Background: Murine SAA1.1 is pathogenic and SAA2.2 is non-pathogenic in AA amyloidosis. Results: SAA1.1 and SAA2.2 exhibit different biophysical properties, including fibrillation kinetics and fibril morphology. Conclusion:The distinct biophysical properties of highly homologous SAA proteins may contribute to their different pathogenicity during chronic inflammation. Significance: Structural and kinetic factors, more than their intrinsic amyloidogenicity, may determine the diverse pathogenicity among nearly identical SAA isoforms.

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This study reports a facile biomineralization route for gold microplates (GMPs) synthesis using bovine serum albumin (BSA) as a reductant and stabilizing agent. Adding BSA to HAuCl4 solution yields spontaneous versatile anisotropic and partially hollow GMPs upon aging. We hypothesize that the instantaneous protein denaturation at low pH enabled access to serine and threonine hydroxyl, and sulfhydryl groups of BSA, which act as a reductant and stabilizer, respectively. This reaction could be hastened by increasing the temperature well beyond 65 °C. Transmission electron microscopy/X-ray diffraction studies revealed highly crystalline and anisotropic structures (triangle, pentagon, and rectangle). Atomic force microscopy/scanning electron microscopy analyses demonstrated unique morphology of microplates with a partially void core and BSA mineralized edge structure. RAW 264.7 mice peritoneal macrophage-microplate interaction studies using live cell confocal imaging reveal that cells are capable of selectively internalizing smaller GMPs. Large GMPs are preferentially picked with sharp vertices but cannot be internalized and exhibit frustrated phagocytosis-like phenomenon. We explored particle phagocytosis as an actin mediated process that recruits phagosome-like acidic organelles, shown by a lysosensor probe technique. The biocompatible GMPs exhibited ∼70% paclitaxel (PCL) loading and sustained release of PCL, showing antitumor activity with the MCF-7 cell line, and could be a novel drug carrier for breast cancer therapy.

The fibrillation of Serum Amyloid A (SAA) – a major acute phase protein – is believed to play a role in the disease Amyloid A (AA) Amyloidosis. To better understand the amyloid formation pathway of SAA, we characterized the oligomerization, misfolding, and aggregation of a disease-associated isoform of human SAA – human SAA1.1 (hSAA1.1) – using techniques ranging from circular dichroism spectroscopy to atomic force microscopy, fluorescence spectroscopy, immunoblot studies, solubility measurements, and seeding experiments. We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C. Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway. A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

Bispecific antibodies are a growing class of therapeutic molecules. Many of the current bispecific formats require DNA engineering to convert the parental monoclonal antibodies into the final bispecific molecules. We describe here a method to generate bispecific molecules from hybridoma IgGs in 3-4 d using chemical conjugation of antigen-binding fragments (Fabs) (bisFabs). Proteolytic digestion conditions for each IgG isotype were analyzed to optimize the yield and quality of the final conjugates. The resulting bisFabs showed no significant amounts of homodimers or aggregates. The predictive value of murine bisFabs was tested by comparing the T-cell redirected cytotoxic activity of a panel of antibodies in either the bisFab or full-length IgG formats. A variety of antigens with different structures and expression levels was used to extend the comparison to a wide range of binding geometries and antigen densities. The activity observed for different murine bisFabs correlated with those observed for the fulllength IgG format across multiple different antigen targets, supporting the use of bisFabs as a screening tool. Our method may also be used for the screening of bispecific antibodies with other mechanisms of action, allowing for a more rapid selection of lead therapeutic candidates.