Clint B. Smith

Francisella tularensis 2-C-Methyl-D-Erythritol 4-Phosphate Cytidylyltransferase: Kinetic Characterization and Phosphoregulation

Deliberate and natural outbreaks of infectious disease, the prevalence of antibiotic resistant strains, and the ease by which antibiotic resistant bacteria can be intentionally engineered all underscore the necessity of effective vaccines and continued development of novel antimicrobial/antiviral therapeutics. Isoprenes, a group of molecules fundamentally involved in a variety of crucial biological functions, are derived from either the mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathway. While mammals utilize the MVA pathway, many bacteria utilize the MEP pathway, highlighting the latter as an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP cytidylyltransferase, a MEP pathway enzyme and potential target for antibiotic development. Size exclusion chromatography indicates the protein exists as a dimer in solution. Enzyme assays produced an apparent , , , , and a . The enzyme exhibits a strict preference for Mg+2 as a divalent cation and CTP as the nucleotide. Titanium dioxide chromatography-tandem mass spectrometry identified Thr141 as a site of phosphorylation. T141D and T141E site-directed mutants are catalytically inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP cytidylyltransferase is an excellent target for the development of novel antibiotics against F. tularensis.

In situ Surface-Etched Bacterial Spore Detection Using Dipicolinic Acid—Europium—Silica Nanoparticle Bioreporters:

A basic approach was optimized for the synthesis of highly selective and sensitive in situ mesoporous (MCM) type imprinted silica polymers for the detection of dipicolinic acid (DPA) using europium as a reporter. DPA is a ubiquitous biochemical marker available during the germination event of endospore-forming bacteria such as Bacillus. Additionally, an MCM-MIP (molecularly imprinted polymeric phenomena) detector and a companion MCM–non-surface–MIP detector were synthesized using europium reporters for the sensing of DPA under optimized laboratory conditions. Our results showed that the in situ molecular imprinting process enabled rapid, selective detection of DPA with high sensitivity compared to MCM-MIP (imprinted for DPA; no DPA present), MCM-Non-MIP (no imprint present), and MCM-SR-MIP (imprinted with DPA present) detectors. The lower detection limit observed for DPA concentration is 5.49 × 10−10 mol dm−3 for MCM-MIP. The performance of the sensor in high-salt-water conditions, under photo-bleaching, and its reusability were also evaluated. The synthesized in situ MCM-MIP material should permit the detection of DPA for field assays related to suspect bacterial sporulation events.

Fluorescence Resonance Energy Transfer Based MCM-EDTA-Tb3+-MES Sensor

An in situ mesopourous surface imprinted polymeric (SIP) sensor was synthesized for a highly sensitive, selective, and kinetically faster detection of the high-vapor-pressure nerve gas surrogate methyl salicylate (MES). Visual detection occurred on the filtrate thin films at 25 pM. Other nerve gas surrogates, TP, DMP, DMMP, PMP, and 1,4-thioxane, were tested and showed a decrease in sensitivity compared to MES. In addition, 2,6-dipicolinic acid (DPA), a biological indicator, was also investigated and showed a decrease in sensitivity compared to MES. Finally, the detection plateau was reached at 40 s and at 1.5 × 10−4 M from pH 6–11.

Utilizing upconverting phosphors for the detection of TNT

Herein we purpose to utilize upconverting phosphors to detect explosives. To detect TNT, antibodies specific to TNT are conjugated to the surface. The role of the antibodies is two fold; to bind a quencher and to accept TNT. The quencher is a bifunctional molecule, with one end containing a TNT analog and the other end being a dark fluorescent quenching dye. The dye is chosen so that the luminescence from the phosphor will be absorbed preventing it from emitting, reducing luminescence from the phosphor. However, in the presence of TNT the quencher that is bound with DNT will be displaced. With the quencher displaced the phosphor will be able to emit light indicating TNT is present in the select area.

Detection of selected solar-stimulated fluorophores with a Fraunhofer line radiometer

In this paper we report the use of Fraunhofer Line Radiometry and the Fraunhofer Line Depth (FLD) method to measure the passive fluorescence of select, high quantum yield (> 0.7) fluorophores. The measurements we describe involved direct solar illumination of both a non-fluorescent standard material (NIST, Spectralon), and the fluorescent target materials (in solution) within several relevant Fraunhofer lines: 486 nm (H-beta), 518 nm (Mg), 589 nm (Na), and 656 nm (H-alpha). Of the selected labels, Rhodamine B demonstrated the highest fluorescence coefficient of 2.2, at 589 nm, and possessed a fluorescence intensity of 1.1 x 106 cps at an optical density of 1.0. These results show fluorescent labels can be selectively measured and observed passively in relevant Fraunhofer lines. The implications for such findings indicate the potential daytime measurement of environmental labels and probes as a promising sensing technology.

