls to chlorophyll a is relatively high and insensitive to the particular xanthophyll that is present. A small effect of the different xanthophylls is observed on the extent of quenching of Chl fluorescence. The data provide the precise wavelengths of the absorption and fluorescence features of the bound pigments in the highly congested spectral profiles from these light-harvesting complexes. This information is important in assessing the mechanisms by which higher plants dissipate excess energy in light-harvesting proteins.

Mechanism of nonphotochemical quenching in green plants: energies of the lowest excited singlet states of violaxanthin and zeaxanthin.

The xanthophyll cycle is an enzymatic, reversible process through which the carotenoids violaxanthin, antheraxanthin, and zeaxanthin are interconverted in response to the need to balance light absorption with the capacity to use the energy to drive the reactions of photosynthesis. The cycle is thought to be one of the main avenues for safely dissipating excitation energy absorbed by plants in excess of that needed for photosynthesis. One of the key factors needed to elucidate the molecular mechanism by which the potentially damaging excess energy is dissipated is the energy of the lowest excited singlet (S(1)) state of the xanthophyll pigments. Absorption from the ground state (S(0)) to S(1) is forbidden by symmetry, making a determination of the S(1) state energies of these molecules by absorption spectroscopy very difficult. Fluorescence spectroscopy is potentially the most direct method for obtaining the S(1) state energies. However, because of problems with sample purity, low emission quantum yields, and detection sensitivity, fluorescence spectra from these molecules, until now, have never been reported. In this work these technical obstacles have been overcome, and S(1) --> S(0) fluorescence spectra of violaxanthin and zeaxanthin are presented. The energies of the S(1) states deduced from the fluorescence spectra are 14 880 +/- 90 cm(-)(1) for violaxanthin and 14 550 +/- 90 cm(-)(1) for zeaxanthin. The results provide important insights into the mechanism of nonphotochemical dissipation of excess energy in plants.

[Diagnostic usefulness of the questionnaire "Alcohol Use Disorders Identification Test" (AUDIT) to detect conditions associated with alcohol in hospitalized patients]. / Utilidad diagnóstica del cuestionario "Alcohol Use Disorders Identification Test" (AUDIT) para detectar trastornos relacionados con el alcohol en pacientes hospitalizados.

OBJECTIVE: To evaluate the diagnostic usefulness of AUDIT (Alcohol Use Disorders Identification Test) for detection of alcohol-related problems (ARP) among hospitalized patients, to assess the potential differences according to age or sex and to compare its diagnostic value with that of some other conventionally used measures (CAGE questionnaire and biological markers). MATERIAL AND METHODS: This is a cross-sectional study for evaluation of diagnostic tests including 179 hospitalized patients in a Medicine Unit. Data about weekly alcohol intake were collected through a semistructured interview. AUDIT and CAGE questionnaires were administered and blood levels of GGT, MCV, AST, ALT, alkaline phosphatase, platelet count, trylicerides and uric acid were determined. RESULTS: AUDIT sensitivity in detecting ARP was of 98%, specificity was of 94% and area under ROC was 0.99 (95% CI: 0.98-1). Its sensitivity was shown to be lower both in the female group (94% vs. 99%) and in age group under 60 years (97% vs. 100%). CAGE showed a sensitivity of 78% and a specificity of 99%. Among biological markers GGT and MCV should be highlighted with sensitivities of 83% and 74% and specificities of 53% and 74% respectively. CONCLUSIONS: AUDIT is an effective tool for detection of ARP among hospitalized patients. Its diagnostic usefulness being lower for females, similar for both age groups considered and clearly higher than that of other commonly used measures.

[Functional social support perceived by patients in a program of home care in rural and urban settings]. / Apoyo social funcional percibido por los pacientes en programa de atención domiciliaria en los medios rural y urbano.

OBJECTIVE: To determine functional social support in patients included in programme of care for chronic patients confined to their homes, and to compare rural area with urban area. DESIGN: Descriptive and crossover study. SETTING: Urban and rural health centres. PATIENTS: 103 patients were interviewed in their homes (41 of them were in a rural area and 62 in a urban area). All patients were included in programme of care for chronic patients confined to their homes. Patients with neurological o psychiatric disorders who were not able to answer coherently were excluded. MEASUREMENTS AND MAIN RESULTS: We used the DUKE-UNC questionnaire which measured the functional social support in two sub-scales: confidential support and affective support. We didn't find any relation between the functional social support and age, sex, civil condition or health problems. Patients of the rural area perceive less confidential support than patients of the urban area. We didn't find any difference in the perception of the affective support between both areas. CONCLUSIONS: In the rural area, the patients included in the programme of care for chronic patients confined to their homes perceive less possibility to communicate problems, important events or conflictive situations that require comprehension and help.

In vitro viability of mouse oocytes vitrified in an ethylene glycol-based solution.

