Fluorescent annulated imidazo[4,5-c]isoquinolines via a GBB-3CR/imidoylation sequence - DNA-interactions in pUC-19 gel electrophoresis mobility shift assay.

Herein we report the development of a sequential synthesis route towards annulated imidazo[4,5-c]isoquinolines comprising a GBB-3CR, followed by an intramolecular imidoylative cyclisation. X-Ray crystallography revealed a flat 3D structure of the obtained polyheterocycles. Thus, we evaluated their interactions with double-stranded DNA by establishing a pUC-19 plasmid-based gel electrophoresis mobility shift assay, revealing a stabilising effect on ds-DNA against strand-break inducing conditions.

Guideline-compliant prescription of biologicals and possible barriers in dermatological practices in Bavaria.

INTRODUCTION: Psoriasis and urticaria are chronic inflammatory skin diseases, which account for a substantial socioeconomic burden and severely affect patients' quality of life. According to the respective German guidelines, biologicals can be used for the treatment of severe forms of these diseases. However, only a minority of patients receive this advised treatment. OBJECTIVE: To analyse the prescription of biologicals according to the German national guidelines for psoriasis and chronic spontaneous urticaria and to assess possible barriers to prescription. MATERIALS AND METHODS: Cross-sectional, questionnaire-based study including all Bavarian dermatologists based in private practices. Linear and logistic regression models were used to identify significant influencing factors on the perception of possible barriers. RESULTS: Between January 2017 and February 2017, a total of 137 (of 499) dermatologists participated. Of all patients with moderate to severe psoriasis and chronic spontaneous urticaria, participating dermatologists indicated treating 14.2% and 6.9% with biologicals, respectively. The most prevalent barriers to prescription were the high cost of the therapy, the low reimbursement and the fear of recourse. Analysis showed that age, years spent working in a dermatological hospital and the number of patients treated with moderate to severe psoriasis affect the perception of many barriers. Furthermore, age and barriers related to physician factors and external factors were identified as modifiers to the prescription scheme of dermatologists. CONCLUSIONS: The role of clinical education and the importance of external and economic barriers in comparison with medical barriers have to be emphasised. Guideline-compliant use of biologicals has to be optimised. Further research is needed to ascertain not only a barrier pattern for Bavaria but also for wider settings. Actions based on this for psoriasis are needed to achieve the goal of the WHO Global Psoriasis Report to strengthen the role of patient-centred care and improve the quality of life of affected patients. Analogue, this applies also for urticaria.

Do outdoor workers know their risk of NMSC? Perceptions, beliefs and preventive behaviour among farmers, roofers and gardeners.

BACKGROUND: Non-melanoma skin cancer (NMSC) was officially recognized in 2015 as an occupational disease for outdoor workers in Germany. Together with the enormous socioeconomic impact of NMSC, this has led to the continuous demand of evidence-based prevention. However, studies assessing the perceptions and beliefs along with risk behaviour of outdoor workers as an essential prerequisite for prevention are rare. OBJECTIVE: To assess perceptions, beliefs, barriers, risk and preventive behaviours towards non-melanoma skin cancer among different outdoor groups as a basis for the development of sustainable prevention programmes. PATIENTS AND METHODS: Cross-sectional study among outdoor workers of three different occupational groups (farmer, gardener, roofer) using a 20-question online survey on NMSC awareness, risk and preventive behaviours. RESULTS: Between March and April 2016, 353 outdoor workers participated in the study. Of these, 153 (43.4%) reported never to use sunscreen during work. Wearing headgear and long pants were the most common sun protection measures. Poor use of sunscreen was more likely in males and farmers. A low perceived skin cancer risk was significantly associated with poor use of sunscreen, long-sleeved shirts, sunglasses and headgear. CONCLUSIONS: Despite great evidence on NMSC risk in outdoor professions throughout the literature, high-risk groups in fact are not yet aware of the topic. Sustainable target group-oriented awareness prevention programmes are needed to lower the immense burden of NMSC.

[The link between food intolerance and weight: a new perspective in the treatment of obesity].

In the article is presented the correlation between body weight and food intolerance to certain products, often associated with the development of food intolerances.

