Automated dairy cattle lameness detection utilizing the power of artificial intelligence; current status quo and future research opportunities.

Lameness represents a major welfare and health problem for the dairy industry across all farming systems. Visual mobility scoring, although very useful, is labour-intensive and physically demanding, especially in large dairies, often leading to inconsistencies and inadequate uptake of the practice. Technological and computational advancements of artificial intelligence (AI) have led to the development of numerous automated solutions for livestock monitoring. The objective of this study was to review the automated systems using AI algorithms for lameness detection developed to-date. These systems rely on gait analysis using accelerometers, weighing platforms, acoustic analysis, radar sensors and computer vision technology. The lameness features of interest, the AI techniques used to process the data as well as the ground truth of lameness selected in each case are described. Measures of accuracy regarding correct classification of cows as lame or non-lame varied with most systems being able to classify cows with adequate reliability. Most studies used visual mobility scoring as the ground truth for comparison with only a few studies using the presence of specific foot pathologies. Given the capabilities of AI, and the benefits of early treatment of lameness, longitudinal studies to identify gait abnormalities using automated scores related to the early developmental stages of different foot pathologies are required. Farm-specific optimal thresholds for early intervention should then be identified to ameliorate cow health and welfare but also minimise unnecessary inspections.

Initial validation of an intelligent video surveillance system for automatic detection of dairy cattle lameness.

Introduction: Lameness is a major welfare challenge facing the dairy industry worldwide. Monitoring herd lameness prevalence, and early detection and therapeutic intervention are important aspects of lameness control in dairy herds. The objective of this study was to evaluate the performance of a commercially available video surveillance system for automatic detection of dairy cattle lameness (CattleEye Ltd). Methods: This was achieved by first measuring mobility score agreement between CattleEye and two veterinarians (Assessor 1 and Assessor 2), and second, by investigating the ability of the CattleEye system to detect cows with potentially painful foot lesions. We analysed 6,040 mobility scores collected from three dairy farms. Inter-rate agreement was estimated by calculating percentage agreement (PA), Cohen's kappa (κ) and Gwet's agreement coefficient (AC). Data regarding the presence of foot lesions were also available for a subset of this dataset. The ability of the system to predict the presence of potentially painful foot lesions was tested against that of Assessor 1 by calculating measures of accuracy, using lesion records during the foot trimming sessions as reference. Results: In general, inter-rater agreement between CattleEye and either human assessor was strong and similar to that between the human assessors, with PA and AC being consistently above 80% and 0.80, respectively. Kappa agreement between CattleEye and the human scorers was in line with previous studies (investigating agreement between human assessors) and within the fair to moderate agreement range. The system was more sensitive than Assessor 1 in identifying cows with potentially painful lesions, with 0.52 sensitivity and 0.81 specificity compared to the Assessor's 0.29 and 0.89 respectively. Discussion: This pilot study showed that the CattleEye system achieved scores comparable to that of two experienced veterinarians and was more sensitive than a trained veterinarian in detecting painful foot lesions.

End-of-life decision-making of dairy cattle and calves: A survey of British farmers and veterinary surgeons.

Background: The study aim was to characterise issues faced by farmers and veterinary surgeons when making end-of-life decisions for dairy cattle. Methods: Online surveys were distributed to British dairy farmers and veterinary surgeons for 20 weeks from November 2020. Results: There were 83 responses (37 farmers, 46 veterinary surgeons). Among youngstock, the risk of unassisted/natural death (2.6% ± 0.3%) was almost double the risk of euthanasia (1.4% ± 0.3%; p = 0.003). The opposite, however, was true in the milking herd: the risk of euthanasia (2.3% ± 0.3%) was greater than unassisted/natural death (1.6% ± 0.2%; p = 0.05). A fallen stock collector (62%) typically performed euthanasia and most farms (66%) did not have anyone trained to perform euthanasia. Most deaths within the milking herd were attributed to 'unknown or not recorded' (median 15% of deaths). The factors that farmers most frequently reported as strongly influencing their decision of when to euthanase an animal relative to the onset of disease were 'failure to respond to treatment' (89%), 'veterinary advice' (89%) and 'severity of disease' (88%). On average, veterinarians had moderate or high confidence that 60% of dairy farm clients 'are performing euthanasia in a timely manner so as to prevent unnecessary suffering'. Veterinary surgeons had variable agreement on the time to euthanasia for various conditions. Conclusions: The survey highlighted end-of life decision-making successes and areas for improvement on dairy farms. An evidence-based, decision-support framework may help end-of-life decision-making, particularly for complex diseases.

