The cross-reactivity of cannabinoid analogs (delta-8-THC, delta-10-THC and CBD), their metabolites and chiral carboxy HHC metabolites in urine of six commercially available homogeneous immunoassays.

There has been an exponential surge in the presence and use of cannabinoids since the federal legalization of hemp (Agricultural Improvement Act of 2018). This growth is attributed to delta-9-tetrahydrocannabinol (delta-9-THC) and cannabidiol (CBD), the most abundant phytocannabinoid components of cannabis and hemp, respectively, but with many other emerging THC analogs. Structurally, these analogs are similar to delta-9-THC, yet very little information is available about their potency and even less information is available regarding their detectability using commercially available cannabinoid screening kits. Due to their structural similarity, current cannabinoid homogeneous immunoassay screening methods may be able to detect these emerging cannabinoid analogs and their metabolites. Six urine immunoassay kits (Abbott Cannabinoids-Abbott Diagnostics, LZI Cannabinoids (cTHC) Enzyme Immunoassay-Lin-Zhi International, DRI® Cannabinoid Assay and CEDIA™ THC-Thermo Fisher Scientific, ONLINE DAT Cannabinoid II-Roche Diagnostics and Syva EMIT®II Plus-Siemens Healthineers) were evaluated at two different cutoff concentrations: 50 ng/mL and 20 or 25 ng/mL, assay dependent. The analysis was performed on an Abbott Architect Plus c4000 (Abbott Diagnostics). Delta-8-THC, CBD, olivetol and their major metabolites, and delta-10-THC and HHC carboxylic acid chiral analogs were evaluated. The cross-reactivity was evaluated by preparing each analyte at 20, 50, 100 and 1,000 ng/mL in urine. Analytes that did not cross-react at 1,000 ng/mL for a cutoff were considered not detectable. If detected, the lowest concentration was used as the decision point to determine the precision at the immunoassay's cutoff. The six commercially available urine cannabinoid homogeneous immunoassay screening kits cross reacted with the following analogs: delta-8-THC, 11-OH-delta-8-THC, 11-COOH-delta-8-THC, 6-OH-CBD, 7-OH-CBD, all delta-10-THC and HHC carboxylic acid chiral analogs and olivetol with varying selectivity depending on the screening kit and cutoff concentration. The kits did not cross-react with the following analogs: CBD, 7-COOH-CBD, Abnormal CBD, CBDA-A and olivetolic acid.

The preanalytical stability of emerging cannabinoid analogs (delta-8 THC and its metabolites, delta-10 THC and carboxy-HHC) in urine.

There has been a surge in the presence and use of cannabinoids since the federal legalization of hemp (Agricultural Improvement Act of 2018). This increase is attributed not only to the use of ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol, the most abundant phytocannabinoid components of cannabis and hemp, respectively, but also to the use of many other emerging THC analogs. Structurally, these analogs are similar to ∆9-THC. Urine specimens for drug analysis are often collected offsite and transported to a laboratory for analysis. Screening assays are usually the first step in urine drug testing. These assays are usually qualitative and automated, which for negative specimens, reduce cost and reporting time. The stability of ∆9-THC and its metabolites has been known for some time; however, the stability of emerging analogs has not been elucidated, and therefore, assuming equivalent storage stability can be erroneous. Previous work assessed the cross-reactivity of ∆8-THC and its major metabolites, the ∆10-THC chiral analogs and the chiral 11-COOH-hexahydrocannabinol analogs. Stability was assessed for each analyte at a concentration two times greater than the analytes' determined decision point. Samples were prepared in drug-free urine at three different pHs (4.5, 7 and 9) and stored at three different temperatures (4°C, 20°C and 45°C) in triplicate. Samples were analyzed utilizing the Lin-Zhi International Cannabinoids Enzyme Immunoassay cannabinoid screening kit calibrated at the 25 ng/mL cut-off. Overall, the cannabinoid analogs produced diminishing instrument responses depending on pH and temperature. The parent analogs were not detected after a single day at 45°C regardless of pH. In general, carboxylic acid analogs at the acidic pH (4.5) produced diminished instrument responses when compared to their counterparts stored at neutral (7) and basic (9) pH. The time, storage temperature and pH of urine specimens may affect the screening results of specimens collected for cannabinoid drug screening.

