Nathan Boggs

Exploring the boundaries of additivity: mixtures of NADH: quinone oxidoreductase inhibitors.

The activity of mitochondrial complex I of the electron transport chain (ETC) is known to be affected by an extraordinarily large number of diverse xenobiotics, and dysfunction at complex I has been associated with a variety of disparate human diseases, including those with potentially environmentally relevant etiologies. However, the risks associated with mixtures of complex I inhibitors have not been fully explored, and this warrants further examination of potentially greater than additive effects that could lead to toxicity. A potential complication for the prediction of mixture effects arises because mammalian mitochondrial complex I has been shown to exist in two distinct dynamic conformations based upon substrate availability. In this study, we tested the accepted models of additivity as applied to mixtures of rotenone, deguelin, and pyridaben, with and without substrate limitation. These compounds represent both natural and synthetic inhibitors of complex I of the ETC, and experimental evidence to date indicates that these inhibitors share a common binding domain with partially overlapping binding sites. Therefore, we hypothesized that prediction of their mixtures effects would follow dose addition. Using human hepatocytes, we analyzed the effects of these mixtures at doses between 0.001 and 100 μM on overall cellular viability. Analysis of the dose-response curves resulting from challenge with all possible binary and ternary mixtures revealed that the appropriate model was not clear. All of the mixtures tested were found to be in agreement with response addition, but only rotenone plus deguelin and the ternary mixture followed dose addition. To determine if conformational regulation via substrate limitation could improve model selection and our predictions, we tested the models of additivity for the binary and ternary mixtures of inhibitors when coexposed with 2-deoxy-d-glucose (2-DG), which limits NADH via upstream inhibition of glycolysis. Coexposure of inhibitors with 2-DG did facilitate model selection: Rotenone plus pyridaben and the ternary mixture were in sole agreement with dose addition, while deguelin plus pyridaben was in sole agreement with response addition. The only ambiguous result was the agreement of both models with the mixture of rotenone plus deguelin with 2-DG, which may be explained by deguelins well-known affinity for protein kinase B (Akt) in addition to complex I. Thus, our findings indicate that predictive models for mixtures of mitochondrial complex I inhibitors appear to be compound specific, and our research highlights the need to control for dynamic conformational changes to improve our mechanistic understanding of additivity with these inhibitors.

Manganese-Enhanced Magnetic Resonance Imaging as a Diagnostic and Dispositional Tool after Mild-Moderate Blast Traumatic Brain Injury.

Traumatic brain injury (TBI) caused by explosive munitions, known as blast TBI, is the signature injury in recent military conflicts in Iraq and Afghanistan. Diagnostic evaluation of TBI, including blast TBI, is based on clinical history, symptoms, and neuropsychological testing, all of which can result in misdiagnosis or underdiagnosis of this condition, particularly in the case of TBI of mild-to-moderate severity. Prognosis is currently determined by TBI severity, recurrence, and type of pathology, and also may be influenced by promptness of clinical intervention when more effective treatments become available. An important task is prevention of repetitive TBI, particularly when the patient is still symptomatic. For these reasons, the establishment of quantitative biological markers can serve to improve diagnosis and preventative or therapeutic management. In this study, we used a shock-tube model of blast TBI to determine whether manganese-enhanced magnetic resonance imaging (MEMRI) can serve as a tool to accurately and quantitatively diagnose mild-to-moderate blast TBI. Mice were subjected to a 30 psig blast and administered a single dose of MnCl2 intraperitoneally. Longitudinal T1-magnetic resonance imaging (MRI) performed at 6, 24, 48, and 72 h and at 14 and 28 days revealed a marked signal enhancement in the brain of mice exposed to blast, compared with sham controls, at nearly all time-points. Interestingly, when mice were protected with a polycarbonate body shield during blast exposure, the marked increase in contrast was prevented. We conclude that manganese uptake can serve as a quantitative biomarker for TBI and that MEMRI is a minimally-invasive quantitative approach that can aid in the accurate diagnosis and management of blast TBI. In addition, the prevention of the increased uptake of manganese by body protection strongly suggests that the exposure of an individual to blast risk could benefit from the design of improved body armor.

Refractive index measurement of biological particles in visible region

Optical cross-sections of biological warfare simulants, killed agents, and live agents are needed to assess the standoff detection performance of active lidar and passive FTIR systems. To aid in this investigation, Johns Hopkins University Applied Physics Laboratory (JHU/APL) has developed a technique to determine the index of refraction of biological materials in the visible region using a combination of transmission measurements and anomalous diffraction theory (ADT). The spectral measurements using a dual beam grating spectrometer provide a basis for calculating the optical cross section of suspended particles. ADT is then used to convert the cross section result into index of refraction. A summary of this procedure is described along with the results for silica microspheres and Bacillus globijii (BG). A comparison of these results to published data is also presented.

Water absorption in a refractive index model for bacterial spores

The complexity of biological agents can make it difficult to identify the important factors impacting scattering characteristics among variables such as size, shape, internal structure and biochemical composition, particle aggregation, and sample additives. This difficulty is exacerbated by the environmentally interactive nature of biological organisms. In particular, bacterial spores equilibrate with environmental humidity by absorption/desorption of water which can affect both the complex refractive index and the size/shape distributions of particles - two factors upon which scattering characteristics depend critically. Therefore accurate analysis of experimental data for determination of refractive index must take account of particle water content. First, spectral transmission measurements to determine visible refractive index done on suspensions of bacterial spores must account for water (or other solvent) uptake. Second, realistic calculations of aerosol scattering cross sections should consider effects of atmospheric humidity on particle water content, size and shape. In this work we demonstrate a method for determining refractive index of bacterial spores bacillus atropheus (BG), bacillus thuringiensis (BT) and bacillus anthracis Sterne (BAs) which accounts for these effects. Visible index is found from transmission measurements on aqueous and DMSO suspensions of particles, using an anomalous diffraction approximation. A simplified version of the anomalous diffraction theory is used to eliminate the need for knowledge of particle size. Results using this approach indicate the technique can be useful in determining the visible refractive index of particles when size and shape distributions are not well known but fall within the region of validity of anomalous dispersion theory.

