Synergistic Treatment of Multidrug-Resistant Bacterial Biofilms Using Silver Nanoclusters Incorporated into Biodegradable Nanoemulsions.

Multidrug resistance (MDR) in bacteria is a critical global health challenge that is exacerbated by the ability of bacteria to form biofilms. We report a combination therapy for biofilm infections that integrates silver nanoclusters (AgNCs) into polymeric biodegradable nanoemulsions (BNEs) incorporating eugenol. These Ag-BNEs demonstrated synergistic antimicrobial activity between the AgNCs and the BNEs. Microscopy studies demonstrated that Ag-BNEs penetrated the dense biofilm matrix and effectively disrupted the bacterial membrane. The Ag-BNE vehicle also resulted in more effective silver delivery into the biofilm than AgNCs alone. This combinacional system featured disruptionof biofilms by BNEs and enhanced delivery of AgNCs for synergy to provide highly efficient killing of MDR biofilms.

Preliminary Capillary Flow Experiments with Amyloid-ß, Possible Needle and Capillary Aß Adsorption, and a Proposal for Drug Evaluation Under Shear Conditions.

Amyloid-ß (Aß) solution injections into an aqueous mobile phase moving through narrow bore stainless-steel capillary tubing results in adsorption of at least 99% Aß within the tubing or injection valve. However, if flow is stopped for a period of 5-10 minutes, then started, wall desorption yields Aß-containing molecules in the new effluent. The amount of desorbed Aß-containing effluent depends on flow rate, period of flow cessation, and number of successive Aß injections into the same tube without cleaning between injections. Unexpected multiple chromatographic peaks in these experiments seem to imply "separation" of released, previously adsorbed Aß-containing products in the empty capillary tubing. These preliminary experiments raise questions about possible errors in Alzheimer's disease (AD) spinal tap analyses, which use stainless-steel needles of approximately the same inner diameter and encounter similar flow rates as those in our capillary experiments. Microliter syringes and HPLC connectors also contain stainless-steel tubing that have similar inner diameter dimensions and similar flow rates. The capillary system involved in these experiments has previously been proposed as a model system for studying the effects of shear on Aß within the brain because it offers a research environment that provides highly restrictive flow through very small dimension channels. A suggestion is made for the use of this system in exploratory anti-amyloid drug studies in which both the drug and Aß are injected in the same solution so that both drug and Aß are subjected to the same shear environment. Reduction in adsorbed Aß is suggested as an indicator of effective anti-Aß drugs.

Effect of Al2O3 nanoparticles on bacterial membrane amphiphilic biomolecules.

Lipopolysaccharide (LPS) and l-α-phosphatidyl-ethanolamine (PE) are amphiphilic biomolecules that are key constituents in the outer cell membranes of Gram-negative bacteria. In this study, micelles and vesicles of LPS/PE were used as cell membrane models to evaluate nanoparticle (NP) effects on membrane structure. Using atomic force microscopy and sorption experiments, we observed that LPS vesicles were dispersed by Al(2)O(3) NPs because of the strong attachment between LPS polysaccharide chain and oxide surface. LPS coated on the Al(2)O(3) NPs, and formed a layer of tens of nanometer as shown from atom force microscopy (AFM) images. High pH, ionic strength and sulfate concentration inhibited the adsorption of LPS molecules on NPs. The features of PE vesicles were changed after exposing to Al(2)O(3) NPs, inducing large round vesicles with detectable thickness as revealed by AFM, resulting in the increase of vesicle diameter. The aqueous stability of PE was disturbed when adding Al(2)O(3) NPs, but was enhanced by increasing pH. The interaction between NPs and membrane amphiphilic biomolecules may affect membrane fluidity, integrity and lateral organization, which is important for NP safety evaluation and for new NP designs in biological and biomedical applications.

Structure of the preamyloid dimer of beta-2-microglobulin from covalent labeling and mass spectrometry.

Beta-2-microglobulin (beta2m) self-associates into fibrillar amyloid deposits in the musculoskeletal system of patients undergoing hemodialysis treatment. Previous studies have shown that stoichiometric amounts of Cu(II) at near physiological conditions can cause beta2m to organize into native-like dimers prior to forming amyloid fibrils. Here, we report the results from selective covalent labeling reactions combined with mass spectrometry that provide insight into the amino acid residues that mediate dimer formation in the wild-type protein. Using three complementary covalent labeling reagents, we find that the dimer interface is formed by the antiparallel stacking of ABED beta-sheets from two beta2m monomers. In addition, our data clearly indicate that a dimer interface involving the interactions of D-D strands from separate protein units as seen in the recent crystal structures of two mutant beta2m oligomers is unlikely.

Identification of the copper(II) coordinating residues in the prion protein by metal-catalyzed oxidation mass spectrometry: evidence for multiple isomers at low copper(II) loadings.

While the Cu(II) binding sites of the prion protein have been well studied under Cu-saturation conditions, the identity of the residues involved in coordinating Cu(II) at low stoichiometries and the order in which the binding sites load with Cu(II) remain unresolved. In this study, we have used two mass spectrometry based methods to gather insight into Cu(II)-prion binding under different stoichiometric loadings of Cu(II). The first method uses metal-catalyzed oxidation reactions to site specifically modify the residues bound to Cu(II) in solution, and the second method determines Cu binding sites based on the protection of His from modification by diethyl pyrocarbonate when this residue binds Cu(II) in solution. For both methods, the residues that are labeled by these reactions can then be unambiguously identified using tandem mass spectrometry. Upon applying these two complementary methods to a construct of the prion protein that contains residues 23-28 and 57-98, several noteworthy observations are made. Coordination of Cu(II) by multiple His imidazoles is found at 1:1 and 1:2 PrP:Cu(II) ratios. Notably, there appear to be four to seven isomers of this multiple histidine coordination mode in the 1:1 complex. Furthermore, our data clearly show that His96 is the dominant Cu(II) binding ligand, as in every isomer His96 is bound to Cu(II). The individual octarepeat binding sites begin to fill at ratios of 1:3 PrP:Cu(II) with no clear preference for the order in which they load with Cu(II), although the His77 octarepeat appears to saturate last. The existence of several "degenerate" Cu binding modes at low PrP:Cu(II) ratios may allow it to more readily accept additional Cu(II) ions, thus allowing PrP to transition from a singly Cu(II) bound state to a multiply Cu(II) bound state as a function of cellular Cu(II) concentration.