Combination of Reverse and Chemical Genetic Screens Reveals Angiogenesis Inhibitors and Targets

We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/ T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis

High Expression of Wee1 Is Associated with Poor Disease-Free Survival in Malignant Melanoma: Potential for Targeted Therapy

Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G2/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21Cip1/WAF1 (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239WTp53, WM45.1MUTp53 and LOXWTp53, further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G1/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents

On assessment methods related to pain in dogs with osteoarthritis

There is a need of valid and reliable assessment methods that are clinically applicable in canine rehabilitation practice. The aim of this thesis was to psychometrically evaluate measurement properties in assessment methods related to pain in naturally occurring canine osteoarthritis. Assessment methods developed for heart rate variability analysis, i.e. Polar heart rate monitor, and owner-reported perceptions of pain severity and pain interference with functionality, i.e. Canine Brief Pain Inventory, were tested. Methods: Four observational studies were conducted. Study I was a cross-sectional study consisting of two groups of consecutively recruited dogs. The Canine Brief Pain Inventory was administered to owners of dogs with naturally occurring osteoarthritis (n=61) and clinically sound dogs (n=21). Study II was a descriptive and correlative cross-sectional study based on the same sample of dogs with osteoarthritis (n=71), assessing chronic pain behavior and associations between explanatory variables and chronic pain behavior. Study III and IV were correlative studies, assessing Polar heart rate monitor measuring interbeat intervals and time- and frequency-based heart rate variability parameters, compared to simultaneously recorded electrocardiogram in dogs (n=11). Results: High internal consistencies and ability to discriminate sound dogs from osteoarthritis dogs were found. The hypothesis of the presented two-factor structure of the Canine Brief Pain Inventory was rejected. Owners reported higher proportions of chronic pain behavior in items targeting physical activities, e.g. getting up, moving after rest and moving after major exercise. A minor proportion of dogs with osteoarthritis showed no owner-perceived behavioural signs of chronic pain. Owner observations were not associated with ongoing antiinflammatory medications. In Study III and IV, 595 errors (12.3%) were identified in Polar data. The number of errors were unequally distributed among the dogs. Interbeat intervals and heart rate variability parameters from electrocardiogram and Polar were strongly associated. Standard error of measurements were high among some heart rate variability parameters in Polar and electrocardiogram. In conclusion, this thesis contributes to our knowledge about assessment methods related to diverse components of pain in dogs with osteoarthritis, allowing improved pain management in clinical practice

On assessment methods related to pain in dogs with osteoarthritis

There is a need of valid and reliable assessment methods that are clinically applicable in canine rehabilitation practice. The aim of this thesis was to psychometrically evaluate measurement properties in assessment methods related to pain in naturally occurring canine osteoarthritis. Assessment methods developed for heart rate variability analysis, i.e. Polar heart rate monitor, and owner-reported perceptions of pain severity and pain interference with functionality, i.e. Canine Brief Pain Inventory, were tested. Methods: Four observational studies were conducted. Study I was a cross-sectional study consisting of two groups of consecutively recruited dogs. The Canine Brief Pain Inventory was administered to owners of dogs with naturally occurring osteoarthritis (n=61) and clinically sound dogs (n=21). Study II was a descriptive and correlative cross-sectional study based on the same sample of dogs with osteoarthritis (n=71), assessing chronic pain behavior and associations between explanatory variables and chronic pain behavior. Study III and IV were correlative studies, assessing Polar heart rate monitor measuring interbeat intervals and time- and frequency-based heart rate variability parameters, compared to simultaneously recorded electrocardiogram in dogs (n=11). Results: High internal consistencies and ability to discriminate sound dogs from osteoarthritis dogs were found. The hypothesis of the presented two-factor structure of the Canine Brief Pain Inventory was rejected. Owners reported higher proportions of chronic pain behavior in items targeting physical activities, e.g. getting up, moving after rest and moving after major exercise. A minor proportion of dogs with osteoarthritis showed no owner-perceived behavioural signs of chronic pain. Owner observations were not associated with ongoing antiinflammatory medications. In Study III and IV, 595 errors (12.3%) were identified in Polar data. The number of errors were unequally distributed among the dogs. Interbeat intervals and heart rate variability parameters from electrocardiogram and Polar were strongly associated. Standard error of measurements were high among some heart rate variability parameters in Polar and electrocardiogram. In conclusion, this thesis contributes to our knowledge about assessment methods related to diverse components of pain in dogs with osteoarthritis, allowing improved pain management in clinical practice

