bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cannabinoid metabolites as potential inhibitors of major CYP450 enzymes. Implication for cannabis‐drug interactions.
Authors: Nasrin Shamema , Watson Christy J. , Chen Gang , Lazarus Philip 
Published: April 2020

The recent legalization of marijuana in many parts of the United States and other countries has led to an increased need for a more comprehensive understanding of the metabolism of marijuana constituents, specifically in regard to potential drug‐drug interactions (DDIs) with cannabinoids and their numerous metabolites. To understand the DDI potential of cannabis, we are evaluating cannabis constituents and cannabinoid metabolites as possible inhibitors of phase I metabolizing enzymes. We hypothesize that inhibition of major drug metabolizing enzymes by cannabinoids metabolites [(−)‐trans‐Δ‐9‐ tetrahydrocannabinol (THC), 11‐hydroxy Δ9‐tetra hydrocannabinol(11‐OH‐THC), 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannbinol (THC‐COOH), THC‐COO‐glucuronide, cannabidiol (CBD) and cannabinol (CBN] can lead to an increase in hepatic and systemic exposure to certain drugs. In vitro assays demonstrated that THC, its major metabolites 11‐OH‐THC, THC‐COOH and THC‐COO‐glucuronide, CBD and CBN inhibit several major CYP enzymes. THC (IC50= 10 ± 4.7 μM), 11‐OH‐THC (IC50= 2.8 ± 1.4 μM), THC‐COO‐glucuronide (IC50= 7.6 ± 4.8 μM), and CBD (2.8 ± 1.2 μM) all inhibited the dextromethorphan‐O‐demethylase activity of recombinant CYP2D6. THC (IC50= 8.3 ± 3.2 μM), THC‐COO‐glucuronide (IC50= 7.2 ± 4.1 μM), CBD (IC50= 8.1 ± 4.3 μM) and CBN (IC50= 5.5 ± 3.1 μM) all inhibited the omeprazole‐5‐hydroxylase activity of recombinant CYP2C19, while 11‐OH‐THC (IC50= 5.02 ± 2.3 μM), THC‐COO‐glucuronide (IC50= 5.2 μM ± 3.5 μM), CBD (IC50= 0.8 ± 0.5 μM), and CBN (IC50= 1.5 ± 0.9 μM) all inhibited the bupropion hydroxylation activity of recombinant CYP2B6. In addition, CBD and CBN inhibited the chlorzoxazone‐6‐hydroxylase activity of recombinant CYP2E1 (IC50 were 0.9 μM ± 0.5 μM and 0.6 μM ± 0.4 μM, respectively). These data indicate that cannabinoids and their major metabolites are able to inhibit the activities of multiple CYP enzymes, suggesting a strong possibility for DDI with many exogenous compounds including a variety of drugs and carcinogens.Support or Funding InformationThis investigation was supported in part by funds provided by the State of Washington Initiative Measure No. 502
A Fluorescent Based System to Monitor 2‐micron Copy Number in Saccharomyces cerevisiae
Authors: Howe Max , Butler Scott , Callahan Kevin 
Published: April 2020

The 2‐micron plasmid is a small genetic element that persists in Saccharomyces cerevisiae with chromosome like stability. It encodes for four proteins (Rep1, Rep2, Raf1, and Flp) that appear to provide no advantage for the host cell, but instead have a role in replicating and maintaining a steady state concentration of the plasmid‐‐
1. Flp recombinase is known to promote amplification of the 2‐micron; disruption of the FLP gene is a valid way of preventing the necessary amplification of the plasmid to pass itself on. The 2‐micron is a selfish DNA element that is able to maintain a copy number of 40–60, however its ability to maintain these levels is not completely understood. To study this regulation we have established a quantitative and efficient means to monitor copy number. We used CRISPR‐Cas9 to replace ADE2 (encoded on 2‐micron/ADE22) with mCherry, enabling copy number to be monitored using flow cytometry. Preparation of the strains required no additional staining, allowing for quick processing and analysis. To establish the sensitivity of the system, mCherry expression was monitored in strains that had ectopic expression of the flp‐H305L mutant, which destabilizes the 2‐micron plasmid
2. We observed a decrease in mCherry expression in these strains, consistent with the role Flp plays in 2‐micron amplification. This reduction in copy number was confirmed using quantitative PCR to examine expression of mCherry from the 2‐micron. Current studies are employing this system to monitor how copy number is affected by two long non‐coding RNAs (lncRNA), which are expressed from the plasmid and regulated by Rrp6p and the TRAMP complex, components of the nuclear RNA surveillance pathway.Support or Funding InformationSJFC internal funds
Progesterone prevents oxidative stress and respiratory dysfunctions in female rats exposed to intermittent hypoxia.
Authors: Bairam Aida , Laouafa Sofien , Marcouiller François , Pialoux Vincent , Joseph Vincent 
Published: April 2020

