Toxoplasma gondii usually causes an asymptomatic and then latent infection in human adults; however, a potentially fatal disseminated form can occur in immunocompromised patients. Given that the diagnosis of acute Toxoplasma infection, as opposed to latent disease, relies on finding direct evidence of T. gondii infection in tissue, pathologic examination is critical. There have only been a few reports describing the cytomorphology of Toxoplasma in exfoliative cytology, and no reports of the findings in Thin Prep. In this report, we describe a fatal case of toxoplasmosis in a cardiac transplant patient that was diagnosed by respiratory cytopathology. Although the extracellular organisms were well visualized on the Wright‐Giemsa stained cytospin, they were only faintly seen on the Pap‐stained cytospin trapped within mucin and were not easily appreciated on the ThinPrep slides nor the H&E stained cell block sections. An immunohistochemical stain for Toxoplasma performed on the cell block was strongly positive, and an autopsy performed on the patient confirmed disseminated infection. Our case illustrates that the diagnosis of Toxoplasma in exfoliative cytology specimens can be challenging since organisms are not well visualized on ThinPrep or Pap‐stained material; therefore, Wright‐Giemsa stained material can be particularly helpful. Diagn. Cytopathol. 2012. © 2011 Wiley Periodicals, Inc.

The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers --> monomers --> partially unfolded monomers --> oligomeric aggregates --> fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.

The effect of a low strength oscillating electric field on the conformation of Bovine Serum Albumin (BSA) and Lysozyme in solution has been measured. A purpose built cell has been used to measure the real time autofluorescence and Circular Dichroism of the protein solutions exposed to electric fields of differing strength and frequency. Exposure to the electric fields results in protein unfolding for both Lysozyme and BSA. The applied field strengths are extremely small compared to the protein inter-chain intra-molecular forces. We propose a model whereby the electrophoretic motion of the proteins leads to a frictional force that results in protein unfolding. For BSA and Lysozyme in the electric fields used in this study, the shear rates at the protein surface under electrophoretic motion are of order 10(3) and 10(4) s(-1) respectively. Prolonged electric field exposure results in significant frictional energy dissipation in the proteins. The energy dissipated in the proteins results in protein unfolding, which is a critical initial step for protein aggregation and potentially amyloid fibril formation.

We have applied a uniform, shear-driven flow field (Couette flow) to study the effect of shear on the structure and conformation of aqueous bovine insulin, in situ and in real time, using intrinsic Tyr fluorescence and circular dichroism (CD) spectroscopy. The morphology of post-shear insulin samples was analyzed using atomic force microscopy (AFM). Both fluorescence and CD data show a shear-dependent deformation of bovine insulin in Couette flow. The shear effect is more pronounced with increasing shear rate. AFM images show large aggregates for insulin samples sheared at 200 and 400 s(-1), whereas samples sheared at 600 s(-1) contained fibrillar forms. We hypothesize that helical segments unfold upon extensional strain in the deformation flow field, resulting in unstructured, aggregation-prone insulin molecules. The occurrence of rotational diffusion in the direction of flow facilitates the coalescence of deformed insulin molecules into oligomeric aggregates. The size of the insulin aggregates diminished with increasing shear rate. This shows that the deformation cycle in fast flow fields retards the formation of large aggregates and promotes the ordering of deformed insulin molecules into the more stable fibrillar forms.

The fibrillogenesis of Abeta1-40 proceeds via three main stages: (i) formation of aggregates from monomers, (ii) linear association of these aggregates to form "beaded" protofibrils, and (iii) fusion and structural reorganization of protofibrils into mature fibrils. We have studied the effect of shear on the rate of each of these steps through a combination of fluorescence, atomic force microscopy, and circular dichroism experiments. We find that shear increases the rate of the first two stages (aggregation and protofibril formation) and inhibits the third. Our hypothesis is that in the first stage shear mechanically perturbs the peptide from its native state inducing aggregation via hydrophobic interactions; in the second stage, shear enhances the linear alignment of aggregates due to minimization of drag in the shear flow field; in the third stage, exposure to constant and uniform shear inhibits the formation of mature fibrils.

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Amyloid fibrils are large ordered fibrillar aggregates formed from mis-folded proteins. Fibril formation is inhibited using a generic macromolecular structure.