Analysis of In Vitro Aptamer Selection Parameters.

Nucleic acid aptamers are novel molecular recognition tools that offer many advantages compared to their antibody and peptide-based counterparts. However, challenges associated with in vitro selection, characterization, and validation have limited their wide-spread use in the fields of diagnostics and therapeutics. Here, we extracted detailed information about aptamer selection experiments housed in the Aptamer Base, spanning over two decades, to perform the first parameter analysis of conditions used to identify and isolate aptamers de novo. We used information from 492 published SELEX experiments and studied the relationships between the nucleic acid library, target choice, selection methods, experimental conditions, and the affinity of the resulting aptamer candidates. Our findings highlight that the choice of target and selection template made the largest and most significant impact on the success of a de novo aptamer selection. Our results further emphasize the need for improved documentation and more thorough experimentation of SELEX criteria to determine their correlation with SELEX success.

"Out-gel" tryptic digestion procedure for chemical cross-linking studies with mass spectrometric detection.

SDS-PAGE is one of the most powerful protein separation techniques, and in-gel digestion is the leading method for converting proteins separated by SDS-PAGE into peptides suitable for mass spectrometry-based proteomic studies. In in-gel digestion, proteins are digested within the gel matrix, and the resulting peptides are extracted into an appropriate buffer. Transfer of the digested peptides to the liquid phase for subsequent mass spectrometric analysis, however, may be hampered by peptide-specific characteristics, including size, shape, poor solubility, adsorption to the polyacrylamide, and-in the case of cross-linking applications-by the branched structure of the peptides produced. This can be a limitation in cross-linking studies where efficient recoveries of the cross-linked peptides are critical. To overcome this limitation, we have developed a modification to the standard in-gel digestion procedure for SDS-PAGE-separated cross-linked proteins, based on older passive diffusion methods. By omitting the gel staining and gel fixation steps, intact proteins or cross-linked protein complexes can move through the gel and into the buffer solution. Digestion of the entire protein in the buffer outside the gel increases the probability that most of the proteolytic peptides produced will be present in the final digest solution. The resulting peptide mixture is then freed of SDS and concentrated using SCX (strong cation exchange) zip-tips and analyzed by mass spectrometry. For standard protein identification studies and the recovery of noncross-linked peptides, the in-gel procedure outperformed the out-gel procedure, but for cross-linking studies with enrichable cross-linkers (such as CBDPS), the standard out-gel procedure allowed the recoveries of cross-links not recovered via the in-gel method. Labeling experiments showed that, with an enrichable cross-linker, 93% of the cross-links showed better or equal recoveries with the out-gel procedure, as compared to the in-gel procedure. It should be noted that this method is not designed to replace in-gel digestion for most proteomics applications. However, by using the out-gel method, we were able to detect twice as many interprotein CBDPS cross-links from the histone H2A/H2B complex as were found in the in-gel digested sample.

Modulation of type 1 cannabinoid receptor activity by cannabinoid by-products from Cannabis sativa and non-cannabis phytomolecules.

Cannabis sativa contains more than 120 cannabinoids and 400 terpene compounds (i.e., phytomolecules) present in varying amounts. Cannabis is increasingly available for legal medicinal and non-medicinal use globally, and with increased access comes the need for a more comprehensive understanding of the pharmacology of phytomolecules. The main transducer of the intoxicating effects of Cannabis is the type 1 cannabinoid receptor (CB1R). ∆9-tetrahydrocannabinolic acid (∆9-THCa) is often the most abundant cannabinoid present in many cultivars of Cannabis. Decarboxylation converts ∆9-THCa to ∆9-THC, which is a CB1R partial agonist. Understanding the complex interplay of phytomolecules-often referred to as "the entourage effect"-has become a recent and major line of inquiry in cannabinoid research. Additionally, this interest is extending to other non-Cannabis phytomolecules, as the diversity of available Cannabis products grows. Here, we chose to focus on whether 10 phytomolecules (∆8-THC, ∆6a,10a-THC, 11-OH-∆9-THC, cannabinol, curcumin, epigallocatechin gallate, olivetol, palmitoylethanolamide, piperine, and quercetin) alter CB1R-dependent signaling with or without a co-treatment of ∆9-THC. Phytomolecules were screened for their binding to CB1R, inhibition of forskolin-stimulated cAMP accumulation, and ßarrestin2 recruitment in Chinese hamster ovary cells stably expressing human CB1R. Select compounds were assessed further for cataleptic, hypothermic, and anti-nociceptive effects on male mice. Our data revealed partial agonist activity for the cannabinoids tested, as well as modulation of ∆9-THC-dependent binding and signaling properties of phytomolecules in vitro and in vivo. These data represent a first step in understanding the complex pharmacology of Cannabis- and non-Cannabis-derived phytomolecules at CB1R and determining whether these interactions may affect the physiological outcomes, adverse effects, and abuse liabilities associated with the use of these compounds.

In-situ TD-GCMS measurements of oxidative products of monoterpenes at typical vaping temperatures: implications for inhalation exposure to vaping products.

Vaping is gaining in popularity. However, there is still much that remains unknown about the potential risk and harms of vaping. Formation of oxidative products is one of such areas that are not well understood. In this study, we used an in-situ thermal desorption GC/MS method to investigate the formation of oxidative products of several monoterpenes at or below typical vaping temperatures. Among the five tested monoterpenes, the unchanged portion of the parent compound in the vapour varied from 97 to 98% for myrcene to 11-28% for terpinolene. The majority of formed oxidative products in the vapour have a molecular weight of 134 (loss of two hydrogens), 150 (insertion of one oxygen and loss of two hydrogen atoms) or 152 (insertion of one oxygen atom). Three products, likely to be p-(1-propenyl)-toluene, ß-pinone and fenchol were also observed. This is the first in-situ thermal desorption GC/MS study to investigate the possible formation of oxidative products of monoterpenes, one of the major components in vaping liquids, at temperatures that are relevant to the vaping process. Although the toxicity of inhaling these oxidative products is not clear yet, allergic and irritation reactions associated with oxidized monoterpene oils are well documented. Therefore, potential adverse effects of inhaling these oxidative products during vaping could be investigated to help support human risk assessment.