SARS-CoV-2 spike protein reduces burst activities in neurons measured by micro-electrode arrays.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) caused a large-scale global pandemic between 2020 and 2022. Despite efforts to understand its biological and pathogenic mechanisms, the viral impact on the neurological systems remains unclear. The main goal of this study was to quantify the neurological phenotypes induced by the SARS-CoV-2 spike protein in neurons, as measured by in-vitro multiwell micro-electrode arrays (MEAs). Materials and methods: The authors extracted the whole-brain neurons from the newborn P1 mice and plated them on multiwell MEAs and administered purified recombinant spike proteins (both S1 and S2 subunits) from the SARS-CoV-2 virus. The signals from the MEAs were transmitted from an amplifier to a high-performance computer for recording and analysis using an in-house developed algorithm to quantify neuronal phenotypes. Results: Primary among the phenotypic features analyzed, we discovered that neuronal treatment with spike 1 protein (S1) protein from SARS-CoV-2 decreased the mean burst numbers observed on each electrode, an effect that could be rescued with an anti-S1 antibody. Conversely, this mean burst number decrease was not observed with spike 2 protein (S2) treatment. Finally, our data strongly suggest that the receptor binding domain of S1 is responsible for the reduction in neuronal burst activity. Conclusion: Overall, our results strongly indicate that spike proteins may play an important role in altering neuronal phenotypes, specifically the burst patterns, when neurons are exposed during early development.

FT-6876, a Potent and Selective Inhibitor of CBP/p300, is Active in Preclinical Models of Androgen Receptor-Positive Breast Cancer.

BACKGROUND: Patients with triple-negative breast cancer (TNBC) expressing the androgen receptor (AR) respond poorly to neoadjuvant chemotherapy, although AR antagonists have shown promising clinical activity, suggesting these tumors are AR-dependent. cAMP responsive element binding protein (CREB)-binding protein (CBP) and p300 are transcriptional co-activators for the AR, a key driver of AR+ breast and prostate cancer, and may provide a novel therapeutic target in AR+ TNBC. OBJECTIVES: The aim of this study was to determine the therapeutic potential of FT-6876, a new CBP/p300 bromodomain inhibitor, in breast cancer models with a range of AR levels in vitro and in vivo. METHODS: Effects of FT-6876 on the CBP/p300 pathway were determined by combining chromatin immunoprecipitation (ChIP) with precision run-on sequencing (PRO-seq) complemented with H3K27 acetylation (Ac) and transcriptional profiling. The antiproliferative effect of FT-6876 was also measured in vitro and in vivo. RESULTS: We describe the discovery of FT-6876, a potent and selective CBP/p300 bromodomain inhibitor. The combination of ChIP and PRO-seq confirmed the reduction in H3K27Ac at specific promoter sites concurrent with a decrease in CBP/p300 on the chromatin and a reduction in nascent RNA and enhancer RNA. This was associated with a time- and concentration-dependent reduction in H3K37Ac associated with a decrease in AR and estrogen receptor (ER) target gene expression. This led to a time-dependent growth inhibition in AR+ models, correlated with AR expression. Tumor growth inhibition was also observed in AR+ tumor models of TNBC and ER+ breast cancer subtypes with consistent pharmacokinetics and pharmacodynamics. CONCLUSION: Our findings demonstrate FT-6876 as a promising new CBP/p300 bromodomain inhibitor, with efficacy in preclinical models of AR+ breast cancer.

Detection of 58 fentanyl analogs using ARK fentanyl II and Immunalysis fentanyl immunoassays.

