Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution

Live Cell Imaging and High Throughput Screening are rapidly evolving techniques and have found many applications in recent years. Modern microscopy enables the visualisation of internal changes in the cell through the use of fluorescently tagged proteins which can be targeted to specific cellular components. A system is presented here which is designed to track cells at low temporal resolution within large populations, and to extract fluorescence data which allows relative expression rates of tagged proteins to be monitored. Cell detection and tracking are performed as separate steps, and several methods are evaluated for suitability using timeseries images of Hoechst-stained C2C12 mouse mesenchymal stem cells. The use of Hoechst staining ensures cell nuclei are visible throughout a time-series. Dynamic features, including a characteristic change in Hoechst fluorescence intensity during chromosome condensation, are used to identify cell divisions and resulting daughter cells. The ability to detect cell division is integrated into the tracking, aiding lineage construction. To establish the efficiency of the method, synthetic cell images have been produced and used to evaluate cell detection accuracy. A validation framework is created which allows the accuracy of the automatic segmentation and tracking systems to be measured and compared against existing state of the art software, such as CellProfiler. Basic tracking methods, including nearest-neighbour and cell-overlap, are provided as a baseline to evaluate the performance of more sophisticated methods. The software is demonstrated on a number of biological systems, starting with a study of different control elements of the Msx1 gene, which regulates differentiation of mesenchymal stem cells. Expression is followed through multiple lineages to identify asymmetric divisions which may be due to cell differentiation. The lineage construction methods are applied to Schizosaccharomyces pombe time-series image data, allowing the extraction of generation lengths for individual cells. Finally a study is presented which examines correlations between the circadian and cell cycles. This makes use of the recently developed FUCCI cell cycle markers which, when used in conjunction with a circadian indicator such as Rev-erbα-Venus, allow simultaneous measurements of both cycles

Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution

Live Cell Imaging and High Throughput Screening are rapidly evolving techniques and have found many applications in recent years. Modern microscopy enables the visualisation of internal changes in the cell through the use of fluorescently tagged proteins which can be targeted to specific cellular components. A system is presented here which is designed to track cells at low temporal resolution within large populations, and to extract fluorescence data which allows relative expression rates of tagged proteins to be monitored. Cell detection and tracking are performed as separate steps, and several methods are evaluated for suitability using timeseries images of Hoechst-stained C2C12 mouse mesenchymal stem cells. The use of Hoechst staining ensures cell nuclei are visible throughout a time-series. Dynamic features, including a characteristic change in Hoechst fluorescence intensity during chromosome condensation, are used to identify cell divisions and resulting daughter cells. The ability to detect cell division is integrated into the tracking, aiding lineage construction. To establish the efficiency of the method, synthetic cell images have been produced and used to evaluate cell detection accuracy. A validation framework is created which allows the accuracy of the automatic segmentation and tracking systems to be measured and compared against existing state of the art software, such as CellProfiler. Basic tracking methods, including nearest-neighbour and cell-overlap, are provided as a baseline to evaluate the performance of more sophisticated methods. The software is demonstrated on a number of biological systems, starting with a study of different control elements of the Msx1 gene, which regulates differentiation of mesenchymal stem cells. Expression is followed through multiple lineages to identify asymmetric divisions which may be due to cell differentiation. The lineage construction methods are applied to Schizosaccharomyces pombe time-series image data, allowing the extraction of generation lengths for individual cells. Finally a study is presented which examines correlations between the circadian and cell cycles. This makes use of the recently developed FUCCI cell cycle markers which, when used in conjunction with a circadian indicator such as Rev-erbα-Venus, allow simultaneous measurements of both cycles.EThOS - Electronic Theses Online ServiceEngineering and Physical Sciences Research Council (EPSRC)GBUnited Kingdo