The occurrence of sleep apnea (SA) increases after menopause in women, which is partly related to the decreased levels of ovarian hormones. Because progesterone (Prog) is a neuroprotective hormone and a potent respiratory stimulant, we tested the hypothesis that Prog reduces the apnea frequency and the oxidative stress induced by IH. We used OVX female rats implanted with an osmotic pump delivering vehicle (OVX‐Veh) or prog (4mg/kg/day). Two weeks following the surgery, rats were exposed to room air (RA), or IH for 7 days (nadir 10% O2, 10 cycles/hour, 8 hours/day). We studied three groups: OVX‐Veh‐RA; OVX‐Veh‐IH; and OVX‐Prog‐IH. At the end of exposure period, we measured the mean arterial pressure (tail cuff) and assessed the frequency of apneas during sleep and the ventilatory responses to hypoxia using whole body plethysmography. The brain was quickly harvested to measure the activity of the pro‐oxidant enzyme NADPH oxidase (NOX), and the antioxidant enzymes (mitochondrial and cytosolic superoxide dismutase ‐ SOD) in the cerebral cortex and brainstem. IH exposure increased the mean arterial blood pressure, the frequency of apneas during sleep, and the hypoxic ventilatory response. Prog did not prevent the elevation of blood pressure, but effectively reduced the frequency of apneas during sleep and prevented the elevation of the hypoxic ventilatory response. In the cerebral cortex, IH increased NOX activity, and decreased the activity of the cytosolic and mitochondrial SOD, these effects were prevented by Prog. None of these enzyme activities were altered by IH or Prog supplementation in the brainstem. We conclude that Prog prevents the respiratory alterations induced by IH, and reduces oxidative stress in the brain cortex. These studies are useful to better understand the responses to IH and sleep apnea in women particularly after menopause.Support or Funding InformationSupported by CIHR.
Oxygen availability and mitochondrial respiration: Impact on permeabilized and isolated in vitro preparations
Authors: Park Soung Hun , Broxterman Ryan M. , Craig Jesse C. , Richardson Russell S. 
Published: April 2020

Skeletal muscle mitochondrial respiration assessed in vitro is typically performed in hyperoxia utilizing either permeabilized fibers or isolated mitochondria preparations. However, although it remains to be comprehensively assessed, growing evidence supports that mitochondrial respiration is likely influenced by oxygen availability and this may have a different impact in these two preparations. Therefore, we tested the hypotheses that hyperoxia would 1) increase peak mitochondrial respiration (Vmax) in permeabilized fibers, but not in isolated mitochondria, without altering submaximal respiration in either preparation, and 2) not alter the mitochondrial critical partial pressure of O2 (PO2) in either preparation.MethodsFiber permeablization and mitochondrial isolation techniques were performed on muscle samples, and mitochondrial respiration was measured by high‐resolution respirometery (O2k) in both moderate (MOD, 100–125 mmHg) and high (HIGH, 250–300 mmHg) initial PO2 conditions, for both preparations. Respiration and chamber PO2 were continuously measured until the cessation of respiration, allowing for the assessment of Vmax, submaximal respiration, and critical PO2.ResultsVmax was significantly greater in HIGH than MOD with permeabilized fibers (~57%, p<0.05, effect size (d) =2.8), but was not significantly different between conditions with isolated mitochondria (~1%, p>0.05, d=0.07). Submaximal respiration was not significantly different between HIGH and MOD conditions with permeabilized fibers (~4%, p>0.05, d=0.1), but was significantly lower in HIGH with isolated mitochondria (~33%, p<0.05, d=2.4). The critical PO2 with isolated mitochondria was lower in HIGH (0.48±0.25 mmHg) compared to MOD (0.97±0.08 mmHg, p=0.1, d=1.5). In contrast, in the permeabilized fibers, in both MOD and HIGH, there was no precipitous fall in respiration with falling oxygen availability, and respiration ceased despite 3–10 mmHg PO2 remaining in the chamber.ConclusionThese findings reveal differing effects of oxygen availability on mitochondrial respiration, dependent upon the mitochondrial preparation, which need to be considered when making such measurements.Support or Funding InformationNational Heart, Lung, and Blood Institute at the National Institute of Health (PO1 HL1091830) and Veterans Administration Rehabilitation Research and Development Service (E6910‐R, E1697‐R, E1433‐P, E9275‐L and E1572‐P)
Concomitant Improvement of Synaptic Decline and NASH using Fermentable Fiber Inulin
Authors: Wan Yu-Jui Yvonne , Jena Prasant , Lucente Jacopo , Jin Lee-Way 
Published: April 2020

