The Three Rs: The Way Forward

This is the report of the eleventh of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM), which was established in 1991 by the European Commission. ECVAM\u27s main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation. and the potential for the possible incorporation of replacement alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward

A closely linked DNA marker for facioscapulohumeral disease on chromosome 4q.

Close linkage of a hypervariable DNA probe on chromosome 4q (pH30, locus D4S139) has been found with the locus for facioscapulohumeral disease. Three recombinants were identified in a total of 140 meioses, giving a maximum lod score of 36.77 at a recombination fraction of 0.02. All but two of the families studied proved informative with this probe; all informative families showed evidence of linkage (except one family with a single scorable meiosis), making genetic heterogeneity unlikely from our data. The close linkage and highly informative nature of the probe will make it suitable for clinical application in presymptomatic and prenatal diagnosis. We have also confirmed loose linkage with the marker (Mfd22, locus D4S171) used to establish the initial assignment of the disorder to chromosome 4

A Cross-Sectional Survey of Near-Infrared Spectroscopy Use in Pediatric Cardiac ICUs in the United Kingdom, Ireland, Italy, and Germany

Objectives: Despite the increasing use of near-infrared spectroscopy across pediatric cardiac ICUs, there is significant variability and equipoise with no universally accepted management algorithms. We aimed to explore the use of near-infrared spectroscopy in pediatric cardiac ICUs in the United Kingdom, Ireland, Italy, and Germany. Design: A cross-sectional multicenter, multinational electronic survey of one consultant in each pediatric cardiac ICU. Setting: Pediatric cardiac ICUs in the United Kingdom and Ireland (n = 13), Italy (n = 12), and Germany (n = 33). Interventions: Questionnaire targeted to establish use, targets, protocols/thresholds for intervention, and perceived usefulness of near-infrared spectroscopy monitoring. Results: Overall, 42 of 58 pediatric cardiac ICUs (72%) responded: United Kingdom and Ireland, 11 of 13 (84.6%); Italy, 12 of 12 (100%); and Germany, 19 of 33 (57%, included all major centers). Near-infrared spectroscopy usage varied with 35% (15/42) reporting that near-infrared spectroscopy was not used at all (7/42) or occasionally (8/42); near-infrared spectroscopy use was much less common in the United Kingdom (46%) when compared with 78% in Germany and all (100%) in Italy. Only four units had a near-infrared spectroscopy protocol, and 18 specifically used near-infrared spectroscopy in high-risk patients; 37 respondents believed that near-infrared spectroscopy added value to standard monitoring and 23 believed that it gave an earlier indication of deterioration, but only 19 would respond based on near-infrared spectroscopy data alone. Targets for absolute values and critical thresholds for intervention varied widely between units. The reasons cited for not or occasionally using near-infrared spectroscopy were expense (n = 6), limited evidence and uncertainty on how it guides management (n = 4), difficulty in interpretation, and unreliability of data (n = 3). Amongst the regular or occasional near-infrared spectroscopy users (n = 35), 28 (66%) agreed that a multicenter study is warranted to ascertain its use. Conclusions: Although most responding units used near-infrared spectroscopy for high-risk patients, the majority (31/35 [88%]) did not have any protocols or guidelines for intervention. Target thresholds and intervention algorithms are needed to support the use of near-infrared spectroscopy in pediatric cardiac ICUs; an international multicenter study is warranted. (Pediatr Crit Care Med 2016; 17:36–44

The Three Rs: The Way Forward

This workshop report outlines the current status of the Three Rs, and contains recommendations aimed at achieving greater acceptance of the concept of humane experimental techniques and, in the interests of both scientific excellence and the highest standards of animal wellfare, the more active implementation of reduction alternatives, refinement alternatives and replacement alternatives.JRC.(EI)-Environment Institut

Calmodulin is Required for Paraflagellar Rod Assembly and Flagellum-Cell Body Attachment in Trypanosomes

In the flagellum of the African sleeping sickness parasite Trypanosoma brucei calmodulin (CaM) is found within the paraflagellar rod (PFR), an elaborate extra-axonemal structure, and the axoneme. In dissecting mechanisms of motility regulation we analysed CaM function using RNAi. Unexpectedly CaM depletion resulted in total and catastrophic failure in PFR assembly; even connections linking axoneme to PFR failed to form following CaM depletion. This provides an intriguing parallel with the role in the green alga Chlamydomonas of a CaM-related protein in docking outer-dynein arms to axoneme outer-doublet microtubules. Absence of CaM had no discernible effect on axoneme assembly, but the failure in PFR assembly was further compounded by loss of the normal linkage between PFR and axoneme to the flagellum attachment zone of the cell body. Thus, flagellum detachment was a secondary, time-dependent consequence of CaM RNAi, and coincided with the loss of normal trypomastigote morphology, thereby linking the presence of PFR architecture with maintenance of cell form, as well as cell motility. Finally, wider comparison between the flagellum detachment phenotypes of RNAi mutants for CaM and the FLA1 glycoprotein potentially provides new perspective into the function of the latter into establishing and maintaining flagellum-cell body attachment