Detecting Pathogen Exposure During the Non-symptomatic Incubation Period Using Physiological Data: Proof of Concept in Non-human Primates.

Background and Objectives: Early warning of bacterial and viral infection, prior to the development of overt clinical symptoms, allows not only for improved patient care and outcomes but also enables faster implementation of public health measures (patient isolation and contact tracing). Our primary objectives in this effort are 3-fold. First, we seek to determine the upper limits of early warning detection through physiological measurements. Second, we investigate whether the detected physiological response is specific to the pathogen. Third, we explore the feasibility of extending early warning detection with wearable devices. Research Methods: For the first objective, we developed a supervised random forest algorithm to detect pathogen exposure in the asymptomatic period prior to overt symptoms (fever). We used high-resolution physiological telemetry data (aortic blood pressure, intrathoracic pressure, electrocardiograms, and core temperature) from non-human primate animal models exposed to two viral pathogens: Ebola and Marburg (N = 20). Second, to determine reusability across different pathogens, we evaluated our algorithm against three independent physiological datasets from non-human primate models (N = 13) exposed to three different pathogens: Lassa and Nipah viruses and Y. pestis. For the third objective, we evaluated performance degradation when the algorithm was restricted to features derived from electrocardiogram (ECG) waveforms to emulate data from a non-invasive wearable device. Results: First, our cross-validated random forest classifier provides a mean early warning of 51 ± 12 h, with an area under the receiver-operating characteristic curve (AUC) of 0.93 ± 0.01. Second, our algorithm achieved comparable performance when applied to datasets from different pathogen exposures - a mean early warning of 51 ± 14 h and AUC of 0.95 ± 0.01. Last, with a degraded feature set derived solely from ECG, we observed minimal degradation - a mean early warning of 46 ± 14 h and AUC of 0.91 ± 0.001. Conclusion: Under controlled experimental conditions, physiological measurements can provide over 2 days of early warning with high AUC. Deviations in physiological signals following exposure to a pathogen are due to the underlying host's immunological response and are not specific to the pathogen. Pre-symptomatic detection is strong even when features are limited to ECG-derivatives, suggesting that this approach may translate to non-invasive wearable devices.

Planning Military Drinking Water Needs: Development of a User-Friendly Smart Device Application.

Potable water is essential to maintain health and sustain military operations, but carrying and transporting water is a major logistical burden. Planning for group drinking water needs is complex, requiring understanding of sweat losses on the basis of intensity of activity, clothing biophysical parameters, and environmental conditions. Use of existing prediction equations is limited to tabled doctrine (e.g., Technical Bulletin, Medical 507) or to individuals with extensive expertise in thermal biophysics. In the present project, we translated the latest updated equations into a user-friendly Android application (Soldier Water Estimation Tool, SWET) that provides estimated drinking water required from 5 simple inputs based upon a detailed multiparametric sensitivity analysis. Users select from multiple choice inputs for activity level, clothing, and cloud cover, and manually enter exact values for temperature and relative humidity. Total drinking water needs for a unit are estimated in the Mission Planner tool on the basis of mission duration and number of personnel. In preliminary user acceptability testing, responses were overall positive in terms of ease of use and military relevance. Use of SWET for water planning will minimize excessive load (water) carriage in training and mission settings, and will reduce the potential for dehydration and/or hyponatremia to impair Warfighter health and performance.

Ultrasound-mediated gastrointestinal drug delivery.

There is a significant clinical need for rapid and efficient delivery of drugs directly to the site of diseased tissues for the treatment of gastrointestinal (GI) pathologies, in particular, Crohn's and ulcerative colitis. However, complex therapeutic molecules cannot easily be delivered through the GI tract because of physiologic and structural barriers. We report the use of ultrasound as a modality for enhanced drug delivery to the GI tract, with an emphasis on rectal delivery. Ultrasound increased the absorption of model therapeutics inulin, hydrocortisone, and mesalamine two- to tenfold in ex vivo tissue, depending on location in the GI tract. In pigs, ultrasound induced transient cavitation with negligible heating, leading to an order of magnitude enhancement in the delivery of mesalamine, as well as successful systemic delivery of a macromolecule, insulin, with the expected hypoglycemic response. In a rodent model of chemically induced acute colitis, the addition of ultrasound to a daily mesalamine enema (compared to enema alone) resulted in superior clinical and histological scores of disease activity. In both animal models, ultrasound treatment was well tolerated and resulted in minimal tissue disruption, and in mice, there was no significant effect on histology, fecal score, or tissue inflammatory cytokine levels. The use of ultrasound to enhance GI drug delivery is safe in animals and could augment the efficacy of GI therapies and broaden the scope of agents that could be delivered locally and systemically through the GI tract for chronic conditions such as inflammatory bowel disease.

Novel electrodes for underwater ECG monitoring.