Oxidative stress is associated with liver damage, inflammatory status, and corticosteroid therapy in patients with systemic lupus erythematosus.

Oxidative stress exerts an important role on the pathophysiological mechanisms of systemic lupus erythematosus (SLE). This study investigated oxidative stress in patients with SLE and its correlation with disease activity, corticosteroid therapy, and liver function biomarkers. The study included 58 patients with SLE and 105 healthy volunteers. Patients showed oxidative stress increase evaluated by tert-butyl hydroperoxide-initiated chemiluminescence (CL-LOOH), advanced oxidation protein products (AOPP), and nitric oxide metabolites. C-reactive protein (CRP) was associated with CL-LOOH and with AOPP. Aspartate aminotransferase correlated significantly with CL-LOOH and with AOPP. Patients with disease activity showed an inverse significant correlation of daily prednisone doses and CL-LOOH and a direct correlation with total antioxidant capacity. In conclusion, patients with SLE have persistent lipoperoxidation and protein oxidation even with inactive disease or mild disease activity. The significant correlation between oxidative stress and CRP suggests that, despite clinical remission, the persistence of an inflammatory condition favors oxidative stress. Oxidative stress was associated with liver enzymes, and this relationship seems to support the hypothesis of drug-induced oxidative stress with consequent liver injury. In relation to non-active disease, patients with active SLE did not present oxidative stress and antioxidant capacity changes, due to the antioxidant drugs used in SLE treatment, especially prednisone.

Surgical hand disinfection using alcohol: the effects of alcohol type, mode and duration of application.

Due to their strong antimicrobial activity, rapid action, good dermal tolerance and ease of application, alcohol-based hand rubs are recommended for pre-operative preparation of the surgical team's hands. Using the EN 12791 protocol, three commercial products containing either mixtures of propan-1-ol and propan-2-ol or ethanol at total alcohol concentrations (w/w) between 73% (propanols) and 78.2% (ethanol), as the main active agents, were tested with a shortened application of 1.5 min rather than the usual 3 min. Preparation A containing 30% propan-1-ol and 45% propan-2-ol not only passed the test at this short application but even exceeded, though not significantly, the efficacy of the reference disinfection procedure in EN 12791 when applied for 3 min. Preparation B containing 45% propan-1-ol and 28% propan-2-ol fulfilled the required standard whereas the ethanol (78.2%)-based product C did not (P<0.1). This demonstrates that some, but not all, alcohol-based hand rubs pass the test even within 1.5 min, emphasising the importance of validation before a product is introduced into clinical practice. In another series with both preparation A and 60% v/v propan-1-ol, it was demonstrated that the additional inclusion of the forearms into the disinfection procedure, not required by EN 12791 but normal practice in surgical hand disinfection, does not significantly interfere with the antimicrobial efficacy of either hand rub. Therefore, the mode of test procedure in EN 12791 does not need specific adaptation for hand disinfection by surgical teams.

Evaluation of a novel internally controlled real-time PCR assay targeting the 16S rRNA gene for confirmation of Neisseria gonorrhoeae infections.

A novel HybProbe real-time LightCycler PCR assay was developed for confirmation of Neisseria gonorrhoeae in samples positive according to the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR assay. The new assay amplifies 375 bp of the N. gonorrhoeae 16S rRNA gene and includes an internal amplification control introduced during DNA purification. The assay had 100% specificity because of the high specificity of the HybProbes and primers. Other Neisseria spp. failed to generate positive crossing-point values and melting peaks. The analytical sensitivity for N. gonorrhoeae DNA was 0.5 fg/PCR, corresponding to 0.3 CFU/PCR. Sensitivity was not impaired in the presence of higher DNA concentrations (>or=1000-fold) from Neisseria spp. other than N. gonorrhoeae. The sensitivity was similar to that reported for the COBAS AMPLICOR assay with cervical swab samples. To assess its clinical applicability as a confirmatory test, 38 (2.9%) of 1313 swabs that were positive according to the COBAS AMPLICOR assay were tested using the new in-house assay and the commercially available GenFlow Neisseria test. Twenty-one samples negative according to COBAS AMPLICOR also underwent confirmatory testing. Both confirmatory tests yielded identical results; the 21 negative samples remained negative, and only 11 (28.9%) of the samples positive according to COBAS AMPLICOR were positive after retesting, suggesting a low prevalence (0.84%) of N. gonorrhoeae infection in the study population. These data suggest that the novel real-time PCR assay is an excellent and easy to interpret confirmatory test for the existing COBAS AMPLICOR assay for N. gonorrhoeae.