On assessment methods related to pain in dogs with osteoarthritis

There is a need of valid and reliable assessment methods that are clinically applicable in canine rehabilitation practice. The aim of this thesis was to psychometrically evaluate measurement properties in assessment methods related to pain in naturally occurring canine osteoarthritis. Assessment methods developed for heart rate variability analysis, i.e. Polar heart rate monitor, and owner-reported perceptions of pain severity and pain interference with functionality, i.e. Canine Brief Pain Inventory, were tested. Methods: Four observational studies were conducted. Study I was a cross-sectional study consisting of two groups of consecutively recruited dogs. The Canine Brief Pain Inventory was administered to owners of dogs with naturally occurring osteoarthritis (n=61) and clinically sound dogs (n=21). Study II was a descriptive and correlative cross-sectional study based on the same sample of dogs with osteoarthritis (n=71), assessing chronic pain behavior and associations between explanatory variables and chronic pain behavior. Study III and IV were correlative studies, assessing Polar heart rate monitor measuring interbeat intervals and time- and frequency-based heart rate variability parameters, compared to simultaneously recorded electrocardiogram in dogs (n=11). Results: High internal consistencies and ability to discriminate sound dogs from osteoarthritis dogs were found. The hypothesis of the presented two-factor structure of the Canine Brief Pain Inventory was rejected. Owners reported higher proportions of chronic pain behavior in items targeting physical activities, e.g. getting up, moving after rest and moving after major exercise. A minor proportion of dogs with osteoarthritis showed no owner-perceived behavioural signs of chronic pain. Owner observations were not associated with ongoing antiinflammatory medications. In Study III and IV, 595 errors (12.3%) were identified in Polar data. The number of errors were unequally distributed among the dogs. Interbeat intervals and heart rate variability parameters from electrocardiogram and Polar were strongly associated. Standard error of measurements were high among some heart rate variability parameters in Polar and electrocardiogram. In conclusion, this thesis contributes to our knowledge about assessment methods related to diverse components of pain in dogs with osteoarthritis, allowing improved pain management in clinical practice

The Immediate Effect of Parachute-Resisted Gallop on Heart Rate, Running Speed and Stride Frequency in Dogs

Simple Summary Physical fitness is needed for canine athletes and working dogs to optimize their performance in various disciplines. Application of resistance on movements causes biomechanical and cardiorespiratory responses to physical exercise. However, there is still a lack of research on the effects of high-intensity resistance exercise on cardiorespiratory fitness components such as heart rate in canine athletes. In this article, we investigate the short-term effects of parachute-resisted galloping on heart rate, running speed and stride frequency. Healthy dogs of various breeds were extensively studied in five experimental single cases. The dogs ran on a straight 200 m course with and without resistive drag force applied by a parachute attached to their harness while heart rate, running speed and stride frequency were measured. Subsequently, the measurements were compared to baseline phases at rest. In the present trials we found that heart rate increases similarly with and without parachute-resistance while dogs galloped at lower speeds and with increased stride frequency with applied drag force. Our findings lead us to suggest that parachute-resisted galloping is a clinically applicable exercise in healthy dogs to achieve instant cardiorespiratory response. Physical fitness is required for canine athletes and working dogs to optimize performance in various disciplines. There is a lack of research on the effects of resistance exercise on cardiorespiratory variables in dogs. The aim of this study was to investigate the immediate effects of parachute-resisted (PR) gallop on heart rate, running speed and stride frequency compared to unresisted (UR) gallop in dogs. Five N-of-1 trials RCTs with alternating interventions were implemented. Dogs ran on a 200 m course with and without resistive force applied by a parachute attached to their harness while cardiac inter-beat intervals (IBI), running speed and stride frequency were measured. The results were visually displayed and interpreted in graphs and percentage of non-overlapping data estimated effect size. Both interventions showed large effects on heart rate compared to resting values. Mean IBI increased (10-17%) during PR gallop compared to UR gallop although this change was small relative to decreased running speed (19-40%) and increased stride frequency (18-63%). Minimum IBI showed no difference between interventions indicating similar maximum heartbeat per minute. In conclusion, parachute-resistance resulted in dogs galloping at lower speeds at the same cardiorespiratory level of intensity, which may be useful in canine physical rehabilitation and fitness training

Dog Handler Beliefs regarding Barriers and Facilitators to Canine Health Promotion and Injury Prevention in Swedish Working Dog Trials and Competitions

Dog trials and competitions involve various sport disciplines, e.g., obedience, agility, working dog trials and rally obedience. Dog handlers navigate their dogs through physically and mentally demanding tasks. The purpose of this study was to gain a better understanding of barriers and facilitators to canine health promotion and injury prevention described by dog handlers. Methods: Qualitative inductive content analysis was applied to systematically organize and interpret narrative data from 654 respondents' answers to open-ended questions in an anonymous online inquiry. Results: Two categories, with seven sub-categories, emerged from the analysis: (1) Challenges in applying the regulations in dog trials and competitions, and (2) Implementation of animal welfare and canine well-being approaches. Respondents described the challenges in applying regulations in dog trials and competitions and lack of scientific research as barriers to their intent to prevent injuries in their dogs. Implementation of animal welfare and canine well-being approaches were described as facilitators. Conclusion: The findings imply that the stakeholders continuously need to work on bridging possible gaps between the canine welfare criteria and the scientific and empirical knowledge in canine sports and performance medicine