Fluorogenic protein labeling using a genetically encoded unstrained alkene.

We developed a new fluorogenic bioorthogonal reaction that is based on the inverse electron-demand Diels-Alder reaction between styrene (an unstrained alkene) and a simple tetrazine. The reaction forms a new fluorophore with no literature precedent. We have identified an aminoacyl-tRNA synthetase/tRNA pair for the efficient and site-specific incorporation of a styrene-containing amino acid into proteins in response to amber nonsense codon. Fluorogenic labeling of purified proteins and intact proteins in live cells were demonstrated. The fluorogenicity of the styrene-tetrazine reaction can be potentially applied to the study of protein folding and function under physiological conditions with low background fluorescence interference.

[Formal prescribing errors are substantially reduced in electronic prescribing and after teaching sessions]. / Formale Fehler bei der Rezeptschreibung sind bei Nutzung elektronischer Verordnung und Schulung erheblich reduziert.

INTRODUCTION: Prescription forms enable the communication between physicians and pharmacists. Hence, incorrectly issued prescriptions may result in delay of health-care delivery, additional workload, and potentially adverse patient outcomes. We aimed to evaluate the formal prescription quality in our outpatient clinics (OC) before and after performing teaching sessions and using an electronic prescription system to replace handwritten prescriptions. METHODS: All OCs of a university hospital were offered a short teaching session on how to issue prescriptions correctly and how to use the electronic prescription system. During four weeks before and after the teaching, we anonymously collected all prescriptions of the OCs in 20 surrounding community pharmacies and assessed whether they were error-free, required an intervention by the pharmacist, additional clarification by the OC, or had to be reissued. RESULTS: After the intervention, the absolute fraction of formally error-free prescriptions increased by 12.9% from 52.9% (516/976) to 65.8% (713/1084, p < 0.001; d = 12,9% 95% confidence interval [8,7%; 17,1%]). Largest improvements were seen in prescriptions requiring clarification by the OC (224/976 prescriptions at baseline versus 93/1084 post-intervention, p < 0.001). The fraction of electronic prescriptions increased from 34.9% (341/976) to 46.9% (509/1084, p < 0.001, d = 12,0% 95% confidence interval [7,8%; 16,2%]) with electronic prescriptions consistently being of higher formal quality than handwritten prescriptions. CONCLUSION: After increased use of electronic prescribing and teaching courses, formal prescription quality was significantly improved.

Voltammetric determination of Ioxynil and 2-methyl-3-nitroaniline using C(18) modified carbon paste electrodes.

The stripping voltammetric behaviour of Ioxynil and 2-methyl-3-nitroaniline has been studied by means of a C(18) modified carbon paste electrode. The analytes are preconcentrated under open-circuit conditions (Ioxynil at pH 3.9; 2-methyl-3-nitroaniline at pH 10). For the determination of Ioxynil and 2-methyl-3-nitroaniline 0.01 mol/l HCl and 1 mol/l KOH, respectively, have been used as supporting electrolyte. Under optimized conditions detection limits up to 0.1 microg/ml Ioxynil and 0.3 microg/ml 2-methyl-3-nitroaniline have been obtained. The methods have been applied to the determination of Ioxynil and 2-methyl-3-nitroaniline in drinking water.

Studies on the potential mutagenicity of p-phenylenediamine in oxidative hair dye mixtures.

