Anthony E. Butterfield

Size and shape characterization of hydrated and desiccated exosomes

Exosomes are stable nanovesicles secreted by cells into the circulation. Their reported sizes differ substantially, which likely reflects the difference in the isolation techniques used, the cells that secreted them, and the methods used in their characterization. We analyzed the influence of the last factor on the measured sizes and shapes of hydrated and desiccated exosomes isolated from the serum of a pancreatic cancer patient and a healthy control. We found that hydrated exosomes are close-to-spherical nanoparticles with a hydrodynamic radius that is substantially larger than the geometric size. For desiccated exosomes, we found that the desiccated shape and sizing are influenced by the manner in which drying occurred. Isotropic desiccation in aerosol preserves the near-spherical shape of the exosomes, whereas drying on a surface likely distorts their shapes and influences the sizing results obtained by techniques that require surface fixation prior to analysis.

Comparison of Surfactants Used to Prepare Aqueous Perfluoropentane Emulsions for Pharmaceutical Applications

Perfluoropentane (PFP), a very hydrophobic, nontoxic, noncarcinogenic fluoroalkane, has generated much interest in biomedical applications, including occlusion therapy and controlled drug delivery. For most of these applications, the dispersion within aqueous media of a large quantity of PFP droplets of the proper size is critically important. Surprisingly, the interfacial tension of PFP against water in the presence of surfactants used to stabilize the emulsion has rarely, if ever, been measured. In this study, we report the interfacial tension of PFP in the presence of surfactants used in previous studies to produce emulsions for biomedical applications: polyethylene oxide-co-polylactic acid (PEO-PLA) and polyethylene oxide-co-poly-epsilon-caprolactone (PEO-PCL). Because both of these surfactants are uncharged diblock copolymers that rely on the mechanism of steric stabilization, we also investigate for comparisons sake the use of the small-molecule cationic surfactant cetyl trimethyl ammonium bromide (CTAB) and the much larger protein surfactant bovine serum albumin (BSA). The results presented here complement previous reports of the PFP droplet size distribution and will be useful for determining to what extent the interfacial tension value can be used to control the mean PFP droplet size.

Bidirectional Power Stroke by Ncd Kinesin

Optical trapping experiments reveal details of molecular motor dynamics. In noisy data, temporal structure within the power stroke of motors can be analyzed by ensemble averaging, but this obscures infrequent subcategories of events. We have here developed an analysis method that uses Kalman filtering of measurements, model-based estimation of the power strokes produced by the motor head, and automatic event classification to discriminate between different types of motor events. This method was applied to optical trap measurements of power strokes of the Drosophila kinesin-14 ncd in a three-bead geometry. We found the majority of events to be consistent with the previously discovered minus-end directed power stroke of ncd, occurring with ATP binding. Unexpectedly, 30% of apparent power strokes were plus-directed and 6% of binding events did not terminate in a discernible stroke. Ensemble averaging for each event category revealed that plus- and minus-directed strokes have different size and occur at different instants within the ncd-MT attachment sequence.

Temporally resolved molecular motor dynamics: A case of ncd-microtubule interactions

Molecular motors and the associated microfilaments play an important role in cell division, intracellular transport and signaling, and are fundamental to such neuronal functions as bidirectional protein transport to and from the periphery of neural cells. A number of neurological diseases have been linked to the abnormal function of molecular motors acting upon microtubules and an improved understanding of the role of various molecular motors has been recently cited as one of the new frontiers of neuroscience. Details of molecular motor-microtubule interactions on a single molecular level can be revealed during optical trapping experiments. Until recently, low signal-to-noise ration of optical trap data required analysis by ensemble averaging, which obscures interaction details and masks infrequent sub-categories of interaction events. In this note, we summarize a recently developed method that allowed us to reveal the details of single molecular motor-microtubule interactions as a function of time based on a time series of the measurements without averaging. This method was applied to Kinesin-14 Ncd in three-bead optical trap geometry and it was found that, contrary to previous results obtained by ensemble averaging, the ncd power strokes are bidirectional, with the majority of strokes directed towards the minus end of the microtubule. The developed capability to analyze the molecular motor dynamics without averaging and in real time opens the possibility to study the mechanism of the intracellular transport, cytoskeletal organization, and therapeutic effects on a single molecular level.

Classification of single molecular motor events

Abstrucf-This paper describes the development of two methods for automatic classification of molecular events. The methods are applied to experimental results obtained to study the interaction of a single microtubule with a single Ncd molecular motor. The data were previously obtained using optical trap assay, and have a very low signal-to-noise ratio of approximately 0.1. The first method can be viewed as a syntactic classification. In an alternative approach. radial basis neural networks, trained with simulated data generated by a system of stochastic, Langevin-like differential equations, are used for classification. Following classification. the molecular events are ensemble averaged separately for different types of events. The analysis of the results suggests the existence of Ncd-microtubule interaction events, which deviate from the traditional view on the kinetics of the Ncd-microtubule interactions. Tnis paper employs a novel paradigm, which emphasizes the importance of the modelbased, time-resolved filtering of the experimental data on molecular interactions, especially for the case of biological molecules, followed by the analysis of different goups of events, automatically segregated according to features revealed during the single realization analysis of the experimental results.