Ovulated mouse oocytes denuded of their cumulus cells, were vitrified in a solution containing 7 M ethylene glycol as the sole cryoprotectant using one or two steps of exposure before vitrification and were diluted in 1 M sucrose solution in 5 or 10 min after warming. The results proved that the viability of oocytes are detrimentally affected by exposure to the vitrification solution even without vitrification. At 5 min dilution time, the two-step exposure was superior to the one-step in terms of the post-warming recovery rate of vitrified oocytes with normal morphology and their subsequent development to the blastocyst stage (p < 0.01) after fertilization in vitro. At 10 min dilution time, no significant difference between one- or two-step exposure was found. The effect of the addition of 0.5 M sucrose to the vitrification solution was also determined and did not result in a significant improvement in the viability of oocytes vitrified in one-step and diluted for 10 min. In conclusion, the results in this study indicate that oocytes can be vitrified with 7 M ethylene glycol as the sole cryoprotectant in the vitrification solution, and that the recovery of normal oocytes after one-step exposure in the vitrification solution can be improved by 10 min dilution time. However, the improvement in the recovery rate of oocytes with normal morphology and their subsequent developmental in vitro was not improved by the addition of 0.5 M sucrose to the vitrification solution.

Current status of vitrification of embryos and oocytes in domestic animals: ethylene glycol as an emerging cryoprotectant of choice.

The cryopreservation of mammalian embryos has become an integral part of methods to control animal reproduction. Numerous vitrification solutions have been formulated with ethylene glycol in combination with macromolecules, sugars and other cryoprotective agents. These indicate that a study of ethylene glycol as a cryoprotectant of choice in vitrification studies would be promising. To understand the cryobiology of ethylene glycol, several factors have to be studied. These are: cryoprotectant toxicity, osmotic stress and temperature at exposure. Understanding these factors could lead to the formulation of vitrification protocols that would lead to higher viability rates after cooling. First, ethylene glycol must be used as the sole cryoprotectant in a solution without macromolecules and sugars. Second, partial dehydration and permeation prior to cooling to subzero temperatures must be studied to achieve accurate exposure and a one-step dilution method. Third, the toxic effects of ethylene glycol must be overcome without sacrificing its vitrification properties by combining step-wise exposure at appropriate temperatures, low concentration and decreased volume. Fourth, the long-term effects of ethylene glycol on exposed or vitrified embryos must be determined. Lastly, the influence of culture on the viability of vitrified embryos must be studied to improve viability rates after warming.

In vitro viability of mouse zygotes vitrified in ethylene glycol.

A study was made to determine if mouse zygotes can be effectively vitrified in 7 M ethylene glycol in modified Dulbecco's phosphate buffered saline (PB1) and to find out if the development of vitrified-warmed zygotes in vitro can be improved by renewing the culture medium. The results showed that without medium change, vitrification reduced the development of zygotes to the expanded blastocyst stage (p < 0.01). With medium change, the development rate of vitrified-warmed zygotes exposed in 7 M ethylene glycol for 1 or 2 min was similar to that of unvitrified zygotes. However, prolonged exposure (5 min) markedly reduced the development rates of vitrified-warmed zygotes to the expanded blastocyst stage (p < 0.05). When the zygotes were vitrified in 7 M ethylene glycol and diluted at 18 degrees C to 22 degrees C, a slower efflux of ethylene glycol from the cell might have occurred, leading to a toxic effect of ethylene glycol in culture. The development rates of vitrified embryos cultured with medium change at 24 hr did not significantly differ from the untreated control (89.0% vs 96.5%). In conclusion, this study showed that mouse zygotes can be vitrified in 7 M ethylene glycol in PB1 and that changing the culture medium can improve the in vitro development rates of vitrified-warmed zygotes to the expanded blastocyst stage.

In vitro viability of mouse 8-cell embryos vitrified in a simple solution of ethylene glycol.

A study was made to determine if ethylene glycol (EG) can be used in a simple solution to vitrify mouse 8-cell embryos and to determine the critical factors that affect its success. Mouse 8-cell embryos were vitrified after exposure to 2M and 7M EG prepared in Dulbecco's phosphate buffered saline (PBS) with 10% heat-inactivated calf serum (CS). Mouse 8-cell embryos exposed to 2M EG for 2, 5 and 10 min, and to 7M EG for 2 and 5 min had survival rates similar to the untreated controls (93.3-100%). No significant difference in their survival rates in vitro was observed. Higher room temperatures (> 24 degrees C) at exposure before cooling resulted in poor development rates to the blastocyst stage. The survival rates of embryos vitrified after 2 min in 7M EG at 18-22 degrees C room temperature did not differ significantly from the control, but embryos vitrified after 5 min had significantly lower survival rates (p < 0.0001). In conclusion, effective vitrification of mouse 8-cell embryos can be achieved by initial exposure to 2M EG for 2-10 min followed by equilibration in 7M EG for 2 min at 18-22 degrees C room temperature. This study has shown that 7M EG in PBS with 10% CS is sufficient to provide cryoprotection of vitrified mouse 8-cell embryos and that exposure of the embryos to the vitrification solution at temperatures over 24 degrees C is critical to their subsequent development in vitro.