Electroencephalographic assessment of concussive non-penetrative captive bolt stunning of turkeys.

1. The aim of this study was to evaluate the electroencephalographic (EEG) and behavioural responses of turkeys stunned with three different concussive non-penetrative captive-bolt guns prior to slaughter. 2. A total of 31 slaughter weight ex-breeding female turkeys (29 weeks of age; mean body weight 13.32 ± SD 0.65 kg) were stunned with the Cash Poultry Killer (CPK) (n = 10), Turkey Euthanasia Device (TED) (n = 10) and Zephyr EXL (n = 11). 3. Mean peak kinetic energy was highest for the CPK compared to the TED and Zephyr EXL (75.9 ± 4.5, 28.4 ± 0.4 and 24.4 ± 0.7 J, respectively). 4. A total of 29 (94%) of the turkeys were rendered unconscious following captive bolt stunning, with total power of the EEG (Ptot) significantly reduced from baseline values (reductions of 67% CPK, 84% TED and 76% Zephyr EXL, p < 0.01) and waveforms becoming isoelectric after periods of transitional EEG. However, two birds shot with the CPK and Zephyr EXL had periods of behavioural/reflexes (rhythmic respiration, nictitating membrane reflex, neck tension) and EEG activity (43-47 and 36-60 + s after the shot, respectively) indicating incomplete concussion and return of consciousness. In one bird, the shot was incorrectly positioned (Zephyr EXL), while the other appeared to be related to a defective cartridge (CPK). 5. In conclusion, all three captive bolt gun models were effective in producing unconsciousness in turkeys, provided they were positioned correctly and power loads performed according to their specifications.

The economics of animal welfare.

This paper examines four examples of animal welfare issues, demonstrating the interactions between welfare and economic principles. Welfare issues associated with purebred companion animals are examined in terms of predicted inherited diseases, highlighting the power of supply and demand in perpetuating traits in pets that compromise their well-being. The livestock industry is presented from the point of view of pig production and the impact that a major disease (pleurisy) has on production and the animals' welfare. The authors investigate the conflicting and complementary demands of animal welfare and economic gains during the transport and slaughter of livestock and poultry. Finally, wildlife species are considered in terms of their prevalence as pests, and the different types of economic analysis that have been conducted to understand the losses caused by these organisms. Also included in this example are decisions made about cost effectiveness and opportunity costs, and regulatory and financial barriers to the development of humane control agents. In conclusion, animal welfare is illustrated as a central factor in the benefits that humans enjoy from the role played by animals in society. There are, however, tradeoffs between optimal animal welfare and meeting the needs of modern human society.

Les auteurs analysent les effets réciproques du bien-être animal et des principes de l'économie à travers quatre exemples. La problématique du bienêtre des animaux de compagnie de race est examinée en lien avec les maladies à prédisposition génétique, ce qui permet de souligner l'influence de l'offre et de la demande dans la perpétuation de traits génétiques particuliers à ces animaux, au péril de leur bien-être. Le secteur de l'élevage est examiné à travers l'exemple de la production porcine en étudiant l'impact d'une maladie majeure (pleurésie) sur la production et le bien-être des porcs. Les auteurs abordent ensuite les exigences antinomiques ou complémentaires du bien-être animal et de la rentabilité économique dans le domaine du transport et de l'abattage des animaux d'élevage et des volailles. Enfin, les espèces sauvages sont examinées du point de vue de leur rôle en tant que nuisibles, en exposant les différentes manières d'expliquer au moyen d'analyses économiques les pertes causées par les nuisibles. Cet exemple aborde également les décisions en matière de rentabilité et les coûts d'opportunité, ainsi que les obstacles réglementaires et financiers à l'utilisation d'agents pouvant servir à contrôler les maladies par des méthodes respectueuses du bien-être animal. En conclusion, le bien-être animal apparaît comme un facteur central des bénéfices que les humains retirent des animaux et de leur rôle dans la société. Il y a néanmoins des compromis à trouver entre l'optimisation du bien-être animal et les exigences d'une société moderne.

Apoyándose en cuatro ejemplos de bienestar animal, los autores ponen de manifiesto cuán imbricados están entre sí los temas de bienestar y los principios económicos. Ante todo examinan los problemas de bienestar que sufren los animales de compañía de pura raza por lo que respecta a sus previsibles enfermedades hereditarias, subrayando el poder de la ley de la oferta y la demanda para perpetuar en ellos una serie de rasgos que comprometen su bienestar. A continuación se detienen en la ganadería industrial, y más concretamente en la producción porcina y la influencia que ejerce una enfermedad importante (la pleuresía) en el bienestar de los animales y en la propia producción. Después exponen los imperativos antagónicos y complementarios que se plantean en clave de bienestar animal y de beneficio económico durante las operaciones de transporte y sacrificio de ganado y aves de corral. Por último, considerando las especies de animales salvajes desde el punto de vista de su prevalencia como plagas, exponen los distintos tipos de análisis económico que se han realizado para aprehender las pérdidas resultantes de las plagas. Valiéndose de este ejemplo examinan también las decisiones adoptadas en materia de rentabilidad y de costos de oportunidad, así como las barreras reglamentarias y económicas que dificultan un funcionamiento más compasivo de los agentes de control. El bienestar animal, en conclusión, aparece como un factor central de los beneficios que extrae el ser humano de la función que cumplen los animales en la sociedad. Sin embargo, es preciso hallar un compromiso entre los niveles óptimos de bienestar animal y la satisfacción de las necesidades de la sociedad humana moderna.

Assessment of the effectiveness of head only and back-of-the-head electrical stunning of chickens.

The study assesses the effectiveness of reversible head-only and back-of-the-head electrical stunning of chickens using 130-950 mA per bird at 50 Hz AC. Three trials were conducted to compare both stunning systems: (a) behavioural assessment of return of consciousness, (b) insensibility to thermal pain, and (c) assessment of return of brain activity with visually evoked potentials (VEPs). Assessment of behaviour suggested that the period of unconsciousness following head-only electrical stunning was shorter in hens compared to broilers. Stunning across the back-of-the-head delayed the time to return of brainstem function compared to stunning with standard head-only electrodes. Additionally, back-of-the-head stunning produced a more prolonged period of electroanalgesia compared to head-only. Based on examination of return of brain function with VEPs in hens, back-of-the-head stunning produced a shorter-lasting stun than standard head-only. However, even for standard head-only, the stun was notably shorter than previously reported. In some birds, brain function had returned within 9 s after the end of stunning. The results suggest that some birds may recover consciousness prior to or during the neck cut. Based on these findings, back-of-the-head stunning and standard head-only stunning of hens should not be recommended without further development.

The identification of short linear motif-mediated interfaces within the human interactome.

MOTIVATION: Eukaryotic proteins are highly modular, containing multiple interaction interfaces that mediate binding to a network of regulators and effectors. Recent advances in high-throughput proteomics have rapidly expanded the number of known protein-protein interactions (PPIs); however, the molecular basis for the majority of these interactions remains to be elucidated. There has been a growing appreciation of the importance of a subset of these PPIs, namely those mediated by short linear motifs (SLiMs), particularly the canonical and ubiquitous SH2, SH3 and PDZ domain-binding motifs. However, these motif classes represent only a small fraction of known SLiMs and outside these examples little effort has been made, either bioinformatically or experimentally, to discover the full complement of motif instances. RESULTS: In this article, interaction data are analysed to identify and characterize an important subset of PPIs, those involving SLiMs binding to globular domains. To do this, we introduce iELM, a method to identify interactions mediated by SLiMs and add molecular details of the interaction interfaces to both interacting proteins. The method identifies SLiM-mediated interfaces from PPI data by searching for known SLiM-domain pairs. This approach was applied to the human interactome to identify a set of high-confidence putative SLiM-mediated PPIs. AVAILABILITY: iELM is freely available at http://elmint.embl.de CONTACT: toby.gibson@embl.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The articles.ELM resource: simplifying access to protein linear motif literature by annotation, text-mining and classification.

