bluuebunny/crossref_metadata_2025_split huggingface dataset:
PRKAR1A deficiency delays postnatal heart growth
Authors: Liu Yuening , Xia Peng , Chen Jingrui , Bandettini W. Patricia , Kirschner Lawrence S. , Stratakis Constantine A. , Cheng Zhaokang 
Published: April 2020

AimsProtein kinase A (PKA) activity is pivotal for proper functioning of the human heart, and its dysregulation has been implicated in a variety of cardiac pathologies. PKA regulatory subunit 1α (R1α, encoded by the PRKAR1A gene) is highly expressed in the heart, and controls PKA kinase activity by sequestering the catalytic subunits. Patients with PRKAR1A mutations are often diagnosed with Carney complex (CNC) in young adults and may die prematurely from cardiac complications such as heart failure. However, no cardiac defect has been reported in adult animal models of PRKAR1A deficiency.Methods and ResultsTo investigate the impact of PRKAR1A deficiency on the heart, we generated cardiac‐specific PRKAR1A heterozygous knockout (cPRKAR1A+/−) mice by breeding the floxed PRKAR1A mice with the Mlc2v‐Cre mice. We also studied a cohort of young CNC patients with PRKAR1A mutations or deletions. Ablation of the PRKAR1A gene in mice increased cardiac PKA activity, reduced heart weight to body weight ratio (Control: 5.3±0.19, cPRKAR1A+/−: 4.7±0.11 mg/g, P<0.05) and cardiomyocyte size (Control: 298.5±9.8 μm2, cPRKAR1A+/−: 191.3±4.5 μm2, P<0.005) without altering contractile function at 3 months of age. Importantly, left ventricular mass was reduced in young patients diagnosed with CNC (Female control: 80.6 ± 12.8g, CNC: 70.8 ± 8.1, P<0.05). Cardiomyocyte hypertrophy in response to activation of the α1‐adrenergic receptor, which is necessary for heart growth after birth, was completely abolished by silencing of PRKAR1A, or stimulation with the PKA activator forskolin. Mechanistically, depletion of PRKAR1A provoked PKA‐dependent phosphorylation of the mitochondrial fission protein Drp1 at S637, leading to suppression of mitochondrial fission and inhibition of cardiomyocyte hypertrophy.ConclusionsCombined with previous findings that mitochondrial fission is required for hypertrophic growth of cardiomyocytes, our results suggest that PRKAR1A deficiency impedes postnatal myocardial development and physiological hypertrophy through modulation of mitochondrial dynamics. These findings provide a potential novel mechanism for the cardiac manifestations associated with CNC.Support or Funding InformationThis study was supported by intramural program of National Institute of Child Health and Human Development (NICHD), National Institutes of Health, National Heart, Lung, and Blood Institute (NIH R00HL119605, R56HL145034); WSU College of Pharmacy and Pharmaceutical Sciences.
Characterization of the Developing Mouse Dopamine Neuron Phenotype
Authors: Barker Samantha , Partington Heath , Nutter Makenzie , Eells Jeffrey B. 
Published: April 2020

Previous research in adult mice has suggested that neurons are capable of changing their phenotype, long after neurogenesis has come to a halt. In the midbrain, the changing dopamine (DA) neuron phenotype may have implications in neurological disorders such as Parkinson’s disease, addiction, attention deficit hyperactivity disorder and schizophrenia. Using a transgenic mouse model with Cre recombinase expression under control of dopamine transporter (DAT) and yellow fluorescent protein (YFP) Cre reporter, we are able to differentiate between former and current DA neurons in the ventral midbrain. Current DA neurons are identified using tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis; whereas former DA neurons are YFP positive but TH negative suggesting that they expressed dopamine transporter at some point in their lifecycle but are not currently synthesizing dopamine. Using this model in adult transgenic mice, previous research in our lab has revealed that the DA neuron phenotype is regulated by changes in the environment. In this study, mice transgenic for DATCre/YFP were analyzed at different time points during development to better understand when the DA neuron phenotype becomes susceptible to changes in the environment. Preliminary data suggests that in newborn mice, approximately 95% of all dopamine neurons identified in the substantia nigra (SN) were classified as current DA neurons with very few YFP positive, TH negative neurons. In adult mice, however, approximately 41% of DA neurons in the SN were both YFP and TH positive suggesting that as mice age, DA neurons change their phenotype in response to the environment. Generating a developmental timeline will provide a new starting point for further understanding how the changing DA neuron phenotype is implicated in dopamine‐related disorders such as Parkinson’s disease.Support or Funding InformationBrody School of Medicine at East Carolina University
Targeting the O‐GlcNAc Transferase to Specific Proteins Using RNA Aptamers
Authors: Zhu Yi , Hart Gerald W. 
Published: April 2020

