Imaging techniques applied for detection of secretion, transgene delivery and G proteincoupled receptors in reproductive and endocrine cells in vivo and in vitro

Post-testicular sperm maturation occurs in the epididymis. The ion concentration and proteins secreted into the epididymal lumen, together with testicular factors, are believed to be responsible for the maturation of spermatozoa. Disruption of the maturation of spermatozoa in the epididymis provides a promising strategy for generating a male contraceptive. However, little is known about the proteins involved. For drug development, it is also essential to have tools to study the function of these proteins in vitro. One approach for screening novel targets is to study the secretory products of the epididymis or the G protein-coupled receptors (GPCRs) that are involved in the maturation process of the spermatozoa. The modified Ca2+ imaging technique to monitor release from PC12 pheochromocytoma cells can also be applied to monitor secretory products involved in the maturational processes of spermatozoa. PC12 pheochromocytoma cells were chosen for evaluation of this technique as they release catecholamines from their cell body, thus behaving like endocrine secretory cells. The results of the study demonstrate that depolarisation of nerve growth factor -differentiated PC12 cells releases factors which activate nearby randomly distributed HEL erythroleukemia cells. Thus, during the release process, the ligands reach concentrations high enough to activate receptors even in cells some distance from the release site. This suggests that communication between randomly dispersed cells is possible even if the actual quantities of transmitter released are extremely small. The development of a novel method to analyse GPCR-dependent Ca2+ signalling in living slices of mouse caput epididymis is an additional tool for screening for drug targets. By this technique it was possible to analyse functional GPCRs in the epithelial cells of the ductus epididymis. The results revealed that, both P2X- and P2Y-type purinergic receptors are responsible for the rapid and transient Ca2+ signal detected in the epithelial cells of caput epididymides. Immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) analyses showed the expression of at least P2X1, P2X2, P2X4 and P2X7, and P2Y1 and P2Y2 receptors in the epididymis. Searching for epididymis-specific promoters for transgene delivery into the epididymis is of key importance for the development of specific models for drug development. We used EGFP as the reporter gene to identify proper promoters to deliver transgenes into the epithelial cells of the mouse epididymis in vivo. Our results revealed that the 5.0 kb murine Glutathione peroxidase 5 (GPX5) promoter can be used to target transgene expression into the epididymis while the 3.8 kb Cysteine-rich secretory protein-1 (CRISP-1) promoter can be used to target transgene expression into the testis. Although the visualisation of EGFP in living cells in culture usually poses few problems, the detection of EGFP in tissue sections can be more difficult because soluble EGFP molecules can be lost if the cell membrane is damaged by freezing, sectioning, or permeabilisation. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilise EGFP may also destroy its usefulness as a fluorescent reporter. We therefore developed a novel tissue preparation and preservation techniques for EGFP. In addition, fluorescence spectrophotometry with epididymal epithelial cells in suspension revealed the expression of functional purinergic, adrenergic, cholinergic and bradykinin receptors in these cell lines (mE-Cap27 and mE-Cap28). In conclusion, we developed new tools for studying the role of the epididymis in sperm maturation. We developed a new technique to analyse GPCR dependent Ca2+ signalling in living slices of mouse caput epididymis. In addition, we improved the method of detecting reporter gene expression. Furthermore, we characterised two epididymis-specific gene promoters, analysed the expression of GPCRs in epididymal epithelial cells and developed a novel technique for measurement of secretion from cells.

First records and potential palaeoecological significance of Dianella (Xanthorrhoeaceae), an extinct representative of the native flora of Rapa Nui (Easter Island).

Easter Island, a remote island in the Pacific Ocean, is currently primarily covered by grasslands, but palaeoecological studies have shown the former presence of different vegetation. Much of its original biota has been removed during the last two millennia, most likely by human activities, and little is known about the native flora.Macrofossil and pollen analyses of a sediment core from the Raraku crater lake have revealed the occurrence of a plant that is currently extinct from the island: Dianella cf. intermedia/adenanthera (Xanthorrhoeaceae), which grew and disappeared at the Raraku site long before human arrival. The occurrence of Dianella within the Raraku sedimentary sequence (between 9.4 and 5.4 cal. kyr B.P.) could have been linked to the existence of favorable palaeoenvironmental conditions (peatland rather than the present-day lacustrine environment) during the early to mid Holocene. This finding contributes new knowledge about indigenous plant diversity on Easter Island and reinforces the usefulness of further macrofossil and pollen analyses to identify native species on Easter Island and elsewhere.

