The role of DNA microarrays in Toxoplasma gondii research, the causative agent of ocular toxoplasmosis

Ocular toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, is the leading cause of retinochoroiditis. Toxoplasma is an obligate intracellular pathogen that replicates within a parasitophorous vacuole. Infections are initiated by digestion of parasites deposited in cat feces or in undercooked meat. Parasites then disseminate to target tissues that include the retina where they then develop into long-lived asymptomatic tissue cysts. Occasionally, cysts reactivate and growth of newly emerged parasites must be controlled by the host’s immune system or disease will occur. The mechanisms by which Toxoplasma grows within its host cell, encysts, and interacts with the host’s immune system are important questions. Here, we will discuss how the use of DNA microarrays in transcriptional profiling, genotyping, and epigenetic experiments has impacted our understanding of these processes. Finally, we will discuss how these advances relate to ocular toxoplasmosis and how future research on ocular toxoplasmosis can benefit from DNA microarrays

Induced Pluripotent Stem Cells Produced From Cryopreserved Pygmy Sperm Whale (Kogia Breviceps) Lung Tissue

Concern over marine mammal health has risen in the last decade due to increases in mortality, unusual mortality events and previously undetected pathologies. Developing environmental trends, such as global warming and acute environmental insults, such as large-scale oil spills have been implicated. Unfortunately, the limited availability of biological samples hinders basic biological/toxicological studies. To compensate for the limited availability of samples and to make the most of rare samples that do become available, we have pursued methods to establish and expand cultures of primary cell types and reconstituted tissues from marine mammals for long-term use as surrogates for freshly isolated samples. To this end, we first developed a method to cryopreserve tissues from deceased/stranded individuals and thereby initiate establishment of a tissue bank biorepository. We were able to establish conditions and perform this successfully on lung tissue from a Pygmy Sperm Whale (PSW; Kogia breviceps). Using these conditions, we were able to establish cultures of viable primary lung cell types from tissue fragments that had been cryopreserved several months earlier (immediately after the stranding event). We then applied genetic or chemical means for generating induced pluripotent stem (iPS) cells to one of these primary cultures (lung fibroblasts). We observed that the genetic means, involving the forced expression of Klf4, Oct3/4, Sox2 and Myc, did produce PSW cells with long-term expansion and differentiative capability, while the chemical means using valproic acid (driving histone deacetylase inhibition and chromatin decondensation) did not. Finally, we employed specialized culture conditions to differentiate in bulk PSW cells with vascular endothelial and airway epithelial-like properties. The cryopreservation of viable tissue samples and the generation of iPS-like cells with unlimited expansion & pluripotent differentiative capacities from marine mammals is anticipated to have far-reaching impacts on levels ranging from basic biology to environmental policy related to stressors of marine and land mammalian health

Role of the N-Terminal Seven Residues of Surfactant Protein B (SP-B)

Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B) is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the “insertion sequence”. These studies employed a construct of SP-B, SP-B (1–25,63–78), also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state 2H NMR were used to study the structure of SP-B (1–25,63–78) and its interactions with phospholipid bilayers. Comparison of results for SP-B (8–25,63–78) and SP-B (1–25,63–78) demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics