Method for retinal gene repair in neonatal mouse.

Gene correction at the site of the mutation in the chromosome is the absolute way to really cure a genetic disease. The oligonucleotide (ODN)-mediated gene repair technology uses an ODN perfectly complementary to the genomic sequence except for a mismatch at the base that is mutated. The endogenous repair machinery of the targeted cell then mediates substitution of the desired base in the gene, resulting in a completely normal sequence. Theoretically, it avoids potential gene silencing or random integration associated with common viral gene augmentation approaches and allows an intact regulation of expression of the therapeutic protein. The eye is a particularly attractive target for gene repair because of its unique features (small organ, easily accessible, low diffusion into systemic circulation). Moreover therapeutic effects on visual impairment could be obtained with modest levels of repair. This chapter describes in details the optimized method to target active ODNs to the nuclei of photoreceptors in neonatal mouse using (1) an electric current application at the eye surface (saline transpalpebral iontophoresis), (2) combined with an intravitreous injection of ODNs, as well as the experimental methods for (3) the dissection of adult neural retinas, (4) their immuno-labelling, and (5) flat-mounting for direct observation of photoreceptor survival, a relevant criteria of treatment outcomes for retinal degeneration.

O2 consumption by the fick method. Methodologic factors.

This study aimed to compare O2 consumption (VO2) determination by the gas-exchange (VO2GE) and Fick (VO2F) methods in cardiac surgical patients. A total of 10 mechanically ventilated postoperative patients were studied prospectively. Thermodilution was performed using three randomly applied techniques: room temperature saline injected at end expiration, room temperature saline randomly injected in the respiratory cycle, and iced saline injected at end expiration. The influence of the number of thermodilution determinations was assessed by comparing results from 2 and 10 injections. The variability of VO2F was greater than that of VO2GE. There was no bias between VO2GE and VO2F values using injectate at room temperature. Accuracy and precision were not improved by increasing the number of cardiac output determinations from 2 to 10. A significant bias was observed using ice-cold injectate, VO2F being 18.0 +/- 15.4 ml/min/m2 lower than VO2GE (p = 0.001). Published results when comparing VO2F and VO2GE are discrepant. However, a significant bias was found in all studies using cold injectate, with lower VO2F values. We conclude that iced injectate should not be used to assess VO2 in critically ill patients.

Results of computer assisted navigation in Total Knee Arthroplasty (TKA): comparison with the conventional method

The aim of this retrospective study was to compare the clinical and radiographic results after TKA (PFC, DePuy), performed either by computer assisted navigation (CAS, Brainlab, Johnson&Johnson) or by conventional means. Material and methods: Between May and December 2006 we reviewed 36 conventional TKA performed between 2002 and 2003 (group A) and 37 navigated TKA performed between 2005 and 2006 (group B) by the same experienced surgeon. The mean age in group A was 74 years (range 62-90) and 73 (range 58-85) in group B with a similar age distribution. The preoperative mechanical axes in group A ranged from -13° varus to +13° valgus (mean absolute deviation 6.83°, SD 3.86), in group B from -13° to +16° (mean absolute deviation 5.35, SD 4.29). Patients with a previous tibial osteotomy or revision arthroplasty were excluded from the study. Examination was done by an experienced orthopedic resident independent of the surgeon. All patients had pre- and postoperative long standing radiographs. The IKSS and the WOMAC were utilized to determine the clinical outcome. Patient's degree of satisfaction was assessed on a visual analogous scale (VAS). Results: 32 of the 37 navigated TKAs (86,5%) showed a postoperative mechanical axis within the limits of 3 degrees of valgus or varus deviation compared to only 24 (66%) of the 36 standard TKAs. This difference was significant (p = 0.045). The mean absolute deviation from neutral axis was 3.00° (range -5° to +9°, SD: 1.75) in group A in comparison to 1.54° (range -5° to +4°, SD: 1.41) in group B with a highly significant difference (p = 0.000). Furthermore, both groups showed a significant postoperative improvement of their mean IKSS-values (group A: 89 preoperative to 169 postoperative, group B 88 to 176) without a significant difference between the two groups. Neither the WOMAC nor the patient's degree of satisfaction - as assessed by VAS - showed significant differences. Operation time was significantly higher in group B (mean 119.9 min.) than in group A (mean 99.6 min., p <0.000). Conclusion: Our study showed consistent significant improvement of postoperative frontal alignment in TKA by computer assisted navigation (CAS) compared to standard methods, even in the hands of a surgeon well experienced in standard TKA implantation. However, the follow-up time of this study was not long enough to judge differences in clinical outcome. Thus, the relevance of computer navigation for clinical outcome and survival of TKA remains to be proved in long term studies to justify the longer operation time. References 1 Stulberg SD. Clin Orth Rel Res. 2003;(416):177-84. 2 Chauhan SK. JBJS Br. 2004;86(3):372-7. 3 Bäthis H, et al. Orthopäde. 2006;35(10):1056-65.