Shape characteristics of biological spores

Calculation of scattering properties of biological materials has classically been addressed using numerical calculations based on T-matrix theory. These calculations use bulk optical properties, particle size distribution, and a limited selection of shape descriptors to calculate the resulting aerosol properties. However, the most applicable shape available in T-matrix codes, the spheroid, is not the best descriptor of most biological materials. Based on imagery of the spores of Bacillus atrophaeus and Bacillus anthracis, capsule and egg shapes are mathematically described and programmed into the Amsterdam Discrete Dipole Approximation (ADDA). Spectrally dependent cross sections and depolarization ratios are calculated and a comparison made to spheroidal shapes of equivalent sizes.

Extinction and backscatter cross sections of biological materials

Aerosol backscatter and extinction cross-sections are required to model and evaluate the performance of both active and passive detection systems. A method has been developed by which begins with laboratory measurements of thin films and suspensions of biological material to obtain the complex index refraction of the film from the UV to the LWIR. Using that result with particle size distribution and shape information as inputs to T-matrix calculations yields the extinction cross-section and backscatter cross section as a function of wavelength. These are important inputs to the lidar equation. In a continuing effort to provide validated optical cross-sections, measurements have been made on a number of high purity biological species in the laboratory as well as measurements of material released at recent field tests. The resulting observed differences aid in distinguishing between intrinsic and extrinsic effects, which can affect the characteristic signatures of important biological aerosols. A variety of biological aerosols are examined.

Spectral Refractive Index and Extinction Cross-Section of BG Spores

Despite the wide spread need for optical cross-section data on single spore bio-aerosols, available databases are sparse and unreliable. Information reported is based on short path measurements on high concentration media containing particle clusters. This represents an upper bound to the single spore cross-section. Measurements on single spore aerosolized media demand long path lengths and moderate particle concentration. Transmittance measurements need to be in the single scatter limit as well. These requirements are often difficult to meet. We present a procedure that leads to aerosol extinction and backscatter cross-sections in a straightforward manner. Transmittance measurements of thin films of bio-aerosols are used to obtain the bulk refractive index. This result and the measured size distribution can be used in a T-matrix calculation to yield the desired cross-sections. To illustrate this technique, infrared cross-sections are obtained for Bacillus globigii.

Manganese-Enhanced MRI as a diagnostic and dispositional tool after mild-moderate blast TBI.

Traumatic brain injury (TBI) caused by explosive munitions, known as blast TBI, is the signature injury in recent military conflicts in Iraq and Afghanistan. Diagnostic evaluation of TBI, including blast TBI, is based on clinical history, symptoms, and neuropsychological testing, all of which can result in misdiagnosis or underdiagnosis of this condition, particularly in the case of TBI of mild-to-moderate severity. Prognosis is currently determined by TBI severity, recurrence, and type of pathology, and also may be influenced by promptness of clinical intervention when more effective treatments become available. An important task is prevention of repetitive TBI, particularly when the patient is still symptomatic. For these reasons, the establishment of quantitative biological markers can serve to improve diagnosis and preventative or therapeutic management. In this study, we used a shock-tube model of blast TBI to determine whether manganese-enhanced magnetic resonance imaging (MEMRI) can serve as a tool to accurately and quantitatively diagnose mild-to-moderate blast TBI. Mice were subjected to a 30 psig blast and administered a single dose of MnCl2 intraperitoneally. Longitudinal T1-magnetic resonance imaging (MRI) performed at 6, 24, 48, and 72 h and at 14 and 28 days revealed a marked signal enhancement in the brain of mice exposed to blast, compared with sham controls, at nearly all time-points. Interestingly, when mice were protected with a polycarbonate body shield during blast exposure, the marked increase in contrast was prevented. We conclude that manganese uptake can serve as a quantitative biomarker for TBI and that MEMRI is a minimally-invasive quantitative approach that can aid in the accurate diagnosis and management of blast TBI. In addition, the prevention of the increased uptake of manganese by body protection strongly suggests that the exposure of an individual to blast risk could benefit from the design of improved body armor.

Models to support active sensing of biological aerosol clouds

Elastic backscatter LIght Detection And Ranging (LIDAR) is a promising approach for stand-off detection of biological aerosol clouds. Comprehensive models that explain the scattering behavior from the aerosol cloud are needed to understand and predict the scattering signatures of biological aerosols under varying atmospheric conditions and against different aerosol backgrounds. Elastic signatures are dependent on many parameters of the aerosol cloud, with two major components being the size distribution and refractive index of the aerosols. The Johns Hopkins University Applied Physics Laboratory (JHU/APL) has been in a unique position to measure the size distributions of released biological simulant clouds using a wide assortment of aerosol characterization systems that are available on the commercial market. In conjunction with the size distribution measurements, JHU/APL has also been making a dedicated effort to properly measure the refractive indices of the released materials using a thin-film absorption technique and laboratory characterization of the released materials. Intimate knowledge of the size distributions and refractive indices of the biological aerosols provides JHU/APL with powerful tools to build elastic scattering models, with the purpose of understanding, and ultimately, predicting the active signatures of biological clouds.