Probing the effects of TbHK2 on Trypanosoma brucei growth, social behavior, and inhibitor response

In sub-Saharan Africa the protozoan parasite, Trypanosoma brucei, continues to be of major concern for the health and economic development of the region. This parasite is known to cause human African trypanosomiasis (HAT or African sleeping sickness) and nagana in livestock such as cattle. Social behaviors, such as colonization and migration, are important in the study of T. brucei because of the way the parasite infects its mammalian host. During the fly bloodmeal, the parasite first passes into the gut but then eventually migrates to the fly salivary glands where it will continue to develop before transmission as a parasitic form able to infect and cause disease in humans and livestock. Past research has shown that the social motility, the ability of the multitude of parasites in an infection to move in a coordinated fashion, is affected by the removal of the T. brucei hexokinase 2 (TbHK2) gene or expression of excess copies of the TbHK2 protein. In exploring social motility phenotypes of TbHK2-deficient insect stage (procylic form, PF)T. brucei parasites and parental forms complemented with excess TbHK2 gene, this project aims to understand more about the role of TbHK2 in social motility of T. brucei. Additionally, in order to understand how hexokinase 2 could be targeted by enzyme inhibitors, known hexokinase 1 inhibitors are explored for their effects on TbHK2 complemented cells compared to the parental strain parasites

Probing the Effects of TbHK2 on Trypanosoma brucei Growth, Social Growth, and Inhibitor Response

In sub-Saharan Africa the protozoan parasite, Trypanosoma brucei, continues to be of major concern for the health and economic development of the region. This parasite is known to cause human African trypanosomiasis (HAT or African sleeping sickness) and nagana in livestock such as cattle. Social behaviors, such as colonization and migration, are important in the study of T. brucei because of the way the parasite infects its mammalian host. During the fly bloodmeal, the parasite first passes into the gut but then eventually migrates to the fly salivary glands where it will continue to develop before transmission as a parasitic form able to infect and cause disease in humans and livestock. Past research has shown that the social motility, the ability of the multitude of parasites in an infection to move in a coordinated fashion, is affected by the removal of the T. brucei hexokinase 2 (TbHK2) gene or expression of excess copies of the TbHK2 protein. In exploring social motility phenotypes of TbHK2-deficient insect stage (procylic form, PF)T. brucei parasites and parental forms complemented with excess TbHK2 gene, this project aims to understand more about the role of TbHK2 in social motility of T. brucei. Additionally, in order to understand how hexokinase 2 could be targeted by enzyme inhibitors, known hexokinase 1 inhibitors are explored for their effects on TbHK2 complemented cells compared to the parental strain parasites

Integrated, Continuous Emulsion Creamer

Automated and reproducible sample handling is a key requirement for high-throughput compound screening and currently demands heavy reliance on expensive robotics in screening centers. Integrated droplet microfluidic screening processors are poised to replace robotic automation by miniaturizing biochemical reactions to the droplet scale. These processors must generate, incubate, and sort droplets for continuous droplet screening, passively handling millions of droplets with complete uniformity, especially during the key step of sample incubation. Here, we disclose an integrated microfluidic emulsion creamer that packs (“creams”) assay droplets by draining away excess oil through microfabricated drain channels. The drained oil coflows with creamed emulsion and then reintroduces the oil to disperse the droplets at the circuit terminus for analysis. Creamed emulsion assay incubation time dispersion was 1.7%, 3-fold less than other reported incubators. The integrated, continuous emulsion creamer (ICEcreamer) was used to miniaturize and optimize measurements of various enzymatic activities (phosphodiesterase, kinase, bacterial translation) under multiple- and single-turnover conditions. Combining the ICEcreamer with current integrated microfluidic DNA-encoded library bead processors eliminates potentially cumbersome instrumentation engineering challenges and is compatible with assays of diverse target class activities commonly investigated in drug discovery