Validation of an analytical method for nitrous oxide (N2O) laughing gas by headspace gas chromatography coupled to mass spectrometry (HS-GC-MS): Forensic application to a lethal intoxication.

Drug abuse is a widespread problem affecting both teenagers and adults. Nitrous oxide is becoming increasingly popular as an inhalation drug, causing harmful neurological and hematological effects. Some gas chromatography-mass spectrometry (GC-MS) methods for nitrous oxide measurement have been previously described. The main drawbacks of these methods include a lack of sensitivity for forensic applications; including an inability to quantitatively determine the concentration of gas present. The following study provides a validated method using HS-GC-MS which incorporates hydrogen sulfide as a suitable internal standard allowing the quantification of nitrous oxide. Upon analysis, sample and internal standard have similar retention times and are eluted quickly from the molecular sieve 5Å PLOT capillary column and the Porabond Q column therefore providing rapid data collection whilst preserving well defined peaks. After validation, the method has been applied to a real case of N2O intoxication indicating concentrations in a mono-intoxication.

Nest distribution varies with dispersal method and familiarity-mediated aggression for two sympatric ants

Dispersal mechanisms and competition together play a key role in the spatial distribution of a population. Species that disperse via fission are likely to experience high levels of localized competitive pressure from conspecifics relative to species that disperse in other ways. Although fission dispersal occurs in many species, its ecological and behavioural effects remain unclear. We compared foraging effort, nest spatial distribution and aggression of two sympatric ant species that differ in reproductive dispersal: Streblognathus peetersi, which disperse by group fission, and Plectroctena mandibularis, which disperse by solitary wingless queens. We found that although both species share space and have similar foraging strategies, they differ in nest distribution and aggressive behaviour. The spatial distribution of S. peetersi nests was extremely aggregated, and workers were less aggressive towards conspecifics from nearby nests than towards distant conspecifics and all heterospecific workers. By contrast, the spatial distribution of P. mandibularis nests was overdispersed, and workers were equally aggressive towards conspecific and heterospecific competitors regardless of nest distance. Finally, laboratory experiments showed that familiarity led to the positive relationship between aggression and nest distance in S. peetersi. While unfamiliar individuals were initially aggressive, the level of aggression decreased within 1 h of contact, and continued to decrease over 24 h. Furthermore, individuals from near nests that were not aggressive could be induced to aggression after prolonged isolation. Overall, these results suggest that low aggression mediated by familiarity could provide benefits for a species with fission reproduction and an aggregated spatial distribution.

Acute endotoxemia inhibits microvascular nitric oxide-dependent vasodilation in humans.

Nitric oxide (NO) is crucial for the microvascular homeostasis, but its role played in the microvascular alterations during sepsis remains controversial. We investigated NO-dependent vasodilation in the skin microcirculation and plasma levels of asymmetric dimethylarginine (ADMA), a potent endogenous inhibitor of the NO synthases, in a human model of sepsis. In this double-blind, randomized, crossover study, microvascular NO-dependent (local thermal hyperemia) and NO-independent vasodilation (post-occlusive reactive hyperemia) assessed by laser Doppler imaging, plasma levels of ADMA, and l-arginine were measured in seven healthy obese volunteers, immediately before and 4 h after either a i.v. bolus injection of Escherichia coli endotoxin (LPS; 2 ng/kg) or normal saline (placebo) on two different visits at least 2 weeks apart. LPS caused the expected systemic effects, including increases in heart rate (+43%, P < 0.001), cardiac output (+16%, P < 0.01), and rectal temperature (+1.4°C, P < 0.001), without change in arterial blood pressure. LPS affected neither baseline skin blood flow nor post-occlusive reactive hyperemia but decreased the NO-dependent local thermal hyperemia response, l-arginine, and, to a lesser extent, ADMA plasma levels. The changes in NO-dependent vasodilation were not correlated with the corresponding changes in the plasma levels of ADMA, l-arginine, or the l-arginine/ADMA ratio. Our results show for the first time that experimental endotoxemia in humans causes a specific decrease in endothelial NO-dependent vasodilation in the microcirculation, which cannot be explained by a change in ADMA levels. Microvascular NO deficiency might be responsible for the heterogeneity of tissue perfusion observed in sepsis and could be a therapeutic target.