OBJECTIVES: The ability to detect fentanyl analogs in urine aids in patient management. Little is published about the new ARK™ Fentanyl II Assay formulation's ability to detect fentanyl analogs. Norfentanyl (fentanyl metabolite) cross-reactivity with the ARK II assays is 7%, while the Immunalysis SEFRIA assay norfentanyl cross-reactivity is approximately 0.005%. The purpose of this study was to determine the new ARK II and SEFRIA fentanyl assays' detection of 58 fentanyl analogs. DESIGN & METHODS: Drug-free urine was fortified with 0-100 ng/mL (0-0.297 µmol/L) of the fentanyl analog and analyzed using the previously evaluated immunoassays. Results were compared to molecular structure. Of the 58 analogs tested at ≤ 100 ng/mL (0-0.297 µmol/L), the ARK II and SEFRIA assays produced 51 and 57 positive results respectively. The cross-reactivity of the assay was predominantly determined by the location of the modification. Most modifications to the aniline ring and/or amide group did not affect the ARK II or SEFRIA assay. Modifications to the piperidine ring decreased detection by ARK II assay. Of the 7 compounds which were undetected by the ARK II assay, all had modifications to the N-alkyl chain. Norsufentanil was not detected by either assay and was the only analog not detected by the SEFRIA assay. CONCLUSIONS: The ARK II and Immunalysis fentanyl immunoassays can detect a range of fentanyl analogs with acryl, butyryl, or furanyl modifications to the amide group or aniline ring of the molecule. N-alkyl chain and piperidine ring modifications significantly affect the ARK II assay's ability to detect the analogs, while the SEFRIA assay appeared less affected and detected all analogs tested except for norsufentanil, which was also not detected by the ARK II assay.

Potential Impact of Pharmacogenomic Single Nucleotide Variants in a Rural Caucasian Population.

BACKGROUND: In the US adverse drug reactions (ADRs) are estimated to cause 100 000 fatalities and cost over $136 billion annually. A patient's genes play a significant role in their response to a drug. Pharmacogenomics aims to optimize drug choice and dose for individual patients by characterizing patients' pharmacologically relevant genes to identify variants of known impact. METHODS: DNA was extracted from randomly selected remnant whole blood samples from Caucasian patients with previously performed complete blood counts. Samples were genotyped by mass spectrometry using a customized pharmacogenomics panel. A third-party result interpretation service used genotypic results to predict likely individual responses to frequently prescribed drugs. RESULTS: Complete genotypic and phenotypic calls for all tested Cytochrome P450 isoenzymes and other genes were obtained from 152 DNA samples. Of these 152 unique genomic DNA samples, 140 had genetic variants suggesting dose adjustment for at least one drug. Cardiovascular and psychiatry drugs had the highest number of recommendations, which included United States Food and Drug Administration warnings for highly prescribed drugs metabolized by CYP2C19, CYP2C9, CYP2D6, HLA-A, and VKORC1. CONCLUSIONS: Risk for each drug:gene pairing primarily depends upon the degree of predicted enzyme impairment or activation, width of the therapeutic window, and whether parent compound or metabolite is pharmacologically active. The resulting metabolic variations range from risk of toxicity to therapeutic failure. Pharmacogenomic profiling likely reduces ADR potential by allowing up front drug/dose selection to fit a patient's unique drug-response profile.

Evaluation of Strontium Interference in Calcium Measurement Procedures and Content in Supplements as Measured by ICP-MS.

BACKGROUND: Bone health supplements containing strontium are available without prescription, however, the effects of strontium interference on clinical laboratory calcium measurement procedures are unknown. METHODS: To evaluate strontium interference on total calcium measurements, plasma pools with exogenously added strontium were measured by 3 total calcium measurement procedures. For ionized calcium measurements, whole blood pools prepared with exogenously added strontium were measured by 2 ionized calcium measurement procedures. An inductively coupled plasma mass spectrometry assay (ICP-MS) was validated for research measurements of strontium content in commercially available supplements. RESULTS: Exogenous strontium addition to plasma caused positive bias for total calcium measurements. Strontium concentrations of 1.0 mg/dL (0.114 mmol/L), 2.5 mg/dL (0.284 mmol/L), and 5.0 mg/Dl (0.568 mmol/L) resulted in mean biases of 1.9% to 3.5%, 4.9% to 9.0%, and 10.8% to 19.2%, respectively, for total calcium measurement procedures. Biases for ionized calcium measurements were less than 4.5% for a strontium concentration of 5.0 mg/dL (0.568 mmol/L). An in-house-developed ICP-MS assay for strontium in commercially available supplements exhibited within-laboratory and within-run coefficients of variation of less than 3%, and a linear response was obtained over the assay analytical measurement range of 10 to 100 000 ng/mL (0.0001 to 1.141 mmol/L). Strontium recovery for the ICP-MS assay was 97.1% to 105.3%. The largest amount of strontium measured in dietary supplements was 395 mg in a 1054 mg tablet. CONCLUSIONS: Some dietary supplements contain larger amounts of strontium than indicated on the product label. High concentrations of strontium may cause significant interference for total calcium measurement procedures, but ionized calcium measurement procedures are not significantly affected.

