Recent developments in the UltraScan-III software make it possible to model multi-speed analytical ultracentrifugation sedimentation velocity experiments using finite-element solutions of the Lamm ...equation. Using simulated data, we demonstrate here how these innovations can be used to enhance the resolution of sedimentation velocity experiments when compared to single-speed experiments. Using heterogeneous systems covering as much as five orders of magnitude in molar mass and fivefold in anisotropy, we compare results from runs performed at multiple speeds to those obtained from single-speed experiments, fitted individually and analyzed globally over multiple speeds, and quantify resolution for sample heterogeneous in size and anisotropy. We also provide guidance on the design of multi-speed experiments and offer a program that can be used to deduce optimal spacing of rotor speeds and speed step durations when a few parameters from the experiment can be estimated. These include the meniscus position, the sedimentation coefficient of the largest species in a mixture, and a solute distribution. Our results show that errors observed in the determination of hydrodynamic parameters for system with great heterogeneity are markedly reduced when multi-speed analysis is employed.
Here we report on the identification and applications of an aqueous 29-atom silver cluster stabilized with 12 lipoate ligands, i.e. Ag29(R-α–LA)12 or (29,12), wherein R-α–LA = R-α-lipoic acid, a ...natural dithiolate. Its uniformity is checked by HPLC-ESI-MS and analytical ultracentrifugation, which confirms its small dimension (∼3 nm hydrodynamic diameter). For the first time, this cluster has been detected intact via electrospray ionization mass spectrometry, allowing one to confirm its composition, its 3- charge-state, and the 8-electron shell configuration of its metallic silver core. Its electronic structure and bonding, including T-symmetry and profound chirality in the outer shell, have been analyzed by DFT quantum-chemical calculations, starting from the known structure of a nonaqueous homologue. The cluster is effective against Methicillin-Resistant Staphylococcus aureus bacteria (MRSA) at a minimum inhibitory concentration (MIC) of 0.6 mg-Ag/mL. A preformed Candida albicans fungal biofilm, impermeable to other antifungal agents, was also inhibited by aqueous solutions of this cluster, in a dose–response manner, with a half-maximal inhibitory concentration (IC50) of 0.94 mg-Ag/mL. Scanning electron micrographs showed the post-treatment ultrastructural changes on both MRSA and C. albicans that are characteristic of those displayed after treatment by larger silver nanoparticles.
DM64 is a toxin-neutralizing serum glycoprotein isolated from
, an ophiophagous marsupial naturally resistant to snake envenomation. This 64 kDa antitoxin targets myotoxic phospholipases A
, which ...account for most local tissue damage of viperid snakebites. We investigated the noncovalent complex formed between native DM64 and myotoxin II, a myotoxic phospholipase-like protein from
venom. Analytical ultracentrifugation (AUC) and size exclusion chromatography indicated that DM64 is monomeric in solution and binds equimolar amounts of the toxin. Attempts to crystallize native DM64 for X-ray diffraction were unsuccessful. Obtaining recombinant protein to pursue structural studies was also challenging. Classical molecular modeling techniques were impaired by the lack of templates with more than 25% sequence identity with DM64. An integrative structural biology approach was then applied to generate a three-dimensional model of the inhibitor bound to myotoxin II. I-TASSER individually modeled the five immunoglobulin-like domains of DM64. Distance constraints generated by cross-linking mass spectrometry of the complex guided the docking of DM64 domains to the crystal structure of myotoxin II, using Rosetta. AUC, small-angle X-ray scattering (SAXS), molecular modeling, and molecular dynamics simulations indicated that the DM64-myotoxin II complex is structured, shows flexibility, and has an anisotropic shape. Inter-protein cross-links and limited hydrolysis analyses shed light on the inhibitor's regions involved with toxin interaction, revealing the critical participation of the first, third, and fifth domains of DM64. Our data showed that the fifth domain of DM64 binds to myotoxin II amino-terminal and beta-wing regions. The third domain of the inhibitor acts in a complementary way to the fifth domain. Their binding to these toxin regions presumably precludes dimerization, thus interfering with toxicity, which is related to the quaternary structure of the toxin. The first domain of DM64 interacts with the functional site of the toxin putatively associated with membrane anchorage. We propose that both mechanisms concur to inhibit myotoxin II toxicity by DM64 binding. The present topological characterization of this toxin-antitoxin complex constitutes an essential step toward the rational design of novel peptide-based antivenom therapies targeting snake venom myotoxins.
The Edmonton Symptom Assessment System (Revised) (ESAS-r) contains 9 questions pertaining to symptoms/well-being. It is a standardized patient-reported assessment instrument, but inconsistently used ...in palliative care. Thus, a problem exists in knowledge translation regarding routine use of the ESAS-r in palliative practice. The objective was to understand clinicians' perspectives on the use of the ESAS-r in palliative care in hospitals and at home. Qualitative focus groups (n = 14 with 46 clinicians) and interviews (n = 24) elicited views regarding use of the ESAS-r in palliative practice. Interpretive description was used as a general approach to this qualitative analysis focused on understanding clinicians' views. Palliative clinicians presented multiple perspectives of the ESAS-r pertaining to their (1) underlying values, (2) disparate purposes, and (3) incommensurate responses toward use in daily practice. Benefits and challenges supported diversity within these themes, highlighting divergence among perspectives and complexity of integrating a standardized tool in patient care. Integration of the ESAS-r in palliative care requires (1) educational support for developing competence; (2) consideration of clinicians' existing, heterogeneous beliefs regarding the use of standardized assessment instruments; and (3) Consultation with multidisciplinary practitioners about optimal ways that ESAS-r results can be used in a person-centered approach to palliative care.
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