Comparative Performance of the Luminex NxTAG Respiratory Pathogen Panel, GenMark eSensor Respiratory Viral Panel, and BioFire FilmArray Respiratory Panel.

This study compares three of the most inclusive and widely used panels for respiratory syndromic testing in the United States, namely, Luminex NxTAG Respiratory Pathogen Panel (RPP), BioFire FilmArray Respiratory Panel (RP), and GenMark eSensor Respiratory Viral Panel (RVP). We compared the three assays using nasopharyngeal swab samples (n = 350) collected from symptomatic patients (n = 329) in the pre-coronavirus disease 2019 (COVID-19) era. There was no significant difference in the overall accuracies of BioFire and Luminex assays (P = 0.6171); however, significant differences were found between BioFire and GenMark (P = 0.0003) and between GenMark and Luminex (P = 0.0009). The positive percent agreement of the BioFire RP assay was 94.1%, compared to 97.3% for GenMark RVP and 96.5% for Luminex RPP. Overall negative percent agreement values were high for all three assays, i.e., 99.9% for BioFire and Luminex and 99.5% for GenMark. The three assays were equivalent for adenovirus, human metapneumovirus, influenza A, and respiratory syncytial virus. Increased false-positive results were seen with BioFire for the endemic coronaviruses and with GenMark for influenza B and the parainfluenza viruses. IMPORTANCE Clinical laboratories have multiple choices when it is comes to syndromic respiratory testing. Here, the Luminex NxTAG RPP is compared to the BioFire FilmArray RP and GenMark eSensor RVP for overall and per-target accuracy. As new tests come to market, it is important to ascertain their performance characteristics, compared to other widely used in vitro diagnostic products.

In Situ Electrochemical Monitoring of Caged Compound Photochemistry: An Internal Actinometer for Substrate Release.

Caged compounds are molecules that release a protective substrate to free a biologically active substrate upon treatment with light of sufficient energy and duration. A notable limitation of this approach is difficulty in determining the degree of photoactivation in tissues or opaque solutions because light reaching the desired location is obstructed. Here, we have addressed this issue by developing an in situ electrochemical method in which the amount of caged molecule photorelease is determined by fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. Using p-hydroxyphenyl glutamate (pHP-Glu) as our model system, we generated a linear calibration curve for oxidation of 4-hydroxyphenylacetic acid (4HPAA), the group from which the glutamate molecule leaves, up to a concentration of 1000 µM. Moreover, we are able to correct for the presence of residual pHP-Glu in solution as well as the light artifact that is produced. A corrected calibration curve was constructed by photoactivation of pHP-Glu in a 3 µL photoreaction vessel and subsequent analysis by high-performance liquid chromatography. This approach has yielded a linear relationship between 4HPAA concentration and oxidation current, allowing the determination of released glutamate independent of the amount of light reaching the chromophore. Moreover, we have successfully validated the newly developed method by in situ measurement in a whole, intact zebrafish brain. This work demonstrates for the first time the in situ electrochemical monitoring of caged compound photochemistry in brain tissue with FSCV, thus facilitating analyses of neuronal function.

5-Fluorouracil impairs attention and dopamine release in rats.

Chemotherapy related cognitive impairment (CTRC; "chemobrain") is a syndrome that is associated with the impairment of various aspects of cognition, including executive function, processing speed, and multitasking. The role of neurotransmitter release in the expression of cognitive impairments is not well known. In this work we employed a newly developed behavioral paradigm to measure attentional shifting, a fundamental component of executive function, in rats treated with 5-fluorouracil (5-FU), a commonly used cancer chemotherapy agent. We found that one and two weeks of 5-FU treatment significantly impaired attentional shifting compared to baseline, while saline treatment had no effect. Post-mortem analysis of these rats revealed that 5-FU caused a significant overall decrease in dopamine release as well. Collectively, these results demonstrate the feasibility of our attentional shifting paradigm for evaluating the cognitive effects of chemotherapy treatment. Moreover, these results support the need for additional studies to determine if impaired dopamine release plays a role in chemobrain.

Regional differences in dopamine release in the R6/2 mouse caudate putamen.