Determining soil moisture at moderately high resolution via Soil Moisture Active Passive (SMAP) and Landsat using inferred learning

This paper reviews an approach to increase soil moisture resolution over a sample region over Australia using the Soil Moisture Active Passive (SMAP) sensor and Landsat 8 only. This approach uses an inductive localized approach, eschewing the need to obtain a resilient and definitive model in favor of a temporary model that utilizes current conditional inputs only. For the purposes of this analysis, the SMAP 36 km soil moisture product is considered fully valid and accurate. Landsat bands coinciding in collection date with a SMAP capture are down sampled to match the resolution of the SMAP product. A series of indices describing the Soil-Vegetation-Atmosphere Triangle (SVAT) relationship are then produced using the down sampled Landsat bands. These indices are then related to the local coincident SMAP values to identify a series of rules or trees to identify the local rules defining the relationship between soil moisture and the aforementioned indices. This paper uses a random forest due to its highly accurate learning against local ground truth data. The defined rules are then applied to the Landsat image in the native Landsat resolution to determine local soil moisture. Ground truth comparison is done via a series of grids using time dielectric impedance and airborne Lband Multi-beam Radiometer (PLMR) observations done under the SMAPEx-5 campaign (Panciera 2013). The predictive power of the inferred learning soil moisture algorithm (ILSMA) did well with a mean absolute error of 0.054 over an airborne L-band retrieved surface over the same region. DISCLAIMER: The contents of this report are not to be used for advertising, publication, or promotional purposes. Citation of trade names does not constitute an official endorsement or approval of the use of such commercial products. All product names and trademarks cited are the property of their respective owners. The findings of this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. DESTROY THIS REPORT WHEN NO LONGER NEEDED. DO NOT RETURN IT TO THE ORIGINATOR. ERDC/GRL TR-18-1 iii

Photogrammetric point cloud compression for tactical networks

We report progress toward the development of a compression schema suitable for use in the Army’s Common Operating Environment (COE) tactical network. The COE facilitates the dissemination of information across all Warfighter echelons through the establishment of data standards and networking methods that coordinate the readout and control of a multitude of sensors in a common operating environment. When integrated with a robust geospatial mapping functionality, the COE enables force tracking, remote surveillance, and heightened situational awareness to Soldiers at the tactical level. Our work establishes a point cloud compression algorithm through image-based deconstruction and photogrammetric reconstruction of three-dimensional (3D) data that is suitable for dissimination within the COE. An open source visualization toolkit was used to deconstruct 3D point cloud models based on ground mobile light detection and ranging (LiDAR) into a series of images and associated metadata that can be easily transmitted on a tactical network. Stereo photogrammetric reconstruction is then conducted on the received image stream to reveal the transmitted 3D model. The reported method boasts nominal compression ratios typically on the order of 250 while retaining tactical information and accurate georegistration. Our work advances the scope of persistent intelligence, surveillance, and reconnaissance through the development of 3D visualization and data compression techniques relevant to the tactical operations environment.

Laser induced fluorescence lifetime characterization of Bacillus endospore species using time correlated single photon counting analysis with the multi-exponential fit method

Rapid detection of biological material is critical for determining presence/absence of bacterial endospores within various investigative programs. Even more critical is that if select material tests positive for bacillus endospores then tests should provide data at the species level. Optical detection of microbial endospore formers such as Bacillus sp. can be heavy, cumbersome, and may only identify at the genus level. Data provided from this study will aid in characterization needed by future detection systems for further rapid breakdown analysis to gain insight into a more positive signature collection of Bacillus sp. Literature has shown that fluorescence spectroscopy of endospores could be statistically separated from other vegetative genera, but could not be separated among one another. Results of this study showed endospore species separation is possible using laser-induce fluorescence with lifetime decay analysis for Bacillus endospores. Lifetime decays of B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain were investigated. Using the Multi-Exponential fit method data showed three distinct lifetimes for each species within the following ranges, 0.2-1.3 ns; 2.5-7.0 ns; 7.5-15.0 ns, when laser induced at 307 nm. The four endospore species were individually separated using principle component analysis (95% CI).

Optical bar code recognition of methyl salicylate (MES) for environmental monitoring using fluorescence resonance energy transfer (FRET) on thin films

Fluorescence resonance energy transfer (FRET) is a process in which energy is transferred nonradiatively from one fluorophore (the donor) in an excited electron state to another, the chromophore (the acceptor). FRET is distinctive in its ability to reveal the presence of specific recognition of select targets such as the nerve agent stimulant Methyl Salicylate (MES) upon spectroscopic excitation. We introduce a surface imprinted and non-imprinted thin film that underwent AC-Electrospray ionization for donor-acceptor pair(s) bound to InGaP quantum dots and mesoporous silicate nanoparticles. The donor-acceptor pair used in this investigation included MES (donor) and 6-(fluorescein-5-(and-6)- carboxamido) hexanoic acid, succinimidyl ester bound to InGaP quantum dots (acceptor). MES was then investigated as a donor to various acceptor fluorophore: InGaP: mesoporous silicate nanoparticle layers.