Clinical utility of a molecular test in adjuvant treatment decision making in women with endocrine-sensitive early stage breast cancer: a retrospective, single center one year experience

Background. Molecular tests for breast cancer (BC) risk assessment are reimbursed by health insurances in Switzerland since the beginning of year 2015. The main current role of these tests is to help oncologists to decide about the usefulness of adjuvant chemotherapy in patients with early stage endocrine-sensitive and human epidermal growth factor receptor 2 (HER2)-negative BC. These gene expression signatures aim at predicting the risk of recurrence in this subgroup. One of them (OncotypeDx/OT) also predicts distant metastases rate with or without the addition of cytotoxic chemotherapy to endocrine therapy. The clinical utility of these tests -in addition to existing so-called "clinico-pathological" prognostic and predictive criteria (e.g. stage, grade, biomarkers status)-is still debated. We report a single center one year experience of the use of one molecular test (OT) in clinical decision making. Methods. We extracted from the CHUV Breast Cancer Center data base the total number of BC cases with estrogen-receptor positive (ER+), HER2-negative early breast cancer (node negative (pN0) disease or micrometastases in up to 3 lymph nodes) operated between September 2014 and August 2015. For the cases from this group in which a molecular test had been decided by the tumor board, we collected the clinicopathologic parameters, the initial tumor board decision, and the final adjuvant systemic therapy decision. Results. A molecular test (OT) was done in 12.2% of patients with ER + HER2 negative early BC. The median age was 57.4 years and the median invasive tumor size was 1.7 cm. These patients were classified by ODX testing (Recurrence Score) into low-, intermediate-, and high risk groups, respectively in 27.2%, 63.6% and 9% of cases. Treatment recommendations changed in 18.2%, predominantly from chemotherapyendocrine therapy to endocrine treatment alone. Of 8 patients originally recommended chemotherapy, 25% were recommended endocrine treatment alone after receiving the Recurrence Score result. Conclusions. Though reimbursed by health insurances since January 2015, molecular tests are used moderately in our institution as per the decision of the multidisciplinary tumor board. It's mainly used to obtain a complementary confirmation supporting the decision of no chemotherapy. The OncotypeDx Recurrence Score results were in the intermediate group in 66% of the 9 tested cases but contributed to avoid chemotherapy in 2 patients during the last 12 months.

The importance of distinguishing information from evidence/observations when formulating propositions

The value of forensic results crucially depends on the propositions and the information under which they are evaluated. For example, if a full single DNA profile for a contemporary marker system matching the profile of Mr A is assessed, given the propositions that the DNA came from Mr A and given it came from an unknown person, the strength of evidence can be overwhelming (e.g., in the order of a billion). In contrast, if we assess the same result given that the DNA came from Mr A and given it came from his twin brother (i.e., a person with the same DNA profile), the strength of evidence will be 1, and therefore neutral, unhelpful and irrelevant 1 to the case at hand. While this understanding is probably uncontroversial and obvious to most, if not all practitioners dealing with DNA evidence, the practical precept of not specifying an alternative source with the same characteristics as the one considered under the first proposition may be much less clear in other circumstances. During discussions with colleagues and trainees, cases have come to our attention where forensic scientists have difficulty with the formulation of propositions. It is particularly common to observe that results (e.g., observations) are included in the propositions, whereas-as argued throughout this note-they should not be. A typical example could be a case where a shoe-mark with a logo and the general pattern characteristics of a Nike Air Jordan shoe is found at the scene of a crime. A Nike Air Jordan shoe is then seized at Mr A's house and control prints of this shoe compared to the mark. The results (e.g., a trace with this general pattern and acquired characteristics corresponding to the sole of Mr A's shoe) are then evaluated given the propositions 'The mark was left by Mr A's Nike Air Jordan shoe-sole' and 'The mark was left by an unknown Nike Air Jordan shoe'. As a consequence, the footwear examiner will not evaluate part of the observations (i.e., the mark presents the general pattern of a Nike Air Jordan) whereas they can be highly informative. Such examples can be found in all forensic disciplines. In this article, we present a few such examples and discuss aspects that will help forensic scientists with the formulation of propositions. In particular, we emphasise on the usefulness of notation to distinguish results that forensic scientists should evaluate from case information that the Court will evaluate.