Huntington's disease (HD) is a fatal neurodegenerative disorder that is characterized by degeneration of the striatum. Here, fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used to uncover regional differences in dopamine (DA) release in the caudate putamen of R6/2 and wild-type control mice. We found a decreasing ventral-to-dorsal gradient in DA release, evoked by a single electrical stimulus pulse, in aged R6/2 mice. Moreover, under more intense stimulation conditions (120 pulses), DA release was significantly attenuated in the dorsal, but not in the ventral caudate. Autoradiography measurements using [3H]WIN 35,428 revealed that the overall density of DA transporter (DAT) protein molecules was significantly less in R6/2 mice compared to WT control mice; however, quadrants of the caudate putamen were not differentially altered in the R6/2 mice. These data collectively suggest that DA release in the dorsal caudate region is more vulnerable with age progression compared to the ventral region.

KU32 prevents 5-fluorouracil induced cognitive impairment.

Chemotherapy induced cognitive impairment (i.e. chemobrain) involves acute and long-term deficits in memory, executive function, and processing speed. Animal studies investigating these cognitive deficits have had mixed results, potentially due to variability in the complexity of behavioral tasks across experiments. Further, common chemotherapy treatments such as 5-fluorouracil (5-FU) break down myelin integrity corresponding to hippocampal neurodegenerative deficits and mitochondrial dysfunction. There is little evidence, however, of pharmacological treatments that may target mitochondrial dysfunction. Using a differential reinforcement of low rates (DRL) task combining spatial and temporal components, the current study evaluated the preventative effects of the pharmacological agent KU32 on the behavior of rats treated with 5-FU (5-FU+Saline vs. 5FU+KU32). DRL performance was analyzed the day after the first set of injections (D1), the day after the second set of injections (D7) and the last day of the experiment (D14). The 5FU+KU32 group earned significantly more reinforcers on the DRL task at D7 and D14 than the 5FU+Saline group. Further, the 5FU+KU32 group showed significantly better temporal discrimination. The 5FU+KU32 showed within-group improvement in temporal discrimination from D7 to D14. No significant differences were observed in spatial discrimination, however, those in the 5FU+Saline group responded more frequently on T3 compared to the 5FU+KU32 group, highlighting temporal discrimination differences between groups. The current data suggest that KU32 shows promise in the prevention of chemotherapy induced impairments in temporal discrimination.

Impaired Brain Dopamine and Serotonin Release and Uptake in Wistar Rats Following Treatment with Carboplatin.

Chemotherapy-induced cognitive impairment, known also as "chemobrain", is a medical complication of cancer treatment that is characterized by a general decline in cognition affecting visual and verbal memory, attention, complex problem solving skills, and motor function. It is estimated that one-third of patients who undergo chemotherapy treatment will experience cognitive impairment. Alterations in the release and uptake of dopamine and serotonin, central nervous system neurotransmitters that play important roles in cognition, could potentially contribute to impaired intellectual performance in those impacted by chemobrain. To investigate how chemotherapy treatment affects these systems, fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to measure dopamine and serotonin release and uptake in coronal brain slices containing the striatum and dorsal raphe nucleus, respectively. Measurements were taken from rats treated weekly with selected doses of carboplatin and from control rats treated with saline. Modeling the stimulated dopamine release plots revealed an impairment of dopamine release per stimulus pulse (80% of saline control at 5 mg/kg and 58% at 20 mg/kg) after 4 weeks of carboplatin treatment. Moreover, Vmax, the maximum uptake rate of dopamine, was also decreased (55% of saline control at 5 mg/kg and 57% at 20 mg/kg). Nevertheless, overall dopamine content, measured in striatal brain lysates by high performance liquid chromatography, and reserve pool dopamine, measured by FSCV after pharmacological manipulation, did not significantly change, suggesting that chemotherapy treatment selectively impairs the dopamine release and uptake processes. Similarly, serotonin release upon electrical stimulation was impaired (45% of saline control at 20 mg/kg). Measurements of spatial learning discrimination were taken throughout the treatment period and carboplatin was found to alter cognition. These studies support the need for additional neurochemical and behavioral analyses to identify the underlying mechanisms of chemotherapy-induced cognitive disorders.

Simultaneous measurement and quantitation of 4-hydroxyphenylacetic acid and dopamine with fast-scan cyclic voltammetry.

