bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bitter taste receptor agonists induced chronotropic effects on rat heart
Authors: Yuan Guanyin , Jing Yang , Wang Ting , Fernandes Vitor Samuel Leite , Xin Wenkuan 
Published: April 2020

BackgroundBitter taste receptors (Tas2rs), the members of the G‐protein‐coupled receptors, mediate the bitter taste and express in extra‐oral tissues. Previous studies have shown that Tas2r mRNAs are expressed in the whole heart and cultured cardiomyocytes of neonatal rat.AimThis study investigates the expression of Tas2rs in the sinoatrial node (SA node) and left ventricle of rat heart, and the mechanism of bitter compounds regulation of the heart rate.MethodsRats were excised by thoracotomy and the sinoatrial node and left ventricle tissues were isolated. These tissues were processed to study the mRNA expression levels of Tas2rs and their coupled G‐protein subunits by real time quantitative PCR (qPCR). Langendorff isolated heart models were used to record the heart rate and electrocardiogram. SA node beating recordings were used to measure the beating rate in combination with bitter compounds, quinine and chloroquine. The statistic dada are expressed as mean ± SEM.ResultsWe detected the mRNA expression of 7 subtypes of Tas2rs (Tas2r108, 120, 121, 126, 135, 137, 143) in the left ventricle and 8 subtypes in the SA node including Tas2r38 in addition to the 7 subtypes in the left ventricle, in which the mRNA level of Tas2r120 is the highest among them followed by Tas2r143 and 121. In addition, Tas2r coupled G‐protein subunits also expressed in the SA node and left ventricle.Tas2r agonists, quinine (1–50 μM) and chloroquine (1–20 μM), decreased the heart rate and increased the RR interval and QRS duration in Langendoff‐perfused isolated rat hearts; they reduced the spontaneous beating rate of isolated SA nodes with pEC50 values of 4.907 ± 0.045 and 4.968 ± 0.030, respectively.The blockade of Tas2r108 with abscisic acid (500 μM), the inhibition of PDEs with IBMX (50 μM), or the selective inhibition of PDE3 and PDE4 with a cocktail of cilostamide (0.5 μM) and rolipram (10 μM), attenuated the negative chronotropic effects of quinine and chloroquine on the SA node. Furthermore, quinine (20 μM) and chloroquine (10 μM) suppressed the tachycardia effect of isoprenaline on the SA node and shifted the concentration‐response curve of isoprenaline rightward.ConclusionTas2r agonists, quinine and chloroquine, induce negative ionotropic effects in rat hearts. Our data provide mechanistic evidence that Tas2r agonists decreased the heart rate by prolonging ventricular depolarization, and by attenuating the SA node pace in a PDE‐dependent manner. Tas2r agonists can counteract with β‐adrenergic receptor activation and eliminate isoprenaline‐induced tachycardia, suggesting Tas2rs could be the potential targets of drugs for treating sinus or ventricular tachycardia and elevated heart rate related to heart failure.Support or Funding InformationThis work was supported by Funding from Southwest University [Grant number SWU 115086, 104290/22300503] and Administration of Foreign Experts Affairs of China [Grant number BC2018042].
Differential phosphorylation modulation of GluN2B at tyrosine 1336 in two types of chemical‐induced LTP in hippocampal slices
Authors: Zhang Bin , Lu Wen , Ma Wu , Xue Fusheng , Zhang Yongchao , Fang Weiqing 
Published: April 2020