Development of an Innovative Online Dietary Assessment Tool for France: Adaptation of myfood24.

myfood24 is an innovative dietary assessment tool originally developed in English for use in the United Kingdom. This online 24 h recall, a tool commonly used in nutritional epidemiology, has been developed into different international versions. This paper aims to describe the creation of its French version. We used a consistent approach to development, aligned with other international versions, using similar methodologies. A nutritional database (food item codes, portion groups and accompaniments, etc.) was developed based on commonly used French food composition tables (CIQUAL 2017). Portion sizes were adapted to French dietary habits (estimation, photographs of French portion sizes, assessment of the photograph series and their angle (aerial vs. 45 degrees)). We evaluated the new tool, which contained nearly 3000 food items with 34 individuals using the System Usability Scale. We validated the French food portion picture series using EFSA criteria for bias and agreement. The results of the picture evaluation showed that the angle with which photos are taken had limited impact on the ability to judge portion size. Estimating food intake is a challenging task. Evaluation showed "good" usability of the system in its French version. myfood24 France will be a useful addition to nutritional epidemiology research in France.

Temperature and Season Influence Phagocytosis by B1 Lymphocytes in the Mojave Desert Tortoise.

Lymphocytes are usually interpreted as functioning in adaptive immunity despite evidence that large proportions of these cells (B1 lymphocytes) have innate immune functions, including phagocytosis, in the peripheral blood of ectothermic vertebrates. We used a recently optimized assay to assess environmental influences on phagocytic activity of lymphocytes isolated from the Mojave desert tortoise (Gopherus agassizii). Previous studies suggest that lymphocytes in this species are associated with reduced pathogen loads, especially in cooler climates, and that lymphocyte numbers fluctuate seasonally. Thus, we evaluated thermal dependence of phagocytic activity in vitro and across seasons. While B1 lymphocytes appeared to be cold-adapted and always increased phagocytosis at cool temperatures, we also found evidence of thermal acclimation. Tortoises upregulated these lymphocytes during cooler seasons in the fall as their preferred body temperatures dropped, and phagocytosis also increased in efficiency during this same time. Like many other ectothermic species, populations of desert tortoises are in decline, in part due to a cold-adapted pathogen that causes chronic respiratory disease. Future studies, similarly focused on the function of B1 lymphocytes, could serve to uncover new patterns in thermal acclimation of immune functions and disease ecology across taxa of ectothermic vertebrates.

Local connectivity and synaptic dynamics in mouse and human neocortex.

We present a unique, extensive, and open synaptic physiology analysis platform and dataset. Through its application, we reveal principles that relate cell type to synaptic properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Synaptic properties are heterogeneous in most subclass-to-subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, whereas the strength axis accounts for substantial heterogeneity within the subclass. In the human cortex, excitatory-to-excitatory synaptic dynamics are distinct from those in the mouse cortex and vary with depth across layers 2 and 3.

Negligible Nux Vomica: Homeopathic Nux Vomica remedies don't contain strychnine?