Prospectus, October 31, 1979

FROM POLIDENT TO EFFERVESCENT: DOWNTOWN TO BE VINTAGE; Week in Review: Across the globe, In the nation, Throughout the state, Around the town; Poltergeists bump at night; Elam wins state CC; Correction…; Wilson thanks Ziggy fans; Briefs: Forum discusses survey, Brazilian pianist at Monticello Nov. 4, Debate: has America failed?, Art field trip features Toulouse-Lautrec, NHB sponsors student awards, One woman is \u27nine Women\u27, EMT workshop at PC Nov. 17, Parkland presents Survival Program; Weekly Calendar; Preppy yet potent: Heads progress the hard way; Letters to the Editor: Rep. Johnson opposes veto; When weather break thieves break in; Foreigners complain again; Treaters shouldn\u27t \u27trick\u27; Amittyville: fact or fiction?; Survey handed out next week; Classifieds; Checks cashable in Nov.; College Day at PC Nov. 7; Model govt. needs plans; Earn less than $6,000? Elgible for higher grant; Parkland offers Folkore; Harmful materials to be discusses; Knee surgery ousts Short; Home no help for golfers; You\u27ve waited long enough: shape up; Soccer club wins; Freddy heading toward poverty; Fast Freddy Contesthttps://spark.parkland.edu/prospectus_1979/1006/thumbnail.jp

Prospectus, October 24, 1979

SURVEY MAKES WAVES; The Week in Review: Across the globe, In the nation, Throughout the state, Around the town, Etc….; Briefs: College to host math contest, Parkland Players present \u27Dracula\u27, Summary of board listed, Champaign council approves bonds, Women\u27s program seeks young blood, Monticello council to discuss tax rate, Program on strokes to be presented, Arthritis program to be presented, Future educators program tomorrow, Nutritionm health, disease are topics, Store celebrates 1st; Letter to editor: Dean congratulates foreigners; Weekly Calendar; Area high schools to visit campus; S.T.O. raffle winners are announced; Give a little blood, then see blood; Women at Home on Tuesday; Editorial: Censorship at WPCD?; PATH raises grievances; Pumpkins and costumes in contest next week; Lincoln Square has art show; Oktoberfest today: a taste of German; Reviews: Ending to the beat has better taste, B-52s: a glimpse of future and past; Classifieds; Music club has enthusiasm; Athletic and rec fields near completion; V-ball gains momentum; Golf team advances to state; Elam blows off competition: harriers 3rd; PC hosts golf, gets 2nd; Fast Freddy loses again; Fast Freddy Contesthttps://spark.parkland.edu/prospectus_1979/1007/thumbnail.jp

