Simultaneous High-performance Liquid Chromatography-tandem Mass Spectrometry (HPLC-MS-MS) Analysis of Cyanide and Thiocyanate from Swine Plasma

An analytical procedure for the simultaneous determination of cyanide and thiocyanate in swine plasma was developed and validated. Cyanide and thiocyanate were simultaneously analyzed by high-performance liquid chromatography tandem mass spectrometry in negative ionization mode after rapid and simple sample preparation. Isotopically labeled internal standards, Na13C15N and NaS13C15N, were mixed with swine plasma (spiked and nonspiked), proteins were precipitated with acetone, the samples were centrifuged, and the supernatant was removed and dried. The dried samples were reconstituted in 10 mM ammonium formate. Cyanide was reacted with naphthalene-2,3-dicarboxaldehyde and taurine to form N-substituted 1-cyano[f]benzoisoindole, while thiocyanate was chemically modified with monobromobimane to form an SCN-bimane product. The method produced dynamic ranges of 0.1–50 and 0.2–50 μM for cyanide and thiocyanate, respectively, with limits of detection of 10 nM for cyanide and 50 nM for thiocyanate. For quality control standards, the precision, as measured by percent relative standard deviation, was below 8 %, and the accuracy was within ±10 % of the nominal concentration. Following validation, the analytical procedure successfully detected cyanide and thiocyanate simultaneously from the plasma of cyanide-exposed swine

Simultaneous Determination of Cyanide and Thiocyanate in Plasma by Chemical Ionization Gas Chromatography Mass-spectrometry (CI-GC-MS)

An analytical method utilizing chemical ionization gas chromatography-mass spectrometry was developed for the simultaneous determination of cyanide and thiocyanate in plasma. Sample preparation for this analysis required essentially one-step by combining the reaction of cyanide and thiocyanate with pentafluorobenzyl bromide and simultaneous extraction of the product into ethyl acetate facilitated by a phase-transfer catalyst, tetrabutylammonium sulfate. The limits of detection for cyanide and thiocyanate were 1 μM and 50 nM, respectively. The linear dynamic range was from 10 μM to 20 mM for cyanide and from 500 nM to 200 μM for thiocyanate with correlation coefficients higher than 0.999 for both cyanide and thiocyanate. The precision, as measured by %RSD, was below 9 %, and the accuracy was within 15 % of the nominal concentration for all quality control standards analyzed. The gross recoveries of cyanide and thiocyanate from plasma were over 90 %. Using this method, the toxicokinetic behavior of cyanide and thiocyanate in swine plasma was assessed following cyanide exposure

Force transmission in a packing of pentagonal particles

We perform a detailed analysis of the contact force network in a dense confined packing of pentagonal particles simulated by means of the contact dynamics method. The effect of particle shape is evidenced by comparing the data from pentagon packing and from a packing with identical characteristics except for the circular shape of the particles. A counterintuitive finding of this work is that, under steady shearing, the pentagon packing develops a lower structural anisotropy than the disk packing. We show that this weakness is compensated by a higher force anisotropy, leading to enhanced shear strength of the pentagon packing. We revisit "strong" and "weak" force networks in the pentagon packing, but our simulation data provide also evidence for a large class of "very weak" forces carried mainly by vertex-to-edge contacts. The strong force chains are mostly composed of edge-to-edge contacts with a marked zig-zag aspect and a decreasing exponential probability distribution as in a disk packing

Cyanide Toxicokinetics: The Behavior of Cyanide, Thiocyanate and 2-Amino-2-Thiazoline-4-Carboxylic Acid in Multiple Animal Models

Cyanide causes toxic effects by inhibiting cytochrome c oxidase, resulting in cellular hypoxia and cytotoxic anoxia, and can eventually lead to death. Cyanide exposure can be verified by direct analysis of cyanide concentrations or analyzing its metabolites, including thiocyanate (SCN−) and 2-amino-2-thiazoline-4-carboxylic acid (ATCA) in blood. To determine the behavior of these markers following cyanide exposure, a toxicokinetics study was performed in three animal models: (i) rats (250–300 g), (ii) rabbits (3.5–4.2 kg) and (iii) swine (47–54 kg). Cyanide reached a maximum in blood and declined rapidly in each animal model as it was absorbed, distributed, metabolized and eliminated. Thiocyanate concentrations rose more slowly as cyanide was enzymatically converted to SCN−. Concentrations of ATCA did not rise significantly above the baseline in the rat model, but rose quickly in rabbits (up to a 40-fold increase) and swine (up to a 3-fold increase) and then fell rapidly, generally following the relative behavior of cyanide. Rats were administered cyanide subcutaneously and the apparent half-life (t1/2) was determined to be 1,510 min. Rabbits were administered cyanide intravenously and the t1/2 was determined to be 177 min. Swine were administered cyanide intravenously and the t1/2 was determined to be 26.9 min. The SCN−t1/2 in rats was 3,010 min, but was not calculated in rabbits and swine because SCN−concentrations did not reach a maximum. The t1/2 of ATCA was 40.7 and 13.9 min in rabbits and swine, respectively, while it could not be determined in rats with confidence. The current study suggests that cyanide exposure may be verified shortly after exposure by determining significantly elevated cyanide and SCN− in each animal model and ATCA may be used when the ATCA detoxification pathway is significant

Behavioral economic methods to inform infectious disease response: Prevention, testing, and vaccination in the COVID-19 pandemic

