A simple tool for stereological assessment of digital images: the STEPanizer

STEPanizer is an easy-to-use computer-based software tool for the stereological assessment of digitally captured images from all kinds of microscopical (LM, TEM, LSM) and macroscopical (radiology, tomography) imaging modalities. The program design focuses on providing the user a defined workflow adapted to most basic stereological tasks. The software is compact, that is user friendly without being bulky. STEPanizer comprises the creation of test systems, the appropriate display of digital images with superimposed test systems, a scaling facility, a counting module and an export function for the transfer of results to spreadsheet programs. Here we describe the major workflow of the tool illustrating the application on two examples from transmission electron microscopy and light microscopy, respectively.

Comparison of three strip-type tests and two laboratory methods for salivary buffering analysis

This study evaluated the correlation between three strip-type, colorimetric tests and two laboratory methods with respect to the analysis of salivary buffering. The strip-type tests were saliva-check buffer, Dentobuff strip and CRT(®) Buffer test. The laboratory methods included Ericsson's laboratory method and a monotone acid/base titration to create a reference scale for the salivary titratable acidity. Additionally, defined buffer solutions were prepared and tested to simulate the carbonate, phosphate and protein buffer systems of saliva. The correlation between the methods was analysed by the Spearman's rank test. Disagreement was detected between buffering capacity values obtained with three strip-type tests that was more pronounced in case of saliva samples with medium and low buffering capacities. All strip-type tests were able to assign the hydrogencarbonate, di-hydrogenphosphate and 0.1% protein buffer solutions to the correct buffer categories. However, at 0.6% total protein concentrations, none of the test systems worked accurately. Improvements are necessary for strip-type tests because of certain disagreement with the Ericsson's laboratory method and dependence on the protein content of saliva.

Disruption of glucocorticoid action by environmental chemicals: potential mechanisms and relevance

Glucocorticoids play an essential role in the regulation of key physiological processes, including immunomodulation, brain function, energy metabolism, electrolyte balance and blood pressure. Exposure to naturally occurring compounds or industrial chemicals that impair glucocorticoid action may contribute to the increasing incidence of cognitive deficits, immune disorders and metabolic diseases. Potentially, "glucocorticoid disruptors" can interfere with various steps of hormone action, e.g. hormone synthesis, binding to plasma proteins, delivery to target cells, pre-receptor regulation of the ratio of active versus inactive hormones, glucocorticoid receptor (GR) function, or export and degradation of glucocorticoids. Several recent studies indicate that such chemicals exist and that some of them can cause multiple toxic effects by interfering with different steps of hormone action. For example, increasing evidence suggests that organotins disturb glucocorticoid action by altering the function of factors that regulate the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) pre-receptor enzymes, by direct inhibition of 11beta-HSD2-dependent inactivation of glucocorticoids, and by blocking GR activation. These observations emphasize on the complexity of the toxic effects caused by such compounds and on the need of suitable test systems to assess their effects on each relevant step.

Evaluation of two fully automated novel enzyme-linked immunosorbent assays for the determination of human adiponectin in serum

BACKGROUND: In contrast to RIA, recently available ELISAs provide the potential for fully automated analysis of adiponectin. To date, studies reporting on the diagnostic characteristics of ELISAs and investigating on the relationship between ELISA- and RIA-based methods are rare. METHODS: Thus, we established and evaluated a fully automated platform (BEP 2000; Dade-Behring, Switzerland) for determination of adiponectin levels in serum by two different ELISA methods (competitive human adiponectin ELISA; high sensitivity human adiponectin sandwich ELISA; both Biovendor, Czech Republic). Further, as a reference method, we also employed a human adiponectin RIA (Linco Research, USA). Samples from 150 patients routinely presenting to our cardiology unit were tested. RESULTS: ELISA measurements could be accomplished in less than 3 h, measurement of RIA had a duration of 24 h. The ELISAs were evaluated for precision, analytical sensitivity and specificity, linearity on dilution and spiking recovery. In the investigated patients, type 2 diabetes, higher age and male gender were significantly associated with lower serum adiponectin concentrations. Correlations between the ELISA methods and the RIA were strong (competitive ELISA, r=0.82; sandwich ELISA, r=0.92; both p<0.001). However, Deming regression and Bland-Altman analysis indicated lack of agreement of the 3 methods preventing direct comparison of results. The equations of the regression lines are: Competitive ELISA=1.48 x RIA-0.88; High sensitivity sandwich ELISA=0.77 x RIA+1.01. CONCLUSIONS: Fully automated measurement of adiponectin by ELISA is feasible and substantially more rapid than RIA. The investigated ELISA test systems seem to exhibit analytical characteristics allowing for clinical application. In addition, there is a strong correlation between the ELISA methods and RIA. These findings might promote a more widespread use of adiponectin measurements in clinical research.

