DeadEasy Mito-Glia: Automatic Counting of Mitotic Cells and Glial Cells in Drosophila

Cell number changes during normal development, and in disease (e.g., neurodegeneration, cancer). Many genes affect cell number, thus functional genetic analysis frequently requires analysis of cell number alterations upon loss of function mutations or in gain of function experiments. Drosophila is a most powerful model organism to investigate the function of genes involved in development or disease in vivo. Image processing and pattern recognition techniques can be used to extract information from microscopy images to quantify automatically distinct cellular features, but these methods are still not very extended in this model organism. Thus cellular quantification is often carried out manually, which is laborious, tedious, error prone or humanly unfeasible. Here, we present DeadEasy Mito-Glia, an image processing method to count automatically the number of mitotic cells labelled with anti-phospho-histone H3 and of glial cells labelled with anti-Repo in Drosophila embryos. This programme belongs to the DeadEasy suite of which we have previously developed versions to count apoptotic cells and neuronal nuclei. Having separate programmes is paramount for accuracy. DeadEasy Mito-Glia is very easy to use, fast, objective and very accurate when counting dividing cells and glial cells labelled with a nuclear marker. Although this method has been validated for Drosophila embryos, we provide an interactive window for biologists to easily extend its application to other nuclear markers and other sample types. DeadEasy MitoGlia is freely available as an ImageJ plug-in, it increases the repertoire of tools for in vivo genetic analysis, and it will be of interest to a broad community of developmental, cancer and neuro-biologists

Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases

Challenging compliance with international intellectual property norms in investor-state dispute settlement

Enforcing intellectual property (IP) rights abroad is not easy – not least because international IP treaties do not create global rights that can invoked in national courts. International investment law offers potential routes for overcoming these hurdles. Whenever investment treaties include IP rights as an investment and allow for investor-state dispute settlement (ISDS), investors can challenge host state measures affecting their IP rights in ISDS proceedings. As this article will show, this in turn offers a unique opportunity for invoking the standards of protection under international investment agreements (IIAs) to challenge host state compliance with international IP treaties. While challenging national IP regimes is an attractive option for right holders, these challenges potentially amount to a sea-change for the international IP regime and cause serious concern for host states. I however argue that most of the routes pursued by right holders under IIAs are unlikely to be successful. Investment protection standards such as fair and equitable treatment, umbrella clauses and most-favored nation treatment should not be construed to allow invoking alleged breaches of international IP norms in ISDS. Some IIAs however contain clauses that subject expropriation claims against compulsory licenses and other IP limitations to a test of consistency with the international IP rules governing these limitations. As they offer the only feasible route for investors to challenge host state compliance with international IP treaties, I review the implications of these clauses, recent reform proposals and suggest alternative mechanisms for aligning international IP and investment protection based on general international law.This is the author accepted manuscript. The final version is available from Oxford University Press via http://dx.doi.org/10.1093/jiel/jgw00