The role of human behavior to thwart transmission of infectious diseases like COVID-19 is evident. Psychological and behavioral science are key areas to understand decision-making processes underlying engagement in preventive health behaviors. Here we adapt well validated methods from behavioral economic discounting and demand frameworks to evaluate variables (e.g., delay, cost, probability) known to impact health behavior engagement. We examine the contribution of these mechanisms within a broader response class of behaviors reflecting adherence to public health recommendations made during the COVID-19 pandemic. Four crowdsourced samples (total N = 1,366) completed individual experiments probing a response class including social (physical) distancing, facemask wearing, COVID-19 testing, and COVID-19 vaccination. We also measure the extent to which choice architecture manipulations (e.g., framing, opt-in/opt-out) may promote (or discourage) behavior engagement. We find that people are more likely to socially distance when specified activities are framed as high risk, that facemask use during social interaction decreases systematically with greater social relationship, that describing delay until testing (rather than delay until results) increases testing likelihood, and that framing vaccine safety in a positive valence improves vaccine acceptance. These findings collectively emphasize the flexibility of methods from diverse areas of behavioral science for informing public health crisis management

Local Suppression of T Cell Responses by Arginase-Induced L-Arginine Depletion in Nonhealing Leishmaniasis

The balance between T helper (Th) 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-γ, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy

The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage

Heat shock protein 90 (HSP90) is a molecular chaperone responsible for the conformational maintenance of a number of client proteins that play key roles in cell cycle arrest, DNA damage repair and apoptosis following radiation. HSP90 inhibitors exhibit antitumor activity by modulating the stabilisation and activation of HSP90 client proteins. We sought to evaluate NVP-AUY922, the most potent HSP90 inhibitor yet reported, in preclinical radiosensitization studies.NVP-AUY922 potently radiosensitized cells in vitro at low nanomolar concentrations with a concurrent depletion of radioresistance-linked client proteins. Radiosensitization by NVP-AUY922 was verified for the first time in vivo in a human head and neck squamous cell carcinoma xenograft model in athymic mice, as measured by delayed tumor growth and increased surrogate end-point survival (p = <0.0001). NVP-AUY922 was shown to ubiquitously inhibit resolution of dsDNA damage repair correlating to delayed Rad51 foci formation in all cell lines tested. Additionally, NVP-AUY922 induced a stalled mitotic phenotype, in a cell line-dependent manner, in HeLa and HN5 cell lines irrespective of radiation exposure. Cell cycle analysis indicated that NVP-AUY922 induced aberrant mitotic entry in all cell lines tested in the presence of radiation-induced DNA damage due to ubiquitous CHK1 depletion, but resultant downstream cell cycle effects were cell line dependent.These results identify NVP-AUY922 as the most potent HSP90-mediated radiosensitizer yet reported in vitro, and for the first time validate it in a clinically relevant in vivo model. Mechanistic analysis at clinically achievable concentrations demonstrated that radiosensitization is mediated by the combinatorial inhibition of cell growth and survival pathways, ubiquitous delay in Rad51-mediated homologous recombination and CHK1-mediated G(2)/M arrest, but that the contribution of cell cycle perturbation to radiosensitization may be cell line specific

Decelerating Spread of West Nile Virus by Percolation in a Heterogeneous Urban Landscape

Vector-borne diseases are emerging and re-emerging in urban environments throughout the world, presenting an increasing challenge to human health and a major obstacle to development. Currently, more than half of the global population is concentrated in urban environments, which are highly heterogeneous in the extent, degree, and distribution of environmental modifications. Because the prevalence of vector-borne pathogens is so closely coupled to the ecologies of vector and host species, this heterogeneity has the potential to significantly alter the dynamical systems through which pathogens propagate, and also thereby affect the epidemiological patterns of disease at multiple spatial scales. One such pattern is the speed of spread. Whereas standard models hold that pathogens spread as waves with constant or increasing speed, we hypothesized that heterogeneity in urban environments would cause decelerating travelling waves in incipient epidemics. To test this hypothesis, we analysed data on the spread of West Nile virus (WNV) in New York City (NYC), the 1999 epicentre of the North American pandemic, during annual epizootics from 2000–2008. These data show evidence of deceleration in all years studied, consistent with our hypothesis. To further explain these patterns, we developed a spatial model for vector-borne disease transmission in a heterogeneous environment. An emergent property of this model is that deceleration occurs only in the vicinity of a critical point. Geostatistical analysis suggests that NYC may be on the edge of this criticality. Together, these analyses provide the first evidence for the endogenous generation of decelerating travelling waves in an emerging infectious disease. Since the reported deceleration results from the heterogeneity of the environment through which the pathogen percolates, our findings suggest that targeting control at key sites could efficiently prevent pathogen spread to remote susceptible areas or even halt epidemics

Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals

Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca(2+)] ([Ca(2+)](o)) indirectly regulates a non-selective cation channel (NSCC) in neocortical nerve terminals via an unknown [Ca(2+)](o) sensor. Here, we identify the first component in a presynaptic calcium signaling pathway.By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca(2+)](o) than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents.Our experiments identify CaSR as the first component in the [Ca(2+)](o) sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca(2+)](o) will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca(2+)](o) following its entry via voltage-activated Ca(2+) channels. CaSR may detects such falls in [Ca(2+)](o) and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca(2+)](o) on release probability. CaSR is positioned to detect the dynamic changes of [Ca(2+)](o) and provide presynaptic feedback that will alter brain excitability