CompAS: A new approach to commonality and variability analysis with applications in computer assisted orthopaedic surgery

In rapidly evolving domains such as Computer Assisted Orthopaedic Surgery (CAOS) emphasis is often put first on innovation and new functionality, rather than in developing the common infrastructure needed to support integration and reuse of these innovations. In fact, developing such an infrastructure is often considered to be a high-risk venture given the volatility of such a domain. We present CompAS, a method that exploits the very evolution of innovations in the domain to carry out the necessary quantitative and qualitative commonality and variability analysis, especially in the case of scarce system documentation. We show how our technique applies to the CAOS domain by using conference proceedings as a key source of information about the evolution of features in CAOS systems over a period of several years. We detect and classify evolution patterns to determine functional commonality and variability. We also identify non-functional requirements to help capture domain variability. We have validated our approach by evaluating the degree to which representative test systems can be covered by the common and variable features produced by our analysis.

A novel comparative research platform designed to determine the functional significance of tree species diversity in European forests

One of the current advances in functional biodiversity research is the move away from short-lived test systems towards the exploration of diversity-ecosystem functioning relationships in structurally more complex ecosystems. In forests, assumptions about the functional significance of tree species diversity have only recently produced a new generation of research on ecosystem processes and services. Novel experimental designs have now replaced traditional forestry trials, but these comparatively young experimental plots suffer from specific difficulties that are mainly related to the tree size and longevity. Tree species diversity experiments therefore need to be complemented with comparative observational studies in existing forests. Here we present the design and implementation of a new network of forest plots along tree species diversity gradients in six major European forest types: the FunDivEUROPE Exploratory Platform. Based on a review of the deficiencies of existing observational approaches and of unresolved research questions and hypotheses, we discuss the fundamental criteria that shaped the design of our platform. Key features include the extent of the species diversity gradient with mixtures up to five species, strict avoidance of a dilution gradient, special attention to community evenness and minimal covariation with other environmental factors. The new European research platform permits the most comprehensive assessment of tree species diversity effects on forest ecosystem functioning to date since it offers a common set of research plots to groups of researchers from very different disciplines and uses the same methodological approach in contrasting forest types along an extensive environmental gradient. (C) 2013 Elsevier GmbH. All rights reserved.

Nano Aerosol Chamber for In-Vitro Toxicity (NACIVT) studies.

Abstract Inhalation of ambient air particles or engineered nanoparticles (NP) handled as powders, dispersions or sprays in industrial processes and contained in consumer products pose a potential and largely unknown risk for incidental exposure. For efficient, economical and ethically sound evaluation of health hazards by inhaled nanomaterials, animal-free and realistic in vitro test systems are desirable. The new Nano Aerosol Chamber for in-vitro Toxicity studies (NACIVT) has been developed and fully characterized regarding its performance. NACIVT features a computer-controlled temperature and humidity conditioning, preventing cellular stress during exposure and allowing long-term exposures. Airborne NP are deposited out of a continuous air stream simultaneously on up to 24 cell cultures on Transwell® inserts, allowing high-throughput screening. In NACIVT, polystyrene as well as silver particles were deposited uniformly and efficiently on all 24 Transwell® inserts. Particle-cell interaction studies confirmed that deposited particles reach the cell surface and can be taken up by cells. As demonstrated in control experiments, there was no evidence for any adverse effects on human bronchial epithelial cells (BEAS-2B) due to the exposure treatment in NACIVT. The new, fully integrated and transportable deposition chamber NACIVT provides a promising tool for reliable, acute and sub-acute dose-response studies of (nano)particles in air-exposed tissues cultured at the air-liquid interface.