Evaluation of a protocol for molecular broad-range diagnosis of culture-negative bacterial infections in clinical routine diagnosis.

AIM: Introduction of a protocol for broad-range diagnosis of bacterial infections, which remain negative in culture. METHODS AND RESULTS: The new TaqMan real-time PCR assay amplifies part of the 16S rRNA gene. Species are identified by subsequent sequencing and phylogenetic blast analysis. The analytical sensitivity showed to be 50 fg DNA per PCR. The lowest detectable bacterial cell concentration in blood was 1000 CFU per 200 mul EDTA-blood. The utility in clinical routine diagnosis was evaluated by testing 136 clinical specimens. Bacterial pathogens were detected in 33 samples (24.3%) either by culture or molecular diagnosis. In 10 culture negative cases, pathogens such as Mycoplasma timone/orale, Ureaplasma parvum/urealyticum, Treponema pallidum, different streptococci and staphylococci were identified by molecular diagnosis only. CONCLUSIONS: The introduced broad-range real-time PCR protocol showed to be useful in the clinical routine in cases where bacterial infection was highly anticipated but culture remained negative. However, the obtained data have to be always interpreted with caution and in conjunction with the clinical data, crossing-point values and with the Blast result of both the sample and the controls. SIGNIFICANCE AND IMPACT OF THE STUDY: This work introduces a new and well-evaluated broad-range real-time PCR protocol for diagnosis of bacterial infections.

Simultaneous measurement of magnetization and magnetostriction in 50 T pulsed high magnetic fields.

To simultaneously perform magnetization and magnetostriction measurements in high magnetic fields, a miniaturized device was developed that combines an inductive magnetometer with a capacitive dilatometer and, therefore, it is called "dilamagmeter." This combination of magnetic and magnetoelastic investigations is a new step to a complex understanding of solid state properties. The whole system can be mounted in a 12 mm clear bore of any cryostat usually used in nondestructive pulsed high field magnets. The sensitivity of both methods is about 10(-5) A m(2) for magnetization and 10(-5) relative changes in length for striction measurements. Measurements on a GdSi single crystal, which are corrected by the background signal of the experimental setup, agree well with the results of steady field experiments. All test measurements, which are up until now performed in the temperature range of 4-100 K, confirm the perfect usability and high stability in pulsed fields up to 50 T with a pulse duration of 10 ms.

Definition, operationalization and quality assurance of psychotherapy. An investigation with the behavior therapy-competence-checklist (btcc).

BACKGROUND: Quality assurance is an important issue in psychotherapy research and practice, which is methodologically still in development. Apart from unstructured supervision, reliable assessment instruments are needed. OBJECTIVE: A theoretical concept is outlined that allows to define the main techniques which constitute behavior therapy. An assessment instrument is introduced, which allows to measure whether a therapist applies these standards in therapy and to evaluate their quality. METHOD: The development of the "Behavior Therapy-Competence-Checklist (BTCC)" is described. 182 tape-recorded therapy sessions of 14 behavioral therapists who were working full time in routine care were evaluated. RESULTS: The most often used behavior therapy techniques were preparation of homework assignments (71.4%) and modification of cognitions (41.6%). Most rare were macro-behavioral-analysis (19.5%) and problem solving (19.5%). The mean-total score of the BTCC was 3.4 on a scale that ranges from 1 (bad) to 7 (very good). The lowest scores were found for "problem solving" (2.7). The highest scores were found for "establishment of a therapeutic relationship" (5.2). CONCLUSIONS: Therapeutic competency can be operationalized and measured with the BTCC. The quality of behavior therapists under conditions of routine is moderate. They show the tendency to prefer certain therapeutic options and do not use the full spectrum of therapeutic options. Such findings point to areas in need of training.

Simplifying the WHO 'how to hand rub' technique: three steps are as effective as six-results from an experimental randomized crossover trial.

