Deployment of the 1st Area Medical Laboratory in a Split-Based Configuration During the Largest Ebola Outbreak in History.

BACKGROUND: The U.S. Army 1st Area Medical Laboratory (1st AML) is currently the only deployable medical CBRNE (Chemical, Biological, Radiological, Nuclear, and Explosives) laboratory in the Army's Forces Command. In support of the United States Agency for International Development Ebola response, the U.S. military initiated Operation United Assistance (OUA), and deployed approximately 2,500 service members to support the Government of Liberia's Ebola control efforts. Due to its unique molecular diagnostic and expeditionary capabilities, the 1st AML was ordered to deploy in October of 2014 in support of OUA via establishment of Ebola testing laboratories. To meet the unique mission requirements of OUA, the unit was re-organized to operate in a split-based configuration and sustain four separate Ebola testing laboratories. METHODS: This article is a review of the 1st AML's OUA participation in a split-based configuration. Topics highlighted include pre-deployment planning/training, operational/logistical considerations in fielding/withdrawing laboratories, laboratory testing results, disease and non-battle injuries, and lessons learned. FINDINGS: Fielding the 1st AML in a split-based configuration required careful pre-deployment planning, additional training, optimal use of personnel, and the acquisition of additional laboratory equipment. Challenges in establishing and sustaining remote laboratories in Liberia included: difficulties in transportation of equipment due to poor road infrastructure, heavy equipment unloading, and equipment damage during transit. Between November 26, 2014 and February 18, 2015 the four 1st AML labs successfully tested blood samples from patients and oral swabs collected by burial teams in rural Liberia. The most significant equipment malfunction during laboratory operations was generators powering the labs, with the same problem impacting headquarters. Generator failures delayed laboratory operations/result reporting, and put temperature sensitive reagents at risk. None of the 22 1st AML soldiers (at remote labs or headquarters) had an Ebola exposure, none were infected with malaria or other tropical diseases, and none required evacuation from the time deployed to remote sites. The primary medical condition encountered was acute gastroenteritis, and within the first week of arrival to Liberia, 19 (86%) soldiers were affected. DISCUSSION/IMPACT/RECOMMENDATIONS: With proper planning and training, the 1st AML can successfully conduct split-based operations in an outbreak setting, and this capability can be utilized in future operations. The performance of the 1st AML during the current Ebola outbreak highlights the value of this asset, and the need to continue its evolution to support U.S. military operations.

Identification of an actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties.

Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin and developed an alternative model to better define this actin-binding surface, using negative-stain electron microscopy (EM), discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes that promote actin bundling.

Lipid binding to the tail domain of vinculin: specificity and the role of the N and C termini.

Vinculin is a highly conserved and abundant cytoskeletal protein involved in linking the actin cytoskeleton to the cell membrane at sites of cellular adhesion. At these sites of adhesion, vinculin plays a role in physiological processes such as cell motility, migration, development, and wound healing. Loss of normal vinculin function has been associated with cancer phenotypes, cardiovascular disease, and lethal errors in embryogenesis. The tail domain of vinculin (Vt) binds to acidic phospholipids and has been proposed to play a role in vinculin activation and focal adhesion turnover. To better characterize Vt-lipid specificity, we conducted a series of lipid co-sedimentation experiments and find that Vt shows specific association with phosphatidylinositol 4,5-bisphosphate (PIP2), compared with phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), or phosphatidylinositol (PI) in the context of mixed lipid vesicles. The C terminus of Vt has been proposed to be important for PIP2 association, as various mutations and deletions within the C-terminal reduce PIP2 association. Lipid co-sedimentation and NMR analyses indicate that removal of the hydrophobic hairpin does not alter Vt structure or PIP2 association. However, more extensive deletions within the C-terminal introduce Vt structural perturbations and reduce PIP2 binding. Intriguingly, a significant increase in PIP2 binding was observed for multiple Vt variants that perturb interactions between the N-terminal strap and helix bundle, suggesting that a rearrangement of this N-terminal strap may be required for PIP2 binding.

Vinculin tail conformation and self-association is independent of pH and H906 protonation.

Vinculin is a highly conserved cytoskeletal protein that localizes to sites of cell adhesion. The tail domain of vinculin (Vt) forms tight autoinhibitory interactions with the head domain and down-regulates vinculin function by obscuring ligand binding sites. Ligand binding is required for both vinculin activation and function, and one of vinculin's primary roles as a cell adhesion protein involves its ability to link the Actin cytoskeleton to the cell membrane. Vt can bind F-Actin and phosphoinositol 4,5-bisphosphate, and association with these ligands has been reported to cause a conformational change in Vt. Moreover, a single histidine residue, H906, was reported to be critical for both a pH dependent conformational change and pH dependent self-association. In this study, we investigate the role of pH on Vt structure and self-association. In contrast to earlier observations, our studies do not support a significant alteration in Vt conformation over this pH range. Moreover, while we identify a site of Vt dimerization, similar to that observed previously by X-ray crystallography, the weak K(d) (approximately 300 microM) determined for Vt self-association does not differ significantly between pH 5.5 and pH 7.5.

Backbone 1H, 13C, and 15N NMR assignments of the tail domain of vinculin.

Vinculin is an essential protein involved in linking the actin cytoskeleton to sites of cell-cell and cell-matrix adhesion. Here we report the majority of the backbone 1H(N), 15N, 13C(alpha), 13CO, and side chain 13C(beta) NMR resonance assignments of the actin binding tail domain of vinculin (Vt).