Comparative enzymology of 11beta-hydroxysteroid dehydrogenase type 1 from six species.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1), catalyzing the intracellular activation of cortisone to cortisol, is currently considered a promising target to treat patients with metabolic syndrome; hence, there is considerable interest in the development of selective inhibitors. For preclinical tests of such inhibitors, the characteristics of 11beta-HSD1 from the commonly used species have to be known. Therefore, we determined differences in substrate affinity and inhibitor effects for 11beta-HSD1 from six species. The differences in catalytic activities with cortisone and 11-dehydrocorticosterone were rather modest. Human, hamster and guinea-pig 11beta-HSD1 displayed the highest catalytic efficiency in the oxoreduction of cortisone, while mouse and rat showed intermediate and dog the lowest activity. Murine 11beta-HSD1 most efficiently reduced 11-dehydrocorticosterone, while the enzyme from dog showed lower activity than those from the other species. 7-ketocholesterol (7KC) was stereospecifically converted to 7beta-hydroxycholesterol by recombinant 11beta-HSD1 from all species analyzed except hamster, which showed a slight preference for the formation of 7alpha-hydroxycholesterol. Importantly, guinea-pig and canine 11beta-HSD1 displayed very low 7-oxoreductase activities. Furthermore, we demonstrate significant species-specific variability in the potency of various 11beta-HSD1 inhibitors, including endogenous compounds, natural chemicals and pharmaceutical compounds. The results suggest significant differences in the three-dimensional organization of the hydrophobic substrate-binding pocket of 11beta-HSD1, and they emphasize that species-specific variability must be considered in the interpretation of results obtained from different animal experiments. The assessment of such differences, by cell-based test systems, may help to choose the appropriate animal for safety and efficacy studies of novel potential drug candidates.

Comparative Cytotoxicity Study of Silver Nanoparticles (AgNPs) in a Variety of Rainbow Trout Cell Lines (RTL-W1, RTH-149, RTG-2) and Primary Hepatocytes.

Among all classes of nanomaterials, silver nanoparticles (AgNPs) have potentially an important ecotoxicological impact, especially in freshwater environments. Fish are particularly susceptible to the toxic effects of silver ions and, with knowledge gaps regarding the contribution of dissolution and unique particle effects to AgNP toxicity, they represent a group of vulnerable organisms. Using cell lines (RTL-W1, RTH-149, RTG-2) and primary hepatocytes of rainbow trout (Oncorhynchus mykiss) as in vitro test systems, we assessed the cytotoxicity of the representative AgNP, NM-300K, and AgNO3 as an Ag+ ion source. Lack of AgNP interference with the cytotoxicity assays (AlamarBlue, CFDA-AM, NRU assay) and their simultaneous application point to the compatibility and usefulness of such a battery of assays. The RTH-149 and RTL-W1 liver cell lines exhibited similar sensitivity as primary hepatocytes towards AgNP toxicity. Leibovitz's L-15 culture medium composition (high amino acid content) had an important influence on the behaviour and toxicity of AgNPs towards the RTL-W1 cell line. The obtained results demonstrate that, with careful consideration, such an in vitro approach can provide valuable toxicological data to be used in an integrated testing strategy for NM-300K risk assessment.

Development of a novel fMRI compatible visual perception prototype battery to test older people with and without dementia

Background: Visuoperceptual deficits in dementia are common and can reduce quality of life. Testing of visuoperceptual function is often confounded by impairments in other cognitive domains and motor dysfunction. We aimed to develop, pilot, and test a novel visuocognitive prototype test battery which addressed these issues, suitable for both clinical and functional imaging use. Methods: We recruited 23 participants (14 with dementia, 6 of whom had extrapyramidal motor features, and 9 age-matched controls). The novel Newcastle visual perception prototype battery (NEVIP-B-Prototype) included angle, color, face, motion and form perception tasks, and an adapted response system. It allows for individualized task difficulties. Participants were tested outside and inside the 3T functional magnetic resonance imaging (fMRI) scanner. Functional magnetic resonance imaging data were analyzed using SPM8. Results: All participants successfully completed the task inside and outside the scanner. Functional magnetic resonance imaging analysis showed activation regions corresponding well to the regional specializations of the visual association cortex. In both groups, there was significant activity in the ventral occipital-temporal region in the face and color tasks, whereas the motion task activated the V5 region. In the control group, the angle task activated the occipitoparietal cortex. Patients and controls showed similar levels of activation, except on the angle task for which occipitoparietal activation was lower in patients than controls. Conclusion: Distinct visuoperceptual functions can be tested in patients with dementia and extrapyramidal motor features when tests use individualized thresholds, adapted tasks, and specialized response systems.

