Future activities: ECVAM and the quality control of biologicals

ECVAM's activities in the field of biologicals have contributed in many ways to the successful incorporation of Three Rs methods, as summarised elsewhere in these proceedings. The progress achieved is impressive, but large numbers of animals are still needed in order to meet the requirements stipulated by various regulations. ECVAM's activities in this area should therefore be continued and extended. Besides the well-established organisation of ECVAM workshops and contributions to conferences, further prevalidation and validation studies should be funded. In addition, studies on refinement, and training courses on validated and well-established Three Rs methods, could be initiated. There is a need for more communication and information exchange, especially between regulators and industry concerning the Three Rs. ECVAM could provide a suitable forum for such activities. An ECVAM Biologicals Task Force should be established in order to define a list of priorities.     

A proposed higher education institution-based Three Rs advisory service

The University of Oxford's Ethical Review Process (ERP) is promoting a pilot scheme for a collaborative UK higher education institution-based Three Rs advisory service. It is believed that there is scope for UK academia to make a significant contribution, through the spread of its science base and the availability of high-quality library and IT services. A collaborative approach based on the concept of the critically appraised topic is envisaged, to minimise duplication and to ensure that limited resources are used to good effect. The initial objective is to identify, research, and validate refinement and replacement alternatives, drawing on both the skills of the information professional and the analogy of evidence-based health care. The results would be disseminated among member institutions by way of web-based systems, which could also offer on-line training in search strategies. The service would provide assistance to university-based project licence applicants, and would contribute to achieving the aims of the ERP, as an initiative "leading to the widest possible application of the Three Rs". It is hoped to develop the scheme in partnership with research council initiatives now under way, as the councils play a key role as funders, not only of research, but also of research student training. Universities are uniquely positioned, as centres of scientific education and, through continuing education, of continuing professional development, to provide a base for training in good practice. A successful pilot project could provide a foundation for a similar approach to reduction strategies and experimental design.     

Information: needs for the future

The four central questions surrounding the use of information are: where to find it; how to find it; how to present it; and how to maintain information availability and information literacy. It is usually assumed that the main source of information for most scientists is the peer-reviewed journal literature. Traditional journal publishing is beset with a number of problems. Although electronic publishing might possibly solve some of these, it in turn introduces new problems. Further problems arise with respect to secondary sources which, in some cases, are being supplemented by electronic archives of full-text documents. One fundamental question that arises when considering any large collection of documents or of records about documents is whether or not to index them, and how to index them. The pros and cons of free-text searching versus the use of controlled vocabularies are discussed, as is the importance of harmonising the Three Rs-related terminology of existing and proposed thesauri. However, there is a further problem that documents pertinent to the Three Rs are not always indexed from this point of view. Authors need to be made aware that, if the information is not provided in the abstract, there is no easy way to identify and retrieve this document from a database. Small specialised databases on the Three Rs in relation to specific subject areas could provide a further solution, especially if they provide references to conference proceedings and book chapters, which are not usually found in the large bibliographical databases. The provision of training in the use of information resources, and the establishment and maintenance of these resources require investment of money and professional skills. Finally, the future of Three Rs information depends on a recognition that this in an important topic which deserves more than lip service.     

The interpretation and application of the three Rs by animal ethics committee members

The Three Rs form the basis of review of animal-use protocols by Animal Ethics Committees (AECs), but little research has examined how AECs actually interpret and implement the Three Rs. This topic was explored through in-depth, open-ended interviews with 28 members of AECs at four Canadian universities. In describing protocol review, AEC members rarely mentioned the Three Rs, but most reported applying some aspects of the basic concepts. Comments identified several factors that could impede full application of the Three Rs: incomplete understanding of the Three Rs (especially Refinement), trust that researchers implement Replacement and Reduction themselves, belief by some members that granting agency review covers the Three Rs, focus on sample size rather than experimental design to achieve Reduction, focus on harm caused by procedures to the exclusion of housing and husbandry, and lack of consensus on key issues, notably on the nature and moral significance of animal pain and suffering, and on whether AECs should minimise overall harm to animals. The study suggests ways to achieve more consistent application of the Three Rs, by providing AECs with up-to-date information on the Three Rs and with access to statistical expertise, by consensus-building on divisive issues, and by training on the scope and implementation of the Three Rs.     