Synaptic plasticity, i.e., long‐term potentiation (LTP) and long‐term depression (LTD), has been extensively studied as a cellular mechanism of learning and memory. Chemcal‐induced LTP, which is dependent on activation of NMDA receptor, was widely accepted as neuronal model for exploring synaptic plasticity in brain slice or neuronal cultures. However, the phosphorylation regulation of GluN2B, a critical modulatory subunit of NMDA receptor chemical‐induced LTP remains poorly understood. In the present study, we examined the regulation of GluN2B at tyrosine 1336 in two types of chemical‐induced LTP, either by forskolin & rolipram or by glycine‐treated acute hippocampal slices. Elevated phosphorylation level of tyrosine 1336 at GluN2B in forskolin & rolipram‐induced LTP rather than glycine‐induced LTP was observed. Moreover, a similar increment of Src activity was detected, evidenced by higher phosphorylation level of tyrosine 416 at Src. Taken together, our results reveal a diassociated regulation of tyrosine 1336 at GluN2B and tyrosine 416 at Src in forskolin & rolipram‐induced LTP, uncovering phosphorylation site specific modulation in two types of chemical‐induced LTP, thus broadening our understanding of the role of GluN2B in learning and memory.Support or Funding InformationThis work was funded by National Natural Science Foundation of China Grant No. 31700882 (to B.Z.) and 31600863 (to W.L.), Zhejiang Provincial Natural Science Foundation of China LY20C090004 ( to B.Z) and LY18H090010 to (H.A.), the start‐up fund of Hainan Medical University (to W.L.).
Astrocyte Regional Heterogeneity Revealed Through Machine Learning‐based Glial Neuroanatomical Assays
Authors: Blackburn Jessica , Alves Michele Joana , Zhao Jing , Czeisler Catherine M. , Otero José Javier 
Published: April 2020

Astrocytes undergo activation during brain injury through an ill‐defined process known as gliosis. Recent work focused on elucidating the molecular underpinnings of glial response to injury. However, astrocytes exist in a unique three‐dimensional space within the brain, and this location is highly relevant to the astrocyte’s function. Therefore, generating neuroanatomical assays, which maintain CNS positional information of astrocytes relative to their neural networks, represent optimal modalities to assess astrocyte function and reactivity. The majority of astrocytic neuroanatomical assays in the scientific literature measure mean fluorescence intensity of GFAP or ALDH1L1. Modern advances in computer vision and machine learning algorithms have now given us the tools with which we can extract a multitude of features from microscopic imaging data. By segmenting astrocytes from an image into numerous GFAP or ALDH1L1 positive “objects”, extracted numerical values indicate object location, object intensities, object shapes, as well as object textures from the original image. We tested the hypothesis that morphological changes in astrocyte reaction to injury represents a heterogeneous response in different brain regions. To test this hypothesis, we developed an automated segmentation algorithm for both GFAP and ALDH1L1 immunostained mouse brain sections treated with vehicle or systemic lipopolysaccharide to induce neuroinflammation. These images were then captured on a laser scanning confocal microscope and features characterizing shape, fluorescent intensity, and fluorescent texture. We then utilized a covariance matrix, random forest‐based boruta algorithm, cluster analysis and principal component analysis to evaluate the differences between reactive astrocytes and baseline astrocytes. By implementing a support vector machine algorithm, we show classification identification methods of reactive versus baseline astrocytes. Machine learning models developed to classify neuroanatomical images provides a highly insightful methodology to discover new processes by which astrocytes react to injury.Support or Funding Information3R01HL132355
Molecular Mechanisms of Stem Cell Reprogramming by CD47 Antagonists in Primary Human Cells
Authors: Cicalese Kyle V. , Kaur Sukhbir , Roberts David D. 
Published: April 2020