An aqueous mixture of p-phenylenediamine dihydrochloride, resorcinol and hydrogen peroxide, incubated for 30 min at 37 degrees C, was not mutagenic in the Ames test and in the mouse lymphoma assay and did not produce chromosome aberrations in human lymphocytes, whereas the same mixture without resorcinol was mutagenic in the Ames test and the chromosome aberration assay, probably due to the formation of Bandrowski's base. The formation of mutagenic Bandrowski's base and other reactive products could be demonstrated by thin-layer chromatography and subsequent Ames testing. Preincubation for 0-7 h of 5 different oxidative mixtures of p-phenylenediamine dihydrochloride and various couplers at 37 degrees C resulted in mutagenicities in the Ames test ranging from 'very strong effects' at 0 or 0.5 h for 2,4-diaminoanisole dihydrochloride to 'not mutagenic' at up to 7 h preincubation time for a recently developed alternative coupler. A comparable ranking of mutagenic potencies of the same mixtures could be obtained by Ames tests with dyed buffalo hair strands, either tested directly on the agar plates or by testing the solvent extracts of the colored strands. The optimal combination without any sign of mutagenic tendency was finally tested in a commercially available hair dye formulation. This complex mixture was also not mutagenic in the Ames test. Our main conclusions are as follows. (I) Oxidative hair dye mixtures of p-phenylenediamine dihydrochloride and non-mutagenic couplers are not mutagenic if tested under normal conditions of use; the formation of mutagenic reaction products (such as Bandrowski's base) can be prevented if the oxidative reaction time is limited to 30 min and if about equimolar proportions of para to meta compounds are used. (II) Optimal non-mutagenic combinations of primary intermediates (such as p-phenylenediamine dihydrochloride) and couplers can be evaluated in the Ames test by either time-dependent preincubation of the mixtures or the described test protocol with hair strands.

Predictive ability of reconstructed human epidermis equivalents for the assessment of skin irritation of cosmetics.

The aim of this study was to examine the concordance between human in vivo and in vitro skin irritation classifications of cosmetic products and to evaluate the correlations between the different parameters. For that purpose, 22 formulations from product development test series, covering the full range of in vivo scores and representing different cosmetic product classes, were tested in vivo (modified Frosch-Kligman Soap Chamber Patch Test with repetitive occlusive application) and in vitro using two epidermis equivalents commercially available as kits (EpiDerm and EPISKIN) and one in-house model (Cosmital). In vivo, skin reactions (erythema, dryness and fissures) were visually evaluated and, in addition, skin redness and transepidermal water loss (TEWL) were measured by means of technical instruments. The parameters measured in vitro were the percent cell viability in the MTT reduction assay, with ET(50) determination, and the extracellular release of the pro-inflammatory mediator IL-1alpha and of the cytosolic enzyme lactate dehydrogenase (LDH), into the culture medium collected after topical application of the products for different exposure times (time-course assay). In general, good Spearman rank correlations could be observed between the different in vivo parameters (with the exception of TEWL and dryness at day 2). Furthermore, high correlation coefficients were obtained by comparing the different in vitro parameters (except for LDH release) and different models, which allowed to conclude that the results obtained with the different reconstructed epidermis models were very similar. A comparison between in vivo and in vitro parameters resulted in the best rank correlation for ET(50), then in decreasing order, for the percent MTT viability at 16 h, the IL-1alpha release and finally, for LDH release, where the correlation was generally low. A direct comparison of the mean total scores (sum of erythema, dryness and fissures at day 5) of the 22 products with the best predictor, ET(50) obtained with the three reconstructed epidermis models, using simple linear regression analysis resulted in a coefficient of correlation R=0.94 for EpiDerm, R=0.90 for Cosmital and R=0.84 for EPISKIN. Multivariate descriptive statistics showed that the in vitro parameters, MTT viability evaluated after the 16-h exposure and ET(50), as well as the in vivo parameters, sum of visual scores at day 5 and chromameter value, were the best endpoints to discriminate between irritant and non-irritant products. Using the in vivo mean total scores at day 5 with a cut-off value at 2 and the in vitro percent MTT viability after the 16-h exposure with a cut-off value at 50% to classify the products, the same two-by-two contingency table was obtained for all the three reconstructed epidermis models with sensitivity=92%, specificity=100% and observed concordance=95% (kappa=0.91; 95% confidence interval 0.74-1.08). This classification system was a satisfactory and relevant approach to discriminate the "irritant" from the "non-irritant" cosmetic products in this study. In conclusion, this study demonstrated the usefulness of reconstructed human epidermis equivalents for the in vitro assessment of the irritation potential of a series of cosmetic products. These models allow the measurement of quantifiable and objective endpoints relevant to in vivo irritative phenomena.

Cosmetics Europe multi-laboratory pre-validation of the EpiOcular™ reconstituted human tissue test method for the prediction of eye irritation.