Le Q-sort, un outil pour la recherche en soins le cas des représentations chez les infirmiers en psychiatrie de l'âge avancé [Q-sort, a useful research method in care in cases of psychiatric nursing of the aged]

Q-sort is a research method which allows defining profiles of attitudes toward a set of statements, ordered in relation to each other. Pertaining to the Q Methodology, the qualitative analysis of the Q-sorts is based on quantitative techniques. This method is of particular interest for research in health professions, a field in which attitudes of patients and professionals are very important. The method is presented in this article, along with an example of application in nursing in old age psychiatry.

I have faith in my thing, you know what I mean? Mixed method elements on individual investments on religious markets.

In contemporary society, religious signification and secular systems mix and influence each other. Holistic conceptions of a world in which man is integrated harmoniously with nature meet representations of a world run by an immanent God. On the market of the various systems, the individual goes from one system to another, following his immediate needs and expectations without necessarily leaving any marks in a meaningful long term system. This article presents the first results of an ongoing research in Switzerland on contemporary religion focusing on (new) paths of socialization of modern that individuals and the various (non-) belief systems that they simultaneously develop

Innovative method for carbon dioxide determination in human postmortem cardiac gas samples using headspace-gas chromatography-mass spectrometry and stable labeled isotope as internal standard.

A novel approach to measure carbon dioxide (CO2) in gaseous samples, based on a precise and accurate quantification by (13)CO2 internal standard generated in situ is presented. The main goal of this study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable in the routine determination of CO2. The main drawback of the GC methods discussed in the literature for CO2 measurement is the lack of a specific internal standard necessary to perform quantification. CO2 measurement is still quantified by external calibration without taking into account analytical problems which can often occur considering gaseous samples. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in situ an internal labeled standard gas ((13)CO2) on the basis of the stoichiometric formation of CO2 by the reaction of hydrochloric acid (HCl) with sodium hydrogen carbonate (NaH(13)CO3). This method allows a precise measurement of CO2 concentration and was validated on various human postmortem gas samples in order to study its efficiency.

Modified 'dumbbell' technique: a simple and intuitive method to position balloon-expandable stent valves.

Intraoperative cardiac imaging plays a key role during transcatheter aortic valve replacement. In recent years, new techniques and new tools for improved image quality and virtual navigation have been proposed, in order to simplify and standardize stent valve positioning and implantation. But routine performance of the new techniques may require major economic investments or specific knowledge and skills and, for this reason, they may not be accessible to the majority of cardiac centres involved in transcatheter valve replacement projects. Additionally, they still require injections of contrast medium to obtain computed images. Therefore, we have developed and describe here a very simple and intuitive method of positioning balloon-expandable stent valves, which represents the evolution of the 'dumbbell' technique for echocardiography-guided transcatheter valve replacement without angiography. This method, based on the partial inflation of the balloon catheter during positioning, traps the crimped valve in the aortic valve orifice and, consequently, very near to the ideal landing zone. It does not require specific echocardiographic knowledge; it does not require angiographies that increase the risk of postoperative kidney failure in elderly patients, and it can be also performed in centres not equipped with a hybrid operating room.

Validation and long-term evaluation of a modified on-line chiral analytical method for therapeutic drug monitoring of (R,S)-methadone in clinical samples.