Vertical Migration of Adult Plecoptera and Trichoptera above Forested Headwater Streams.

Stream insects are essential components of aquatic and terrestrial ecosystem structure and function. Terrestrial stages are important components of terrestrial food webs, and flight-capable individuals are responsible for long-distance dispersal. Horizontal migrations by flying or crawling adults away from stream channels that link insects to riparian food webs and movements across catchment boundaries are well established through empirical research, but studies examining vertical migration of adult stream insects into forest canopies are generally lacking. This study focused on differences in adult Plecoptera and Trichoptera abundance at ground level versus the riparian canopy and differences in abundances among summer and autumn sampling periods to empirically demonstrate use of canopy ecosystems by stream insects. Malaise traps at ground level and canopy traps placed 8 to 10 m above the stream at four sites in the Mosquito Creek watershed (Pennsylvania) were used to examine vertical migration. Larval assemblages were collected and compared to adult assemblage to investigate patterns of local migration in the catchment. We found significantly more stream insects at ground level than in the forest canopy for Trichoptera, Plecoptera, and all individual plecopteran families, but a meaningful number of individuals were found in the riparian canopy. Canopy abundances were similar to abundances captured in adjacent ground-level habitats in other studies. Comparisons of adult and larval abundances among sites, taxa, and stages indicated site- and taxon-specific patterns for vertical movement into riparian canopies. Demonstrating that adult stream insects utilize riparian forest canopies indicates that riparian forest conservation should be prioritized over reforestation and that several potential research questions exist to inform riparian management.

Quantitative Trait Loci Mapping for Lameness Associated Phenotypes in Holstein-Friesian Dairy Cattle.

Lameness represents a significant challenge for the dairy cattle industry, resulting in economic losses and reduced animal health and welfare. The existence of underlying genomic variation for lameness associated traits has the potential to improve selection strategies by using genomic markers. Therefore, the aim of this study was to identify genomic regions and potential candidate genes associated with lameness traits. Lameness related lesions and digital cushion thickness were studied using records collected by our research team, farm records, and a combination of both. Genome-wide analyses were performed to identify significant genomic effects, and a combination of single SNP association analysis and regional heritability mapping was used to identify associated genomic regions. Significant genomic effects were identified for several lameness related traits: Two genomic regions were identified on chromosome 3 associated with digital dermatitis and interdigital hyperplasia, one genomic region on chromosome 23 associated with interdigital hyperplasia, and one genomic region on chromosome 2 associated with sole haemorrhage. Candidate genes in those regions are mainly related to immune response and fibroblast proliferation. Quantitative trait loci (QTL) identified in this study could enlighten the understanding of lameness pathogenesis, providing an opportunity to improve health and welfare in dairy cattle with the addition of these regions into selection programs.

Effects of Stream Temperature and Substrate Type on Emergence Patterns of Plecoptera and Trichoptera From Northeastern United States Headwater Streams.