Assessing the search for information on Three Rs methods, and their subsequent implementation: a national survey among scientists in the Netherlands

A local survey conducted among scientists into the current practice of searching for information on Three Rs (i.e. Replacement, Reduction and Refinement) methods has highlighted the gap between the statutory requirement to apply Three Rs methods and the lack of criteria to search for them. To verify these findings on a national level, we conducted a survey among scientists throughout The Netherlands. Due to the low response rate, the results give an impression of opinions, rather than being representative of The Netherlands as a whole. The findings of both surveys complement each other, and indicate that there is room for improvement. Scientists perceive searching the literature for information on Three Rs methods to be a difficult task, and specific Three Rs search skills and knowledge of Three Rs databases are limited. Rather than using a literature search, many researchers obtain information on these methods through personal communication, which means that published information on possible Three Rs methods often remains unfound and unused. A solution might be to move beyond the direct search for information on Three Rs methods and choose another approach. One approach that seems rather appropriate is that of systematic review. This provides insight into the necessity for any new animal studies, as well as optimal implementation of available data and the prevention of unnecessary animal use in the future.     

Comments on the sixth report of the Animal Welfare Advisory Committee

The Animal Welfare Advisory Committee (AWAC) was established in July 1996, to consider the care, welfare and use of animals involved in procedures for defence research purposes at Defence and Evaluation Research Agency (DERA) establishments in the UK. Two of the objectives of AWAC are to examine the broad trends in animal use at DERA establishments, and to implement and audit the application of the Three Rs principle. AWAC's sixth report addressed the period from 31 October 2000 to 28 February 2002. The statistics of animal use within the report are briefly examined, and some of the actions undertaken by defence research establishments to facilitate the application of the Three Rs are highlighted. It is recommended that, if possible (subject to security constraints), figures detailing the severity of the procedures undertaken should be included in future issues of the report, in order to provide a more-detailed account. It is concluded that Defence Science and Technology Laboratory establishments have made a contribution to the Three Rs, and that other establishments may be able to incorporate some of their actions into their own research programmes. There was an overall 36% increase in the number of procedures carried out by defence research establishments between 1995 and 2000, from 8,900 to 12,065. This probably reflects alterations in the research programme, which is, in turn, decided primarily by the Ministry of Defence's customers and the progress made with previous research programmes. It is therefore recommended that the UK Government allocates significantly more financial resources for the development and validation of alternatives, in order to maximise the potential for achieving the Three Rs in defence research, and to complement the existing initiatives within the defence research industry.     

Survey of Canadian animal-based researchers' views on the Three Rs: replacement, reduction and refinement

The 'Three Rs' tenet (replacement, reduction, refinement) is a widely accepted cornerstone of Canadian and international policies on animal-based science. The Canadian Council on Animal Care (CCAC) initiated this web-based survey to obtain greater understanding of 'principal investigators' and 'other researchers' (i.e. graduate students, post-doctoral researchers etc.) views on the Three Rs, and to identify obstacles and opportunities for continued implementation of the Three Rs in Canada. Responses from 414 participants indicate that researchers currently do not view the goal of replacement as achievable. Researchers prefer to use enough animals to ensure quality data is obtained rather than using the minimum and potentially waste those animals if a problem occurs during the study. Many feel that they already reduce animal numbers as much as possible and have concerns that further reduction may compromise research. Most participants were ambivalent about re-use, but expressed concern that the practice could compromise experimental outcomes. In considering refinement, many researchers feel there are situations where animals should not receive pain relieving drugs because it may compromise scientific outcomes, although there was strong support for the Three Rs strategy of conducting animal welfare-related pilot studies, which were viewed as useful for both animal welfare and experimental design. Participants were not opposed to being offered "assistance" to implement the Three Rs, so long as the input is provided in a collegial manner, and from individuals who are perceived as experts. It may be useful for animal use policymakers to consider what steps are needed to make replacement a more feasible goal. In addition, initiatives that offer researchers greater practical and logistical support with Three Rs implementation may be useful. Encouragement and financial support for Three Rs initiatives may result in valuable contributions to Three Rs knowledge and improve welfare for animals used in science.     

