Effects of acute cannabidiol on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice.

Background: Some evidence suggests that cannabidiol (CBD) has potential to help alleviate HIV symptoms due to its antioxidant and anti-inflammatory properties. Here we examined acute CBD effects on various behaviors and the endocannabinoid system in HIV Tat transgenic mice. Methods: Tat transgenic mice (female/male) were injected with CBD (3, 10, 30 mg/kg) and assessed for antinociception, activity, coordination, anxiety-like behavior, and recognition memory. Brains were taken to quantify endocannabinoids, cannabinoid receptors, and cannabinoid catabolic enzymes. Additionally, CBD and metabolite 7-hydroxy-CBD were quantified in the plasma and cortex. Results: Tat decreased supraspinal-related nociception and locomotion. CBD and sex had little to no effects on any of the behavioral measures. For the endocannabinoid system male sex was associated with elevated concentration of the proinflammatory metabolite arachidonic acid in various CNS regions, including the cerebellum that also showed higher FAAH expression levels for Tat(+) males. GPR55 expression levels in the striatum and cerebellum were higher for females compared to males. CBD metabolism was altered by sex and Tat expression. Conclusion: Findings indicate that acute CBD effects are not altered by HIV Tat, and acute CBD has no to minimal effects on behavior and the endocannabinoid system.

APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size.

The E4 variant of APOE strongly predisposes individuals to late-onset Alzheimer's disease. We demonstrate that in response to lipogenesis, apolipoprotein E (APOE) in astrocytes can avoid translocation into the endoplasmic reticulum (ER) lumen and traffic to lipid droplets (LDs) via membrane bridges at ER-LD contacts. APOE knockdown promotes fewer, larger LDs after a fatty acid pulse, which contain more unsaturated triglyceride after fatty acid pulse-chase. This LD size phenotype was rescued by chimeric APOE that targets only LDs. Like APOE depletion, APOE4-expressing astrocytes form a small number of large LDs enriched in unsaturated triglyceride. Additionally, the LDs in APOE4 cells exhibit impaired turnover and increased sensitivity to lipid peroxidation. Our data indicate that APOE plays a previously unrecognized role as an LD surface protein that regulates LD size and composition. APOE4 causes aberrant LD composition and morphology. Our study contributes to accumulating evidence that APOE4 astrocytes with large, unsaturated LDs are sensitized to lipid peroxidation, which could contribute to Alzheimer's disease risk.

Effects of acute Δ9-tetrahydrocannabinol on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice.

Cannabis use is highly prevalent especially among people living with HIV (PLWH). Activation of the anti-inflammatory and neuroprotective endocannabinoid system by phytocannabinoids, i.e. Δ9-tetrahydrocannabinol (THC), has been proposed to reduce HIV symptoms. However, THC's effects on HIV-related memory deficits are unclear. Using HIV-1 Tat transgenic mice, the current study investigates acute THC effects on various behavioral outcomes and the endocannabinoid system. For the rodent tetrad model, THC doses (1, 3, 10 mg/kg) induced known antinociceptive effects, with Tat induction increasing antinociceptive THC effects at 3 and 10 mg/kg doses. Only minor or no effects were noted for acute THC on body temperature, locomotor activity, and coordination. Increased anxiety-like behavior was found for females compared to males, but acute THC had no effect on anxiety. Object recognition memory was diminished by acute THC in Tat(-) females but not Tat(+) females, without affecting males. The endocannabinoid system and related lipids were not affected by acute THC, except for THC-induced decreases in CB1R protein expression levels in the spinal cord of Tat(-) mice. Female sex and Tat induction was associated with elevated 2-AG, AEA, AA, CB1R, CB2R, FAAH and/or MAGL expression in various brain regions. Further, AEA levels in the prefrontal cortex of Tat(+) females were negatively associated with object recognition memory. Overall, findings indicate that acute THC exerts differential effects on antinociception and memory, dependent on sex and HIV Tat expression, potentially in relation to an altered endocannabinoid system, which may be of relevance in view of potential cannabis-based treatment options for PLWH.