The timing and spatial distribution of aquatic insect emergence is linked to the abiotic and biotic environment in streams. Studies of aquatic insect emergence are needed to generate baseline data to identify potential shifts in phenology and habitat-related emergence with global change. The purpose of this study was to 1) compare the timing of Plecoptera (stonefly) species emergence between two streams with different thermal regimes and 2) characterize the distribution of emerging Plecoptera and Trichoptera (caddisflies) from wood, rock, gravel, and sand substrates in five forested, headwater streams. Emergence timing and duration varied among Plecoptera species, with Ostrocerca albidipennis (Walker) (Plecoptera: Nemouridae) emerging only in May and four species in the genus Leuctra (Plecoptera: Leuctridae) collectively emerging throughout the summer (May to September). We observed earlier emergence of Amphinemura nigritta (Provancher) (Plecoptera: Nemouridae) and a longer total emergence period for Leuctra ferruginea (Walker) (Plecoptera: Leuctridae) in the stream with ~1.5°C warmer temperatures, which suggested that some insects may experience phenological shifts in streams with subtle differences in temperature. The abundance of plecopteran and trichopteran taxa emerging from wood was generally greater than for gravel or sand, and sand was the least preferred emergence substrate. The results suggest that human actions that decrease large wood and increase fine sedimentation may decrease habitat quality for many insect larvae and limit preferred emergence substrates.

Nicotine-induced and D1-receptor-dependent dendritic remodeling in a subset of dorsolateral striatum medium spiny neurons.

Nicotine is one of the most addictive substances known, targeting multiple memory systems, including the ventral and dorsal striatum. One form of neuroplasticity commonly associated with nicotine is dendrite remodeling. Nicotine-induced dendritic remodeling of ventral striatal medium spiny neurons (MSNs) is well-documented. Whether MSN dendrites in the dorsal striatum undergo a similar pattern of nicotine-induced structural remodeling is unknown. A morphometric analysis of Golgi-stained MSNs in rat revealed a natural asymmetry in dendritic morphology across the mediolateral axis, with larger, more complex MSNs found in the dorsolateral striatum (DLS). Chronic nicotine produced a lasting (at least 21day) expansion in the dendritic complexity of MSNs in the DLS, but not dorsomedial striatum (DMS). Given prior evidence that MSN subtypes can be distinguished based on dendritic morphology, MSNs were segregated into morphological subpopulations based on the number of primary dendrites. Analysis of these subpopulations revealed that DLS MSNs with more primary dendrites were selectively remodeled by chronic nicotine exposure and remodeling was specific to the distal-most portions of the dendritic arbor. Co-administration of the dopamine D1 receptor (D1R) antagonist SCH23390 completely reversed the selective effects of nicotine on DLS MSN dendrite morphology, supporting a causal role for dopamine signaling at D1 receptors in nicotine-induced dendrite restructuring. Considering the functional importance of the DLS in shaping and expressing habitual behavior, these data support a model in which nicotine induces persistent and selective changes in the circuit connectivity of the DLS that may promote and sustain addiction-related behavior.

The effect of subclinical ketosis on activity at estrus and reproductive performance in dairy cattle.

Our aims were to investigate the influence of subclinical ketosis (SCK) on physical activity at estrus using a neck accelerometer device and on future reproductive performance. Two hundred three Holstein-Friesian cows were studied on 3dairy farms in Northwest England between September 2013 and March 2014. Seventeen percent (35 of 203) of the enrolled cows were affected with SCK between 7 and 21d in milk, defined as a blood ß-hydroxybutyrate concentration of 1.2 to 2.9mmol/L. Time to event analyses and multivariable regression analyses were used to assess the effect of SCK on reproductive performance and activity at estrus. The SCK cows exhibited a lower peak activity (measured as the number of standard deviations above mean activity) and shorter duration in activity clusters associated with first estrus and first insemination postpartum, compared with non-SCK cows. Peak activity and cluster duration associated with the insemination that led to a pregnancy were not different between SCK and non-SCK cows. Calving to first estrus, calving to first insemination, and calving to pregnancy intervals were prolonged in SCK cows. First insemination was 4.3 times (95% confidence interval=1.6 to 15.0) less likely to be successful in SCK cows compared with non-SCK cows. Adjusted mean number of inseminations per pregnancy was 2.8 for SCK cows and 2.0 for non-SCK cows. The current study confirms the long-lasting effects of SCK on reproductive efficiency. Furthermore, it is indicated that physical activity around estrus is reduced by SCK in early lactation, but this negative effect appears to diminish as cows progress through lactation.