Cosmetics Europe, The Personal Care Association (known as Colipa before 2012), conducted a program of technology transfer and within/between laboratory reproducibility of MatTek Corporation's EpiOcular™ Eye Irritation Test (EIT) as one of the two human reconstructed tissue test methods. This EIT EpiOcular™ used a single exposure period for each chemical and a prediction model based on a cut-off in relative survival [ ≤60%=irritant (I) (GHS categories 2 and 1); >60%=no classification (NC)]. Test substance single exposure time was 30 min with a 2-h post-exposure incubation for liquids and 90 min with an 18-h post-exposure incubation for solids. Tissue viability was determined by tetrazolium dye (MTT) reduction. Combinations of 20 coded chemicals were tested in 7 laboratories. Standardized laboratory documentation was used by all laboratories. Twenty liquids (11 NC/9 I) plus 5 solids (3 NC/2 I) were selected so that both exposure regimens could be assessed. Concurrent positive (methyl acetate) and negative (water) controls were tested in each trial. In all, 298 independent trials were performed and demonstrated 99.7% agreement in prediction (NC/I) across the laboratories. Coefficients of variation for the% survival for tissues from each treatment group across laboratories were generally low. This protocol has entered in 2010 the experimental phase of a formal ECVAM validation program.

Cosmetics Europe multi-laboratory pre-validation of the SkinEthic™ reconstituted human corneal epithelium test method for the prediction of eye irritation.

Cosmetics Europe, The Personal Care Association, known as Colipa before 2012, conducted a program of technology transfer and assessment of Within/Between Laboratory (WLV/BLV) reproducibility of the SkinEthic™ Reconstituted Human Corneal Epithelium (HCE) as one of two human reconstructed tissue eye irritation test methods. The SkinEthic™ HCE test method involves two exposure time treatment procedures - one for short time exposure (10 min - SE) and the other for long time exposure (60 min - LE) of tissues to test substance. This paper describes pre-validation studies of the SkinEthic™ HCE test method (SE and LE protocols) as well as the Eye Peptide Reactivity Assay (EPRA). In the SE WLV study, 30 substances were evaluated. A consistent outcome with respect to viability measurement across all runs was observed with all substances showing an SD of less than 18%. In the LE WLV study, 44 out of 45 substances were consistently classified. These data demonstrated a high level of reproducibility within laboratory for both the SE and LE treatment procedures. For the LE BLV, 19 out of 20 substances were consistently classified between the three laboratories, again demonstrating a high level of reproducibility between laboratories. The results for EPRA WLV and BLV studies demonstrated that all substances analysed were categorised similarly and that the method is reproducible. The SkinEthic™ HCE test method entered into the experimental phase of a formal ECVAM validation program in 2010.

Reconstructed skin kits: reproducibility of cutaneous irritancy testing.

The aim of this study was to determine the reproducibility of data obtained from in vitro irritation testing using three industrial reconstructed human epidermis models, EpiDerm, Episkin and SkinEthic, and one in-house model developed at Wella/Cosmital. A common protocol was established based on the measurement of cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and of extracellular release of proinflammatory mediators and cytosolic enzymes after a range of exposure times to sodium lauryl sulfate (SLS). This time course protocol was applied to 6 different batches of each skin model using triplicate tissue cultures per test condition. The parameters analyzed for intra- and inter-batch reproducibility were the cell viability determined as MTT reduction capacity and the ET-50 values in the 6 batches, as well as the release of the cytokine IL-1alpha and of the cellular enzymes LDH and GOT in 3 batches only. The MTT viability results showed that EpiDerm was the most resistant to the SLS treatment and at the same time the most reproducible model, SkinEthic was the most sensitive to SLS and the least reproducible, and Episkin and the Cosmital model were intermediate. Measurements of IL-1alpha release showed a relatively high intra- and inter-batch variability in all the skin models. It was not possible to detect the extracellular release of the enzymes LDH and GOT in the Episkin assay medium. With the 3 other models, the release of LDH and GOT varied in about the same range as that of IL-1alpha. For all the parameters in this study, the inter-batch variability was generally greater than the intra-batch variability. A possible reduction in the number of batches and replicates for future applications in routine irritancy testing is discussed on the basis of the results obtained using 6 batches in triplicate.

Characterization and comparison of reconstructed skin models: morphological and immunohistochemical evaluation.