Modern biology produces data at a staggering rate. Yet, much of these biological data is still isolated in the text, figures, tables and supplementary materials of articles. As a result, biological information created at great expense is significantly underutilised. The protein motif biology field does not have sufficient resources to curate the corpus of motif-related literature and, to date, only a fraction of the available articles have been curated. In this study, we develop a set of tools and a web resource, 'articles.ELM', to rapidly identify the motif literature articles pertinent to a researcher's interest. At the core of the resource is a manually curated set of about 8000 motif-related articles. These articles are automatically annotated with a range of relevant biological data allowing in-depth search functionality. Machine-learning article classification is used to group articles based on their similarity to manually curated motif classes in the Eukaryotic Linear Motif resource. Articles can also be manually classified within the resource. The 'articles.ELM' resource permits the rapid and accurate discovery of relevant motif articles thereby improving the visibility of motif literature and simplifying the recovery of valuable biological insights sequestered within scientific articles. Consequently, this web resource removes a critical bottleneck in scientific productivity for the motif biology field. Database URL: http://slim.icr.ac.uk/articles/.

Clustal W and Clustal X version 2.0.

SUMMARY: The Clustal W and Clustal X multiple sequence alignment programs have been completely rewritten in C++. This will facilitate the further development of the alignment algorithms in the future and has allowed proper porting of the programs to the latest versions of Linux, Macintosh and Windows operating systems. AVAILABILITY: The programs can be run on-line from the EBI web server: http://www.ebi.ac.uk/tools/clustalw2. The source code and executables for Windows, Linux and Macintosh computers are available from the EBI ftp site ftp://ftp.ebi.ac.uk/pub/software/clustalw2/

Catalytic RNA and the origin of genetic systems.

The recent discovery of catalytic RNA molecules has provoked enormous interest in the origin of life and has given rise to new speculations about how living systems developed on the primitive earth. Here we outline why the discovery of catalytic RNA molecules has profound evolutionary implications, and then go on to discuss models for the emergence of metabolic complexity and protein synthesis in a primitive 'RNA world', emphasizing the constraints imposed on such models by genetical arguments.

Factors influencing occupant-to-seat belt interaction in far-side crashes.

Seat belt interaction with a far-side occupant's shoulder and thorax is critical to governing excursion towards the struck-side of the vehicle in side impact. In this study, occupant-to-belt interaction was simulated using a modified MADYMO human model and finite element belts. Quasi-static tests with volunteers and dynamic sled tests with PMHS and WorldSID were used for model validation and comparison. Parameter studies were then undertaken to quantify the effect of impact direction, seat belt geometry and pretension on occupant-to-seat belt interaction. Results suggest that lowering the D-ring and increasing pretension reduces the likelihood of the belt slipping off the shoulder. Anthropometry was also shown to influence restraint provided by the shoulder belt. Furthermore, the belt may slip off the occupant's shoulder at impact angles greater than 40 degrees from frontal when no pretension is used. However, the addition of pretension allowed the shoulder to engage the belt in all impacts from 30 to 90 degrees.

Friedreich's ataxia protein: phylogenetic evidence for mitochondrial dysfunction.

Friedreich's ataxia is the most common inherited spinocerebellar ataxia. A decade of linkage and physical mapping studies have culminated in the identification of the Friedreich's ataxia gene. The presence of homologues in purple bacterial genomes, but not in other bacteria, allows us to infer a mitochondrial location for frataxin (Friedreich's ataxia protein) on the basis of bacterial phylogeny. Frataxin possesses a non-globular N-terminus domain providing a candidate mitochondrial targeting peptide. Clues to the function of frataxin are provided by the mitochondrial location, a clinically similar ataxia with vitamin E deficiency, and certain neuropathies with mitochondrial DNA instability caused by mutations in nuclear genes.

Gene2EST: a BLAST2 server for searching expressed sequence tag (EST) databases with eukaryotic gene-sized queries.

Expressed sequence tags (ESTs) are randomly sequenced cDNA clones. Currently, nearly 3 million human and 2 million mouse ESTs provide valuable resources that enable researchers to investigate the products of gene expression. The EST databases have proven to be useful tools for detecting homologous genes, for exon mapping, revealing differential splicing, etc. With the increasing availability of large amounts of poorly characterised eukaryotic (notably human) genomic sequence, ESTs have now become a vital tool for gene identification, sometimes yielding the only unambiguous evidence for the existence of a gene expression product. However, BLAST-based Web servers available to the general user have not kept pace with these developments and do not provide appropriate tools for querying EST databases with large highly spliced genes, often spanning 50 000-100 000 bases or more. Here we describe Gene2EST (http://woody.embl-heidelberg.de/gene2est/), a server that brings together a set of tools enabling efficient retrieval of ESTs matching large DNA queries and their subsequent analysis. RepeatMasker is used to mask dispersed repetitive sequences (such as Alu elements) in the query, BLAST2 for searching EST databases and Artemis for graphical display of the findings. Gene2EST combines these components into a Web resource targeted at the researcher who wishes to study one or a few genes to a high level of detail.

