Pathogenic serum amyloid A 1.1 shows a long oligomer-rich fibrillation lag phase contrary to the highly amyloidogenic non-pathogenic SAA2.2.

Serum amyloid A (SAA) is best known for being the main component of amyloid in the inflammation-related disease amyloid A (AA) amyloidosis. Despite the high sequence identity among different SAA isoforms, not all SAA proteins are pathogenic. In most mouse strains, the AA deposits mostly consist of SAA1.1. Conversely, the CE/J type mouse expresses a single non-pathogenic SAA2.2 protein that is 94% identical to SAA1.1. Here we show that SAA1.1 and SAA2.2 differ in their quaternary structure, fibrillation kinetics, prefibrillar oligomers, and fibril morphology. At 37 °C and inflammation-related SAA concentrations, SAA1.1 exhibits an oligomer-rich fibrillation lag phase of a few days, whereas SAA2.2 shows virtually no lag phase and forms small fibrils within a few hours. Deep UV resonance Raman, far UV-circular dichroism, atomic force microscopy, and fibrillation cross-seeding experiments suggest that SAA1.1 and SAA2.2 fibrils possess different morphology. Both the long-lived oligomers of pathogenic SAA1.1 and the fleeting prefibrillar oligomers of non-pathogenic SAA2.2, but not their respective amyloid fibrils, permeabilized synthetic bilayer membranes in vitro. This study represents the first comprehensive comparison between the biophysical properties of SAA isoforms with distinct pathogenicities, and the results suggest that structural and kinetic differences in the oligomerization-fibrillation of SAA1.1 and SAA2.2, more than their intrinsic amyloidogenicity, may contribute to their diverse pathogenicity.

Design of monodisperse and well-defined polypeptide-based polyvalent inhibitors of anthrax toxin.

The design of polyvalent molecules, presenting multiple copies of a specific ligand, represents a promising strategy to inhibit pathogens and toxins. The ability to control independently the valency and the spacing between ligands would be valuable for elucidating structure-activity relationships and for designing potent polyvalent molecules. To that end, we designed monodisperse polypeptide-based polyvalent inhibitors of anthrax toxin in which multiple copies of an inhibitory toxin-binding peptide were separated by flexible peptide linkers. By tuning the valency and linker length, we designed polyvalent inhibitors that were over four orders of magnitude more potent than the corresponding monovalent ligands. This strategy for the rational design of monodisperse polyvalent molecules may not only be broadly applicable for the inhibition of toxins and pathogens, but also for controlling the nanoscale organization of cellular receptors to regulate signaling and the fate of stem cells.

Evaluation of Antibacterial Activity of Madhuca longifolia (Mahua) Stem Extract Against Streptococcus mutans: An In Vitro Study.

Introduction Madhuca longifolia is one of the important folklore medicinal plants with a plethora of established pharmaceutical properties. Its twigs are used as chewing sticks (toothbrushes), and it is believed that if a person uses it daily, it will make their gum healthy and strong. No study has ever been conducted to evaluate the antibacterial effect of M. longifolia extracts against oral microorganisms. Materials and methods Fresh stem twigs (Madkam Kaarkad) of M. longifolia were collected and dried. The dried stem was cut into small pieces, 5 g of which was mixed with 50 ml distilled water (in the ratio 1:10) and kept for two days for maceration. After two days, the liquid was filtered and the final filtrate was obtained, from which dry pellets were made and stored in the refrigerator at 4°C. Brain heart infusion agar was used as a medium to grow the lyophilized bacteria. Pure strains of Streptococcus mutans 890 were obtained from the Microbial Type Culture Collection (MTCC) and MTCC-suggested protocol was followed for the revival of lyophilized bacteria. The agar well diffusion method was used to determine the zone of inhibition. The extract of stems with different concentrations (10%, 7.5%, 5.0%, and 2.5%) and at different volumes (100 µl, 150 µl, 200 µl, and 250 µl) was transferred to the agar plates. Chlorhexidine 0.2% was used as a control and it was also transferred to agar plates, which were incubated aerobically at 37°C for 24 hours. Antibacterial activity was interpreted from the size of the diameter of zones of inhibition measured in millimeters using a measuring scale in all the agar plates. Results The minimum zone of inhibition of 11 mm at 2.5% concentration and 100 µl volume of M. longifolia extract and the maximum zone of inhibition of 20 mm at 10% concentration and 250 µl volume was notified. While for chlorhexidine at 0.2% concentration, the zone of inhibition obtained was 9.5 mm at 40 µl volume. The minimum inhibitory concentration (MIC) value of M. longifolia was found to be 35 mg/ml. Conclusion M. longifolia showed marked antibacterial activity against S. mutans and has a high MIC value. Therefore, this plant can be considered an effective agent against oral diseases like dental caries.

Characterization of the oligomerization and aggregation of human Serum Amyloid A.

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.