Swiss family physicians' perceptions and attitudes towards knowledge translation practices.

BACKGROUND: Several studies have been performed to understand the way family physicians apply knowledge from medical research in practice. However, very little is known concerning family physicians in Switzerland. In an environment in which information constantly accumulates, it is crucial to identify the major sources of scientific information that are used by family physicians to keep their medical knowledge up to date and barriers to use these sources. Our main objective was to examine medical knowledge translation (KT) practices of Swiss family physicians. METHODS: The population consisted of French- and German-speaking private practice physicians specialised in family medicine. We conducted four interviews and three focus groups (n = 25). The interview guides of the semi-structured interviews and focus groups focused on (a) ways and means used by physicians to keep updated with information relevant to clinical practice; (b) how they consider their role in translating knowledge into practice; (c) potential barriers to KT; (d) solutions proposed by physicians for effective KT. RESULTS: Family physicians find themselves rather ambivalent about the translation of knowledge based on scientific literature, but generally express much interest in KT. They often feel overwhelmed by "information floods" and perceive clinical practice guidelines and other supports to be of limited usefulness for their practice. They often combine various formal and informal information sources to keep their knowledge up to date. Swiss family physicians report considering themselves as artisans, caring for patients with complex needs. CONCLUSION: Improved performance of KT initiatives in family medicine should be tailored to actual needs and based on high quality evidence-based sources.

Guideline adaptation: a methodology to enhance efficiency in guideline development and improve utilization.

BACKGROUND: Developing and updating high-quality guidelines requires substantial time and resources. To reduce duplication of effort and enhance efficiency, we developed a process for guideline adaptation and assessed initial perceptions of its feasibility and usefulness. METHODS: Based on preliminary developments and empirical studies, a series of meetings with guideline experts were organised to define a process for guideline adaptation (ADAPTE) and to develop a manual and a toolkit made available on a website (http://www.adapte.org). Potential users, guideline developers and implementers, were invited to register and to complete a questionnaire evaluating their perception about the proposed process.

The Strengths and Difficulties Questionnaire (SDQ-Fin) among Finnish children and adolescents

Objective: The psychometric properties of The Strengths and Difficulties Questionnaire (SDQ-Fin), a Finnish version of a brief screening instrument were studied. Emotional and behavioural problems of 7- to 15-year-olds measured by the SDQ were reported, as well as the occurrence of self-reported eating disturbance symptoms and alcohol use among adolescents. Methods and samples: The cross-sectional school survey included 25 items of the SDQ-Fin, items about eating disturbance, alchol use and child psychiatric help-seeking. The study consists of three community samples: 1. The SDQ-Fin parent (n = 703) and teacher (n = 376) versions of 7 – 12 –year-olds, and self-report versions (n = 528) of 11 – 16 years-olds were obtained, and 2. the parent (n = 81) and self-report versions of 15-16 –year olds (n = 129) were obtained in Laitila and Pyhäranta. 3. The self-report versions of 13 – 16 – year-olds (n = 1458) in Salo and Rovaniemi were obtained. Results: The psychometric properties of the SDQ-Fin were for the most part comparable with the other European SDQ research results. The internal consistency (Cronbach’s alpha = 0.71 in all informants’ reports) and inter-rater reliability (between the pairs of reports r = 0.38 - 0.44) were adequate. The concurrent validity (r = 0.75 between the SDQ and the CBCL total scores; r = 0.71 between the SDQ and the YSR total scores) was sufficient. Factor analysis of the SDQ self-report generally confirmed the postulated structure for girls and boys, except for the conduct problems scale of boys, which was fused with emotional symptoms and with hyperactivity. The response rates, means and cut-off points of the SDQ self-report scores were similar to those found, e.g. in Norway and in Britain. A high level of psychological problems, especially emotional and conduct problems and hyperactivity-inattention, were associated with high level of eating disturbance symptoms and alcohol use. Conclusion: The results showed that the psychometric properties of the SDQ-Fin are adequate and provide additional confirmation of the usefulness of the SDQ-Fin for, e.g. screening, epidemiological research and clinical purposes.