O‐linked β‐D‐N‐acetylglucosamine (O‐GlcNAc) is an abundant post‐translational modification (PTM) that modifies more than 4,000 nucleocytoplasmic proteins. This PTM regulates multiple protein functions including kinases activity, transcriptional activity and degradation. Abnormal O‐GlcNAcylation is involved in many human diseases, such as cancer, diabetes and neurodegenerative diseases. A major difficulty in the field of O‐GlcNAc research is our inability to alter O‐GlcNAcylation on a single protein at a time. Here, we propose a novel method to regulate O‐GlcNAcylation on an individual protein. We selected an RNA aptamer that binds O‐GlcNAc transferase (OGT) from systematic evolution of ligands by exponential enrichment (SELEX). With this aptamer, we designed series of recombined RNA aptamers that could target OGT to a substrate protein. Expression of these aptamers increased O‐GlcNAcylation on GFP‐β‐catenin without changing global O‐GlcNAcylation. Detailed studies revealed that both the distance and spatial alignment between OGT and the substrate affect the efficacy of these aptamers, thus they are critical considerations for aptamer design. With different aptamers, we were able to promote O‐GlcNAcylation on endogenous β‐catenin, and some other proteins selectively. Furthermore, we observed the interplay between O‐GlcNAcylation and phosphorylation on GFP‐β‐catenin at the degradation‐related sites. Elevated O‐GlcNAcylation inhibited polyubiquitination on GFP‐β‐catenin and stabilized the protein. On endogenous β‐catenin, O‐GlcNAcylation upregulated its transcriptional activity. Our research provides a novel tool to illustrate the regulatory role of O‐GlcNAc on protein functions, establishes a novel method to induce proximity between two cellular components, and sheds light on how O‐GlcNAc regulate the protein of β‐catenin.Support or Funding InformationSupported by NIH P01HL107153, & R01GM116891, the Georgia Research Alliance and the Univ. of Georgia. Dr. Hart receivesa share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.
Pogil as a tool for Biochemistry Instruction
Authors: Srinivasan Rekha , Saluan Quinn , Khanna Anushka , Patel Anjali 
Published: April 2020

A challenge that many students face when learning biochemistry, involves balancing concept learning and concept application in problem‐solving. Too often, students lean towards rote memorization and binge learning routes to learn biochemistry, without understanding the concepts behind them. In this project, we used POGIL inspired worksheets along with peer instruction to facilitate deep learning of biochemical principles. In a novel approach, these worksheets were created by a group of upper‐class former students of the course and the instructor of the course, to address concepts from the perspectives of learner and teacher. The worksheets were administered one out of every three lectures periods every week. Traditional lecturing was also carried out on remaining days to facilitate discussions and to clarify doubts in concepts. A voluntary attitude survey was conducted to observe the pedagogical usefulness of these newly created worksheets, and the results are presented herein.Support or Funding InformationNord Foundation, UCITE, Case Western Reserve University
Effect of bilateral lesions in the amygdala on the maximum running velocity and cardiovascular response in rats during treadmill running
Authors: Tsukioka Kei , Yamanaka Ko , Waki Hidefumi 
Published: April 2020

The high intensity of exercise around/at maximum levels induces psychological distress. It is known that the amygdala in the limbic system is one of the regions of the brain controlling such emotion, as well as regulating the cardiovascular system. Therefore, we hypothesized that the amygdala could play an important role in limiting the high‐intensity exercise performance. To test this, we examined whether bilateral amygdala lesions increased maximal exercise performance with altered cardiovascular responses during exercise. The arterial pressure in rats was chronically measured using radio‐telemetry during treadmill running. The rats were subjected to forced running for 60 min per day (velocity ≤20 m/min) for 3 days to achieve treadmill habituation. Thereafter, an incremental exercise test was conducted until exhaustion (pre‐test). Exercise intensity was initiated at 10 m/min, and the velocity was increased by 2 m/min every 3 min. In the amygdala lesion group, the bilateral lesions in the amygdala were induced by electrical stimulation (direct current 1 mA for 5 s). In the sham group, the tip of the electrode was inserted into the amygdala and immediately removed without electrical stimulation. After surgery, re‐training was conducted similar to the habituation protocol, and the incremental exercise test was repeated (post‐test). In the sham group, there were no significant differences between pre‐ and post‐test in the mean maximum running velocities (pre‐test: 47.2 ± 1.8 m/min, post‐test: 44.0 ± 2.8 m/min) and running time (pre‐test: 56.5 ± 3.2 min; post‐test: 49.9 ± 4.7 min). However, the mean maximum running velocity was greater (pre‐test: 45.5 ± 2.3 m/min; post‐test: 54.0 ± 0.6 m/min; p < 0.05) and the running time was longer (pre‐test: 57.9 ± 3.4 min; post‐test: 70.0 ± 0.8 min; p < 0.05) in the post‐test as compared to the pre‐test of the lesion group. Moreover, we found that the mean arterial pressure in both groups of animals exhibited a gradual increase during submaximal exercise (low/moderate intensity) followed by a drastic increase during high/maximal‐intensity exercise. However, compared to the sham group, the mean arterial pressure response in the amygdala lesion group was significantly lower during submaximal exercise, whereas it was greater during high/maximal‐intensity exercise, suggesting that the amygdala could play a role in limiting the exercise performance by modulating the autonomic cardiovascular control.Support or Funding InformationGrant‐in‐Aid for JSPS Fellows (19J22706)
Histamine‐mediated regulatory loop in mesenteric perilymphatic mast cells
Authors: Gashev Anatoliy A. , Gasheva Olga Yu. , Zawieja David C. , Meininger Cynthia J. , Pal Sarit 
Published: April 2020

