John L. Alls

Pulsed microwave induced bioeffects

High-power pulsed microwave radiation, when applied to solutions containing dissolved carbon dioxide (or bicarbonate), hydrogen peroxide, and the soluble organic semi-conductor diazoluminomelanin, generates sound, pulsed luminescence, and electrical discharge. Microbes exposed to these phenomena experienced damage comparable to short-time, high-temperature insults, even though the average and measurable localized temperatures were insufficient to cause the observed effects.

Directed killing of anthrax spores by microwave-induced cavitation

High-power pulsed-microwave radiation damages anthrax spores by apparent sonoluminescence in aqueous solutions containing the organic semiconductor diazoluminomelanin (DALM). DALM biosynthesized by JM109 E. coli, containing the plasmid pIC2ORNR/sub 1.1/, had a higher affinity for spores of Sterne strain anthrax when compared to several other species of bacilli and enhanced the effect. Upon exposure to pulsed-microwave radiation, anthrax spores showed a maximum of 4 to 5 (i.e., 4.6) logs of kill. The light emitted was typical of plasma gas emissions and the spores, upon scanning electron-microscopic examination, showed enlargement and rupture typical of rapid expansion. Therefore, microwave-induced cavitations localized to the spore surfaces enhanced kill.

Preliminary electrochemiluminescence studies of metal ion–bacterial diazoluminomelanin (DALM) interactions

Electrochemiluminescence (ECL) studies of the chemiluminescent (CL) polymer diazoluminomelanin (DALM) biosynthesized in nitrate reductase transfected Escherichia coli JM109 bacteria revealed noteworthy anodic ECL and even more intense cathodic ECL. Bacterial DALM (BD) ECL was also assessed in the presence of 100 ppm of 33 different metal and non-metal ions which revealed specific anodic, but not cathodic, enhancements of BD ECL with Ag+, Hg2+ and Ru3+. The precursors and intermediate polymers which comprise DALM, such as luminol, 3-amino-L-tyrosine (3-AT), aminomelanin (AM) and diazomelanin (DM) were screened for ECL enhancement against the same set of elemental ions. Significant anodic ECL enhancements were observed for luminol with Hg2+ in the presence of tripropylamine (TPA), but not for any other DALM component in combination with other elemental ions, either anodically or cathodically. Comparison of BD with luminol in the presence and absence of TPA and Hg2+ revealed very different ECL activity patterns and suggested different mechanisms for BD and luminol ECL.

Rapid recovery and identification of anthrax bacteria from the environment.

Abstract: Bacillus anthracis has been recognized as a highly likely biological warfare or terrorist agent. We have designed culture techniques to rapidly isolate and identify “live” anthrax from suspected environmental release. A special medium (3AT medium) allows for discrimination between closely related bacilli and non‐pathogenic strains. Nitrate was found to be a primary factor influencing spore formation in Bacillus anthracis. Nitrate reduction in anthrax is not an adaptation to saprophytic environmental existence, but it is a signal to enhance environmental survival upon the death of the anthrax host, which can be mimicked in culture.

Thermal sensitivity of biowarfare simulants

In developing high temperature incendiary weapons, the temperature and duration required to inactivate spores is needed information. Three common biowarfare simulants, Bacillus anthracis var Sterne, Bacillus thuringiensis var Kurstaki and Bacillus globigii var niger have been studied for their susceptibility to heat. The spores of all three simulants lose viability when exposed to temperatures between 250 and 300 degree(s)C for 1 second. Bacillus globigii is perhaps the most heat resistant of the three simulants studied, with Bacillus anthracis and Bacillus thuringiensis having similar susceptibilities to heat. Low temperature experiments requiring longer durations were also conducted; over a period of days at 90 degree(s)C. Bacillus anthracis spores can be inactivated. Thermodynamic and kinetic analysis were also performed. An important implication for any high temperature incendiary is the amount of heat or energy the spores absorb between ambient temperatures and 100 degree(s)C. A phase transition occurs centered at 184 degree(s)C for Bacillus thuringiensis. This is also the beginning of a massive weight loss from the spores, as well as a point at which the kinetics of the kill seem to change.