Protruding domain of tomato bushy stunt virus coat protein is a hitherto unrecognized class of jellyroll conformation.

The capsid protein of tomato bushy stunt virus (TBSV) has two antiparallel beta-sheet domains with the so-called jellyroll conformation. Contrary to previous analyses, we note that these domains are non-superimposable topologies. The TBSV shell (S) domain topology is common to many other proteins but the protruding (P) domain is a unique conformation so far found in no other protein. The TBSV capsid P domain did not arise from the S domain by a gene duplication event as previously assumed. It is proposed instead that the P domain was acquired from an as yet unidentified cellular protein. The four possible unique jellyroll topologies that might occur in proteins are discussed and illustrated.

Homology-based method for identification of protein repeats using statistical significance estimates.

Short protein repeats, frequently with a length between 20 and 40 residues, represent a significant fraction of known proteins. Many repeats appear to possess high amino acid substitution rates and thus recognition of repeat homologues is highly problematic. Even if the presence of a certain repeat family is known, the exact locations and the number of repetitive units often cannot be determined using current methods. We have devised an iterative algorithm based on optimal and sub-optimal score distributions from profile analysis that estimates the significance of all repeats that are detected in a single sequence. This procedure allows the identification of homologues at alignment scores lower than the highest optimal alignment score for non-homologous sequences. The method has been used to investigate the occurrence of eleven families of repeats in Saccharomyces cerevisiae, Caenorhabditis elegans and Homo sapiens accounting for 1055, 2205 and 2320 repeats, respectively. For these examples, the method is both more sensitive and more selective than conventional homology search procedures. The method allowed the detection in the SwissProt database of more than 2000 previously unrecognised repeats belonging to the 11 families. In addition, the method was used to merge several repeat families that previously were supposed to be distinct, indicating common phylogenetic origins for these families.

Lorist6, a cosmid vector with BamHI, NotI, ScaI and HindIII cloning sites and altered neomycin phosphotransferase gene expression.

Four new features have been included in a phage-lambda-replicon-based cosmid vector, Lorist6. A ScaI restriction site allows insertion of blunt-ended DNA, partially digested with AluI or any other restriction enzyme which creates blunt ends after cleavage. NotI and BamHI sites enable construction of libraries using double digestion with NotI + Sau3AI. The promoter of the neo gene has been substituted by a promoter derived from the tet gene of pBR322, increasing expression of the resistance gene product. A transcriptional terminator has been placed downstream from the neo gene to prevent transcription into insert DNAs.

Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promoters within the insert: effect on DNA yield and cloned insert frequency.

Transcription terminators have been included in a phage-lambda-replicon-based cosmid vector, Lorist2, to insulate vector genes against transcriptional interference from cloned insert DNA. DNA yields of recombinant clones containing Escherichia coli genomic DNA inserts are more even for Lorist2 than with its progenitor LoristB. However, the terminators provide only a partial reduction in the over-representation of r X DNA-containing clones generally observed in cosmid libraries of Caenorhabditis elegans DNA, suggesting that causes other than transcriptional readthrough into the vector contribute to this problem.

The KH domain occurs in a diverse set of RNA-binding proteins that include the antiterminator NusA and is probably involved in binding to nucleic acid.

New findings are presented for the approximately 50 residue KH motif, a domain recently discovered in RNA-binding proteins. The conserved sequence is approximately 10 residues larger than previously reported. Profile searches have revealed new members of this family, including two, E. coli NusA and human GAP-associated p62 phosphoprotein, for which RNA-binding data exists. A nusA homolog was detected in the RNA polymerase gene complex of six archaebacterial species and may encode an antiterminator. All KH-containing proteins are linked with RNA and the KH motif most probably functions as a nucleic acid binding domain.

Dystrophin and utrophin: the missing links!

There is considerable sequence homology between dystrophin and utrophin, both at the protein and DNA level, and consequently it was assumed that their domain structures and functions would be similar. As more of the detailed biochemical and cell biological properties of these two proteins become known, so it becomes clear that there are subtle if not significant differences between them. We review recent findings and present new hypotheses into the structural and functional properties of the actin-binding domain, central coiled-coil region and regulatory/membrane protein-binding regions of dystrophin and utrophin.