Caged compounds have been used extensively to investigate neuronal function in a variety of preparations, including cell culture, ex vivo tissue samples, and in vivo. As a first step toward electrochemically measuring the extent of caged compound photoactivation while also measuring the release of the catecholamine neurotransmitter, dopamine, fast-scan cyclic voltammetry at carbon-fiber microelectrodes (FSCV) was used to electrochemically characterize 4-hydroxyphenylacetic acid (4HPAA) in the absence and presence of dopamine. 4HPAA is a by-product formed during the process of photoactivation of p-hydroxyphenacyl-based caged compounds, such as p-hydroxyphenylglutamate (pHP-Glu). Our data suggest that the oxidation of 4HPAA occurs through the formation of a conjugated species. Moreover, we found that a triangular waveform of -0.4 V to +1.3 V to -0.4 V at 600 V s(-1), repeated every 100 ms, provided an oxidation current of 4HPAA that was enhanced with a limit of detection of 100 nM, while also allowing the detection and quantitation of dopamine within the same scan. Along with quantifying 4HPAA in biological preparations, the results from this work will allow the electrochemical measurement of photoactivation reactions that generate 4HPAA as a by-product as well as provide a framework for measuring the photorelease of electroactive by-products from caged compounds that incorporate other chromophores.

Localized drug application and sub-second voltammetric dopamine release measurements in a brain slice perfusion device.

The use of fast scan cyclic voltammetry (FSCV) to measure the release and uptake of dopamine (DA) as well as other biogenic molecules in viable brain tissue slices has gained popularity over the last 2 decades. Brain slices have the advantage of maintaining the functional three-dimensional architecture of the neuronal network while also allowing researchers to obtain multiple sets of measurements from a single animal. In this work, we describe a simple, easy-to-fabricate perfusion device designed to focally deliver pharmacological agents to brain slices. The device incorporates a microfluidic channel that runs under the perfusion bath and a microcapillary that supplies fluid from this channel up to the slice. We measured electrically evoked DA release in brain slices before and after the administration of two dopaminergic stimulants, cocaine and GBR-12909. Measurements were collected at two locations, one directly over and the other 500 µm away from the capillary opening. Using this approach, the controlled delivery of drugs to a confined region of the brain slice and the application of this chamber to FSCV measurements, were demonstrated. Moreover, the consumption of drugs was reduced to tens of microliters, which is thousands of times less than traditional perfusion methods. We expect that this simply fabricated device will be useful in providing spatially resolved delivery of drugs with minimum consumption for voltammetric and electrophysiological studies of a variety of biological tissues both in vitro and ex vivo.

Conference Report: Pittcon 2013: microfluidics, electrochemistry, and much much more.

The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, also known as Pittcon, is the world's largest annual conference and exposition based on measurement science and instrumentation. Each year, more than 18,000 worldwide participants, coming mostly from academia, industry and government agencies, attend Pittcon to exchange information on the latest analytical techniques, the most advanced instrumentation and the current job market. In 2013, the 64th Pittcon Conference was held at the Pennsylvania Conference Center in Philadephia, PA, USA. Herein, we highlight just a few of the many presentations that describe creative and transformative research efforts aimed at obtaining bioanalytical measurements that enhance knowledge of living systems and improve human health.

Colloidal particles derived from a complex of phosphotungstic Acid and ethoxylated surfactants.

Stable, colloidal sols of submicron size were prepared by titration of aqueous solutions of alkylene oxide surfactants with phosphotungstic acid, H(3)PW(12)O(40) (PTA), followed by neutralization with ammonium or potassium hydroxide. The stoichiometry of the complex between phosphotungstic acid and the ethoxylated surfactant was determined by (1)H and (31)P NMR and was dependent upon the degree of ethoxylation. For example, in the ethoxylated octylphenol having 9-10 ethylene oxide units, Triton X-100, the mole ratio of surfactant to PTA was 4.5. In the ethoxylated octylphenol having 70 ethylene oxide units, Triton X-705, the mole ratio of surfactant to PTA was 1. Prior to nucleation of particles, phosphotungstic acid forms an apparent yellow charge transfer complex with ethoxylated alkylphenol surfactants, typified by Triton X-405. This complex is characterized by an absorption spectrum that is the sum of the spectra of Triton X-405 and PTA with a very weak shoulder at 400-500 nm. Particles were nearly monodisperse and their size was dependent on the nonionic surfactant employed, the heteropolyacid, and the rate of addition of heteropolyacid solution.