Intrinsic and extrinsic regulation of rhabdomyolysis susceptibility by Tango2.

Rhabdomyolysis is a clinical emergency characterized by severe muscle damage, resulting in the release of intracellular muscle components, which leads to myoglobinuria and, in severe cases, acute kidney failure. Rhabdomyolysis is caused by genetic factors linked to increased disease susceptibility in response to extrinsic triggers. Recessive mutations in TANGO2 result in episodic rhabdomyolysis, metabolic crises, encephalopathy and cardiac arrhythmia. The underlying mechanism contributing to disease onset in response to specific triggers remains unclear. To address these challenges, we created a zebrafish model of Tango2 deficiency. Here, we demonstrate that the loss of Tango2 in zebrafish results in growth defects, early lethality and increased susceptibility of skeletal muscle defects in response to extrinsic triggers, similar to TANGO2-deficient patients. Using lipidomics, we identified alterations in the glycerolipid pathway in tango2 mutants, which is critical for membrane stability and energy balance. Therefore, these studies provide insight into key disease processes in Tango2 deficiency and have increased our understanding of the impacts of specific defects on predisposition to environmental triggers in TANGO2-related disorders.

Non-esterified erythrocyte linoleic acid, arachidonic acid, and subjective sleep outcomes.

OBJECTIVE: This study investigated whether non-esterified erythrocyte omega-6 PUFAs were associated with subjective assessment of sleep quality and duration, and risk for obstructive sleep apnea. METHODS: In this secondary analysis of the cross-sectional OPPERA-II study, 538 adults completed the Pittsburgh Sleep Quality Index (PSQI), reported their usual hours of sleep, and answered STOP screening questions for obstructive sleep apnea. Circulating non-esterified erythrocyte concentrations of omega-6 PUFA linoleic acid and arachidonic acid were quantified by liquid chromatography tandem mass spectroscopy. Sleep outcomes were dichotomized as poor (PSQI ≤5) vs good (PSQI ≥6) sleep quality, insufficient or excessive (≤6 or >9 h) vs good (7-9 h) sleep duration, and high (≥2 affirmative responses) vs low (<2 affirmative responses) risk for obstructive sleep apnea. Non-esterified omega-6 PUFAs and the continuous covariates of body mass index, Short Form (SF) 12 Health Survey Physical and Mental Component scores and resting measures of systolic and diastolic blood pressure were standardized for multivariable analysis. Categorical covariates were study site, age, sex, and race/ethnicity. Multivariable-adjusted logistic regression first estimated odds ratios (OR) and 95% confidence limits (CL) for sleep outcomes using linoleic acid as the main exposure. Analysis was then repeated using arachidonic acid as the main exposure. RESULTS: In the multivariable-adjusted model, each standard deviation increase in non-esterified erythrocyte linoleic acid was associated with higher odds of poor sleep quality (OR=1.2, 95% CL: 1.1, 1.5), insufficient or excessive sleep (OR= 1.3, 95% CL: 1.1, 1.6) and high-risk for obstructive sleep apnea (OR=1.3, 95% CL: 1.1, 1.6). Likewise, for each standard deviation increase in non-esterified erythrocyte arachidonic acid, odds increased of poor sleep quality (OR=1.2, 95% CL: 1.1, 1.5), and insufficient or excessive sleep (OR=1.2, 95% CL: 1.1, 1.5). Odds of being high risk for obstructive sleep apnea increased with greater circulating arachidonic acid, but the association did not reach statistical significance (OR=1.1, 95% CL: 0.9, 1.4). CONCLUSION: Non-esterified erythrocyte linoleic acid and arachidonic acid were associated with poor sleep quality and insufficient or excessive sleep duration. Linoleic acid, but not arachidonic acid, was also associated with high risk for obstructive sleep apnea.