Development and Use of Assay Conditions Suited to Screening for and Profiling of SET-Domain-Targeted Inhibitors of the MLL/SET1 Family of Lysine Methyltransferases.

Methylation of histone H3 lysine-4 (H3K4) is an important, regulatory, epigenetic post-translational modification associated with actively transcribed genes. In humans, the principal mediators of this modification are part of the MLL/SET1 family of methyltransferases, which comprises six members, MLLs1-4 and SET1A/SET1B. Aberrations in the structure, expression, and regulation of these enzymes are implicated in various disease states, making them important potential targets for drug discovery, particularly for oncology indications. The MLL/SET1 family members are most enzymatically active when part of a "core complex," the catalytic SET-domain-containing subunits bound to a subcomplex consisting of the proteins WDR5, RbBP5, Ash2L and a homodimer of DPY-30 (WRAD2). The necessity of MLL/SET1 members to bind WRAD2 for full activity is the basis of a particular drug development strategy, which seeks to disrupt the interaction between the MLL/SET1 subunits and WDR5. This strategy is not without its theoretical and practical drawbacks, some of which relate to the ease with which complexes of Escherichia coli-expressed MLL/SET1 and WRAD2 fall apart. As an alternative strategy, we explore ways to stabilize the complex, focusing on the use of an excess of WRAD2 to drive the binding equilibria toward complex formation while maintaining low concentrations of the catalytic subunits. The purpose of this approach is to seek inhibitors that bind the SET domain, an approach proven successful with the related, but inherently more stable, enhancer of zeste homolog 2 (EZH2) complex.

Adolescent nicotine induces persisting changes in development of neural connectivity.

Adolescent nicotine induces persisting changes in development of neural connectivity. A large number of brain changes occur during adolescence as the CNS matures. These changes suggest that the adolescent brain may still be susceptible to developmental alterations by substances which impact its growth. Here we review recent studies on adolescent nicotine which show that the adolescent brain is differentially sensitive to nicotine-induced alterations in dendritic elaboration, in several brain areas associated with processing reinforcement and emotion, specifically including nucleus accumbens, medial prefrontal cortex, basolateral amygdala, bed nucleus of the stria terminalis, and dentate gyrus. Both sensitivity to nicotine, and specific areas responding to nicotine, differ between adolescent and adult rats, and dendritic changes in response to adolescent nicotine persist into adulthood. Areas sensitive to, and not sensitive to, structural remodeling induced by adolescent nicotine suggest that the remodeling generally corresponds to the extended amygdala. Evidence suggests that dendritic remodeling is accompanied by persisting changes in synaptic connectivity. Modeling, electrophysiological, neurochemical, and behavioral data are consistent with the implication of our anatomical studies showing that adolescent nicotine induces persisting changes in neural connectivity. Emerging data thus suggest that early adolescence is a period when nicotine consumption, presumably mediated by nicotine-elicited changes in patterns of synaptic activity, can sculpt late brain development, with consequent effects on synaptic interconnection patterns and behavior regulation. Adolescent nicotine may induce a more addiction-prone phenotype, and the structures altered by nicotine also subserve some emotional and cognitive functions, which may also be altered. We suggest that dendritic elaboration and associated changes are mediated by activity-dependent synaptogenesis, acting in part through D1DR receptors, in a network activated by nicotine. The adolescent nicotine effects reviewed here suggest that modification of late CNS development constitutes a hazard of adolescent nicotine use.

Role of the D3 dopamine receptor in nicotine sensitization.