Using CLUSTAL for multiple sequence alignments.

We have tested CLUSTAL W in a wide variety of situations, and it is capable of handling some very difficult protein alignment problems. If the data set consists of enough closely related sequences so that the first alignments are accurate, then CLUSTAL W will usually find an alignment that is very close to ideal. Problems can still occur if the data set includes sequences of greatly different lengths or if some sequences include long regions that are impossible to align with the rest of the data set. Trying to balance the need for long insertions and deletions in some alignments with the need to avoid them in others is still a problem. The default values for our parameters were tested empirically using test cases of sets of globular proteins where some information as to the correct alignment was available. The parameter values may not be very appropriate with nonglobular proteins. We have argued that using one weight matrix and two gap penalties is too simplistic to be of general use in the most difficult cases. We have replaced these parameters with a large number of new parameters designed primarily to help encourage gaps in loop regions. Although these new parameters are largely heuristic in nature, they perform surprisingly well and are simple to implement. The underlying speed of the progressive alignment approach is not adversely affected. The disadvantage is that the parameter space is now huge; the number of possible combinations of parameters is more than can easily be examined by hand. We justify this by asking the user to treat CLUSTAL W as a data exploration tool rather than as a definitive analysis method. It is not sensible to automatically derive multiple alignments and to trust particular algorithms as being capable of always getting the correct answer. One must examine the alignments closely, especially in conjunction with the underlying phylogenetic tree (or estimate of it) and try varying some of the parameters. Outliers (sequences that have no close relatives) should be aligned carefully, as should fragments of sequences. The program will automatically delay the alignment of any sequences that are less than 40% identical to any others until all other sequences are aligned, but this can be set from a menu by the user. It may be useful to build up an alignment of closely related sequences first and to then add in the more distant relatives one at a time or in batches, using the profile alignments and weighting scheme described earlier and perhaps using a variety of parameter settings. We give one example using SH2 domains. SH2 domains are widespread in eukaryotic signalling proteins where they function in the recognition of phosphotyrosine-containing peptides. In the chapter by Bork and Gibson ([11], this volume), Blast and pattern/profile searches were used to extract the set of known SH2 domains and to search for new members. (Profiles used in database searches are conceptually very similar to the profiles used in CLUSTAL W: see the chapters [11] and [13] for profile search methods.) The profile searches detected SH2 domains in the JAK family of protein tyrosine kinases, which were thought not to contain SH2 domains. Although the JAK family SH2 domains are rather divergent, they have the necessary core structural residues as well as the critical positively charged residue that binds phosphotyrosine, leaving no doubt that they are bona fide SH2 domains. The five new JAK family SH2 domains were added sequentially to the existing alignment of 65 SH2 domains using the CLUSTAL W profile alignment option. Figure 6 shows part of the resulting alignment. Despite their divergent sequences, the new SH2 domains have been aligned nearly perfectly with the old set. No insertions were placed in the original SH2 domains. In this example, the profile alignment procedure has produced better results than a one-step full alignment of all 70 SH2 domains, and in considerably less time. (ABSTRACT TRUNCATED)

Examination of cyanine intercalation dyes for rapid and sensitive detection of DNA fragments by capillary electrophoresis.

Recently, a series of cyanine dyes has been developed that are reported to offer superior fluorescence upon intercalation relative to ethidium bromide (EB). The dimeric cyanine dyes, such as POPO-3 (Molecular Probes, Eugene, OR, U.S.A.), are reported to offer the highest fluorescence enhancement of the cyanine dyes. Although POPO-3 does offer an improvement in sensitivity, achieving reproducible, high-efficiency results by employing pre-column, on-column, and postcolumn labeling strategies proved to be problematic. Evaluation of the labeling rate of these dimeric dyes indicates that perhaps the most limiting factor to using this dye for rapid CE analysis of DNA fragments is the apparent slow rate at which the dye fully intercalates into the DNA structure. Use of the monomeric analog PO-PRO-3 (Molecular Probes) resulted in much better separations and low limits of defection when employed for on-column intercalation. However, we find that the kinetics are too slow to employ this dye in a sheath flow cell for postcolumn derivatization.