OBJECTIVES: The World Health Organization (WHO) issued guidelines on hand hygiene recommending a six-step 'how to hand rub' technique for applying alcohol-based hand rub. However, adherence to all six steps is poor. We assessed a simplified three-step technique and compared it to the conventional WHO six-step technique in terms of bacterial count reduction on healthcare workers' hands. METHODS: Thirty-two participants were randomly assigned to clean their hands following the six-step 'how to hand rub' technique (WHO reference group) or a simplified three-step technique (intervention group). Assignments were reversed after 1 day. The degree of bacterial killing was assessed following the European norm for testing hand hygiene products. Hands were contaminated with Escherichia coli, and the mean logarithmic reduction in bacterial counts was compared between both techniques. RESULTS: Bacterial density before hand hygiene performance did not differ between the WHO reference group (median 6.37 log10 CFU, interquartile range (IQR) 6.19-6.54) and the intervention group (median 6.34 log10 CFU, IQR 6.17-6.60, p 0.513). After hand hygiene, the logarithmic reduction factor was higher in the intervention group (median 4.45, IQR 4.04-5.15) compared to the WHO reference group (median 3.91, IQR 3.69-4.62, p 0.021). CONCLUSIONS: The WHO six-step 'how to hand rub' technique can be simplified to a 3-step procedure based on the reduction of bacterial counts on healthcare workers' hands achieved under experimental conditions. The proposed technique is easier to perform and could improve adherence to the execution of hand hygiene action.

VHL alterations in human clear cell renal cell carcinoma: association with advanced tumor stage and a novel hot spot mutation.

To elucidate the role of somatic alterations for renal cancer etiology and prognosis, we analyzed 227 sporadic renal epithelial tumors for mutations and hypermethylations in the von Hippel-Lindau tumor suppressor gene VHL. Tumors were classified according to the recommendations of the Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Somatic VHL mutations were identified by PCR, single-strand conformation polymorphism analysis, and sequencing, and hypermethylations were identified by restriction enzyme digestion and Southern blotting. Frequencies of VHL alterations were established, and an association with tumor type or tumor type and tumor stage was evaluated. VHL mutations and hypermethylations were identified in 45% of clear cell renal cell carcinomas (CCRCCs) and occasionally (3 of 28) in papillary (chromophilic) renal cell carcinomas (RCCs). Lack of VHL mutations and hypermethylations in chromophobe RCCs and oncocytomas was statistically significant (P = 0.0001 and P = 0.0004, respectively). RCCs carrying VHL alterations showed, in nine cases (12%), mutations at a hot spot involving a thymine repeat (ATT.TTT) in exon 2. Tumor staging was critical to the VHL mutation/hypermethylation detection rate in CCRCCs shown by separate evaluation of patients from medical centers in Munich, Heidelberg, and Mainz. The spectrum of pT1, pT2, and pT3 CCRCCs and the VHL mutation/hypermethylation detection rate varied among these three groups. Altogether, VHL alterations were significantly associated with pT3 CCRCCs (P = 0.009). This is the first evidence of frequent somatic VHL mutations at a particular site within exon 2 and an association of VHL mutations/hypermethylations with a standard prognostic factor.

[The diagnostic and therapeutic challenges of bilateral renal oncocytosis : Illustrative case presentations and a review of the literature]. / Die bilaterale renale Onkozytose als diagnostische und therapeutische Herausforderung : Falldarstellung und Literaturreview.

Bilateral oncocytosis along with multiple tumours in both kidneys represents a very rare pathology that is accompanied by diagnostic and therapeutic challenges. We report the case of a 60-year old male patient who underwent computer tomography with incidental detection of multiple bilateral and contrast enhancing renal tumours of different size. Subsequently the patient underwent nephron-sparing tumor resection, first on the right side and 4 weeks later on the left side. The histology of all removed tumors showed evidence of pure oncocytoma. There were no postoperative complications and renal function reached a stable state within 6 months follow-up. The major challenge regarding diagnostic process and therapy of this pathology is to distinguish benign oncocytoma from chromophobe renal cell carcinoma and hybrid tumours, which can all be associated with renal oncocytosis. Because of limitations concerning imaging processes and biopsy, all patients should undergo nephron-sparing surgery as far as possible. On the other hand alternative therapies should - regarding to therapy-associated morbidity and the basically benign prognosis of oncocytoma - be well discussed to obtain informed consent. In this case report different therapy options and the international literature concerning renal oncocytosis will be discussed.