INTRODUCTION: The St. Ignatius bean of the Strychnos ignatii tree and Nux Vomica homeopathic products presumably could contain the toxic alkaloids strychnine and brucine. This study aimed to determine the amount of these toxic alkaloids in some commercially available Nux Vomica products and the St. Ignatius bean and to determine if overdose of these products could result in clinically significant toxicity. METHODS: Using ultra-performance liquid chromatography-tandem mass spectrometry, various formulations of Nux Vomica products and St. Ignatius beans were analyzed for strychnine, and brucine with detection limits set at 0.1 ng/g. RESULTS: None of the analyzed Nux Vomica products contained any detectable strychnine or brucine, while the expected strychnine dose from a St. Ignatius bean would be < 0.001 mg. CONCLUSIONS: Overall, our study reveals that the amount of strychnine in homeopathic Nux Vomica products or St. Ignatius beans are not likely to result in clinically significant strychnine toxicity.

Developing an online food composition database for an Indigenous population in south-western Uganda.

OBJECTIVE: To develop an online food composition database of locally consumed foods among an Indigenous population in south-western Uganda. DESIGN: Using a community-based approach and collaboration with local nutritionists, we collected a list of foods for inclusion in the database through focus group discussions, an individual dietary survey and markets and shops assessment. The food database was then created using seven steps: identification of foods for inclusion in the database; initial data cleaning and removal of duplicate items; linkage of foods to existing generic food composition tables; mapping and calculation of the nutrient content of recipes and foods; allocating portion sizes and accompanying foods; quality checks with local and international nutritionists; and translation into relevant local languages. SETTING: Kanungu District, south-western Uganda. PARTICIPANTS: Seventy-four participants, 36 Indigenous Batwa and 38 Bakiga, were randomly selected and interviewed to inform the development of a food list prior the construction of the food database. RESULTS: We developed an online food database for south-western Uganda including 148 commonly consumed foods complete with values for 120 micronutrients and macronutrients. This was for use with the online dietary assessment tool myfood24. Of the locally reported foods included, 56 % (n 82 items) of the items were already available in the myfood24 database, while 25 % (n 37 items) were found in existing Ugandan and Tanzanian food databases, 18 % (n 27 items) came from generated recipes and 1 % (n 2 items) from food packaging labels. CONCLUSION: Locally relevant food databases are sparse for African Indigenous communities. Here, we created a tool that can be used for assessing food intake and for tracking undernutrition among the communities living in Kanungu District. This will help to develop locally relevant food and nutrition policies.

Clinical Validation of a 106-SNV MALDI-ToF MS Pharmacogenomic Panel.

BACKGROUND: Laboratorians have the opportunity to help minimize the frequency of adverse drug reactions by implementing pharmacogenomic testing and alerting care providers to possible patient/drug incompatibilities before drug treatment is initiated. Methods combining PCR with MALDI-ToF MS have allowed for sensitive, economical, and multiplexed pharmacogenomic testing results to be delivered in a timely fashion. METHOD: This study evaluated the analytical performance of the Agena Biosciences iPLEX® PGx 74 panel and a custom iPLEX panel on a MassARRAY MALDI-TOF MS instrument in a clinical laboratory setting. Collectively, these panels evaluate 112 SNVs across 34 genes implicated in drug response. Using commercially available samples (Coriell Biorepository) and in-house extracted DNA, we determined ideal reaction conditions and assessed accuracy, precision, and robustness. RESULTS: Following protocol optimization, the Agena PGx74 and custom panels demonstrated 100% concordance with the 1000 Genomes Project Database and clinically validated hydrolysis probe genotyping assays. 100% concordance was also observed in all assessments of assay precision when appropriate QC metrics were applied. CONCLUSIONS: Significant development time was required to optimize sample preparation and instrumental analysis and 3 assays were removed due to inconsistent performance. Following modification of the manufacturer's protocol and instituting manual review of each assay plate, the Agena PGx74 and custom panel constitute a cost-effective, robust, and accurate method for clinical identification of 106 SNVs involved in drug response.

Assessment of biotin interference with qualitative point-of-care hCG test devices.