Matrix effects, which represent an important issue in liquid chromatography coupled to mass spectrometry or tandem mass spectrometry detection, should be closely assessed during method development. In the case of quantitative analysis, the use of stable isotope-labelled internal standard with physico-chemical properties and ionization behaviour similar to the analyte is recommended. In this paper, an example of the choice of a co-eluting deuterated internal standard to compensate for short-term and long-term matrix effect in the case of chiral (R,S)-methadone plasma quantification is reported. The method was fully validated over a concentration range of 5-800 ng/mL for each methadone enantiomer with satisfactory relative bias (-1.0 to 1.0%), repeatability (0.9-4.9%) and intermediate precision (1.4-12.0%). From the results obtained during validation, a control chart process during 52 series of routine analysis was established using both intermediate precision standard deviation and FDA acceptance criteria. The results of routine quality control samples were generally included in the +/-15% variability around the target value and mainly in the two standard deviation interval illustrating the long-term stability of the method. The intermediate precision variability estimated in method validation was found to be coherent with the routine use of the method. During this period, 257 trough concentration and 54 peak concentration plasma samples of patients undergoing (R,S)-methadone treatment were successfully analysed for routine therapeutic drug monitoring.

The management of iron deficiency in inflammatory bowel disease--an online tool developed by the RAND/UCLA appropriateness method.

BACKGROUND: Iron deficiency is a common and undertreated problem in inflammatory bowel disease (IBD). AIM: To develop an online tool to support treatment choice at the patient-specific level. METHODS: Using the RAND/UCLA Appropriateness Method (RUAM), a European expert panel assessed the appropriateness of treatment regimens for a variety of clinical scenarios in patients with non-anaemic iron deficiency (NAID) and iron deficiency anaemia (IDA). Treatment options included adjustment of IBD medication only, oral iron supplementation, high-/low-dose intravenous (IV) regimens, IV iron plus erythropoietin-stimulating agent (ESA), and blood transfusion. The panel process consisted of two individual rating rounds (1148 treatment indications; 9-point scale) and three plenary discussion meetings. RESULTS: The panel reached agreement on 71% of treatment indications. 'No treatment' was never considered appropriate, and repeat treatment after previous failure was generally discouraged. For 98% of scenarios, at least one treatment was appropriate. Adjustment of IBD medication was deemed appropriate in all patients with active disease. Use of oral iron was mainly considered an option in NAID and mildly anaemic patients without disease activity. IV regimens were often judged appropriate, with high-dose IV iron being the preferred option in 77% of IDA scenarios. Blood transfusion and IV+ESA were indicated in exceptional cases only. CONCLUSIONS: The RUAM revealed high agreement amongst experts on the management of iron deficiency in patients with IBD. High-dose IV iron was more often considered appropriate than other options. To facilitate dissemination of the recommendations, panel outcomes were embedded in an online tool, accessible via http://ferroscope.com/.

Multilayer perceptron with local constraint as an emerging method in spatial data analysis

The use of Geographic Information Systems has revolutionalized the handling and the visualization of geo-referenced data and has underlined the critic role of spatial analysis. The usual tools for such a purpose are geostatistics which are widely used in Earth science. Geostatistics are based upon several hypothesis which are not always verified in practice. On the other hand, Artificial Neural Network (ANN) a priori can be used without special assumptions and are known to be flexible. This paper proposes to discuss the application of ANN in the case of the interpolation of a geo-referenced variable.

IQRray, a new method for Affymetrix microarray quality control, and the homologous organ conservation score, a new benchmark method for quality control metrics.

MOTIVATION: Microarray results accumulated in public repositories are widely reused in meta-analytical studies and secondary databases. The quality of the data obtained with this technology varies from experiment to experiment, and an efficient method for quality assessment is necessary to ensure their reliability. RESULTS: The lack of a good benchmark has hampered evaluation of existing methods for quality control. In this study, we propose a new independent quality metric that is based on evolutionary conservation of expression profiles. We show, using 11 large organ-specific datasets, that IQRray, a new quality metrics developed by us, exhibits the highest correlation with this reference metric, among 14 metrics tested. IQRray outperforms other methods in identification of poor quality arrays in datasets composed of arrays from many independent experiments. In contrast, the performance of methods designed for detecting outliers in a single experiment like Normalized Unscaled Standard Error and Relative Log Expression was low because of the inability of these methods to detect datasets containing only low-quality arrays and because the scores cannot be directly compared between experiments. AVAILABILITY AND IMPLEMENTATION: The R implementation of IQRray is available at: ftp://lausanne.isb-sib.ch/pub/databases/Bgee/general/IQRray.R. CONTACT: Marta.Rosikiewicz@unil.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin.

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of -9.9 to +5% and -4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.