IntroductionA Western diet with enriched sugar and fat is implicated in cognitive impairment and the development of non‐alcoholic steatohepatitis (NASH). This study examines the impact of fermentable fiber inulin in shifting gut microbiota and metabolites to affect liver and brain health in Western diet‐fed mice.MethodsC57BL/6 mice were fed a healthy rodent control diet (CD) or a fructose, palmitate, and cholesterol (FPC)‐enriched diet after weaning until they were 3‐months old. Then, FPC diet‐fed mice were supplemented with or without 6% inulin for 5 months. All the mice were euthanized when they were 8 months of age.ResultsLong‐term consumption of the FPC diet induced NASH with elevated IL‐17A signaling. In addition, FPC diet reduced hippocampal long‐term potentiation (LTP) and induced microglia activation. Similarly, FPC diet induced Th17 pathway driven hepatic IL17A production along with increased IL17A inducers including Il1β, Il6, and Tgfβ. In contrast, inulin supplementation reduced these changes. Furthermore, FPC diet intake induced gut dysbiosis by increasing Proteobacteria whereas inulin supplementation prevented such changes. In the microbiota families Coriobacteriaceae and Erysipelotrichaceae, which are implicated in cholesterol metabolism, and the genus of Erysipelotrichaceae and Allobaculum also shifted in the same direction in response to FPC intake and inulin supplementation. Accordingly, cecum cholesterol and zymosterol, a precursor of cholesterol, were increased in FPC diet‐fed mice but reduced in inulin‐supplemented mice. In addition, FPC diet‐fed mice had reduced bile acid receptors TGR5 and FXR signaling, and inulin supplementation reversed such changes.ConclusionThe data suggest that inulin via gut fermentation shifts gut microbes, alters IL‐17A signaling, as well as activates bile acid receptor‐regulated pathways to coordinately ameliorate impaired neuroplasticity and neuroinflammation as well as NASH found in Western diet‐fed mice. Together, diet simultaneously affects brain and liver health.Support or Funding InformationThis study is supported by grants funded by National Institutes of Health U01CA179582 and R01CA222490 and Mouse Metabolic Phenotyping Center (MMPC) Microbiome Program (30835‐60), Subaward A19‐2654.
The protective effect of vasopressin deficiency in liver fibrosis in rats with chronic portocaval anastomosis
Authors: Gonzalez Yesenia Danyeli Navarro , Quintanar-Stephano Andrés , Ventura-Juarez Javier , Muñoz-Ortega Martín Humberto , González-Blas Daniel , Tinajero-Ruelas Manuel 
Published: April 2020

Liver fibrosis occur in liver diseases and its complications is cause of the increased rate of human mortality. The disease is characterized by the gradual accumulation of scar tissue in the liver parenchyma with decrease in metabolic capacity and liver failure. It is known that arginine vasopressin (AVP) participate in the regulation of the immune responses, including the scarring. However, the effect of how AVP regulates this process is not clear. Here we study the role of the AVP deficiency induced by the neurointermediate pituitary lobectomy (NIL) in rats with chronic anastomosis portocaval (APC) model (sixteen weeks). Groups of young male Wistar rats of 8–10 each were prepared: Sham operated (SHAM), APC, LNI and APC+NIL. Blood, plasma and serum biochemical variables were assessed: prothrombin time (TP), blood NH3, transaminases (TGO and TGP), bilirubins and γ‐Glutamyl Transpeptidase (GGT). Liver histological slides were stained with H‐E, Masson, Sirius Red and PAS. Results showed that in the APC group: the TP, NH3, TGO and TGP, bilirubins and GGT were significantly increased, whereas serum glucose levels, cholesterol, triglycerides, albumin, total proteins and the liver glycogen content where significantly augmented. Comparisons between SHAM and NIL groups did not shown significant differences in all these variables. As compared with the APC, the APC+NIL group developed a significant improvement in all these variables. The liver histopathology showed that the APC group developed a significant increase in fibrosis septa, inflammatory infiltrates and diminution of the glycogen content. The NIL group we do not have available data yet. In the NIL+APC group histology data showed a significant liver regeneration; less fibrosis, disappearance of inflammatory infiltrates and liver glycogen restoration.ConclusionsResults indicate the AVP play a role in liver fibrosis (demonstrated by the AVP deficiency). Results also suggest that AVP antagonists may be used for liver fibrosis treatment.Support or Funding InformationUniversidad Autónoma de Aguascalientes PIFF14‐1 andCONACYT 221262. México
White Mountain Expedition 2019: Peaks and Valleys ‐ Oscillatory cerebral blood flow at high altitude
Authors: Anderson Garen K. , Rosenberg Alexander J. , Barnes Haley J. , Bird Jordan , Pentz Brandon , Byman Britta R.M. , Jendzjowsky Nicholas , Wilson Richard J. , Day Trevor A. , Rickards Caroline A. 
Published: April 2020