Reconstructed human skin equivalents are currently being investigated as in vitro models for the prediction of human skin toxicity and irritation responses. Three different industrial reconstructed skin models (EpiDerm, Episkin and SkinEthic) and one in-house equivalent were characterized and compared using light microscopy, immunohistochemistry and reduction of (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT). Their inter- and intra-batch variation was evaluated. Histological examination showed a completely stratified epithelium in all skin models, which closely resembled normal human epidermis. Low intra-batch variation in tissue architecture was observed in all skin models, but moderate to considerable inter-batch variation was noticed. Evaluation of the expression and localization of a number of differentiation-specific protein markers revealed that all skin models showed an aberrant expression of keratin 6, skin-derived antileukoproteinase, small proline rich proteins, involucrin and transglutaminase. Although variation within batches was low, in particular keratin 6, involucrin and skin-derived antileukoproteinase expression demonstrated some inter-batch variation. Reduction of MTT in vehicle-treated cultures showed high similarities between skin models, but marked differences were observed when 1.0% sodium lauryl sulfate was applied topically for 3 or 16 h. Most pronounced effects were noticed in SkinEthic cultures. Intra-batch variations were low and moderate variations were observed between batches. All skin models tested reproduced many of the characteristics of normal human epidermis and therefore provide a morphologically relevant in vitro means to assess skin irritation and other skin-related studies.

Comparison of in vitro cell toxicity with in vivo eye irritation.

The effects of 26 different cosmetic ingredients (e.g., permanent wave and hair dye compounds, emulsifiers, resins, and detergents such as quats) were assessed by four end points indicative for qualitatively and quantitatively different cytotoxicity: (1) neutral red uptake reduction after 24 h of treatment (NR-90 and NR-50); (2) cell detachment from culture dish after 4 h of treatment (CD-25); (3) growth inhibition after 48 h of treatment (GI-50); and (4) membrane permeability measured by fluorescent dye retention (fluorescence shift FS-25) and dye exclusion (viability ratio VR-25). The cytotoxicity potentials of the test agents were ranked for each in vitro test and compared with the in vivo eye irritation in guinea pigs (Draize test) after application of 5 or 2.5% (w/v) solutions of the same test batches. Strong irritants could be easily detected by most of the in vitro tests, but the neutral red uptake assay (especially NR-50) was the only one that was able to distinguish the minimally irritating test agents from strong irritants as well as from nonirritants. (I) All three extremely irritating quaternary ammonia compounds were identified as the strongest cytotoxic agents. (II) Nine out of 12 minimally irritating substances (mainly emulsifiers and resins) were ranked in the intermediate group. (III) Eight out of 11 non-or practically nonirritating chemicals (mainly permanent wave compounds) showed cytotoxic effects at very high concentrations only. The distinction of these three groups was better by means of NR-50 than by NR-90 data. At least two of the other cell tests (CD-25, GI-50, FS-25, and VR-25) had to be considered to allow an adequate interpretation of in vitro cytotoxicity.

Evaluation of an in vitro percutaneous permeation model with two oxidative hair dyes.

Synopsis The percutaneous permeation of two oxidative hair dyes was measured by means of pig skin in a flow-through diffusion cell system entirely constructed from Teflon. Pig skin membranes were prepared by reducing full thickness skin with a dermatome to a more in vivo-like barrier layer and their integrity was checked by measuring the steady-state permeation of tritiated water. Initially, the inter- and intraindividual variability of percutaneous permeation was determined with an aqueous solution of 1-(2'-hydroxyethyl)-amino-3,4-methylenedioxybenzene-hydrochloride, an oxidative hair dye component. In the same way the proper flow rate of elution fluid through the receptor cell was found to be most favourable at 10 ml h(-1), the thickness of permeation membranes was fixed at 1 mm, and it was shown that storage of the skin at -20 degrees C for up to 35 days did not change the permeability. The percutaneous permeation of the same hair dye component and of 4-amino-2-hydroxymethylphenol-hydrochloride was determined after application to pig skin membranes under practical conditions of hair dyeing. The in vitro skin permeation was in the same order of magnitude as results from comparable in vivo skin absorption studies in rats. Perméation percutanée in vitro de colorants d'oxydation pour cheveux.