A comparison of high- and low-resolution gas chromatography-mass spectrometry for herbal product classification: A case study with Ocimum essential oils.

INTRODUCTION: Selection of marker compounds for targeted chemical analysis is complicated when considering varying instrumentation and closely related plant species. High-resolution gas chromatography-mass spectrometry (GC-MS), via orbitrap detection, has yet to be evaluated for improved marker compound selection. OBJECTIVE: This study directly compares high- and low-resolution GC-MS for botanical maker compound selection using Ocimum tenuiflorum L. (OT) and Ocimum gratissimum L. (OG) for botanical ingredient authentication. METHODS: The essential oils of OT and OG were collected via hydrodistillation before untargeted chemical analysis with gas chromatography coupled to single-quadrupole (GC-SQ) and orbitrap (GC-Orbitrap) detectors. The Global Natural Products Social Molecular Networking (GNPS) software was used for compound annotation, and a manual search was used to find the 41 most common Ocimum essential oil metabolites. RESULTS: The GC-Orbitrap resulted in 1.7-fold more metabolite detection and increased dynamic range compared to the GC-SQ. Spectral matching and manual searching were improved with GC-Orbitrap data. Each instrument had differing known compound concentrations; however, there was an overlap of six compounds with higher abundance in OG than OT and three compounds with a higher abundance in OT than OG, suggesting consistent detection of the most variable compounds. An unsupervised principal component analysis (PCA) could not discern the two species with either dataset. CONCLUSION: GC-Orbitrap instrumentation improves compound detection, dynamic range, and feature annotation in essential oil analysis. However, considering both high- and low-resolution data may improve reliable marker compound selection, as GC-Orbitrap analysis alone did not improve unsupervised separation of two Ocimum species compared to GC-SQ data.

APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size.

The E4 variant of APOE strongly predisposes individuals to late-onset Alzheimer's disease. We demonstrate that in response to neutral lipid synthesis, apolipoprotein E (APOE) in astrocytes can avoid translocation into the ER lumen and traffic to lipid droplets (LDs) via membrane bridges at ER-LD contacts. APOE knockdown promotes fewer, larger LDs containing more unsaturated triglyceride. This LD size distribution phenotype was rescued by chimeric APOE that targets only LDs. APOE4 - expressing astrocytes also form a small number of large LDs enriched in unsaturated triglyceride. Additionally, the larger LDs in APOE4 cells exhibit impaired turnover and increased sensitivity to lipid peroxidation. Our data indicate that APOE plays a previously unrecognized role as an LD surface protein that regulates LD size and composition. APOE4 is a toxic gain of function variant that causes aberrant LD composition and morphology. We propose that APOE4 astrocytes with large, unsaturated LDs are sensitized to lipid peroxidation or lipotoxicity, which could contribute to Alzheimer's disease risk. Summary: Windham et al . discover that APOE in astrocytes can traffic to lipid droplets (LDs), where it modulates LD composition and size. Astrocytes expressing the Alzheimer's risk variant APOE4 form large LDs with impaired turnover and increased peroxidation sensitivity.

Scavenger receptor BI attenuates oxidized phospholipid-induced pulmonary inflammation.