Influence of amyloglucosidase in bread crust properties

Enzymes are used in baking as a useful tool for improving the processing behavior or properties of baked products. A number of enzymes have been proposed for improving specific volume, imparting softness, or extend the shelf life of breads, but scarce studies have been focused on bread crust. The aim of this study was to determine the use of amyloglucosidase for modulating the properties of the bread crust and increase its crispness. Increasing levels of enzyme were applied onto the surface of two different partially bake breads (thin and thick crust bread). Amyloglucosidase treatment affected significantly (P<0.05) the color of the crust and decreased the moisture content and water activity of the crusts. Mechanical properties were modified by amyloglucosidase, namely increasing levels of enzyme promoted a decrease in the force (Fm) required for crust rupture and an increase in the number of fracture events (Nwr) related to crispy products. Crust microstructure analysis confirmed that enzymatic treatment caused changes in the bread crust structure, leading to a disruption of the structure, by removing the starchy layer that covered the granules and increasing the number of voids, which agree with the texture fragility.Authors acknowledge the financial support of Spanish Ministry of Economy and Sustainability (Project AGL2011-23802), the European Regional Development Fund (FEDER), Generalitat Valenciana (Project Prometeo 2012/064) and the Consejo Superior de Investigaciones Cientificas (CSIC). R. Altamirano-Fortoul would like to thank her grant to CSIC. The authors also thank Forns Valencians S. A. (Spain) for supplying commercial frozen partially baked breads.Altamirano Fortoul, RDC.; Hernando Hernando, MI.; Molina Rosell, MC. (2014). Influence of amyloglucosidase in bread crust properties. Food and Bioprocess Technology. 7(4):1037-1046. https://doi.org/10.1007/s11947-013-1084-xS1037104674Altamirano-Fortoul R, Hernando I & Rosell CM (2013) Texture of bread crust: puncturing settings effect and its relationship to microstructure. Journal of Texture Studies. doi: 10.1111/j.1745-4603.2012.00368.x .Altamirano-Fortoul, R., Le Bail, A., Chevallier, S., & Rosell, C. M. (2012). Effect of the amount of steam during baking on bread crust features and water diffusion. Journal of Food Engineering, 108, 128–134.Altamirano-Fortoul R & Rosell CM (2010) Alternatives for extending crispiness of crusty breads. In Proceedings of International Conference on Food Innovation, FoodInnova, 25–29 October 2010, Valencia, Spain. ISBN978-84-693-5011-.9.Arimi, J. M., Duggan, E., O’sullivan, M., Lyng, J. G., & O’riordan, E. D. (2010). Effect of water activity on the crispiness of a biscuit (crackerbread): mechanical and acoustic evaluation. Food Research International, 43, 1650–1655.Castro-Prada, E. M., Primo-Martin, C., Meinders, M. B. J., Hamer, R. J., & Van Vliet, T. (2009). Relationship between water activity, deformation speed, and crispness characterization. Journal of Texture Studies, 40, 127–156.Esveld, D. C., Van Der Sman, R. G. M., Van Dalen, G., Van Duynhoven, J. P. M., & Meinders, M. B. J. (2012). Effect of morphology on water sorption in cellular solid foods. Part I: Pore Scale Network Model. Journal of Food Engineering, 109, 301–310.Goedeken, D. L., & Tong, C. H. (1993). Permeability measurements of porous food materials. Journal of Food Science, 58, 1329–1331.Gondek, E., Lewicki, P. P., & Ranachowski, Z. (2006). Influence of water activity on the acoustic properties of breakfast cereals. Journal of Texture Studies, 37, 497–515.Guerrieri, N., Eynard, L., Lavelli, V., & Cerletti, P. (1997). Interactions of protein and starch studied through amyloglucosidase action. Cereal Chemistry, 74, 846–850.ICC. (1994). Standard methods of the International Association for Cereal Science and Technology. Vienna: Austria.Heenan, S. P., Dufour, J. P., Hamid, N., Harvey, W., & Delahunty, C. M. (2008). The sensory quality of fresh bread: descriptive attributes and consumer perceptions. Food Research International, 41, 989–997.Heiniö, R. L., Nordlund, E., Poutanen, K., & Buchert, J. (2012). Use of enzymes to elucidate the factors contributing to bitterness in rye flavor. Food Research International, 45, 31–38.Hug-Iten, S., Escher, F., & Conde-Petit, B. (2003). Staling of bread: role of amylose and amylopectin and influence of starch-degrading enzymes. Cereal Chemistry., 80(6), 