The tumor suppressor p53 is subject to both nuclear import and export, and both are fast, energy-dependent and lectin-inhibited.

Human p53 was expressed in E. coli, purified, labeled with fluorescein iodoacetamide (IAF) and characterized for sequence-specific DNA binding and epitope disposition. Injected into the cytoplasm or nuclei of 3T3 cells IAF-p53 was imported into or exported from nuclei within minutes. Import was inhibited by coinjection of the lectin wheat germ agglutinine (WGA). In contrast, the peptide-protein conjugate NLS-HSA carrying the nuclear localization sequence (NLS) of the SV40 T antigen was only imported but not exported. 3T3 polykaryons were injected with IAF-p53 and photo-bleached by Scanning Microphotolysis in such a manner that only a single nucleus per polykaryon remained non-bleached. IAF-p53 was found to migrate rapidly (halftime 10 min) from non-bleached into bleached nuclei, while NLS-HSA did not. In digitonin permeabilized cells IAF-p53 was imported into nuclei. When removed from the medium after nuclear accumulation IAF-p53 was exported from the nuclei. Nuclear import and export of IAF-p53 both were rapid (halftimes of a few minutes, 22 C) and strongly inhibited by WGA or incubation on ice. NLS-HSA was only imported but not exported. We conclude that the nucleocytoplasmic transport of p53, in contrast to that of NLS-HSA, is bidirectional and that transport in both directions is carrier mediated and energy dependent. These results suggest that p53 contains nuclear export signals (NES) in addition to import signals (NLS) and thus open new views on the potential regulation of p53 cellular fractions.

Effect of T-R conformational change on sickle-cell hemoglobin interactions and aggregation.

We compare the role of a conformational switch and that of a point mutation in the thermodynamic stability of a protein solution and in the consequent propensity toward aggregation. We study sickle-cell hemoglobin (HbS), the beta6 Glu-Val point mutant of adult human hemoglobin (HbA), in its R (CO-liganded) conformation, and compare its aggregation properties to those of both HbS and HbA in their T (unliganded) conformation. Static and dynamic light scattering measurements performed for various hemoglobin concentrations showed critical divergences with mean field exponents as temperature was increased. This allowed determining spinodal data points T(S)(c) by extrapolation. These points were fitted to theoretical expressions of the T(S)(c) spinodal line, which delimits the region where the homogeneous solution becomes thermodynamically unstable against demixing in two sets of denser and dilute mesoscopic domains, while remaining still liquid. Fitting provided model-free numerical values of enthalpy and entropy parameters measuring the stability of solutions against demixing, namely, 93.2 kJ/mol and 314 J/ degrees K-mol, respectively. Aggregation was observed also for R-HbS, but in amorphous form and above physiological temperatures close to the spinodal, consistent with the role played in nucleation by anomalous fluctuations governed by the parameter epsilon = (T - T(S))/T(S). Fourier transform infrared (FTIR) and optical spectroscopy showed that aggregation is neither preceded nor followed by denaturation. Transient multiple interprotein contacts occur in the denser liquid domains for R-HbS, T-HbS, and T-HbA. The distinct effects of their specific nature and configurations, and those of desolvation on the demixing and aggregation thermodynamics, and on the aggregate structure are highlighted.

Echocardiographic advances in atrial fibrillation and supraventricular arrhythmias.

Advances in echocardiography have paved the way for the development of intracardiac catheters with ultrasound transducers mounted on its tip. With this technology it has become possible for the interventional electrophysiologist to perform continuous echocardiographic examination during a procedure without the need for general anaesthesia or additional staff. Intracardiac echocardiography (ICE) allows the monitoring of catheter movement in real-time, assessment of catheter-tissue contact and potentially prevents and recognizes complications like thrombus formation and pericardial effusion. In addition recent technologies allow acquiring the full spectrum of Doppler-imaging permitting evaluation of haemodynamic data during the procedure. All these advances have made ICE an ideal tool for the interventional electrophysiologist, serving as a diagnostic and imaging tool during invasive electrophysiological procedures. This review will summarize currently available technology of ICE and its indications and applications in electrophysiological procedures.