External Validation of the Prestroke Independence, Sex, Age, National Institutes of Health Stroke Scale (ISAN) Score for Predicting Stroke-Associated Pneumonia in the Athens Stroke Registry.

BACKGROUND AND PURPOSE: The Prestroke Independence, Sex, Age, National Institutes of Health Stroke Scale (ISAN) score was developed recently for predicting stroke-associated pneumonia (SAP), one of the most common complications after stroke. The aim of the present study was to externally validate the ISAN score. METHODS: Data included in the Athens Stroke Registry between June 1992 and December 2011 were used for this analysis. Inclusion criteria were the availability of all ISAN score variables (prestroke independence, sex, age, National Institutes of Health Stroke Scale score). Receiver operating characteristic curves and linear regression analyses were used to determine the discriminatory power of the score and to assess the correlation between actual and predicted pneumonia in the study population. Separate analyses were performed for patients with acute ischemic stroke (AIS) and intracerebral hemorrhage (ICH). RESULTS: The analysis included 3204 patients (AIS: 2732, ICH: 472). The ISAN score demonstrated excellent discrimination in patients with AIS (area under the curve [AUC]: .83 [95% confidence interval {CI}: .81-.85]). In the ICH group, the score was less effective (AUC: .69 [95% CI: .63-.74]). Higher-risk groups of ISAN score were associated with an increased relative risk of SAP; risk increase was more prominent in the AIS population. Predicted pneumonia correlated very well with actual pneumonia (AIS group: R(2) = .885; β-coefficient = .941, P < .001; ICH group: R(2) = .880, β-coefficient = .938, P < .001). CONCLUSIONS: In our external validation in the Athens Stroke Registry cohort, the ISAN score predicted SAP very accurately in AIS patients and demonstrated good discriminatory power in the ICH group. Further validation and assessment of clinical usefulness would strengthen the score's utility further.

Estudio de los tumores óseos mediante el tratamiento de imágenes digitalizadas

Aquest informe tècnic mostra la classificació, incidència, característiques i diagnòstic dels tumors ossis primaris i secundaris metastàsics més freqüents a partir de 145 radiografies digitalitzades

Interrater variability of EEG interpretation in comatose cardiac arrest patients.

OBJECTIVE: EEG is widely used to predict outcome in comatose cardiac arrest patients, but its value has been limited by lack of a uniform classification. We used the EEG terminology proposed by the American Clinical Neurophysiology Society (ACNS) to assess interrater variability in a cohort of cardiac arrest patients included in the Target Temperature Management trial. The main objective was to evaluate if malignant EEG-patterns could reliably be identified. METHODS: Full-length EEGs from 103 comatose cardiac arrest patients were interpreted by four EEG-specialists with different nationalities who were blinded for patient outcome. Percent agreement and kappa (κ) for the categories in the ACNS EEG terminology and for prespecified malignant EEG-patterns were calculated. RESULTS: There was substantial interrater agreement (κ 0.71) for highly malignant patterns and moderate agreement (κ 0.42) for malignant patterns. Substantial agreement was found for malignant periodic or rhythmic patterns (κ 0.72) while agreement for identifying an unreactive EEG was fair (κ 0.26). CONCLUSIONS: The ACNS EEG terminology can be used to identify highly malignant EEG-patterns in post cardiac arrest patients in an international context with high reliability. SIGNIFICANCE: The establishment of strict criteria with high transferability between interpreters will increase the usefulness of routine EEG to assess neurological prognosis after cardiac arrest.