CD47 is a ubiquitously expressed transmembrane receptor whose interaction with signal regulatory protein alpha on macrophages initiates anti‐phagocytic signaling. Additionally, thrombospondin‐1 (THBS1) binding to CD47 induces cell‐autonomous signaling that regulates cell proliferation, differentiation, and recovery from genotoxic stress. Previous studies showed that CD47‐ or THBS1‐null primary lung endothelial cells exposed to ionizing radiation have higher viability than primary wildtype cells. The mechanisms that enhance the viability of CD47‐null primary cells include enhanced autophagy, anabolic metabolism, and increased expression of c‐Myc and other stem cell transcription factors. The latter enable null cells to spontaneously form embryoid bodies. These results suggest that loss of CD47 may generally facilitate cell reprogramming and self‐renewal. Induced pluripotent stem cells (iPS) can be induced by introducing Yamanaka stem cell factors (MYC, KLF4, SOX2, and OCT4) into primary somatic cells, but iPS cells can form teratomas and thus pose risk of cancer when used for regenerative medicine. Therefore, alternative approaches are needed to overcome these limitations. We propose that CD47 gene knock out (KO) via CRISPR or treating with CD47 antagonists from primary human cells could be a new, simpler, and more effective way to develop multipotent stem cells. To investigate this, we have studied the effects of CD47 knockout and antagonists on embryoid body formation and mRNA expression of stem cell factors using fibroblasts, adipocytes, and human umbilical vein cells (HUVEC). Reprogramming was measured by quantifying embryoid body formation, cell viability, and mRNA expression using real‐time PCR. Preliminary results with HUVEC, fibroblast, and adipocyte primary cells treated with CD47 knockout or antagonists have shown enhanced cell proliferation and expression of stem cell factors similar to murine cd47‐null lung endothelial cells. These results suggest that CD47 plays a key regulatory role in stem cell reprogramming of human primary cells.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIH/National Cancer Institute.
Competition and Identification of Anaerobic Organisms Isolated from Apis mellifera Gut Microbiota
Authors: Knier Rochelle Helen 
Published: April 2020

Apis mellifera, the most common species of honeybee, has been suffering the epidemic of colony collapse disorder (CCD). This is defined by the sudden and unexplainable death of an entire colony of bees, and populations worldwide dwindle further every year. Although research continues, there exists no lasting or effective treatment. One underdeveloped area of study has been the option to treat bees with probiotics (
Audisio, 2017; Baffoni et al., 2015). There have been numerous studies within other organisms and honeybees that show that a well‐maintained gut microbiome is a key component of health, especially when the pathogen is endemic to the intestines – as in the case of Nosema pathogen, which has been implicated in CCD (
Raymann, 2018; Shreiner, 2015). The hypothesis being investigated is that microbes cultivated from the gut of normal bees will suppress the growth of known honey bee pathogens. Identification of such normal flora may serve as a probiotic for treatment of bees experiencing CCD due to infectious agents.Support or Funding InformationResearch Grant from the University of Wisconsin ‐ Stout
Skeletal Muscle ULK1 and ULK2 Jointly Couple Muscle Mass with Force and Are Required for Survival Under Low Nutrient Availability
Authors: Fuqua Jordan D. , Hughes Julia , Kuennen Dylan , Harris Matthew , Turner Kristen , Kronemberger Ana , Ebert Scott , Dierdorff Jason , Zhu Zhiyong , Zingman Leonid , Adams Christopher , Lira Vitor A. 
Published: April 2020

The protein kinases Unc‐51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2) have evolved from a single yeast gene, autophagy related 1 (Atg1). Although ULK1 and ULK2 are thought to redundantly stimulate autophagy, other functions are emerging. Interestingly, skeletal muscle expresses both ULK1 and ULK2 at high levels (i.e., >2‐fold the median expression of 91 mouse tissues and cell lines) suggesting an important, yet unknown role for their combined elevated expression in muscle. Therefore, to investigate the joint role of ULK1 and ULK2 on muscle homeostasis and whole‐body metabolism we generated mice with skeletal muscle‐specific knockout of these genes (ULK2/1 mKO). Here, we demonstrate that ULK2/1 mKO mice have larger muscles (~12%) that are actually weaker (~21% decrease in relative force) in comparison with wild type littermates. Loss of ULK2/1 in skeletal muscle also affects whole‐body metabolic homeostasis as indicated by decreased body fat mass (~20% at 7wk of age), attenuated fat mass gains with aging (~40% at 21mo. of age), and life‐threatening inability to maintain euglycemia under fasting conditions (~32% reduction of blood glucose compared to WT). Mechanistically, loss of ULK 2/1 not only impairs skeletal muscle autophagy but also AMPK function as indicated by accumulation of LC3, p62, NBR1, and ubiquitinated proteins, as well as reduced phosphorylation of ACC (a surrogate of AMPK activity), respectively. These results identify a previously unknown role for ULK2 and ULK1 in jointly coupling skeletal muscle size with force. They also reveal skeletal muscle ULK2 and ULK1 as fundamental proteins for survival under conditions of reduced nutrient availability.Support or Funding InformationThis work is funded by:AHA 16SDG30360001 (V.A.L.). and NASA Iowa Space Grant Graduate Fellowship Program
Neuroprotective effect of PEA‐OXA on oxaliplatin‐Induced neuropathic pain.
Authors: Cuzzocrea Salvatore , Lanza Marika , Campolo Michela , Casili Giovanna , Paterniti Irene , Filippone Alessia , Esposito Emanuela 
Published: April 2020

Chemotherapy‐induced neuropathy is a common, dose‐dependent adverse effect of several antineoplastics, like oxaliplatin. It can lead to detrimental dose reductions and discontinuation of treatment, and severely affects the quality of life of cancer survivors. N‐Acylethanolamines (NAEs) involve a family of lipid molecules existent in animal and plant, of which is N‐palmitoylethanolamide (PEA) that arouses great attention owing to its anti‐inflammatory, analgesic, and neuroprotective activities. The modulation of specific amidases for NAEs (and in particular NAE‐hydrolyzing acid amidase NAAA, which is more selective for PEA) could be a condition to preserve its levels.So, the aim of the present work was to evaluate the potential beneficial effects of 2‐pentadecyl‐2‐oxazoline (PEA‐OXA) the oxazoline of PEA that reportedly modulates activity of NAAA, in a model of oxaliplatin‐induced peripheral neuropathy. Chemotherapic pain was induced by an intraperitoneally injection of oxaliplatin (oxa) in rats on 5 consecutive days (D0–4) at an injection volume of 0.2 ml for a final cumulative dose of 10 mg/kg. In prophylactic paradigms, PEA‐OXA was given orally 15–20 min prior oxa. Sacrifice was made on day 25.Our results demonstrated that oxaliplatin developed mechano‐hypersensitivity (allodynia and hyperalgesia) in rats; this was associated with the hyperactivation of glia cells and an increased production of pro‐inflammatory cytokines ( IL‐1β and TNF) in the dorsal horn of the spinal cord. Moreover, we showed that oxaliplatin activated NF‐κB pathway and modulated Nrf‐2 ones. The oral injection of PEA‐OXA attenuated inflammatory and oxidative process, decreasing neuropathic pain.Our findings identify PEA‐OXA as a therapeutic target in chemotherapy‐induced painful neuropathy, throughout the biomolecular signaling NF‐κB/Nrf‐2 axis. Therefore, we can consider PEA‐OXA as a promising adjunct to chemoterapy to reduce chronic pain in patients.
Autophagy contributes to the development of salt‐induced hypertension and kidney injury in Dahl S rats
Authors: Kumar Vikash , Kurth Theresa , Cowley Allen W. 
Published: April 2020

Studies were carried to determine the role of autophagy in the development of renal injury in the hypertensive Dahl salt‐sensitive (SS) rat fed a high‐salt (HS; 4.0% NaCl) diet. Autophagy is an essential process (autophagosome formation, maturation, fusion with lysosome, release of macromolecular back into cytosol) required to remove misfolded proteins and damaged organelles (mitochondria, endoplasmic reticulum and peroxisomes) from all cells. It is a dynamic process (“autophagic flux”) and challenged in cells faced with excessive metabolic workloads such as the renal nephrons in kidneys of SS rats fed a HS diet. The tubular mitochondria of SS rats fed a HS produce excess reactive oxygen species (ROS) which we have found can activate mammalian target of rapamycin complex 1 (mTORC1). We have reported that the elevation of renal tissue H2O2 in kidneys of SS rats fed a HS diet can activate mTORC1 and that inhibition of this pathway with rapamycin attenuated salt‐induced hypertension and kidney injury. It is also known that mTORC1 plays a crucial role in regulating autophagy but the extent to which these events contribute to the renal injury observed in the SS rat has not been examined.GoalStudies were carried out in male SS rats to determine the effects of a HS diet (4.0% NaCl) upon the “autophagic flux” of the kidneys. SS rats (n=6 each group) were studied at days 3 or 21 of the HS diet and compared to those receiving a 0.4% NaCl diet. Rat received either the HS diet treated with vehicle, or HS plus daily rapamycin (IP, 1.5 mg/kg per day); Western blot analysis of renal cortical and medullary tissue LCII (microtubule‐associated protein light chain 2) and BECLIN1 protein levels was carried out as an index of autophagic flux.ResultsWhile no changes in the LCII levels were observed at day 3 of HS, a significant reduction was found in both the cortex and medulla after 21 days of HS compared to rats fed 0.4% NaCl. In rapamycin treated SS rats, both the renal cortex and medulla showed significantly greater increases of LCII and BECLIN1 compared with vehicle treated rats suggested an increase of autophagic index. Together, our observations suggest that the attenuation of salt‐induced hypertension and kidney injury in rapamycin treated SS rats may be in part the consequence of increased autophagic influx.Support or Funding InformationHL‐116264
DNA clamp loader subunit DnaX gamma plays a role in the response to DNA damage
Authors: Tashjian Tommy Fay , Chien Peter 
Published: April 2020

The bacterial DNA damage response (DDR) is an important cellular system used to maintain a balance between maintenance of genomic integrity and mutagenesis that drives adaptation in the face of stress. An essential part of this response is the AAA+ protease ClpXP, which degrades several key DDR proteins. Our lab discovered a novel ClpXP substrate in Caulobacter crescentus, DnaX, a conserved component of the DNA replication clamp loader complex. DnaX has two forms: the essential full‐length DnaX tau and a shorter DnaX gamma. DnaX gamma is conserved in bacterial species, yet three distinct mechanism have been found to create it: ribosomal frameshift, transcriptional slippage, and partial ClpXP proteolysis in Caulobacter. Despite being discovered over 30 years ago, the function of DnaX gamma remains unclear.We find that a strain encoding nonproteolyzable version of DnaX (tau‐only), which does not contain any DnaX gamma has no observable growth or morphological defects, but is sensitive to certain types of DNA damage. While the DnaX tau‐only strain is not more sensitive than the wild type to ultra‐violet light, which primarily causes intra‐strand crosslinks, it is significantly more sensitive than wild type to mitomycin C and zeocin, which primarily cause inter‐ and intra‐strand crosslinks and double strand breaks, respectively. To better define the pathways that are affected in the absence of DnaX gamma, we performed transposon (Tn) sequencing analysis and found synthetically lethal and conditionally dispensable genetic interactions with the dnaX‐tau‐only allele. Even in normal growth conditions, we find several synthetic lethal interactions with this allele and DNA damage response genes, especially those involved in double‐strand break repair. We also find that the global transcriptional repressor of the DDR, LexA, is conditionally dispensable in our DnaX tau‐only strain. Together, this suggests a role for DnaX gamma in DNA damage prevention or repair under normal growth conditions and addresses the long‐standing question of why the shorter forms of DnaX are so broadly conserved in bacteria.
Using C. elegans Genetics to Identify Regulators of μ‐Opioid Receptor Signaling
Authors: Wang Dandan , Stoveken Hannah M. , Zucca Stefano , Dao Maria , Orlandi Cesare , Song Chenghui , Masuho Ikuo , Johnston Caitlin , Opperman Karla J. , Giles Andrew C. , Gill Matthew S. , Lundquist Erik A. , Grill Brock , Martemyanov Kirill A. 
Published: April 2020