Evidence That Metal Particles in Cannabis Vape Liquids Limit Measurement Reproducibility.

Cannabis vaping involves the vaporization of a cannabis vaping liquid or solid via a vaping accessory such as a vape pen constructed of various metals or other parts. An increasing number of reports advocate for expansion of the testing and regulation of metal contaminants in cannabis vape liquids beyond the metals typically tested such as arsenic, cadmium, mercury, and lead to reflect the possibility of consumers' exposure to other metal contaminants. Metal contaminants may originate not only from the cannabis itself but also from the vape devices in which the cannabis vape liquid is packaged. However, metal analyses of cannabis vape liquids sampled from cannabis vaping devices are challenged by poor precision and reproducibility. Herein, we present data on the metal content of 12 metals in 20 legal and 21 illegal cannabis vape liquids. The lead mass fraction in several illegal samples reached up to 50 µg g-1. High levels of nickel (max 677 µg g-1) and zinc (max 426 µg g-1) were found in illegal samples, whereas the highest copper content (485 µg g-1) was measured in legal samples. Significant differences in metal mass fractions were observed in the legal cannabis vape liquid taken from two identical devices, even though the liquid was from the same lot of the same cannabis product. Metal particles in the vape liquids were observed by scanning electron microscopy, and laser ablation inductively coupled plasma mass spectrometry confirmed the presence of copper-, zinc-, lead-, and manganese-bearing particles, metals that are in common alloys that may be used to make vape devices. Colocalized particles containing aluminum, silica, and sodium were also detected. These results suggest that metal particles could be a contributing factor to poor measurement precision and for the first time, to the best of our knowledge, provide evidence of metal particles in cannabis vape liquids contained in unused cannabis vape pens.

Assessing the bioactivity of cannabis extracts in larval zebrafish.

BACKGROUND: Whole-plant cannabis extracts are consumed by the public for medical and non-medical ("recreational") purposes but are poorly researched compared to pure cannabinoids. There is emerging evidence that cannabis extracts comprising complex mixtures of cannabinoids may have different biological effects from that of pure cannabinoids. In the current study, we sought to assess the effect of whole-plant cannabis extracts produced from different chemotypes of cannabis on the normal behavior of zebrafish larvae. METHODS: Three cannabis plant chemotypes were used in this study that contained either high amounts of THC, high amounts of CBD, high but equal amounts of THC and CBD, or low but equal amounts of THC and CBD. Following solvent extraction, liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) was performed for the detection and quantitation of target cannabinoids. Larval zebrafish behavioral models were subsequently used to assess the effect of the four different whole-plant cannabis extracts on the normal larval behavior using the DanioVision behavioral tracking systems and software. To compare, changes in the behavior activity levels for 30 min periods were compared to controls using 2-way ANOVA with multiple comparisons followed by a Bonferroni post hoc test. RESULTS: It was found that the whole-plant extracts that contained high levels of THC had similar effects on larval behavior, while the high CBD and low THC:CBD extracts produced distinct effects on normal larval behavior. Exposure of larvae to concentration-matched levels of THC and CBD found in the extracts revealed that a subset of the cannabis extracts tested had similar behavioral profiles to the pure cannabinoids while others did not. CONCLUSIONS: To our knowledge, this is the first study to test and compare the bioactivity of different whole-plant cannabis extracts in larval zebrafish. This work will provide a framework for future studies of distinct cannabis extracts and will be useful for comparing the bioactivity of extracts from different cannabis chemotypes as well as extracts made through various heating processes. It will also act as the first stage of assessment before testing the extracts against zebrafish models of toxicity and disease.

A method for quantification of volatile organic compounds in blood by SPME-GC-MS/MS with broader application: From non-occupational exposure population to exposure studies.

Humans are continuously exposed to volatile organic compounds (VOCs) as these chemicals are ubiquitously present in most indoor and outdoor environments. In order to assess recent exposure to VOCs for population-based studies, VOCs are measured in the blood of participants. This work describes an improved method to detect 12 VOCs by head-space solid-phase microextraction gas chromatography coupled with isotope-dilution mass spectrometry in selected reaction monitoring mode (SPME-GC-MS/MS). This method was applied to the analysis of trihalomethanes, styrene, trichloroethylene, tetrachloroethylene and BTEX (benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene) in a population-based biomonitoring study (Canadian Health Measures Survey). The method showed good linearity (>0.990) in the range of 0.010-10µg/L and detection limits between 0.007 and 0.027µg/L, precision better than 25% and good accuracy (±25%) based on proficiency testing materials. Quality Control data among runs over a 7 month period showed %RSD between 14 and 25% at low levels (∼0.03µg/L) and between 9 and 23% at high levels (∼0.4µg/L). The method was modified to analyze samples from a pharmacokinetic study in which 5 healthy volunteers were exposed to single, binary and quaternary mixtures of CTEX (chloroform, ethylbenzene, toluene and m-xylene), thus the expected concentration in blood was 1 order of magnitude higher than those found in the general population. The method was modified by reducing the sample size (from 3g to 0.5g) and increasing the upper limit of the concentration range to 395µg/L. Good linearity was found in the range of 0.13-395µg/L for toluene and ethylbenzene and 0.20-609µg/L for m/p-xylene. Quality control data among runs over the period of the study (n=13) were found to vary between 7 and 25%.