Adolescent cigarette use is associated with reduced quitting success and continued smoking in adulthood. Interestingly, polymorphisms of the dopamine D3 receptor (DRD3) gene have been associated with smoking behavior, and the receptor is expressed in an age- and brain region-dependent manner that suggests relevance to addiction. Here, we investigate the possible role of dopamine-related receptors, including DRD3 and an intriguing splice variant known as D3nf, in nicotine-induced sensitization. In adolescent and adult male rats, we examined (1) alterations occurring in dopamine receptor-related mRNAs (DRD1, DRD2, DRD3 and D3nf) at two time points during a sensitizing regimen of nicotine and (2) whether DRD3 antagonism either during the initial treatment (induction) or at a later challenge exposure (expression) is able to block nicotine sensitization. Nicotine-induced changes were seen for DRD3 and D3nf mRNAs in the nucleus accumbens shell early in repeated exposure in both age groups. DRD3 antagonism only blocked the induction of sensitization in adolescents and did not block the expression of sensitization in either age group. Adolescents and adults showed opposite DRD1 mRNA responses to nicotine treatment, while no age- and nicotine-related changes in DRD2 mRNA were observed. These data reveal important age-dependent regulation of DRD1- and DRD3-related mRNAs during the course of nicotine exposure. Furthermore, they highlight a requirement for DRD3 signaling in the development of adolescent nicotine sensitization, suggesting it may represent an appropriate target in the prevention of nicotine dependence initiated at this age.

Tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of left-hand-side moiety (Part-2).

Novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. A series of novel oxabicyclooctane-linked NBTIs with new tricyclic-1,5-naphthyridinone left hand side moieties have been described. Compounds with a (R)-hydroxy-1,5-naphthyridinone moiety (7) showed potent antibacterial activity (e.g., Staphylococcus aureus MIC 0.25 µg/mL), acceptable Gram-positive and Gram-negative spectrum with rapidly bactericidal activity. The compound 7 showed intravenous and oral efficacy (ED50) at 3.2 and 27 mg/kg doses, respectively, in a murine model of bacteremia. Most importantly they showed significant attenuation of functional hERG activity (IC50 >170 µM). In general, lower logD attenuated hERG activity but also reduced Gram-negative activity. The co-crystal structure of a hydroxy-tricyclic NBTI bound to a DNA-gyrase complex exhibited a binding mode that show enantiomeric preference for R isomer and explains the activity and SAR. The discovery, synthesis, SAR and X-ray crystal structure of the left-hand-side tricyclic 1,5-naphthyridinone based oxabicyclooctane linked NBTIs are described.

Synthesis and Evaluation of Heterocyclic Catechol Mimics as Inhibitors of Catechol-O-methyltransferase (COMT).

3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg(2+). The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors.

Adolescent nicotine alters dendritic morphology in the bed nucleus of the stria terminalis.

Adolescent nicotine increases dendritic elaboration in several areas associated with the extended amygdala. It also increases anxiety-like behavior in adulthood. An unresolved question is whether adolescent nicotine alters dendritic structure in the bed nucleus of the stria terminalis (BNST), which may contribute to altered anxiety-like behavior. To investigate this possibility, adolescent male Sprague-Dawley rats were administered nicotine (0.5mg/kg/day) 3 days a week for 2 consecutive weeks, starting at postnatal day P (32). 17 days following the end of dosing, brains were processed for Golgi-Cox staining, and neurons were digitally reconstructed in three dimensions. Animals previously treated with nicotine exhibited an increase in the total number of branches and total length of dendrites on BNST neurons. Sholl analysis revealed an increase in the number of intersections with concentric spheres, increased amount of dendritic material within concentric spheres, and an increase of dendritic branching within concentric spheres occurring between 20 and 300 µm from the soma in dendrites. Collectively, our results show that adolescent nicotine alters dendritic structure (by triggering new branch growth), and, by inference, connectivity of the BNST, which may contribute to alterations in behavior induced by adolescent nicotine.