We have developed hydrophobic electrodes that provide all morphological waveforms without distortion of an ECG signal for both dry and water-immersed conditions. Our electrode is comprised of a mixture of carbon black powder (CB) and polydimethylsiloxane (PDMS). For feasibility testing of the CB/PDMS electrodes, various tests were performed. One of the tests included evaluation of the electrode-to-skin contact impedance for different diameters, thicknesses, and different pressure levels. As expected, the larger the diameter of the electrodes, the lower the impedance and the difference between the large sized CB/PDMS and the similarly-sized Ag/AgCl hydrogel electrodes was at most 200 kΩ, in favor of the latter. Performance comparison of CB/PDMS electrodes to Ag/AgCl hydrogel electrodes was carried out in three different scenarios: a dry surface, water immersion, and postwater immersion conditions. In the dry condition, no statistical differences were found for both the temporal and spectral indices of the heart rate variability analysis between the CB/PDMS and Ag/AgCl hydrogel (p > 0.05) electrodes. During water immersion, there was significant ECG amplitude reduction with CB/PDMS electrodes when compared to wet Ag/AgCl electrodes kept dry by their waterproof adhesive tape, but the reduction was not severe enough to obscure the readability of the recordings, and all morphological waveforms of the ECG signal were discernible even when motion artifacts were introduced. When water did not penetrate tape-wrapped Ag/AgCl electrodes, high fidelity ECG signals were observed. However, when water penetrated the Ag/AgCl electrodes, the signal quality degraded to the point where ECG morphological waveforms were not discernible.

Universal process-inert encoding architecture for polymer microparticles.

Polymer microparticles with unique, decodable identities are versatile information carriers with a small footprint. Widespread incorporation into industrial processes, however, is limited by a trade-off between encoding density, scalability and decoding robustness in diverse physicochemical environments. Here, we report an encoding strategy that combines spatial patterning with rare-earth upconversion nanocrystals, single-wavelength near-infrared excitation and portable CCD (charge-coupled device)-based decoding to distinguish particles synthesized by means of flow lithography. This architecture exhibits large, exponentially scalable encoding capacities (>10(6) particles), an ultralow decoding false-alarm rate (<10(-9)), the ability to manipulate particles by applying magnetic fields, and pronounced insensitivity to both particle chemistry and harsh processing conditions. We demonstrate quantitative agreement between observed and predicted decoding for a range of practical applications with orthogonal requirements, including covert multiparticle barcoding of pharmaceutical packaging (refractive-index matching), multiplexed microRNA detection (biocompatibility) and embedded labelling of high-temperature-cast objects (temperature resistance).

Bioactive polyelectrolyte multilayers: hyaluronic acid mediated B lymphocyte adhesion.

A strategy was developed to produce thin, biopolymer-based polyelectrolyte multilayer films, based on hyaluronic acid and chitosan, that are able to effectively bind B lymphocytes. These films explore CD44-hyaluronate interactions and provide a method to make surface-bound B cell arrays without the need for nonselective covalent chemistry. The rational design of these films using solution deposition variables, such as ionic strength and pH, allows one to maximize and fine tune this binding efficiency ex vivo. This work suggests two important conditions for successfully attaching B cells to hyaluronate-containing polyelectrolyte multilayer films: (1) hyaluronic acid is required for the proposed CD44-mediated binding mechanism, and (2) hyaluronic acid deposition conditions that favor loops and tails, such as low pH and with added salt, result in more available CD44 binding ligands and higher cell binding efficiency. Chitosan-terminated films prepared without NaCl in the deposition solutions and hyaluronic acid-terminated films prepared with salt, both under pH 3.0 assembly conditions, presented a similar high lymphocyte binding efficiency. In the former case, however, the binding strength was weaker due to a significant electrostatic contribution to the binding. Bioactive polyelectrolyte multilayers for selective binding of lymphocytes hold great promise in fields ranging from cell-based biosensors to immune system engineering.

Freely suspended cellular "backpacks" lead to cell aggregate self-assembly.

Cellular "backpacks" are a new type of anisotropic, nanoscale thickness microparticle that may be attached to the surface of living cells creating a "bio-hybrid" material. Previous work has shown that these backpacks do not impair cell viability or native functions such as migration in a B and T cell line, respectively. In the current work, we show that backpacks, when added to a cell suspension, assemble cells into aggregates of reproducible size. We investigate the efficiency of backpack-cell binding using flow cytometry and laser diffraction, examine the influence of backpack diameter on aggregate size, and show that even when cell-backpack complexes are forced through small pores, backpacks are not removed from the surfaces of cells.

Layer-by-layer deposited chitosan/silk fibroin thin films with anisotropic nanofiber alignment.

Chitosan/silk fibroin multilayer thin films were assembled using layer-by-layer deposition. The resultant multilayer films contained nanofibers aligned parallel to the dipping direction. Fiber deposition and orientation was enabled uniquely by a judicious choice of solvent and drying conditions and layer-by-layer assembly with chitosan. The deposition of oriented nanofibers was found to be the result of a unique combination of layer-by-layer and Langmuir-Blodgett type processing. Fiber orientation was confirmed by fast Fourier transform (FFT) analysis of optical micrographs and atomic force microscopy (AFM). Bidirectional fiber alignment was realized by rotating the substrate between multilayer deposition steps. Infrared spectroscopy revealed that the silk fibroin adopted the silk II secondary structure in the deposited films. We anticipate that these anisotropic films are able to combine the biocompatibility of a natural polymer system with the mechanical strength of SF, two properties useful in many biological applications including scaffolds suitable for guiding cell attachment and spreading.

Surface functionalization of living cells with multilayer patches.

We demonstrate that functional polyelectrolyte multilayer (PEM) patches can be attached to a fraction of the surface area of living, individual lymphocytes. Surface-modified cells remain viable at least 48 h following attachment of the functional patch, and patches carrying magnetic nanoparticles allow the cells to be spatially manipulated using a magnetic field. The patch does not completely occlude the cellular surface from the surrounding environment; this approach allows a functional payload to be attached to a cell that is still free to perform its native functions, as suggested by preliminary studies on patch-modified T-cell migration. This approach has potential for broad applications in bioimaging, cellular functionalization, immune system and tissue engineering, and cell-based therapeutics where cell-environment interactions are critical.