Lymphatic vessels play a critical role in mounting a proper immune response by trafficking peripheral immune cells to draining lymph nodes. Mast cells (MCs) are well known for their roles in type I hypersensitivity reactions but little is known about their secretory regulation in the lymphatic niche. MCs, as innate sensor and effector cells, reside close to mesenteric lymphatic vessels (MLVs) and their activation and ability to release histamine influences the lymphatic microenvironment in a histamine‐NF‐kB‐dependent manner. Using an established experimental protocol involving surgical isolation of rat mesenteric tissue segments, including MLVs and surrounding perilymphatic tissues, we tested the hypothesis that perilymphatic mesenteric MCs possess histamine receptors (HRs) that bind and respond to histamine released from these same MCs. Under various experimental conditions, including inflammatory stimulation by LPS, we measured histamine in mesenteric perilymphatic tissues, evaluated expression of histidine decarboxylase in MCs along with the degree of MC degranulation, assessed the functional status of HRs in MCs, and evaluated the ability of histamine itself to induce MC activation. Finally, we evaluated the importance of MCs and HRs 1 and 2 for MLV‐directed trafficking of CD11b/c‐positive cells during acute tissue inflammation. Our data indicate the existence of a functionally potent MC‐histamine autocrine regulatory loop, the elements of which are crucially important for acute inflammation‐induced trafficking of the CD11b/c‐positive cells towards MLVs. Our previously published data suggest that MLVs‐released histamine can also trigger the MC‐histamine loop during development of inflammation. Therefore, this MC‐histamine loop serves as a first‐line cellular servo control system, playing a key role in the innate and adaptive immune response as well as NF‐κB‐mediated maintenance of body homeostasis.Support or Funding InformationNIH/NIA R56 AG061097‐01; NIH/NIDDK R01 DK110035; NIH/NHLBI 1U01HL123420; funds from TAMU HSC COM and Dept. of Medical Physiology.
GIPSITUMAB TM, A NOVEL NATURALLY OCCURRING BISPECIFIC ONCOLYTIC/ANTI‐INFECTIVE ANTIBODY IS ABSENT IN TWO AFRICAN‐AMERICAN SUBJECTS
Authors: Nair Padmanabhan Padmanabhan 
Published: April 2020

We have recently reported the discovery of an oncolytic IgA/IgG heterodimeric, bispecific immunoglobulin generated by exfoliated colonic cells isolated from stool samples of free living subjects (EB 2019, Orlando. Florida). We isolated these cells from stool samples (0.5 gm) of a cohort of 60 free‐living subjects using our SCSR technology and labelled them with fluorescently tagged antibodies to IgA/IgG and analyzed them by flow cytometry to detect the co‐expression of the chimeric GIPSITUMABTM. We found two subjects in this group without any evidence of this chimeric antibody. On unblinding the data‐set we found that these two subjects were of African‐American descent. Unlike the cells from a few other individuals in this group, these cells upon culturing in stem cell growth medium confirmed the absence of this antibody in their progeny. Our observations appear to show the existence of a syndrome in which this immunoglobulin is not expressed and may be indicative of a germ‐line deletion signalling the presence of an increased risk for developing a malignancy.Support or Funding InformationSupported in part by US National Institutes of Health, SBIR Grants, R44CA81799 and R44DK56567
Vascular Function in Continuous‐Flow Left Ventricular Assist Device Recipients: Effect of a 6‐Week Pulsatility Treatment Regimen.
Authors: Hydren Jay R. , Gifford Jayson R. , Jarrett Catherine L. , Park Soung Hun , Shields Katherine L. , Broxterman Ryan M. , Kithas Andrew C. , Bisconti Angela Valentina , Thurston Taylor S. , Ratchford Stephen M. , Wray D. Walter , