A Tale of Two Carboxypeptidases

Proopiomelanocortin (Pomc) neurons play a central role in energy homeostasis. Despite the complexity of Pomc posttranslational processing, regulation of Pomc gene expression often takes center stage. Complementary papers that zero in on distinct carboxypeptidases (Plum et al., 2009; Wallingford et al., 2009) now refocus the spotlight on regulated peptide cleavage

Developmental single-cell transcriptomics of hypothalamic POMC neurons reveal the genetic trajectories of multiple neuropeptidergic phenotypes

Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential to regulate food intake and energy balance. However, the ontogenetic transcriptional programs that specify the identity and functioning of these neurons are poorly understood. Here, we use scRNAseq to define the transcriptomes characterizing Pomc-expressing cells in the developing hypothalamus and TRAP-seq to analyze the subsequent translatomes of mature POMC neurons. Our data showed that Pomc-expressing neurons give rise to multiple developmental pathways expressing different levels of Pomc and unique combinations of transcription factors. The predominant cluster, featured by high levels of Pomc and Prdm12 transcripts represents the canonical arcuate POMC neurons. Additional cell clusters expressing medium or low levels of Pomc mature into different neuronal phenotypes featured by distinct sets of transcription factors, neuropeptides, processing enzymes, cell surface and nuclear receptors. We conclude that the genetic programs specifying the identity and differentiation of arcuate POMC neurons are diverse and generate a heterogeneous repertoire of neuronal phenotypes early in development that continue to mature postnatally.Fil: Yu, Hui. University of Michigan; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. University of Michigan; Estados Unidos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Low, Malcolm J.. University of Michigan; Estados Unido

Failure of Intravenous Morphine to Serve as an Effective Instrumental Reinforcer in Dopamine D2 Receptor Knock-Out Mice

The rewarding effects of opiates are thought to be mediated through dopaminergic mechanisms in the ventral tegmental area, dopamine-independent mechanisms in the nucleus accumbens, or both. The purpose of the present study was to explore the contribution of dopamine to opiate-reinforced behavior using D2 receptor knock-out mice. Wild-type, heterozygous, and D2 knock-out mice were first trained to lever press for water reinforcement and then implanted with intravenous catheters. The ability of intravenously delivered morphine to maintain lever pressing in these mice was studied under two schedules of reinforcement: a fixed ratio 4 (FR4) schedule (saline, 0.1, 0.3, or 1.0 mg/kg, per injection) and a progressive ratio (PR) schedule (1.0 mg/kg, per injection). In the wild-type and heterozygous mice, FR4 behavior maintained by morphine injections was significantly greater than behavior maintained by vehicle injections. Response rate was inversely related to injection dose and increased significantly in the wild-type and heterozygous mice when the animals were placed on the PR schedule. In contrast, the knock-out mice did not respond more for morphine than for saline and did not respond more when increased ratios were required by the PR schedule. Thus, morphine served as a positive reinforcer in the wild-type and heterozygous mice but failed to do so in the knock-out mice. Under this range of doses and response requirements, the rewarding effects of morphine appear to depend critically on an intact D2 receptor systemFil: Elmer, Greg I.. University of Maryland; Estados UnidosFil: Pieper, Jeanne O.. National Institutes of Health; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Low, Malcolm J.. Oregon Health and Sciences University; Estados UnidosFil: Grandy, David K.. Oregon Health and Sciences University; Estados UnidosFil: Wise, Roy A.. National Institutes of Health; Estados Unido

Dysfunctional Light-Evoked Regulation of cAMP in Photoreceptors and Abnormal Retinal Adaptation in Mice Lacking Dopamine D4 Receptors

