Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure

Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.The mesocorticolimbic dopamine (DA) system provides a critical link between the brain regions that process cognitive information and those controlling motor behavior. Precise control of these ventral tegmental area (VTA) projections facilitates seeking rewarding stimuli, retreating from aversive stimuli, constraint of motivational state, and behavioral flexibility necessary for survival. GABAergic signaling provides robust inhibition that gates VTA DA cell excitability (1, 2), and loss of this inhibition leads to pathological dysregulation of mesocorticolimbic circuitry (3, 4).Endocannabinoids (eCBs) regulate DAergic activity through retrograde signaling from DA cell bodies onto presynaptic cannabinoid type 1 (CB₁) receptors expressed on both inhibitory and excitatory inputs. Although both 2-arachidonoylglycerol (2-AG) and anandamide (AEA) function as endogenous CB₁ agonists in the brain (5–7), these lipids exhibit distinct pharmacological profiles in vivo (8, 9) and mediate differential behavioral effects (10, 11). Endocannabinoids are produced and degraded on-demand, and the primary enzymes responsible for eCB degradation have been well-characterized using selective pharmacological tools that inactivate monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH) (11–13). However, a complete evaluation of the influence of eCB signaling in the brain has been hampered by the lack of appropriate corresponding tools for selectively inactivating on-demand eCB biosynthesis.Substantial evidence implicates eCB signaling in the etiology of nicotine addiction, and recent work demonstrates that chronic nicotine exposure (CNE) selectively enhances nicotine-induced increases in VTA 2-AG formation (14). The present study investigated the possible contribution of this effect to aberrant VTA DA cell excitation present following CNE (15). We find that sensitized nicotine-induced 2-AG release (14) strongly correlates with a loss of nicotine-induced GABA release, which may contribute to impaired inhibitory constraint of VTA DA cell excitation following CNE. To test this hypothesis, we characterized a series of selective inhibitors of 2-AG biosynthesis by diacylglycerol lipase α and β (DAGLα and DAGLβ; hereafter referred to as DAGL) (16–18) and 2-AG degradation by α/β-hydrolase domain 6 (ABHD6) and MAGL (11, 12, 19), and used these compounds to investigate the functional impact of enhanced 2-AG recruitment on GABAergic signaling at VTA synapses and nicotine self-administration.     

The Widened Pipe Model of plant hydraulic evolution

Shaping global water and carbon cycles, plants lift water from roots to leaves through xylem conduits. The importance of xylem water conduction makes it crucial to understand how natural selection deploys conduit diameters within and across plants. Wider conduits transport more water but are likely more vulnerable to conduction-blocking gas embolisms and cost more for a plant to build, a tension necessarily shaping xylem conduit diameters along plant stems. We build on this expectation to present the Widened Pipe Model (WPM) of plant hydraulic evolution, testing it against a global dataset. The WPM predicts that xylem conduits should be narrowest at the stem tips, widening quickly before plateauing toward the stem base. This universal profile emerges from Pareto modeling of a trade-off between just two competing vectors of natural selection: one favoring rapid widening of conduits tip to base, minimizing hydraulic resistance, and another favoring slow widening of conduits, minimizing carbon cost and embolism risk. Our data spanning terrestrial plant orders, life forms, habitats, and sizes conform closely to WPM predictions. The WPM highlights carbon economy as a powerful vector of natural selection shaping plant function. It further implies that factors that cause resistance in plant conductive systems, such as conduit pit membrane resistance, should scale in exact harmony with tip-to-base conduit widening. Furthermore, the WPM implies that alterations in the environments of individual plants should lead to changes in plant height, for example, shedding terminal branches and resprouting at lower height under drier climates, thus achieving narrower and potentially more embolism-resistant conduits.