Damage associated molecular patterns (DAMPs) are molecules released from dead/dying cells following toxicant and/or environmental exposures that activate the immune response through binding of pattern recognition receptors (PRRs). Excessive production of DAMPs or failed clearance leads to chronic inflammation and delayed inflammation resolution. One category of DAMPs are oxidized phospholipids (oxPLs) produced upon exposure to high levels of oxidative stress, such as following ozone (O3) induced inflammation. OxPLs are bound by multiple classes of PRRs that include scavenger receptors (SRs) such as SR class B-1 (SR-BI) and toll-like receptors (TLRs). Interactions between oxPLs and PRRs appear to regulate inflammation; however, the role of SR-BI in oxPL-induced lung inflammation has not been defined. Therefore, we hypothesize that SR-BI is critical in protecting the lung from oxPL-induced pulmonary inflammation/injury. To test this hypothesis, C57BL/6J (WT) female mice were dosed with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (oxPAPC) by oropharyngeal aspiration which increased pulmonary SR-BI expression. Following oxPAPC exposure, SR-BI deficient (SR-BI-/-) mice exhibited increased lung pathology and inflammatory cytokine/chemokine production. Lipidomic analysis revealed that SR-BI-/- mice had an altered pulmonary lipidome prior to and following oxPAPC exposure, which correlated with increased oxidized phosphatidylcholines (PCs). Finally, we characterized TLR4-mediated activation of NF-κB following oxPAPC exposure and discovered that SR-BI-/- mice had increased TLR4 mRNA expression in lung tissue and macrophages, increased nuclear p65, and decreased cytoplasmic IκBα. Overall, we conclude that SR-BI is required for limiting oxPAPC-induced lung pathology by maintaining lipid homeostasis, reducing oxidized PCs, and attenuating TLR4-NF-κB activation, thereby preventing excessive and persistent inflammation.

Obesity reprograms the pulmonary polyunsaturated fatty acid-derived lipidome, transcriptome, and gene-oxylipin networks.

Obesity exacerbates inflammation upon lung injury; however, the mechanisms by which obesity primes pulmonary dysregulation prior to external injury are not well studied. Herein, we tested the hypothesis that obesity dysregulates pulmonary PUFA metabolism that is central to inflammation initiation and resolution. We first show that a high-fat diet (HFD) administered to C57BL/6J mice increased the relative abundance of pulmonary PUFA-containing triglycerides and the concentration of PUFA-derived oxylipins (particularly prostaglandins and hydroxyeicosatetraenoic acids), independent of an increase in total pulmonary PUFAs, prior to onset of pulmonary inflammation. Experiments with a genetic model of obesity (ob/ob) generally recapitulated the effects of the HFD on the pulmonary oxylipin signature. Subsequent pulmonary next-generation RNA sequencing identified complex and unique transcriptional regulation with the HFD. We found the HFD increased pathways related to glycerophospholipid metabolism and immunity, including a unique elevation in B cell differentiation and signaling. Furthermore, we conducted computational integration of lipidomic with transcriptomic data. These analyses identified novel HFD-driven networks between glycerophospholipid metabolism and B cell receptor signaling with specific PUFA-derived pulmonary oxylipins. Finally, we confirmed the hypothesis by demonstrating that the concentration of pulmonary oxylipins, in addition to inflammatory markers, were generally increased in mice consuming a HFD upon ozone-induced acute lung injury. Collectively, these data show that a HFD dysregulates pulmonary PUFA metabolism prior to external lung injury, which may be a mechanism by which obesity primes the lungs to respond poorly to infectious and/or inflammatory challenges.

Defining the Expression, Production, and Signaling Roles of Specialized Metabolites during Bacillus subtilis Differentiation.