Crop Updates 2002 - Oilseeds

This session covers twenty seven papers from different authors: 1. Forward and acknowledgements, Dave Eksteen, ACTING MANAGER OILSEEDS PRODUCTIVITY AND INDUSTRY DEVELOPMENT Department of Agriculture PLENARY SESSION 2. GMO canola - Track record in Canada, K. Neil Harker and George W. Clayton,Agriculture and Agri-Food Canada, Lacombe Research Centre, Lacombe, Alberta, R. Keith Downey, Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, Saskatchewan 3. GMO canola – Prospects in Western Australia farming systems, Keith Alcock, Crop Improvement Institute, Department of Agriculture 4. Diamondback moth (DBM) in canola, Kevin Walden, Department of Agriculture CANOLA AGRONOMY 5. Getting the best out of canola in the low rainfall central wheatbelt, Bevan Addison and Peter Carlton, Elders Ltd 6. Canola variety performance in Western Australia, Kevin Morthorpe, Stephen Addenbrooke and Alex Ford, Pioneer Hi-Bred Australia P/L 7. Relative performance of new canola varieties in Department of Agriculture variety trials in 2000 and 2001, S. Hasan Zaheer, GSARI, Department of Agriculture, G. Walton, Crop Improvement Institute, Department of Agriculture 8. Which canola cultivar should I sow? Imma Farré, CSIRO Plant Industry, Floreat, Bill Bowden,Western Australia Department of Agriculture 9. The effect of seed generation and seed source on yield and quality of canola, Paul Carmody, Department of Agriculture 10. The accumulation of oil in Brassica species, J.A. Fortescue and D.W. Turner, Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, B. Tan, PO Box 1249, South Perth 11. Potential and performance of alternative oilseeds in WA, Margaret C. Campbell, Centre for Legumes in Mediterranean Agriculture 12. Comparison of oilseed crops in WA, Ian Pritchard and Paul Carmody, Department of Agriculture, Centre for Cropping Systems, Margaret Campbell, Centre for Legumes in Mediterranean Agriculture 13. Identifying constraints to canola production, Dave Eksteen, Canola Development Officer, Department of Agriculture 14. Boron – should we be worried about it? Richard W. BellA, K. FrostA, Mike WongB, and Ross BrennanC , ASchool of Environmental Science, Murdoch University, BCSIRO Land and Water, CDepartment of Agriculture PEST AND DISEASE 15. Yield losses caused when Beet Western Yellows Virus infects canola, Roger Jones and Jenny Hawkes, Department of Agriculture, and Centre for Legumes in Mediterranean Agriculture 16. Influence of climate on aphid outbreaks and virus epidemics in canola, Debbie Thackray, Jenny Hawkes and Roger Jones, Centre for Legumes in Mediterranean Agriculture and Department of Agriculture 17. The annual shower of blackleg ascospores in canola: Can we predict and avoid it? Moin U. Salam, Ravjit K. Khangura, Art J. Diggle and Martin J. Barbetti, Department of Agriculture 18. Environmental influences on production and release of ascospores of blackleg and their implications in blackleg management in canola, Ravjit K. Khangura, Martin J. Barbetti , Moin U. Salam and Art J. Diggle, Department of Agriculture 19. WA blackleg resistance ratings on canola varieties form 2002, Ravjit Khangura, Martin J. Barbetti and Graham Walton, Department of Agriculture 20. Bronzed field beetle management in canola, Phil Michael, Department of Agriculture 21. DBM control in canola: Aerial versus boom application, Paul Carmody, Department of Agriculture 22. Effect of single or multiple spray trearments on the control of Diamondback moth (Plutella xylostella) and yield of canola at Wongan Hills, Françoise Berlandier, Paul Carmody and Christiaan Valentine, Department of Agriculture ESTABLISHMENT 23. GrainGuardÔ - A biosecurity plan for the canola industry, Greg Shea, Department of Agriculture 24. Large canola seed is best, particularly for deep sowing, Glen Riethmuller, Rafiul Alam, Greg Hamilton and Jo Hawksley, Department of Agriculture 25. Canola establishment with seed size, tines and discs, with and without stubble, Glen Riethmuller, Rafiul Alam, Greg Hamilton and Jo Hawksley, Department of Agriculture WEEDS 26. Role of Roundup ReadyÒ canola in the farming system, Art Diggle1, Patrick Smith2, Paul Neve3, Felicity Flugge4, Amir Abadi5, Stephen Powles3 1Department of Agriculture, 2CSIRO, Sustainable Ecosystems, 3Western Australian Herbicide Resistance Initiative, University of Western Australia, 4Centre for Legumes in Mediterranean Agriculture, University of Western Australia, 5Touchstone Consulting, Mt Hawthorn FEED 27. Getting value from canola meals in the animal feed industries: Aquaculture, Brett Glencross and John Curnow, Department of Fisheries - Government of Western Australia and Wayne Hawkins, Department of Agricultur

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

Background: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. Methods: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. Findings: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96–1·28). Interpretation: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. Funding: National Institute for Health Research Health Services and Delivery Research Programme

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

BACKGROUND: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. METHODS: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. FINDINGS: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96-1·28). INTERPRETATION: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. FUNDING: National Institute for Health Research Health Services and Delivery Research Programme

Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution

The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell types and fluorescent markers

Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle

Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer. Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle.. Available from: https://www.researchgate.net/publication/264641131_Phase_locking_and_multiple_oscillating_attractors_for_the_coupled_mammalian_clock_and_cell_cycle [accessed Apr 8, 2015]