Water transport through plants is a key driver of the carbon and other biogeochemical cycles (1–3) and is a crucial link in plant adaptation to climate and vegetation response to climate change (4–9). The water conducting cells of plants, xylem conduits, widen with distance from the stem tip, and, therefore, taller plants have wider conduits (6, 10–12). Xylem conduits are of two main types: tracheids, found in most gymnosperms, and vessels, found in most flowering plants. Tracheids have intact cell membranes, so water must flow from cell to cell through these membranes. Vessels are made up of cells aligned vertically end to end, with the cell membranes dissolved between successive members, forming a tube. Whatever their differences in structure, wider conduits are beneficial because they conduct more water. Tip-to-base widening is expected to help maintain conductance per unit leaf area constant as an individual plant grows taller, counterbalancing the resistance that would otherwise accrue with increasing conductive path length the individual grows (2, 13). Wider conduits, however, are more vulnerable to embolisms caused by cold and likely drought (8, 14–18) and cost more in terms of carbon for a plant (ref. 1; cf. ref. 19). Embolisms in the xylem even affect transport of photosynthates in the phloem (8, 20). This means that as trees grow taller, conductance, embolism vulnerability, and carbon costs must interrelate in a delicate evolutionary balance.Because of the importance of this balance in plant hydraulic evolution and in forest reactions to climate change (3, 6, 21–23), an important goal of plant biology is to construct models that predict how and why plants deploy conduit diameters throughout their bodies (1, 2, 17, 24–26). Some models predict that conduits should be of uniform diameter (27, 28), while others predict that they should widen tip to base (1, 2, 13, 24, 29, 30). But even current models include untested assumptions and large numbers of parameters, making it difficult to identify the biological causes of the predictions they make. For example, some invoke Da Vinci’s rule, the largely untested assumption that the summed wood area of the twigs is the same as that at the base (24, 26). Other models depict plant conduits as branching as they do in mammalian circulatory systems, but whether this happens along the entire stem in plants is unclear (30–33). There is an expectation that conduit diameter D should widen with distance from the stem tip L following a power-law (D ∝ L^b), but there is no agreement on the value of b, the conduit widening exponent (1, 2). Furthermore, even though within-individual tip-to-base conduit widening has been confirmed in a handful of species (34–36), and the scaling of conduit diameter with plant size across species is consistent with it (6, 10–12, 34), the expectation that conduits should widen similarly within stems across terrestrial vascular plant lineages and habits has yet to be empirically confirmed. Here we present the Widened Pipe Model (WPM), which correctly predicts the form of tip-to-base conduit widening across the span of plant size, life form, and habitat across the terrestrial plant phylogeny.     

Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors

Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery.     

Genetic contributors toward increased risk for ischemic heart disease

Cardiovascular disease is a leading cause of mortality in the United States, and is a significant cause of death worldwide. In 2002, it accounted for 38.0% of all deaths in the US, and approximately one-third of all global deaths. It has a significant economic impact, with an estimated cost in the US of 393.5 billion US dollars for 2005. The most common form of heart disease is coronary heart disease (CHD)(1)/coronary artery disease (CAD) resulting from atherosclerosis. Thirteen million Americans are affected by CHD annually, with 7.1 million of these experiencing a myocardial infarction (MI). Five to ten percent of new MI's occur in individuals younger than age 50, and the lifetime risk of developing CAD after age 40 ranges from 32% in women to 49% in men. Because of its major impact on morbidity and mortality, as well as its contribution to annual health care costs, it is of the utmost importance that improved strategies for preventing and treating CAD be developed. A promising, but inherently difficult, area of study is the identification of genes that predispose to or directly cause CAD. The identification of these genes may lead to screening tests that will allow persons at risk for developing CAD to be identified early enough that prevention/intervention strategies can be implemented to prevent or ameliorate the disease process, and may also lead to the development of gene therapy mechanisms useful in the treatment of ischemic heart disease (IHD). Because an exhaustive review of all the genes being studied in relation to CAD and MI is difficult within the confines of a review article, this review will focus on describing representative studies investigating the genes considered most likely to potentially contribute toward an increased risk for CAD and MI. Genes resulting in inherited disorders with which an increased risk of CAD and MI is associated will be discussed, as well as a number of candidate genes that may play a role in the multifactorial inheritance of CHD risk.     

The colitis of Behcet's disease: A separate entity?

Summary An additional case of Behcet's disease with colonic involvement has been presented. This association is rare, as only 13 prior cases with adequate data were available for comparison. Controversy exists as to whether these cases represented true involvement of the colon by Behcet's disease, coincidental inflammatory bowel disease and Behcet's disease, or merely autoimmune phenomena associated with inflammatory bowel disease. Unusual colonoscopic lesions noted in our patient and other features enumerated in the text suggested to us that at least some of these cases represented primary Behcet's disease involving the colon.     

A novel assay to detect nucleotide receptor P2X7 genetic polymorphisms influencing numerous innate immune functions

The importance of accessory signaling pathways amplifying endotoxin responses has recently been highlighted by genetic studies describing LPS-hyporesponsive individuals despite carrying the common allele for TLR4. The nucleotide receptor P2X7 modulates the production of numerous LPS-stimulated inflammatory mediators. We have recently described the largest phenotypic screen known for genetic polymorphisms associated with the nucleotide receptor P2X7, a global regulator of leukocyte function. This required the development of a novel monocyte pore assay with numerous advantages over previous methods and with the potential to facilitate rapid (< 3 h), multiplex analysis of clinical samples. This paper addresses aspects pertinent to the development of the monocyte pore assay, briefly summarizes our results suggesting that P2X7 alleles modulate LPS-stimulated cytokine production, and discusses a model wherein P2X7 may serve as an amplification loop of innate immunity.     