Properties of fluorescently labeled Xenopus lamin A in vivo.

Wild type Xenopus lamin A, a lamin A mutant lacking the carboxy-terminal cysteine (C662-->S), and human vimentin were expressed in bacteria and fluorescently labeled with 5-iodoacetamidofluorescein (5-IAF). In vitro reconstitution experiments and microinjection of both lamins into living cells revealed that they were indistinguishable from the non-fluorescently labeled proteins. When the 5-IAF lamin A was microinjected into the cytoplasm of 3T3 cells it was rapidly transported into the nucleus, giving rise within 1 h to a strong lamina fluorescence, whereas the lamin A mutant formed dotlike intranuclear aggregates. 5-IAF lamin A associated with the nuclear envelope of microinjected 3T3 cells and 5-IAF vimentin which was incorporated into the preexisting vimentin filaments of this cell line, were analyzed by photobleaching employing two different methods, (i) scanning microphotolysis using a modified laser scanning microscope, and (ii) the conventional photobleaching technique in which the integral fluorescence of a single spot was measured by photon counting. A low but significant fluorescence recovery was measured within 10 min for both 5-IAF-labeled intermediate filament proteins, lamin A and vimentin, in bleached areas of the nuclear envelope and the cytoplasmic intermediate filaments, respectively.

Confocal laser scanning analysis of the association of fibulin-2 with fibrillin-1 and fibronectin define different stages of skin regeneration.

The fibulins represent a novel family of extracellular matrix proteins. We report the temporo-spatial expression of fibulin-2 in skin regenerating from keratinocyte autografts. In normal dermis, fibulin-2 was associated with the fibrillin-containing microfibrillar apparatus, except for the portion immediately adjacent to the dermo-epidermal junction. In contrast, early regenerating dermis showed numerous fusiform fibrillin-microfibrils along the basement membrane, whereas fibulin-2 was present in a distinct and separate layer below. Both proteins formed independent fibrillar systems also in the reticular dermis without significant colocalization; however, over time both fibril systems became congruent: after 4 mo there was extensive colocalization of fibulin-2/fibrillin in the reticular dermis, after 17 and 24 mo this also occurred in the papillary dermis. Simultaneous visualization of fibulin-2 and fibronectin revealed an inverse pattern: complete colocalization at 7 d and discordant distribution 17-24 mo after grafting. In particular, the fibrillar fibronectin pattern at early time points changed into a faint granular distribution throughout the dermis and along the subbasement membrane region as in normal skin. Dermal fibroblast cultures showed that fibrillin and fibronectin participated in distinct fibrillar systems; however, fibulin-2 colocalized with either protein. We propose that, in regenerating skin, fibulin-2 is a late component of the cutaneous microfibrillar apparatus with an earlier existence in a fibrillar matrix mediated by fibronectin. This suggests interaction of fibulin-2 with both fibronectin fibrils and fibrillin microfibrils, and is consistent with in vitro binding data.

Effects of aerobic exercise and weight loss on riboflavin requirements of moderately obese, marginally deficient young women.

In a previous study, exercise was shown to increase riboflavin requirements of active, normal weight young women. The present study examined the effect of exercise and weight loss on riboflavin status of moderately overweight women. The experiment was designed as a two-period cross-over with an initial base-line period and two 5-wk metabolic periods. The basic diet contained 1200 kcal with a riboflavin concentration of 0.8 mg/1000 kcal. Exercise consisted of a program of dance exercise. Riboflavin depletion, as measured by increased erythrocyte glutathione reductase activity coefficients and decreased urinary excretion of riboflavin, occurred during both nonexercise and exercise periods. Erythrocyte glutathione reductase activity coefficients increased from a base-line mean of 1.28 +/- 0.11 to 1.40 +/- 0.12 during nonexercise and to 1.49 +/- 0.16 during exercise. Urinary excretion of riboflavin fell from 48 +/- 12% of intake during base-line to 30 +/- 13% during nonexercise and to 19 +/- 6% during exercise. Riboflavin depletion was not related to the rate or composition of weight loss or to change in aerobic capacity.