Biotin is commonly used as a dietary supplement for the claimed benefits of promoting healthy hair and nail growth and is available without a prescription at doses up to 10mg/capsule. Biotin-mediated interference in immunoassay testing is an emerging issue for clinical laboratories and previous studies have indicated that biotin at regularly encountered doses may interfere with these assays. In this study, we evaluated the effect of supplemental biotin on seven POC urine hCG test devices using purified biotin and urine collected from four volunteers consuming 10mg biotin/day. Six of the seven devices showed no evidence of biotin interference as each device's control line remained clearly detectable at all biotin concentrations tested. However, the QuickVue device control line demonstrated a marked decrease in intensity when used to test solutions containing >5µg/mL biotin. The absence of a control line during patient testing has the potential to delay care due to the generation of an invalid test result and lead to additional unnecessary testing. It is not realistic to measure urinary biotin concentrations in every patient undergoing qualitative urine hCG testing but biotin supplementation should be considered if repeat testing on a patient sample generates an invalid test result.

Cervical kinematics in patients with vestibular pathology vs. patients with neck pain: A pilot study.

OBJECTIVE: Research has consistently shown cervical kinematic impairments in subjects with persistent neck pain (NP). It could be reasoned that those with vestibular pathology (VP) may also have altered kinematics since vestibular stimulation via head movement can cause dizziness and visual disturbances. However, this has not been examined to date. This pilot study investigated changes in cervical kinematics between asymptomatic control, NP and VP subjects using a Virtual Reality (VR) system. It was hypothesised that there would be altered kinematics in VP subjects, which might be associated with dizziness and visual symptoms. DESIGN: Pilot cross sectional observational study. PARTICIPANTS: Twenty control, 14 VP and 20 NP subjects. INTERVENTION: Not applicable. MAIN OUTCOME MEASURES: Measures included questionnaires (neck disability index, pain on movement, dizziness and pain intensity, visual disturbances) and cervical kinematics (range, peak and mean velocity, smoothness, symmetry, and accuracy of cervical motion) using a virtual reality system. RESULTS: Results revealed significantly decreased mean velocity and symmetry of motion in both planes in those with NP but no differences in accuracy or range of motion. No significant differences were seen between VP subjects and asymptomatic controls. However, correlation analysis showed some moderate correlations between dizziness to selected kinematics in both the NP and the VP groups. CONCLUSIONS: These results support that cervical kinematics are altered in NP patients, with velocity most affected. There is potential for VP subjects to also have altered kinematics, especially those who experience dizziness. More research is required.

Design and Optimization of Benzopiperazines as Potent Inhibitors of BET Bromodomains.

A protein structure-guided drug design approach was employed to develop small molecule inhibitors of the BET family of bromodomains that were distinct from the known (+)-JQ1 scaffold class. These efforts led to the identification of a series of substituted benzopiperazines with structural features that enable interactions with many of the affinity-driving regions of the bromodomain binding site. Lipophilic efficiency was a guiding principle in improving binding affinity alongside drug-like physicochemical properties that are commensurate with oral bioavailability. Derived from this series was tool compound FT001, which displayed potent biochemical and cellular activity, translating to excellent in vivo activity in a mouse xenograft model (MV-4-11).

Exploring G protein-coupled receptor signaling networks using SILAC-based phosphoproteomics.

The type 1 parathyroid hormone receptor (PTH1R) is a key regulator of calcium homeostasis and bone turnover. Here, we employed SILAC-based quantitative mass spectrometry and bioinformatic pathways analysis to examine global changes in protein phosphorylation following short-term stimulation of endogenously expressed PTH1R in osteoblastic cells in vitro. Following 5min exposure to the conventional agonist, PTH(1-34), we detected significant changes in the phosphorylation of 224 distinct proteins. Kinase substrate motif enrichment demonstrated that consensus motifs for PKA and CAMK2 were the most heavily upregulated within the phosphoproteome, while consensus motifs for mitogen-activated protein kinases were strongly downregulated. Signaling pathways analysis identified ERK1/2 and AKT as important nodal kinases in the downstream network and revealed strong regulation of small GTPases involved in cytoskeletal rearrangement, cell motility, and focal adhesion complex signaling. Our data illustrate the utility of quantitative mass spectrometry in measuring dynamic changes in protein phosphorylation following GPCR activation.