A single post-exposure oxime RS194B treatment rapidly reactivates acetylcholinesterase and reverses acute symptoms in macaques exposed to diethylphosphorothioate parathion and chlorpyrifos insecticides.

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticides, significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins that target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with an oxime to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently used oximes either reactivate poorly, do not readily cross the blood-brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure, for example RS194B, have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticides or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms, and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these findings and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos. These OPs require conversion by P450 in the liver of the inactive thions to the active toxic oxon forms, and once again demonstrated RS194B efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1-2 h was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

The impact of solvents on the toxicity of the banned parathion insecticide.

The aerial crop dusting and spraying of fields with the phosphorothioate insecticide parathion in the late 1900s, significantly improved crop yields but resulted in high levels of occupational toxicity in handlers and agricultural workers, as well as cases of intentional self-harm poisoning, culminating in its banning in many western countries by early 2000s. However because of the low solubility and volatility of parathion, most available products were formulated using organic solvents e.g. xylene, to increase the efficacy of the aerosols and dusts. In the present study, the toxicity of parathion was assessed when formulated in an aqueous solvents (ethanol/PBS (1:9)), and delivered to macaques as an aerosol. Doses of 780 µg/kg and 1.56 mg/kg were delivered one day apart, using a modified nebulizer calculated to result in lung deposition of ∼480 µg/kg with a similar or larger amount being swallowed; these doses being similar to the estimated lethal oral dose 286ug/kg - 1.43 mg/kg of formulated parathion in humans. Surprisingly, this dose (a combined amount of ∼14 mg) caused only low AChE inhibition and moderate BChE inhibition with no clinical symptoms, indicating that the use of organic solvents may have previously played a critical role in the severity of parathion toxicity following inhalation exposure. In addition, unlike constitutively toxic OPs, which are highly toxic when inhaled, these results are consistent with the idea that phosphorothioate insecticides appear to be more intoxicating following oral than inhalation exposure. However, this still remains uncertain because the presence of organic solvents in the ingested parathion studies was not always known.

Antioxidant enzymes mediate survival of breast cancer cells deprived of extracellular matrix.

Metastasis by cancer cells relies upon the acquisition of the ability to evade anoikis, a cell death process elicited by detachment from extracellular matrix (ECM). The molecular mechanisms that ECM-detached cancer cells use to survive are not understood. Striking increases in reactive oxygen species (ROS) occur in ECM-detached mammary epithelial cells, threatening cell viability by inhibiting ATP production, suggesting that ROS must be neutralized if cells are to survive ECM-detachment. Here, we report the discovery of a prominent role for antioxidant enzymes, including catalase and superoxide dismutase, in facilitating the survival of breast cancer cells after ECM-detachment. Enhanced expression of antioxidant enzymes in nonmalignant mammary epithelial cells detached from ECM resulted in ATP elevation and survival in the luminal space of mammary acini. Conversely, silencing antioxidant enzyme expression in multiple breast cancer cell lines caused ATP reduction and compromised anchorage-independent growth. Notably, antioxidant enzyme-deficient cancer cells were compromised in their ability to form tumors in mice. In aggregate, our results reveal a vital role for antioxidant enzyme activity in maintaining metabolic activity and anchorage-independent growth in breast cancer cells. Furthermore, these findings imply that eliminating antioxidant enzyme activity may be an effective strategy to enhance susceptibility to cell death in cancer cells that may otherwise survive ECM-detachment.

3D printing of preclinical X-ray computed tomographic data sets.

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.(1) However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.(2) These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. (3, 4) The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Dual tracer imaging of SPECT and PET probes in living mice using a sequential protocol.

Over the past 20 years, multimodal imaging strategies have motivated the fusion of Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) scans with an X-ray computed tomography (CT) image to provide anatomical information, as well as a framework with which molecular and functional images may be co-registered. Recently, pre-clinical nuclear imaging technology has evolved to capture multiple SPECT or multiple PET tracers to further enhance the information content gathered within an imaging experiment. However, the use of SPECT and PET probes together, in the same animal, has remained a challenge. Here we describe a straightforward method using an integrated trimodal imaging system and a sequential dosing/acquisition protocol to achieve dual tracer imaging with (99m)Tc and (18)F isotopes, along with anatomical CT, on an individual specimen. Dosing and imaging is completed so that minimal animal manipulations are required, full trimodal fusion is conserved, and tracer crosstalk including down-scatter of the PET tracer in SPECT mode is avoided. This technique will enhance the ability of preclinical researchers to detect multiple disease targets and perform functional, molecular, and anatomical imaging on individual specimens to increase the information content gathered within longitudinal in vivo studies.

Segmentation and measurement of fat volumes in murine obesity models using X-ray computed tomography.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life. Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated. Several small animal models of obesity have been developed and are employed in a variety of studies. A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions. Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice. Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Imaging and analysis of Pseudomonas aeruginosa swarming and rhamnolipid production.

Many bacteria spread over surfaces by "swarming" in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacterium Pseudomonas aeruginosa. First, we quantify the temporal distribution of P. aeruginosa cells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming of P. aeruginosa and Salmonella enterica serovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of several P. aeruginosa strains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.