654–661.Jakubczyk, E., Marzec, A., & Lewicki, P. P. (2008). Relationship between water activity of crisp bread and its mechanical properties and structure. Polish Journal of Food and Nutrition Sciences, 58(1), 45–51.Luyten, A., Pluter, J. J., & Van Vliet, T. (2004). Crispy/crunchy crusts of cellular solid foods: a literature review with discussion. Journal of Texture Studies, 35, 445–492.Potter, N. N., & Hotchkiss, J. H. (1998). Food dehydration and concentration. In N. N. Potter & J. H. Hotchkiss (Eds.), Food Science (5th ed.). New York: Aspen Publishers.Primo-Martin, C., Van de Pijpekamp, A., Van Vliet, T., Jongh, H. H. J., Plijter, J. J., & Hamer, R. J. (2006). The role of the gluten network in the crispness of bread crust. Journal of Cereal Science, 43, 342–352.Primo-Martin, C., Sozer, N., Hamer, R. J., & Van Vliet, T. (2009). Effect of water activity on fracture and acoustic characteristics of a crust model. Journal of Food Engineering, 90, 277–284.Roudaut, G., Dacremont, C., & Le Meste, M. (1998). Influence of water on the crispness of cereal-based foods: acoustic, mechanical, and sensory studies. Journal of Texture Studies, 29, 199–213.Roudaut, G., Dacremont, C., Pamies, B. V., Colas, B., & Le Meste, M. (2002). Crispness: a critical review on sensory and material science approaches. Trends in Food Science and Technology, 13, 217–227.Rojas JA (2000) Uso combinado de hidrocoloides y alfa-amilasa como mejorantes en panificación. Dissertation PhD Thesis. Universidad Politécnica de ValenciaRosell, C. M. (2007). Vitamin and mineral fortification of bread. In B. Hamaker (Ed.), Technology of functional cereal products. Cambridge: Woodhead Publishing Ltd.Rosell, C. M. (2011). The science of doughs and bread quality. In V. R. Preedy, R. R. Watson, & V. B. Patel (Eds.), Flour and breads and their fortification in health and disease prevention (pp. 3–14). London: Academic.Rosell CM, Altamirano-Fortoul R & Hernando I (2011) Mechanical properties of bread crust by puncture test and the effect of sprayed enzymes. In: Proceedings of 6th International Congress Flour. Bread’11, 8th Croatian Congress of Cereal Technologist, 12–14 October 2011, Opatija, Croatia. ISSN 1848–2562.Sahlström, S., & Brathen, E. (1997). Effects of enzyme preparations for baking, mixing time and resting time on bread quality and bread staling. Food Chemistry, 58, 75–80.Sharma K & Singh J (2010) Enzymes in baking industry. Panesar, P.S.; Marwaha, S.S and Chopra, H.K. (Eds), Enzymes in food processing, fundamentals and potential applications, IK International Publishing House Pvt. Ltd, New Delhi, India.Stokes, D. J., & Donald, A. M. (2000). In situ mechanical testing of dry and hydrated breadcrumb in the environmental scanning electron microscope (ESEM). Journal of Materials Science, 35, 599–607.Tsukakoshi, Y., Naito, S., & Ishida, N. (2008). Fracture intermittency during a puncture test of cereal snacks and its relation to porous structure. Food Research International, 41, 909–917.Van Benschop CHM, Terdu AG & Hille JDR (2012) Baking enzyme composition as SSL replacer. Patent No.US2012164272.Van Eijk JH (1991) Retarding the firming of bread crumb during storage. Patent No. US5023094.Van Hecke, E., Allaf, K., & Bouvier, J. M. (1998). Texture and structure of crispy-puffed food products—part II: mechanical properties in puncture. Journal of Texture Studies, 29, 617–632.Vanin, F. M., Lucas, T., & Trystram, G. (2009). Crust formation and its role during bread baking. Trends in Food Science and Technology, 20, 333–343.Van Nieuwenhuijzen, N. H., Primo-Martin, C., Meinders, M. B. J., Tromp, R. H., Hamer, R. J., & Van Vliet, T. (2008). Water content or water activity: what rules crispy behavior in bread crust? Journal of Agricultural and Food Chemistry, 56, 6432–6438.Van Oort, M. (2010). Enzymes in bread making. In R. J. Whitehurst & M. Van Oort (Eds.), Enzymes in food technology (2nd ed.). Iowa: Wiley-Blackwell.Vidal, F.D., Guerrety, A.B. (1979) Antistaling agent for bakery products. Patent No. US54160848.Wählby, U., & Skjoldebrand, C. (2002). Reheating characteristic of crust formed on buns, and crust formation. Journal of Food Engineering, 53, 177–184.Würsch, P., & Gumy, D. (1994). Inhibition of amylopectin retrogradation by partial beta-amylosis. Carbohydrate Research, 256, 129–137.Xiong, X., Narsimhan, G., & Okos, M. R. (1991). Effect of composition and pore structure on binding energy and effective diffusivity of moisture in porous foods. Journal of Food Engineering, 15, 187–208