Dopamine is a retinal neuromodulator that has been implicated in many aspects of retinal physiology. Photoreceptor cells express dopamine D4 receptors that regulate cAMP metabolism. To assess the effects of dopamine on photoreceptor physiology, we examined the morphology, electrophysiology, and regulation of cAMP metabolism in mice with targeted disruption of the dopamine D4 receptor gene. Photoreceptor morphology and outer segment disc shedding after light onset were normal in D4 knock-out (D4KO) mice. Quinpirole, a dopamine D2/ D3/D4 receptor agonist, decreased cAMP synthesis in retinas of wild-type (WT) mice but not in retinas of D4KO mice. In WT retinas, the photoreceptors of which were functionally isolated by incubation in the presence of exogenous glutamate, light also suppressed cAMP synthesis. Despite the similar inhibition of cAMP synthesis, the effect of light is directly on the photoreceptors and independent of dopamine modulation, because it was unaffected by application of the D4 receptor antagonist L-745,870. Nevertheless, compared with WT retinas, basal cAMP formation was reduced in the photoreceptors of D4KO retinas, and light had no additional inhibitory effect. The results suggest that dopamine, via D4 receptors, normally modulates the cascade that couples light responses to adenylyl cyclase activity in photoreceptor cells, and the absence of this modulation results in dysfunction of the cascade. Dark-adapted electroretinogram (ERG) responses were normal in D4KO mice. However, ERG b-wave responses were greatly suppressed during both light adaptation and early stages of dark adaptation. Thus, the absence of D4 receptors affects adaptation, altering transmission of light responses from photoreceptors to inner retinal neurons. These findings indicate that dopamine D4 receptors normally play a major role in regulating photoreceptor cAMP metabolism and adaptive retinal responses to changing environmental illumination.Fil: Nir, Izhak. The University of Texas Health Science Center; Estados UnidosFil: Harrison, Joseph M.. The University of Texas Health Science Center; Estados UnidosFil: Haque, Rashidul. Emory University School of Medicine; Estados UnidosFil: Low, Malcolm J.. Oregon Health and Science University; Estados UnidosFil: Grandy, David K.. Oregon Health and Science University; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Iuvone, P. Michael. Emory University School of Medicine; Estados Unido

V1b Receptor Antagonist SSR149415 and Naltrexone Synergistically Decrease Excessive Alcohol Drinking in Male and Female Mice

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141424/1/acer13544.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141424/2/acer13544_am.pd

Nuclear transcriptional changes in hypothalamus of Pomc enhancer knockout mice after excessive alcohol drinking

Persistent alterations of proopiomelanocortin (Pomc) and mu‐opioid receptor (Oprm1) activity and stress responses after alcohol are critically involved in vulnerability to alcohol dependency. Gene transcriptional regulation altered by alcohol may play important roles. Mice with genome‐wide deletion of neuronal Pomc enhancer1 (nPE1−/−), had hypothalamic‐specific partial reductions of beta‐endorphin and displayed lower alcohol consumption, compared to wildtype littermates (nPE1+/+). We used RNA‐Seq to measure steady‐state nuclear mRNA transcripts of opioid and stress genes in hypothalamus of nPE1+/+ and nPE1−/− mice after 1‐day acute withdrawal from chronic excessive alcohol drinking or after water. nPE1−/− had lower basal Pomc and Pdyn (prodynorphin) levels compared to nPE1+/+, coupled with increased basal Oprm1 and Oprk1 (kappa‐opioid receptor) levels, and low alcohol drinking increased Pomc and Pdyn to the basal levels of nPE1+/+ in the water group, without significant effects on Oprm1 and Oprk1. In nPE1+/+, excessive alcohol intake increased Pomc and Oprm1, with no effect on Pdyn or Oprk1. For stress genes, nPE1−/− had lowered basal Oxt (oxytocin) and Avp (arginine vasopressin) that were restored by low alcohol intake to basal levels of nPE1+/+. In nPE1+/+, excessive alcohol intake decreased Oxt and Avpi1 (AVP‐induced protein1). Functionally examining the effect of pharmacological blockade of mu‐opioid receptor, we found that naltrexone reduced excessive alcohol intake in nPE1+/+, but not nPE1−/−. Our results provide evidence relevant to the transcriptional profiling of the critical genes in mouse hypothalamus: enhanced opioid and reduced stress gene transcripts after acute withdrawal from excessive alcohol may contribute to altered reward and stress responses.Transcriptional profiling of the critical genes in mouse hypothalamus: enhanced opioid and reduced stress gene transcripts after acute withdrawal from excessive alcohol may contribute to altered reward and stress responses.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152013/1/gbb12600.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152013/2/gbb12600_am.pd