Bacterial specialized (or secondary) metabolites are structurally diverse molecules that mediate intra- and interspecies interactions by altering growth and cellular physiology and differentiation. Bacillus subtilis, a Gram-positive model bacterium commonly used to study biofilm formation and sporulation, has the capacity to produce more than 10 specialized metabolites. Some of these B. subtilis specialized metabolites have been investigated for their role in facilitating cellular differentiation, but only rarely has the behavior of multiple metabolites been simultaneously investigated. In this study, we explored the interconnectivity of differentiation (biofilm and sporulation) and specialized metabolites in B. subtilis. Specifically, we interrogated how development influences specialized metabolites and vice versa. Using the sporulation-inducing medium DSM, we found that the majority of the specialized metabolites examined are expressed and produced during biofilm formation and sporulation. Additionally, we found that six of these metabolites (surfactin, ComX, bacillibactin, bacilysin, subtilosin A, and plipastatin) are necessary signaling molecules for proper progression of B. subtilis differentiation. This study further supports the growing body of work demonstrating that specialized metabolites have essential physiological functions as cell-cell communication signals in bacteria. IMPORTANCE Bacterially produced specialized metabolites are frequently studied for their potential use as antibiotics and antifungals. However, a growing body of work has suggested that the antagonistic potential of specialized metabolites is not their only function. Here, using Bacillus subtilis as our model bacterium, we demonstrated that developmental processes such as biofilm formation and sporulation are tightly linked to specialized metabolite gene expression and production. Additionally, under our differentiation-inducing conditions, six out of the nine specialized metabolites investigated behave as intraspecific signals that impact B. subtilis physiology and influence biofilm formation and sporulation. Our work supports the viewpoint that specialized metabolites have a clear role as cell-cell signaling molecules within differentiated populations of bacteria.

The Chemistry of Kratom [Mitragyna speciosa]: Updated Characterization Data and Methods to Elucidate Indole and Oxindole Alkaloids.

Two separate commercial products of kratom [Mitragyna speciosa (Korth.) Havil. Rubiaceae] were used to generate reference standards of its indole and oxindole alkaloids. While kratom has been studied for over a century, the characterization data in the literature for many of the alkaloids are either incomplete or inconsistent with modern standards. As such, full 1H and 13C NMR spectra, along with HRESIMS and ECD data, are reported for alkaloids 1-19. Of these, four new alkaloids (7, 11, 17, and 18) were characterized using 2D NMR data, and the absolute configurations of 7, 17, and 18 were established by comparison of experimental and calculated ECD spectra. The absolute configuration for the N(4)-oxide (11) was established by comparison of NMR and ECD spectra of its reduced product with those for compound 7. In total, 19 alkaloids were characterized, including the indole alkaloid mitragynine (1) and its diastereoisomers speciociliatine (2), speciogynine (3), and mitraciliatine (4); the indole alkaloid paynantheine (5) and its diastereoisomers isopaynantheine (6) and epiallo-isopaynantheine (7); the N(4)-oxides mitragynine-N(4)-oxide (8), speciociliatine-N(4)-oxide (9), isopaynantheine-N(4)-oxide (10), and epiallo-isopaynantheine-N(4)-oxide (11); the 9-hydroxylated oxindole alkaloids speciofoline (12), isorotundifoleine (13), and isospeciofoleine (14); and the 9-unsubstituted oxindoles corynoxine A (15), corynoxine B (16), 3-epirhynchophylline (17), 3-epicorynoxine B (18), and corynoxeine (19). With the ability to analyze the spectroscopic data of all of these compounds concomitantly, a decision tree was developed to differentiate these kratom alkaloids based on a few key chemical shifts in the 1H and/or 13C NMR spectra.

Identification of adulteration in botanical samples with untargeted metabolomics.

Adulteration remains an issue in the dietary supplement industry, including botanical supplements. While it is common to employ a targeted analysis to detect known adulterants, this is difficult when little is known about the sample set. With this study, untargeted metabolomics using liquid chromatography coupled to ultraviolet-visible spectroscopy (LC-UV) or high-resolution mass spectrometry (LC-MS) was employed to detect adulteration in botanical dietary supplements. A training set was prepared by combining Hydrastis canadensis L. with a known adulterant, Coptis chinensis Franch., in ratios ranging from 5 to 95% adulteration. The metabolomics datasets were analyzed using both unsupervised (principal component analysis and composite score) and supervised (SIMCA) techniques. Palmatine, a known H. canadensis metabolite, was quantified as a targeted analysis comparison. While the targeted analysis was the most sensitive method tested in detecting adulteration, statistical analyses of the untargeted metabolomics datasets detected adulteration of the goldenseal samples, with SIMCA providing the greatest discriminating potential. Graphical abstract.