G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). Although HA-MOP knock-in mice exhibit reduced receptor expression, we found that this approach allowed for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the 387LENLEAETAPLP398 motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all MOP expressed in the mouse brain exhibit the canonical amino acid sequence.

Electronic cigarettes (ECs) are purported to be tobacco harm-reduction products whose degree of harm has been highly debated. EC use is considered less hazardous than smoking but is not expected to be harmless. Following the banning of e-liquid flavors in countries such as the US, Finland, Ukraine, and Hungary, there are growing concerns regarding the safety profile of e-liquid flavors used in ECs. While these are employed extensively in the food industry and are generally regarded as safe (GRAS) when ingested, GRAS status after inhalation is unclear. The aim of this review was to assess evidence from 38 reports on the adverse effects of flavored e-liquids on the respiratory system in both in vitro and in vivo studies published between 2006 and 2021. Data collected demonstrated greater detrimental effects in vitro with cinnamon (9 articles), strawberry (5 articles), and menthol (10 articles), flavors than other flavors. The most reported effects among these investigations were perturbations of pro-inflammatory biomarkers and enhanced cytotoxicity. There is sufficient evidence to support the toxicological impacts of diacetyl- and cinnamaldehyde-containing e-liquids following human inhalation; however, safety profiles on other flavors are elusive. The latter may result from inconsistencies between experimental approaches and uncertainties due to the contributions from other e-liquid constituents. Further, the relevance of the concentration ranges to human exposure levels is uncertain. Evidence indicates that an adequately controlled and consistent, systematic toxicological investigation of a broad spectrum of e-liquid flavors may be required at biologically relevant concentrations to better inform public health authorities on the risk assessment following exposure to EC flavor ingredients.

Oxytocin (OT) is a developmentally important neuropeptide recognized to play a dominant role in social functioning and stress‐related behaviors, in a sex‐dependent manner. Nonetheless, the underlining factors driving OT and OT receptor (OTR) early brain development remain unclear. Recent evidence highlight the critical influence of gut microbiota and its bidirectional interaction with the brain on neurodevelopment via the gut microbiota‐brain axis. Therefore, we aimed to determine the impact of gut microbiota on the OTR system of the rat brain at different developmental stages in a pilot study. Quantitative OTR [125I]‐OVTA autoradiographic binding was carried out in the forebrain of male and female conventional (CON) and germ‐free (GF) rats at postnatal days (PND) 8, 22, and 116–150. OTR binding was also assessed in the eyes of PND 1 and PND 4 GF female rats. Significant “microbiota × sex × region” interaction and age‐dependent effects on OTR binding were demonstrated. Microbiota status influenced OTR levels in males but not females with higher levels of OTR observed in GF versus CON rats in the cingulate, prelimbic, and lateral/medial/ventral orbital cortex, and septum across all age groups, while sex differences were observed in GF, but not in CON rats. Interestingly, OTRs present in the eyes of CON rats were abolished in GF rats. This is the first study to uncover a sex‐specific role of gut microbiota on the central OTR system, which may have implications in understanding the developmental neuroadaptations critical for behavioral regulation and the etiology of certain neurodevelopmental disorders.

μ‐opioid receptors (MOPr) play a critical role in social play, reward and pain, in a sex‐ and age‐dependent manner. There is evidence to suggest that sex and age differences in brain MOPr density may be responsible for this variability; however, little is known about the factors driving these differences in cerebral MOPr density. Emerging evidence highlights gut microbiota's critical influence and its bidirectional interaction with the brain on neurodevelopment. Therefore, we aimed to determine the impact of gut microbiota on MOPr density in male and female brains at different developmental stages. Quantitative [3H]DAMGO autoradiographic binding was carried out in the forebrain of male and female conventional (CON) and germ‐free (GF) rats at postnatal days (PND) 8, 22 and 116–150. Significant ‘microbiota status X sex’, ‘age X brain region’ interactions and microbiota status‐ and age‐dependent effects on MOPr binding were uncovered. Microbiota status influenced MOPr levels in males but not females, with higher MOPr levels observed in GF versus CON rats overall regions and age groups. In contrast, no overall sex differences were observed in GF or CON rats. Interestingly, within‐age planned comparison analysis conducted in frontal cortical and brain regions associated with reward revealed that this microbiota effect was restricted only to PND22 rats. Thus, this pilot study uncovers the critical sex‐dependent role of gut microbiota in regulating cerebral MOPr density, which is restricted to the sensitive developmental period of weaning. This may have implications in understanding the importance of microbiota during early development on opioid signalling and associated behaviours.

Background Electronic cigarettes (ECs) are considered a less hazardous alternative to tobacco smoking but are not harmless. Growing concerns about the safety profiles of flavors in e-liquids underpin the need for this study. Methods Here, we screened 53 nicotine-free flavored e-liquids (across 15 flavor categories) across a 3-point concentration range (0.25%, 0.5%, and 1% v/v) in a high-throughput fashion in human bronchial epithelial (HBEC-3KT) submerged cell cultures to identify ‘toxic hits’ using in vitro endpoint assays comprising cell count, cell viability, and lactate dehydrogenase (LDH). Results We observed significant, dose-dependent adverse effects only with cinnamon, vanilla tobacco, and hazelnut e-liquids compared to media-only control and PG/VG vehicle controls. Hence, we further analyzed these three flavors for their effects on HBEC-3KT proliferation, mitochondrial health, and oxidative stress. A significant decrease in cell proliferation after 36h was observed for each e-liquid toxic hit compared to media-only and PG/VG controls. Hazelnut (at all concentrations) and vanilla tobacco (1%) increased cytoplasmic reactive oxygen species (ROS) generation compared to media-only and PG/VG controls. Conversely, all three flavors at 0.5% and 1% significantly decreased mitochondrial membrane potential (MP) compared to PG/VG and media-only controls. We hypothesized that the cytotoxic effects of cinnamon flavor in e-liquids might be mediated via the transient protein receptor ankyrin subtype 1 (TRPA1); however, TRPA1 antagonist AP-18 (10 µM) did not mitigate these effects, and cinnamon significantly increased TRPA1 transcript levels. Therefore, pathways that mediate cinnamon’s cytotoxicity warrant further investigations. Conclusion This study could inform public health authorities on the relative health risks assessment following exposure to EC flavor ingredients.