No abstract

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 bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable.

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections.

The contamination crisis of 2008 has brought to light several risks associated with use of animal tissue derived heparin. Because the total chemical synthesis of heparin is not feasible, a bioengineered approach has been proposed, relying on recombinant enzymes derived from the heparin/HS biosynthetic pathway and Escherichia coli K5 capsular polysaccharide. Intensive process engineering efforts are required to achieve a cost-competitive process for bioengineered heparin compared to commercially available porcine heparins. Towards this goal, we have used 96-well plate based screening for development of a chitosan-based purification process for heparin and precursor polysaccharides. The unique pH responsive behavior of chitosan enables simplified capture of target heparin or related polysaccharides, under low pH and complex solution conditions, followed by elution under mildly basic conditions. The use of mild, basic recovery conditions are compatible with the chemical Ndeacetylation/N-sulfonation step used in the bioengineered heparin process. Selective precipitation of glycosaminoglycans (GAGs) leads to significant removal of process related impurities such as proteins, DNA and endotoxins. Use of highly sensitive liquid chromatographymass spectrometry and nuclear magnetic resonance analytical techniques reveal a minimum impact of chitosan-based purification on heparin product composition.