Do smoking prevention programs really work? Attrition and the internal and external validity of an evaluation of a refusal skills training program.

This study investigated the effects of a smoking prevention program that emphasized refusal skills training on 1730 adolescents in three high schools and six middle schools. Classes within these schools were randomly assigned to treatment or no-treatment conditions to avoid confounding schools with treatment condition. The effects of attrition on the internal and external validity of the study were examined. Although the results indicated an apparent effect of the program at the 1-year follow-up in deterring continued smoking among those who were smoking at pretest, this result may have been due to a higher rate of attrition among high-rate smokers in the treatment condition than in the control condition. Attrition also affected external validity. Across both conditions, subjects who were smoking at pretest and who were at risk to smoke were more likely to be missing at follow-up. The program did have an effect on the refusal skills of participants and the validity of this effect was not jeopardized by differential attrition.

[Treatment of depression with sleep deprivation and sleep phase advancement]. / Behandlung von Depressionen mit Schlafentzug und Schlafphasenvorverlagerung.

Total sleep deprivation (TSD) exerts beneficial but only transient effects on mood in approximately 60% of the patients with a major depressive disorder (MDD). The positive effect of TSD is generally reversed after the next night of sleep. A pilot study of our group indicated that a consecutive one week phase advance of the sleep phase stabilized mood in more than half of the patients who responded to TSD. However, the majority of patients in our pilot study had been treated concomitantly with antidepressive medication. To exclude a possible synergistic effect of simultaneous antidepressive medication and the sleep-wake manipulation in the present study eleven medicated and sixteen drug-free depressed patients were investigated. In two thirds of the patients relapse into depression after successful TSD could be prevented. This effect seemed to be independent of adjunct antidepressant pharmacotherapy. Ten of these patients were studied polysomnographically prior to and during the treatment. Data analysis revealed that during the advance of the sleep phase no prolonged partial sleep deprivation took place. At the end of the study REM % had even increased and REM latency was still short in spite of clinical improvement, thus contradicting the assumption that REM sleep suppression is a necessary prerequisite for antidepressive therapy. The results support the hypothesis of a "critical phase" in the morning hours during which sleep can reinduce depressive mood and, vice versa, prevention of sleep during this time may act antidepressively.

[Chronotherapeutic approaches in depressive disorders]. / Chronotherapeutische Ansätze bei depressiven Erkrankungen.

Sleep deprivation has been regarded as a beneficial treatment modality for about 60% of patients with Major Depressive Disorder. Unfortunately, however, more than 80% of the improved patients relapse into depression after the next night of sleep. In the present study we tried to prevent relapses into depression after successful total sleep deprivation (TSD) by treating 27 patients suffering from a Major Depressive Disorder according to DSM-III-R with a combination of sleep deprivation with consecutive sleep phase advance for 1 week. Advancing the sleep period to 5.00 p.m. until 12.00 p.m. following TSD and then gradually shifting back by 1 h daily the sleep period to the normal phase position (11.00 p.m. until 6.00 a.m.) prevented 61% of these patients from relapsing into depression. Thus, the combination of sleep deprivation with consecutive sleep phase advance has been proven to be an effective antidepressant treatment besides other well established antidepressive treatment modalities.

How generalizable are the effects of smoking prevention programs? Refusal skills training and parent messages in a teacher-administered program.

This study investigated both substantive and methodological issues associated with school-based smoking prevention programs. Substantive issues included the efficacy of a refusal skills training curriculum and of parent messages mailed to students' homes. Methodological issues included the effects of assigning classrooms versus entire schools to experimental conditions and determination of the effects of attrition on internal and external validity. Results revealed differential impact for different subgroups of adolescents. The refusal skills program produced lower rates of smoking than the control condition for students who were smokers at the pretreatment assessment but may have produced detrimental effects among males who were nonsmokers at pretest. The provision of parent messages did not affect outcome. Method of assignment (schools versus classrooms) failed to produce significant effects, and attrition did not affect internal validity. However, the above differential findings, as well as the impact of attrition on external validity, raise questions concerning the generalizability of smoking prevention programs.