IntroductionCerebral tissue oxygenation can be impaired by decreases in oxygen delivery as a result of reduced cerebral blood flow, and environmental conditions such as ascent to high altitude. Recent evidence suggests that an oscillatory pattern in cerebral blood flow (at ~0.1 Hz) may protect cerebral oxygenation under conditions of cerebral hypoperfusion. In this study, we hypothesized that inducing oscillations in cerebral blood flow at 0.1 Hz would protect cerebral blood flow and cerebral tissue oxygen saturation during exposure to combined simulated hemorrhage and sustained hypobaric hypoxia (ascent and partial acclimatization to high altitude).Methods8 healthy human subjects (4 M, 24.7 ± 4.1 y; 4 F, 34.3 ± 8.3 y) participated in two experiments at high altitude (White Mountain, California, USA; altitude, 3800 m): 1) a control condition (CTRL) where lower body negative pressure (LBNP) was used to induce central hypovolemia by reducing chamber pressure to −60 mmHg for 10‐min, and 2) oscillatory LBNP (OLBNP) where chamber pressure was reduced to −60 mmHg, then oscillated every 5‐s between −30 mmHg and −90 mmHg for 10‐min (0.1 Hz). Measurements included internal carotid artery (ICA) blood flow via duplex Doppler ultrasound, middle cerebral artery velocity (MCAv) via transcranial Doppler ultrasound, and cerebral tissue oxygen saturation via near‐infrared spectroscopy. Frequency analysis (via fast Fourier transform) was performed to verify that oscillations in mean MCAv were generated at ~0.1 Hz. Data were analyzed with a linear mixed‐model. All data are represented as mean ± SE.ResultsLow frequency power (0.07–0.15 Hz) in mean MCAv increased during OLBNP vs. CTRL (P = 0.02). OLBNP did not protect ICA flow (OLBNP: −32.5 ± 4.5 Δ%; CTRL: −19.9 ± 8.9 Δ%; P = 0.18) or mean MCAv (OLBNP: −18.5 ± 3.4 Δ%; CTRL: −15.3 ± 5.4 Δ%; P = 0.58), but cerebral tissue oxygenation was protected (OLBNP: −0.67 ± 1.0 Δ%; CTRL: −4.07 ± 2.0 Δ%; P = 0.004).ConclusionsThese results support our hypothesis that inducing oscillatory blood flow leads to protection of cerebral tissue oxygenation, despite no differences in ICA blood flow or mean MCAv. Overall, these data suggest that therapies using oscillatory perfusion may help preserve cerebral tissue oxygen saturation under conditions of reduced oxygen delivery.Support or Funding InformationAHA 17GRNT33671110
The Functional Expression of the Lamin A/C Mutant Q517X in HL1 Cardiomyocytes Induces Electrophysiological Impairments Through Impaired Ca2+‐Homeostasis at the Endoplasmic Reticulum.
Authors: Gerbino Andrea , De Zio Roberta , Forleo Cinzia , Milano Serena , Procino Giuseppe , Favale Stefano , Svelto Maria , Carmosino Monica 
Published: April 2020