PULSED MICROWAVE INDUCED LIGHT, SOUND, AND ELECTRICAL DISCHARGE ENHANCED BY A BIOPOLYMER

Intense flashes of light were observed in sodium bicarbonate and hydrogen peroxide solutions when they were exposed to pulsed microwave radiation, and the response was greatly enhanced by a microwave-absorbing, biosynthesized polymer, diazoluminomelanin. A FPS-7B radar transmitter, operating at 1.25 GHz provided pulses of 5.73 +/- 0.09 micros in duration at 10.00 +/- 0.03 pulses/s with 2.07 +/- 0.08 MW forward power (mean +/- standard deviation), induced the effect but only when the appropriate chemical interaction was present. This phenomenon involves acoustic wave generation, bubble formation, pulsed luminescence, ionized gas ejection, and electrical discharge. The use of pulsed microwave radiation to generate highly focused energy deposition opens up the possibility of a variety of biomedical applications, including targeting killing of microbes or eukaryotic cells. The full range of microwave intensities and frequencies that induce these effects has yet to be explored and, therefore, the health and safety implications of generating the phenomena in living tissues remain an open question.

Basis for the extraordinary genetic stability of anthrax.

Abstract: Over 500 isolates of anthrax bacillus from around the world represent one of the most genetically homogeneous microbes. There are three possibilities for this genetic stability: (1) anthrax has an extraordinarily high fidelity repair system, (2) genetic damage to anthrax is usually lethal, and/or (3) a highly demanding and selective process exists in its environment that is necessary for the completion of its life cycle. Using probes made from genes selected by growth of an Escherichia coli expression vector Bacillus anthracis library on hypertrophic high nitrate concentration medium, genes unique to B. anthracis were isolated. High nitration conditions generated stable chromosomal mutants that displayed altered morphology and life‐cycle progression. Therefore, life‐cycle progression connected to nitration, associated with host inflammatory response, selects for mutants that show life‐cycle progression tightly coupled to progression of the inflammatory response to anthrax. Significant variation from this coupled progression leads to failure of anthrax to complete its life‐cycle at the death of its host.

Thermochemiluminescence as a technique for radio frequency radiation dosimetry

Abstract Radio frequency radiation (RFR) dosimetry is based on the rate of absorbed energy (specific absorption rate: SAR) per unit mass. It is most conveniently measured by acquiring changes in temperature per unit time and converting the results to joules per second (watts) per kilogram, based on the specific heat of the biological material interacting with the RFR. To date, SAR has been predicted by modeling based on the dielectric properties of tissues, or measured by infrared (IR) thermography or with macroscopic high-resistance thermistors or thermofluorescent macroscopic point probes. Thermochemiluminescence (TCL) was invented to provide a high degree of continuous spatial and temporal thermal resolution in phantoms. It is defined as the steady-state emission of visible light from a peroxidizing mixture based on the temperature of the mixture. The best material for this purpose, to date, is diazoluminomelanin (DALM). Unfortunately, standardization of the synthesis (chemical composition) of this polymer and its thermal response constant (thermal quantum efficiency) has been difficult. This paper presents a biosynthetic method for the large-scale production of the polymer and a computational method for directly determining the SAR from the luminescence.

Feasibility of a DNA-Based Combinatorial Array Recognition Surface (CARS) in a Polyacrylamide Gel Matrix

We report initial attempts at developing a self-assembled combinatorial DNA biosensor array which may be capable of binding and identifying virtually any soluble analyte that binds the array by pattern recognition, in effect making it a universal biosensor surface. Data are presented for differential binding patterns of various analytes to 1-D arrays of combinatorial deoxyribonucleic acid (DNA) concatamer libraries which are spatially separated according to size and charge by electrophoresis in polyacrylamide gels. These DNA concatamer libraries are essentially composed of single-stranded (ss) random DNA 60 mers, which form a ldquosmearrdquo pattern in gels following electrophoresis. When used to bind and detect various analytes or mixtures of analytes in the gel, we refer to the DNA smear as a ldquocombinatorial array recognition surfacerdquo (CARS). Differences in intrinsic fluorescence scanning patterns of CARS gel strips were compared before and after addition of various analytes to the arrays to detect binding patterns. Scans revealed a high level of reproducibility for individual CARS arrays in a given gel with or without bound analytes. Scan patterns between different CARS gel strips were initially less reproducible, but purification of the DNA library using spin columns prior to electrophoresis improved gel-to-gel reproducibility.