Subclinical mastitis in dairy cows in Swiss organic and conventional production systems

The objective was to compare the prevalence of subclinical mastitis (SM) and of udder pathogens in 60 Swiss organic (OP) and 60 conventional production systems (CP). Cows (n=970) were studied for SM prevalence and udder pathogens at median 31 d and 102 d post partum. Cows showing a >/=1+ positive California Mastitis Test (CMT) in at least one quarter were considered to have SM. Cow-level prevalences of SM for visits at 31 d and 102 d post partum (39% and 40% in OP and 34% and 35% in CP) were similar, but quarter-level prevalences of SM were higher (P<0.02) in OP than CP (15% and 18% in OP and 12% and 15% in CP). Median somatic cell counts in milk at 31 d post partum were higher (P<0.05) in OP than CP cows (43000 and 28000 cells/ml, respectively), but were similar at 102 d post partum in OP and CP cows (45000 and 38000 cells/ml, respectively). In milk samples from quarters showing a CMT reaction >/=2+ the prevalences of coagulase negative staphylococci were lower (P<0.05) at 102 d post partum, whereas prevalences of non-agalactiae streptococci were higher (P<0.05) in OP than in CP cows at 31 d and 102 d post partum. In conclusion, under Swiss conditions, subclinical mastitis is a greater problem in organic than in conventional production systems, but differences are not marked.

Test Blueprints - Exposing Side Effects in Execution Traces to Support Writing Unit Tests

Writing unit tests for legacy systems is a key maintenance task. When writing tests for object-oriented programs, objects need to be set up and the expected effects of executing the unit under test need to be verified. If developers lack internal knowledge of a system, the task of writing tests is non-trivial. To address this problem, we propose an approach that exposes side effects detected in example runs of the system and uses these side effects to guide the developer when writing tests. We introduce a visualization called Test Blueprint, through which we identify what the required fixture is and what assertions are needed to verify the correct behavior of a unit under test. The dynamic analysis technique that underlies our approach is based on both tracing method executions and on tracking the flow of objects at runtime. To demonstrate the usefulness of our approach we present results from two case studies.

Effect of tin-chloride pretreatment on bond strength of two adhesive systems to dentin

OBJECTIVES: To determine the effect on resin composite-to-dentin bond strength of incorporation of an acidic tin-chloride pretreatment in two adhesive systems. MATERIALS AND METHODS: Human molars were ground to expose mid-coronal dentin. For microtensile bond strength (μTBS) testing, dentin was treated with Optibond FL or Clearfil SE according to one of six protocols (n = 22/group). Group 1: Phosphoric acid etching, Optibond FL Prime, Optibond FL Adhesive (manufacturer's instructions; control); Group 2: Tin-chloride pretreatment, Optibond FL Prime, Optibond FL Adhesive; Group 3: Phosphoric acid etching, tin-chloride pretreatment, Optibond FL Prime, Optibond FL Adhesive; Group 4: Clearfil SE Primer, Clearfil SE Bond (manufacturer's instructions; control); Group 5: Phosphoric acid etching, Clearfil SE Primer, Clearfil SE Bond; and Group 6: Tin-chloride pretreatment, Clearfil SE Primer, Clearfil SE Bond. The molars were then built up with resin composite (Clearfil Majesty Esthetic). After storage (1 week, 100  % humidity, 37 °C) the μTBS was measured and failure mode was determined. Additionally, pretreated dentin surfaces were evaluated using SEM and EDX. The μTBS results were analyzed statistically by a Welch Two Sample t-test and a Kruskal-Wallis test followed by exact Wilcoxon rank sum tests with Bonferroni-Holm adjustment for multiple testing (α = 0.05). RESULTS: When Optibond FL was used, partial or total replacement of phosphoric acid with tin-chloride decreased μTBS significantly. In contrast, when Clearfil SE was used, inclusion of a tin-chloride pretreatment in the adhesive procedure increased μTBS significantly. CONCLUSIONS: Tin-chloride pretreatment had a beneficial influence on the bond promoting capacity of the MDP-containing adhesive system Clearfil SE.