Mutations in the LMNA gene, which encodes A‐type nuclear Lamins, are among the most frequent genetic cause of dilated cardiomyopathy (DCM). We identified a novel LMNA nonsense mutation (Q517X) co‐segregating with DCM and permanent atrial fibrillation. Aim of this study is to gain insights into the unknown pathogenic mechanism induced by the expression of Q517X at cellular level.HEK293 cells and HL‐1 cardiomyocytes were transiently transfected with either Lamin A or Q517X both tagged with mCherry. Q517X expression, localization and its effects on diverse cellular mechanisms were evaluated by western blotting, laser‐scanning confocal microscopy and real time FRET/imaging analysis in single cell. Also, patch clamp experiments were used to evaluate the effect of Q517X expression on HL‐1 spontaneous action potentials (APs).When expressed in both cell types, Q517X mislocalized within the nucleoplasm where it clustered in aggregates of different sizes. Emerin was also irregularly distributed along the nuclear envelope probably indicating a general impairment of the inner nuclear membrane organization upon Q517X expression. HL‐1 cardiomyocytes expressing the mutant showed a significant reduction in APs frequency induced by an increased duration of both single APDs (APD100) and cycle length (i.e. the distance between two sequential APDs’ thresholds). In addition, after depolarizations events were constantly recorded in cells expressing the mutant. When we measured intracellular Ca2+ level in cardiomyocytes expressing either LMNA wild type or Q517X we found similar patterns of oscillation. However, in the absence of electrical activity at the plasma membrane, cardiomyocytes expressing the mutant showed spontaneous Ca2+ spikes, in free‐extracellular Ca2+, likely induced by abnormal release by the endoplasmic reticulum (ER) as directly confirmed by steady state FRET experiment with the ER Ca2+ probe ERD1.So far, these results depict a pathogenic mechanism in which the exogenous expression of Q517X changed the geometry of emerin within the nucleus and altered the electrical activity at the plasma membrane by probably depleting ER Ca2+ content.Support or Funding InformationThis research was supported by funding from the ‘CLUSTER TECNOLOGICO REGIONALE DICLIMAX’ (project # MTJU9H8)
Hyperglycemia‐Induced Inhibition of the Angiotensin II Type 1 Receptor Pathway in the Vascular Endothelium
Authors: Hoffmann Brian R. , Haspula Dhanush 
Published: April 2020

Hyperglycemia, associated with diabetes, is known to damage blood vessels and contribute to negative cardiovascular health outcomes. However, the etiology behind the negative influence of the exposure to prolonged elevated glucose on endothelial cells is not yet fully understood. Our laboratories’ previous study identified changes in endothelial cell surface glycosylation resulting from hyperglycemia. One such modified protein was the Angiotensin II type 1 receptor (AT1R), which was N‐glycosylated adjacent to the active site. Activation of AT1R by angiotensin II (AngII) at subpressor doses is associated with positive endothelial cell growth, proliferation, and tube formation. However, AngII binding to the Angiotensin II type 2 receptor (AT2R), has been shown to elicit opposing effects. The objective of this study was to further investigate the effects of this hyperglycemia‐induced glycosylation of AT1R. A two‐week high glucose treatment (25 mM) of rat cardiac microvascular endothelial cells (RCMVECs) displayed impaired function by tube formation analysis (TFA) in comparison to a normal glucose treatment (4.5 mM). The decrease tube formation was further exacerbated by the addition of a subpressor dose of AngII. To further test the influence of hyperglycemia‐induced AT1R N‐glycosylation on signaling we treated RCMVECs with the PNGaseF enzyme for N‐glycosylation removal at the cell surface in combination with functional assays and AngII treatment under varying glycemic conditions. The efficacy of this treatment was tested through TFA and measurements of ERK1/2 activation through phosphorylation (known contributor to AT1R signaling). We observed a decrease in tube length, tube area, and number of branch points in a high glucose environment plus Ang‐II and were able to recover functionality with the addition of PNGaseF. While normal glucose treated cells had shown ERK1/2 activation, no observable increases in ERK1/2 were observed in response to AngII in the high glucose conditions. In the high glucose plus AngII treatment group we also observed a significant decrease in Vegfr2 gene expression compared to the normal glucose plus AngII treatment group. Further analysis is currently underway to further validate this mechanism of AT1R signaling impairment, but the data we have so far lead us to believe that hyperglycemia‐induced glycosylation blocks the AT1R active site to decrease functionality. It is possible that the hyperglycemia‐induced glycosylation of AT1R inhibits AngII binding, allowing for a higher rate of binding in the AT2R to further impair endothelial cell functionality.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University.