Opioids exert their analgesic effects by activating the μ‐opioid receptor (MOR), a G protein‐coupled receptor expressed in the nervous system. In addition to clinical pain relief, activation of MOR produces euphoria and leads to dependence. Despite significant process in understanding molecular mechanisms of MOR signaling, the regulatory mechanisms of MOR function are not fully understood. To better understand MOR regulation and to uncover genes influencing MOR signaling, we developed a genetic behavioral platform utilizing nematode C. elegans. Mammalian MOR was transgenically expressed in the nervous system of C. elegans endowing the resulting model (tgMOR) with opioid sensitivity. We validated tgMOR platform showing conservation of pharmacological properties and regulatory mechanisms of MOR mediated behavioral responsiveness. We then performed a large scale forward genetic screen isolating a population of mutants with altered opioid sensitivity. Integrating whole genome sequencing, CRISPR/Cas9 gene editing and transgenic rescue, we identified a set of candidate negative modulators of MOR signaling. One of these genes encoded an orphan GPCR, FRPR‐13. Our phylogenic and functional analysis revealed that mammalian ortholog of FRPR‐13 is GPR139. We found GPR139 to be extensively co‐expressed with MOR in the mammalian nervous system. Knockout of GPR139 in mice enhanced opioid analgesia and reward, but diminished withdrawal. These observations suggest the existence of an “anti‐opioid” system that regulates the extent of MOR signaling in vivo. Our findings further showcase the utility of C. elegans as a scalable platform for genetic discovery of novel GPCR signaling principles.Support or Funding InformationThis work was supported by an NIH Cutting Edge Basic Research Award (R21DA040406) to B.G. and K.A.M., DA036596 to K.A.M., and an NIH Center of Biomedical Research Excellence Grant (P20GM103638) to University of Kansas Genome Sequencing Core.
Dissection of anterograde trafficking and sorting of GPCRs in primary neurons
Authors: Wei Zhe , Naughton Sean X. , Terry Alvin V. , Wu Guangyu 
Published: April 2020

G protein‐coupled receptors (GPCRs) play an important role in regulating the function of the nervous system. However, the molecular mechanisms underlying the targeting and sorting of GPCRs to the functional destination in neurons are not well understood. Here we investigated the role of small GTPase Rab43, via dominant negative inhibition and CRISPR‐Cas9‐mediated knockout, in the transport of several prototypic GPCRs to dendrites and spines and elucidated the possible mechanisms. We demonstrated that inhibition of Rab43 function significantly reduced the surface numbers of α2‐adrenergic receptors (ARs) and β2‐ARs, but not M3‐muscarinic receptors (MRs) in primary cultures of neurons as measured by live neuron radioligand binding. Confocal microscopy revealed that three receptors utilized similar structural determinants, including the highly conserved LL motif in the C‐termini, for their anterograde transport, and that Rab43 affected the expression of α2‐ARs, but not M3‐MRs and vesicular stomatitis virus glycoprotein, at both dendrites and spines. Interestingly, Rab43 only altered β2‐AR expression at spines, but not dendrites. We also found that Rab43 directly and activation‐dependently interacted with α2‐ARs, but not M3‐MRs, and the Rab43‐binding domain was located in the third intracellular loop of the receptor. The chimeric M3‐MRs containing the Rab43‐binding domain of α2‐ARs were subject to Rab43 regulation. These data demonstrate distinct, as yet unidentified pathways that transport different GPCRs to dendrites and spines, in which Rab43 plays a crucial role in the sorting and trafficking of the receptors likely via direct interaction.Support or Funding InformationNIH R01 GM118915