Activation of cells containing estrogen receptor alpha or somatostatin in the medial preoptic area, arcuate nucleus, and ventromedial nucleus of intact ewes during the follicular phase, and alteration after lipopolysaccharide.

Cells in the medial preoptic area (mPOA), arcuate nucleus (ARC), and ventromedial nucleus (VMN) that possess estrogen receptor alpha (ER alpha) mediate estradiol feedback to regulate endocrine and behavioral events during the estrous cycle. A percentage of ER alpha cells located in the ARC and VMN express somatostatin (SST) and are activated in response to estradiol. The aims of the present study were to investigate the location of c-Fos, a marker for activation, in cells containing ER alpha or SST at various times during the follicular phase and to determine whether lipopolysaccharide (LPS) administration, which leads to disruption of the luteinizing hormone (LH) surge, is accompanied by altered ER alpha and/or SST activation patterns. Follicular phases were synchronized with progesterone vaginal pessaries, and control animals were killed at 0, 16, 31, and 40 h (n = 4-6/group) after progesterone withdrawal (PW [time 0]). At 28 h, other animals received LPS (100 ng/kg) and were subsequently killed at 31 h or 40 h (n = 5/group). Hypothalamic sections were immunostained for c-Fos and ER alpha or SST. LH surges occurred only in control ewes with onset at 36.7 ± 1.3 h after PW; these animals had a marked increase in the percentage of ER alpha cells that colocalized c-Fos (%ER alpha/c-Fos) in the ARC and mPOA from 31 h after PW and throughout the LH surge. In the VMN, %ER alpha/c-Fos was higher in animals that expressed sexual behavior than in those that did not. SST cell activation in the ARC and VMN was greater during the LH surge than in other stages in the follicular phase. At 31 or 40 h after PW (i.e., 3 or 12 h after treatment, respectively), LPS decreased %ER alpha/c-Fos in the ARC and the mPOA, but there was no change in the VMN compared to that in controls. The %SST/c-Fos increased in the VMN at 31 h after PW (i.e., 3 h after LPS) with no change in the ARC compared to controls. These results indicate that there is a distinct temporal pattern of ER alpha cell activation in the hypothalamus during the follicular phase, which begins in the ARC and mPOA at least 6-7 h before the LH surge onset and extends to the VMN after the onset of sexual behavior and LH surge. Furthermore, during the surge, some of these ER alpha-activated cells may be SST-secreting cells. This pattern is markedly altered by LPS administered during the late follicular phase, indicating that the disruptive effects of this stressor are mediated by suppressing ER alpha cell activation at the level of the mPOA and ARC and enhancing SST cell activation in the VMN, leading to the attenuation of the LH surge.

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad spectrum antibacterial agents.

Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents. Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to inhibition by diverse classes of inhibitors with alternative and distinct binding sites to quinolone antibiotics, thus enabling the development of agents that lack cross-resistance to quinolones. Described here are novel bacterial topoisomerase inhibitors (NBTIs), which are a new class of gyrase and topo IV inhibitors and consist of three distinct structural moieties. The substitution of the linker moiety led to discovery of potent broad-spectrum NBTIs with reduced off-target activity (hERG IC50 > 18 µM) and improved physical properties. AM8191 is bactericidal and selectively inhibits DNA synthesis and Staphylococcus aureus gyrase (IC50 = 1.02 µM) and topo IV (IC50 = 10.4 µM). AM8191 showed parenteral and oral efficacy (ED50) at less than 2.5 mg/kg doses in a S. aureus murine infection model. A cocrystal structure of AM8191 bound to S. aureus DNA-gyrase showed binding interactions similar to that reported for GSK299423, displaying a key contact of Asp83 with the basic amine at position-7 of the linker.

Anxiety status affects nicotine- and baclofen-induced locomotor activity, anxiety, and single-trial conditioned place preference in male adolescent rats.