The effect of CO2 loading on the properties of normal concrete vs fly ash concrete

Abstract: Purpose – There is an increase in greenhouse gasses and global climate change is frequently reported on. What can be done? Certainly to try and reduce the carbon footprint, which is not a new topic, by encouraging applications and activities for concrete during its lifetime (Portland Cement Association, 2019). This study focuses on introducing CO2 to normal and fly ash concrete and thus investigating the effect on the carbon footprint of the samples and the effectiveness of the CO2 introduction methods, namely carbonated water addition during the mixing process and by means of an infusion pipe directly into the concrete when the samples are casted and have been casted. Design/methodology/approach – The feasibility of carbon dioxide storage within concrete is determined by investigating the effects of introduced carbon dioxide into concrete samples and the effectiveness of the concrete at storing carbon dioxide. The concrete was mixed in a 1:3:3 ratio for the OPC or blended 52.5R cement:sand:stone (22mm) with a 28 day strength of 50MPa. Samples were also prepared containing low grade fly ash cement contents ranging from 15 to 60%. CO2 was introduced to the concrete via carbonated mixing water and an infusion pipe system directly to the hardening concrete cubes. 16g CO2 bicycle carbon dioxide inflators and valve system were used to infuse the concrete over a period of a week until the canister was emptied with valve release on the lowest setting. A compression test was carried out to determine the strength of the concrete cubes with, and without, the introduction of carbon dioxide. Results were also obtained using a Scanning Electron Microscope (SEM) and energy dispersive x-ray spectrometer (EDS) to determine how the carbon dioxide changed the microscopic composition and chemical composition of the concrete. A microcontroller with carbon dioxide sensors was used to gather carbon dioxide emission data for a period of 3 months. Findings - The compressive strength tests show by introducing carbon dioxide to the concrete, the compressive strength has increased by as much as 13.86% as expected from the literature. Furthermore, by infusing carbon dioxide with the fly ash blended cement, will give a higher strength compared to the control with ordinary portland cement. This correlates to an overall reduction in cost for the structure. The optimal fly ash content for the control with minimal strength degradation is 30%. Where the optimal fly ash content for the concrete with carbon dioxide stored within, is 45%. The SEM analysis showed the concrete with sequestered carbon dioxide has significantly more C-S-H gel formation, thus the strength increase. Furthermore, the carbon dioxide emission test showed the concrete with infused carbon dioxide stores carbon dioxide more efficiently compared to the control sample. With the data showing the infused sample releases 11.19% less carbon dioxide compared to the control sample. However, the carbonated water sample releases 20.9% more carbon dioxide when compared to the control sample. Thus the introduction of carbon dioxide by means of infusion is more effective. Originality/value - This is a practical pilot investigation of carbon dioxide introduction via two methods, one being infusion of CO2 into normal concrete and fly ash concrete and two, mixing normal and fly ash concrete with carbonated water. These results show, cheaper cement can be used to achieve equivalent or better strength. This can help in the reduction of the construction industry’s carbon footprint

High-dimensional Objective-based Data Farming

In objective-based data farming, decision variables of the Red Team are evolved using evolutionary algorithms such that a series of rigorous Red Team strategies can be generated to assess the Blue Team\u27s operational tactics. Typically, less than 10 decision variables (out of 1000+) are selected by subject matter experts (SMEs) based on their past experience and intuition. While this approach can significantly improve the computing efficiency of the data farming process, it limits the chance of discovering “surprises” and moreover, data farming may be used only to verify SMEs\u27 assumptions. A straightforward solution is simply to evolve all Red Team parameters without any SME involvement. This modification significantly increases the search space and therefore we refer to it as high-dimensional objective-based data farming (HD-OBDF). The potential benefits of HD-OBDF include: possible better performance and information about more important decision variables. In this paper, several state-of-the-art multi-objective evolutionary algorithms are applied in HD-OBDF to assess their suitability in terms of convergence speed and Pareto efficiency. Following that, we propose two approaches to identify dominant/key evolvable parameters in HD-OBDF - decision variable coverage and diversity spread