Expanding the three Rs to meet new challenges in humane animal experimentation

The Three Rs are the main principles used by Animal Ethics Committees in the governance of animal experimentation, but they appear not to cover some ethical issues that arise today. These include: a) claims that certain species should be exempted on principle from harmful research; b) increased emphasis on enhancing quality of life of research animals; c) research involving genetically modified (GM) animals; and d) animals bred as models of disease. In some cases, the Three Rs can be extended to cover these developments. The burgeoning use of GM animals in science calls for new forms of reduction through improved genetic modification technology, plus continued attention to alternative approaches and cost-benefit analyses that include the large numbers of animals involved indirectly. The adoption of more expanded definitions of refinement that go beyond minimising distress will capture concerns for enhancing the quality of life of animals through improved husbandry and handling. Targeting refinement to the unpredictable effects of gene modification may be difficult; in these cases, careful attention to monitoring and endpoints are the obvious options. Refinement can also include sharing data about the welfare impacts of gene modifications, and modelling earlier stages of disease, in order to reduce the potential suffering caused to disease models. Other issues may require a move beyond the Three Rs. Certain levels of harm, or numbers and use of certain species, may be unacceptable, regardless of potential benefits. This can be addressed by supplementing the utilitarian basis of the Three Rs with principles based on deontological and relational ethics. The Three Rs remain very useful, but they require thoughtful interpretation and expansion in order for Animal Ethics Committees to address the full range of issues in animal-based research.     

Have the 3Rs and alternatives been effectively explored?

Although the concept of the 3Rs was developed in the 1950s,it took many years before it became a central theme when planning and conducting experiments which might involve animals.There are still many ways in which protocols can be improved to increase both animal welfare and the reliability of the scientific data obtained from the studies.This paper gives some practical advice on how the 3Rs can be implemented more thoroughly in animal research.     

Identifying optimal groundwater remediation strategies through a simulation-based PROMETHEE-TOPSIS approach: An application to a naphthalene-contaminated site

Abstract

The aim of this paper is to develop a simulation-aided PROMETHEE-TOPSIS approach for the selection of the most desirable groundwater remediation strategies. The combination methods enables a careful evaluation of the identified remediation alternatives in which their strong and weak points can be detected and a ranking is provided which facilitates the final selection for the decision-maker. The capabilities and effectiveness of the developed method are illustrated through a case study on the remedial alternative selection for a naphthalene contaminated site in Anhui, China. Four attributes (i.e., total pumping rate, total cost, mean residual contaminant concentration and maximum excess life time cancer risk) for fifty remedial alternatives in each duration are considered and analytic hierarchy process is used to determine the weight of attributes importance. The results demonstrates that the developed method could help decision makers obtain the useful ranking information strategies that covering a variety of decision-relevant remediation options, which is beneficial for public health and environmental protection.     

New developments in applying quantum mechanics to proteins

Algorithmic improvements of quantum mechanical methodologies have increased our ability to study the electronic structure of fragments of a biomolecule (e.g. an enzyme active site) or entire biomolecules. Three main strategies have emerged as ways in which quantum mechanics can be applied to biomolecules. The supermolecule approach continues to be utilized, but it is slowly being replaced by the so-called coupled quantum mechanical/molecular mechanical methodologies. An exciting new direction is the continued development and application of linear-scaling quantum mechanical approaches to biomolecular systems.     

Implementation of the three Rs in the human hazard assessment of Brazilian medicinal plants: an evaluation of the cytotoxic and genotoxic potentials of Dipteryx alata Vogel

In Brazil, medicinal plants are widely used by the indigenous people, which leads to a constant requirement for toxicity tests to be performed on the plant extracts. Although the current Brazilian Directive 90/2004 on the preclinical toxicity testing of phytotherapeutics recommends only in vivo tests, some Brazilian researchers would like to change this situation by implementing the Three Rs in the toxicological testing of medicinal plants. The present study evaluated the cytotoxic and genotoxic potentials of bark extracts from Dipteryx alata Vogel, a medicinal plant of the Brazilian cerrado, by using CHO-K1 (Chinese hamster ovary) cells. An IC50 value was obtained, which corresponded to 0.16mg/ml of plant extract, and from this the equivalent LD50 was determined as 705mg/kg. In order to determine the genotoxic potential of the sample, the frequency of micronucleus formation was assessed. CHO-K1 cells were exposed, during targeted mitosis, to different concentrations of plant extract and cytochalasin B, in the presence and absence of an appropriate metabolic activation system (an S9 mix). The results obtained indicated that it might be possible to implement the Three Rs in assessing the potential human hazard of medicinal plants. The publication of such data can increase awareness of the Three Rs by showing how to optimise the management of animal use, if in vivo toxicological experiments are required.     