Adolescents have an increased vulnerability to nicotine and anxiety may play a role in the development of nicotine abuse. One possible treatment for anxiety disorders and substance abuse is the GABAB agonist, baclofen. The aim of the present study was to determine the effect of anxiety-like behavior on single-trial nicotine conditioned place preference in adolescent rats, and to assess the action of baclofen. Baclofen was shown to have effects on locomotor and anxiety-like behavior in rats divided into high-anxiety and low-anxiety groups. Baclofen decreased locomotor behavior in high-anxiety rats. Baclofen alone failed to produce differences in anxiety-like behavior, but nicotine and baclofen + nicotine administration were anxiolytic. High- and low-anxiety groups also showed differences in single-trial nicotine-induced place preference. Only high-anxiety rats formed place preference to nicotine, while rats in the low-anxiety group formed no conditioned place preference. These results suggest that among adolescents, high-anxiety individuals are more likely to show preference for nicotine than low-anxiety individuals.

Using hormones to manage dairy cow fertility: the clinical and ethical beliefs of veterinary practitioners.

In the face of a steady decline in dairy cow fertility over several decades, using hormones to assist reproduction has become common. In the European Union, hormones are prescription-only medicines, giving veterinary practitioners a central role in their deployment. This study explored the clinical and ethical beliefs of practitioners, and provides data on their current prescribing practices. During 2011, 93 practitioners working in England completed a questionnaire (95% response rate). Of the 714 non-organic farms they attended, only 4 farms (0.6%) never used hormones to assist the insemination of lactating dairy cows. Practitioners agreed (>80%) that hormones improve fertility and farm businesses profitability. They also agreed (>80%) that if farmers are able to tackle management issues contributing to poor oestrus expression, then over a five year period these outcomes would both improve, relative to using hormones instead. If management issues are addressed instead of prescribing hormones, practitioners envisaged a less favourable outcome for veterinary practices profitability (p<0.01), but an improvement in genetic selection for fertility (p<0.01) and overall cow welfare (p<0.01). On farms making no efforts to address underlying management problems, long-term routine use at the start of breeding for timing artificial insemination or inducing oestrus was judged "unacceptable" by 69% and 48% of practitioners, respectively. In contrast, practitioners agreed (≥ 90%) that both these types of use are acceptable, provided a period of time has been allowed to elapse during which the cow is observed for natural oestrus. Issues discussed include: weighing quality versus length of cow life, fiscal factors, legal obligations, and balancing the interests of all stakeholders, including the increasing societal demand for food. This research fosters debate and critical appraisal, contributes to veterinary ethics, and encourages the pro-active development of professional codes of conduct.

Assay development for histone methyltransferases.

Epigenetic modifications play a crucial role in human diseases. Unlike genetic mutations, however, they do not change the underlying DNA sequences. Epigenetic phenomena have gained increased attention in the field of cancer research, with many studies indicating that they are significantly involved in tumor establishment and progression. Histone methyltransferases (HMTs) are a large group of enzymes that specifically methylate protein lysine and arginine residues, especially in histones, using S-adenosyl-L-methionine (SAM) as the methyl donor. However, in general, HMTs have no widely accepted high-throughput screening (HTS) assay format, and reference inhibitors are not available for many of the enzymes. In this study, we describe the application of a miniaturized, radioisotope-based reaction system: the HotSpot(SM) platform for methyltransferases. Since this platform employs tritiated SAM as a cofactor, it can be applied to the assay of any HMT. The key advantage of this format is that any substrate can be used, including peptides, proteins, or even nucleosomes, without the need for labeling or any other modifications. Using this platform, we have determined substrate specificities, characterized enzyme kinetics, performed compound profiling for both lysine and arginine methyltransferases, and carried out HTS for a small-library LOPAC against DOT1L. After hit confirmation and profiling, we found that suramin inhibited DOT1L, NSD2, and PRMT4 with IC50 values at a low µM range.