A measure of stability as a criterion for the verification and analysis of simulation models

The aim of this study is to define a new statistic, PVL, based on the relative distance between the likelihood associated with the simulation replications and the likelihood of the conceptual model. Our results coming from several simulation experiments of a clinical trial show that the PVL statistic range can be a good measure of stability to establish when a computational model verifies the underlying conceptual model. PVL improves also the analysis of simulation replications because only one statistic is associated with all the simulation replications. As well it presents several verification scenarios, obtained by altering the simulation model, that show the usefulness of PVL. Further simulation experiments suggest that a 0 to 20 % range may define adequate limits for the verification problem, if considered from the viewpoint of an equivalence test.

Prostate cancer screening in Switzerland: 20-year trends and socioeconomic disparities.

BACKGROUND: Despite important controversy in its efficacy, prostate cancer (PCa) screening has become widespread. Important socioeconomic screening disparities have been reported. However, trends in PCa screening and social disparities have not been investigated in Switzerland, a high risk country for PCa. We used data from five waves (from 1992-2012) of the population-based Swiss Health Interview Survey to evaluate trends in PCa screening and its association with socioeconomic indicators. METHODS: We used multivariable Poisson regression to estimate prevalence ratios (PR) and 95% Confidence Intervals (CI) adjusting for demographics, health status, and use of healthcare. RESULTS: The study included 12,034 men aged ≥50 years (mean age: 63.9). Between 1992 and 2012, ever use of PCa screening increased from 55.3% to 70.0% and its use within the last two years from 32.6% to 42.4% (p-value <0.05). Income, education, and occupational class were independently associated with PCa screening. PCa screening within the last two years was greater in men with the highest (>$6,000/month) vs. lowest income (≤$2,000) (46.5% vs. 38.7% in 2012, PR for overall period =1.29, 95%CI: 1.13-1.48). These socioeconomic disparities did not significantly change over time. CONCLUSIONS: This study shows that about half of Swiss men had performed at least one PCa screening. Men belonging to high socioeconomic status are clearly more frequently screened than those less favored. Given the uncertainty of the usefulness of PCa screening, men, including those with high socioeconomic status, should be clearly informed about benefits and harms of PCa screening, in particular, the adverse effect of over-diagnosis and of associated over-treatment.

Sacral fusion as an aid in age estimation

The degree of fusion at the anterior aspect of the sacral vertebrae has been scored in 242 male and female skeletons from the Lisbon documented collection, ranging in age from 16 to 59 years old. Statistical tests indicate a sex difference towards earlier fusion in young females compared with young males, as well as a clear association between degree of fusion and age. Similar results have been found in documented skeletal samples from Coimbra and Sassari, and the recommendations stated by these authors regarding age estimation have been positively tested in the Lisbon collection. Although more research from geographically diverse samples is required, a general picture of the pattern of sacral fusion and its associations with age and sex is emerging. We also provide a practical example of the usefulness of the sacrum in age estimation in a forensic setting, a mass grave from the Spanish Civil War. It is concluded that the scoring of the degree of fusion of the sacral vertebrae, specially of S1-2, can be a simple tool for assigning skeletons to broad age groups, and it should be implemented as another resource for age estimation in the study of human skeletal remains.

El desarrollo personal y profesional mediante el aprendizaje reflexivo : una experiencia en el grado de Educación Social

In the last years, an increase of the interest to develop educational practices in higher education, based on the approach of the reflective learning, is observed. Nevertheless, we scarcely find in our context researches or experiences that allow knowing students’ perceptions about this teaching and learning approach. We have developed an experience in the bachelor of Social Education at the University of Girona with the aim to contribute to the personal and professional development of future social educators in their initial training, using reflective learning methodology. In this article we present an evaluation of the experience based on students’ perceptions. They assessed the usefulness they think the module has for their training, the methodology and the activities. This evaluation has been carried out through in-depth interviews to 17 students who participated in the module in 2010-11 academic year. The results show that students assess positively the experience, either its general usefulness or the methodology of reflective learning, although they acknowledge some difficulties to carry out such a process which involves a high degree of introspection and a difficulty to set the boundaries in the narration of personal questions. The study also shows some challenges related on the need, but also the difficulty, to include personal and professional development as a powerful axis in the higher education curriculum, as well as elements linked to reflective learning assessment

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.