Rapid simultaneous cloning of drug targets from multiple mammalian species

A method for the routine, rapid and simultaneous cloning of drug targets from multiple mammalian species is described. This expedites the generation of recombinant proteins and cell lines that can provide alternatives to animal experiments. This was achieved by the collection of RNA from a comprehensive range of tissues from a variety of species, and the optimisation of cDNA synthesis. This "zooplate" has been successfully used for the simultaneous amplification and cloning of drug targets from multiple species. These products have subsequently been used to develop in vitro assays that support efficacy and safety studies in new drug discovery programmes. Within the framework of the Three Rs, these reagents can reduce the number of animals required to provide material for ex vivo assays and can refine the in vivo studies that are still necessary.     

Controlling factors of temporal variation of soil respiration in a natural beech forest as revealed by natural incubation experiments

Microorganisms decompose organic substrates to obtain energy. This process releases carbon dioxide (CO₂) and is the main cause of soil carbon release in terrestrial ecosystems. Fluctuations in microbial composition significantly affect net CO₂ emissions in forests. Because factors are cross-correlated, addressing how they affect soil respiration (Rs), both directly and indirectly, is challenging. In this study, Rs-impacting soil properties, including soil organic carbon (SOC), soil organic nitrogen (SON), microbial diversity (Dsim, indicated by Simpson’s diversity index), and total microbial DNA concentration in natural beech forests were examined by structure equation modeling (SEM), which can explicitly evaluate the causal relationships among interacting variables. The results showed that decreasing Dsim, soil temperature, SOC, and SON clearly had direct and indirect effects on Rs under natural conditions. Increasing temperature was a primary factor and promoted a decrease in Rs during the growth season. Dsim was the only parameter with a direct positive effect on Rs, indicating that microbial diversity could accurately predict Rs. Soil nutrient factors indirectly affected Rs through Dsim, which was also affected by soil physical and chemical properties. Significant covariance between SON and Rs (0.42, p < 0.001) indicated multiple interacting variables affecting soil activity. Although the current study suggests that SEM can clarify complex functional processes related to Rs, future studies should consider additional impacting variables such as vegetation properties and enzyme dynamics.     

The EU Physical Agents (EMF) Directive and its impact on MRI imaging in animal experiments: a submission by FRAME to the HSE

The EU Physical Agents (EMF) Directive, Directive 2004/40/EC, which threatens to greatly restrict the use of magnetic resonance imaging (MRI) in both clinical and research situations, will come into force on 30 April 2008. This could severely affect experimental animal welfare and scientific progress, as well as patient care. FRAME made a submission to a Health and Safety Executive round-table discussion about the Directive, held in January 2006, detailing concerns about the implications that the legislation would have on implementing the Three Rs in animal-based research and testing, and the subsequent consequences for animal welfare and the quality of scientific output. The submission is reproduced here, with additional comments on the outcome of the meeting and recommendations for further research into the consequences of the Directive.     

Divergence of melanocortin pathways in the control of food intake and energy expenditure

Activation of melanocortin-4-receptors (MC4Rs) reduces body fat stores by decreasing food intake and increasing energy expenditure. MC4Rs are expressed in multiple CNS sites, any number of which could mediate these effects. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB Mc4r) that can be reactivated by Cre-recombinase. Mice homozygous for the loxTB Mc4r allele do not express MC4Rs and are markedly obese. Restoration of MC4R expression in the paraventricular hypothalamus (PVH) and a subpopulation of amygdala neurons, using Sim1-Cre transgenic mice, prevented 60% of the obesity. Of note, increased food intake, typical of Mc4r null mice, was completely rescued while reduced energy expenditure was unaffected. These findings demonstrate that MC4Rs in the PVH and/or the amygdala control food intake but that MC4Rs elsewhere control energy expenditure. Disassociation of food intake and energy expenditure reveals unexpected divergence in melanocortin pathways controlling energy balance.     

化石直脉蝎蛉科再分类(长翅目)

本文对化石直脉蝎蛉科(Orthophlebiidae)的分属做了再分类,仍保留该科内的 2个属:直脉蝎蛉属 Orthophlebia和原直脉蝎蛉属Protorthophlebia,解体中蝎蛉属Mesopanorpa。中蝎蛉属被解体后,其包含所有的种,如Rs1主干带3支或3支以上支脉,归入直脉蝎蛉属;如Rs1仅带2支支脉,归入原直脉蝎蛉属;原中蝎蛉属以Rs主干两倍长于MA主干作为属的分类根据,但这一属征常常导致分类上的混乱,故本文不作为属或亚属的分类根据。上述保留的2个属,再以Rs分支先于、后于或等于MA的特征,即Rs主干短于、长于或等于MA主干作为亚属的分类根据。以此,在直脉蝎蛉属Orthophlebia和原直脉蝎蛉属Protorthophlebia之下各自设立3个亚属(Hong et Zhang, 2004)。在已建立的6个亚属中,Orthophlebia(Brachyphlebia)Hong et Zhang, 2004和Protorthophlebia(Mecynophlebia)Hong et Zhang,2004分别修改为Orthophlebia(Orthophlebia)Westwood, 1845和Protorthophlebia(Protorthophlebia)Tillyard,1933的新异名。中生代直脉蝎蛉中脉的前支(MA)并非在古生代之后就完全消失。根据对中生代不同时期、不同层位、不同地区直脉蝎蛉和本目其他科在内化石标本的观察,中脉及其前、后支脉(MA、MP)仍然继续存在与发展。基于这种事实,本文认为直脉蝎蛉科脉序命名中沿用已久的Rs后支(Rs3 4)应为中脉的前支(MA),原来沿用的中脉(M)为中脉的后支(MP)。这样,有助于我们对长翅目中脉及其前、后(MA、MP)支脉的准确辨认,尤其是对MA脉在地史上变革、发展,以及与Rs脉的演化关系的认识可能有所裨益。     

Modular DAG-RNN architectures for assembling coarse protein structures.

We develop and test machine learning methods for the prediction of coarse 3D protein structures, where a protein is represented by a set of rigid rods associated with its secondary structure elements (alpha-helices and beta-strands). First, we employ cascades of recursive neural networks derived from graphical models to predict the relative placements of segments. These are represented as discretized distance and angle maps, and the discretization levels are statistically inferred from a large and curated dataset. Coarse 3D folds of proteins are then assembled starting from topological information predicted in the first stage. Reconstruction is carried out by minimizing a cost function taking the form of a purely geometrical potential. We show that the proposed architecture outperforms simpler alternatives and can accurately predict binary and multiclass coarse maps. The reconstruction procedure proves to be fast and often leads to topologically correct coarse structures that could be exploited as a starting point for various protein modeling strategies. The fully integrated rod-shaped protein builder (predictor of contact maps + reconstruction algorithm) can be accessed at http://distill.ucd.ie/.     

Genetic information and insurance: some ethical issues.

Life is risky, and insurance provides one of the best developed ways of controlling risks. By pooling, and so transferring risks, those who turn out to suffer antecedently uncertain harms can be assured in advance that they will be helped if those harms arise; they can then plan their lives and activities with confidence that they are less at the mercy of ill fortune. Both publicly organized and commercial insurance can organize the pooling of risk in ways that are beneficial for all concerned. They provide standard ways of securing fundamental ethical values such as solidarity and mutuality. Although policy holders do not know or contract with one another, each benefits from the contribution of others to a shared scheme for pooling and so controlling risk. Although there is a limit to the degree to which commercially-based insurance, where premiums depend on risk level, can go beyond mutuality towards solidarity, in practice it too often achieves a measure of solidarity by taking a broad brush approach to pooling risk. However, the ordinary practices of insurance, and in particular of commercial insurance, also raise ethical questions. These may be put in simple terms by contrasting the way in which an insurance market discriminates between different people, on the basis of characteristics that (supposedly) determine their risk level, and our frequent abhorrence of discrimination, in particular on the basis on religious, racial and gender characteristics. Are the discriminations on which insurance practice relies upon as standard acceptable or not? The increasing availability of genetic information, which testing (of individuals) and screening (of populations) may provide, could lend urgency to these questions. Genetic information may provide a way of obtaining more accurate assessment of individual risks to health and life. This information could be used to discriminate more finely between the risk levels of different individuals, and then to alter the availability and the costs of health, life and unemployment insurance to them. Since all of these forms of insurance bear very directly on the way most people live, it will matter to them how (if at all) insurers take account of genetic information. Will use of this information improve or damage the capacity of insurance to provide confidence in the face of uncertain harms, and help if they happen? Will it discriminate in acceptable or in unacceptable ways? Will it support or damage the sorts of mutuality and solidarity various sorts of insurance schemes have successfully institutionalized?