Deficiency in insulin secretion and function that characterize type 2 diabetes often requires administration of extraneous insulin, leading to injection-site amyloidosis. Insulin aggregation at neutral pH is not well understood. Although oligomer formation is believed to play an important role, insulin oligomers have not been fully characterized yet. Here, we elucidate similarities and differences between in vitro insulin aggregation at acidic and neutral pH for a range of insulin concentrations (2.5-100 μM) by using kinetic thioflavin T fluorescence, circular dichroism, atomic force and electron microscopy imaging. Importantly, we characterize the size distribution of insulin oligomers at different assembly stages by the application of covalent cross-linking and gel electrophoresis. Our results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, where oligomers up to heptamers are formed in 10 μM insulin. These findings suggest that oligomers may be on- and off-pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, which is required to induce insulin aggregation at neutral pH, is shown to increase fibril formation rate and fibrillar mass both by an order of magnitude. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer-sized aggregates, which may be of physiological relevance and provides insight into injection-site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.

The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266-579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402-494 (6-8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6-8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.

The experiments described in this paper have two goals. The first goal is to show how students can perform simple but fundamental measurements of objects moving through simple liquids (such as water, oil, or honey). In doing so, students can verify Stokes' law, which governs the motion of spheres through simple liquids, and see how it fails at higher object speeds. Moreover, they can qualitatively study fluid patterns at various object speeds (Reynolds numbers). The second goal is to help students make connections between physics and other sciences. Specifically, the results of these experiments can be used to help students understand the role of fluid motion in determining the shape of an organism, or where it lives. At Saint Josephs University we have developed these experiments as part of a newly developed course in biomechanics where both physics and biology undergraduate students bring their ideas and expertise to enrich a shared learning environment.

region, and found significant effects of the single point mutation on the global conformational ensemble. In the present study, we investigate the effects of the presence of the ordered region on this conformational ensemble, as well as the role of the SNP on docking of the disordered prodomain to the ordered region, using fully atomistic Hamiltonian Replica Exchange Simulations. These computational investigations complement previous NMR approaches that were restricted to the isolated prodomain, and serve as model calculations for studying the role of adjacent structured regions on conformations of intrinsically disordered regions.

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