Implementing a Prospective Study of Women Seeking Abortion in the United States: Understanding and Overcoming Barriers to Recruitment

BackgroundThe Turnaway Study is designed to prospectively study the outcomes of women who sought—but did not all obtain—abortions. This design permits more accurate inferences about the health consequences of abortion for women, but requires the recruitment of a large number of women from remote health care facilities to a study a sensitive topic. This paper explores the Turnaway Study's recruitment process.MethodsFrom 2008 to 2010, the staff at 30 abortion-providing facilities recruited eligible female patients. Eight interventions were evaluated using multilevel logistic regression for their impact on eligible patients being approached, approached patients agreeing to go through informed consent by phone, and enrolled patients completing the baseline interview.FindingsAfter site visits, patients had roughly twice the odds of being approached by facility staff and twice the odds of then agreeing to go through informed consent. When all recruitment steps were considered together, the net effect of site visits was to increase the odds that eligible patients participated by nearly a factor of six. After the introduction of a patient gift card incentive, patients had over three times the odds of agreeing to go through informed consent. With each passing month, however, staff demonstrated a 9% reduced odds of approaching eligible patients about the study.ConclusionPrioritizing scientific rigor over the convenience of using existing datasets, the Turnaway Study confronted recruitment challenges common to medical practice-based studies and unique to sensitive services. Visiting sites and communicating frequently with facility staff, as well as offering incentives to patients to hear more about the study before informed consent, may help to increase participation in prospective health studies and facilitate evaluation of sensitive women's health services.     

Safety of repeated hyperpolarized helium 3 magnetic resonance imaging in pediatric asthma patients

Background

Hyperpolarized helium 3 magnetic resonance imaging (³He MRI) is useful for investigating pulmonary physiology of pediatric asthma, but a detailed assessment of the safety profile of this agent has not been performed in children.

Objective

To evaluate the safety of ³He MRI in children and adolescents with asthma.

Materials and methods

This was a retrospective observational study. ³He MRI was performed in 66 pediatric patients (mean age 12.9 years, range 8–18 years, 38 male, 28 female) between 2007 and 2017. Fifty-five patients received a single repeated examination and five received two repeated examinations. We assessed a total of 127 ³He MRI exams. Heart rate, respiratory rate and pulse oximetry measured oxygen saturation (SpO₂) were recorded before, during (2 min and 5 min after gas inhalation) and 1 h after MRI. Blood pressure was obtained before and after MRI. Any subjective symptoms were also noted. Changes in vital signs were tested for significance during the exam and divided into three subject age groups (8–12 years, 13–15 years, 16–18 years) using linear mixed-effects models.

Results

There were no serious adverse events, but three minor adverse events (2.3%; headache, dizziness and mild hypoxia) were reported. We found statistically significant increases in heart rate and SpO₂ after ³He MRI. The youngest age group (8–12 years) had an increased heart rate and a decreased respiratory rate at 2 min and 5 min after ³H inhalation, and an increased SpO₂ post MRI.

Conclusion

The use of ³He MRI is safe in children and adolescents with asthma.

     

Coronary artery bypass graft surgery outcomes among African-Americans and Caucasian patients

There have been few studies to date that investigate the effect of race on outcomes related to coronary artery bypass grafting. The objective of the present study was to investigate race as an independent predictor of outcomes among patients undergoing coronary artery bypass graft (CABG). A nested case-control study from a twelve-year hospitalization cohort (N=9671) in which data were collected prospectively was conducted. Cases were African-American patients undergoing CABG (N=644). Controls were randomly selected Caucasian patients undergoing CABG (N=1932). Controls were matched to cases 3:1 on year of surgery. Fifteen preoperative and intraoperative risk factors and 14 outcomes were examined. The 14 outcomes of interest were length of stay, readmission to ICU, total ICU stay, total hours on ventilator post-op, reoperation for bleeding/tamponade, deep sternal wound infection, neurological complications, pneumonia, other pulmonary complications, renal failure, gastrointestinal complications, atrial fibrillation requiring treatment, in-hospital mortality, and intraoperative complications. Regression analysis was used to control for risk factors. Multivariate analysis revealed African-Americans were at greater risk for renal complications (OR 1.88, 95% CI 1.27-2.77), neurological complications (OR 1.34, 95% CI 1.01-1.77), and pulmonary complications (OR 2.11, 95% CI 1.72-2.59). African Americans had a significantly longer hospitalization post-operatively (OR 0.79, 95% CI 0.66-0.96), but were less likely to experience post-operative atrial fibrillation requiring treatment than Caucasians (OR 0.64, 95% CI 0.49-0.84). Even after multiple adjustments, African-Americans undergoing CABG surgery had significantly greater morbidity compared to Caucasian patients.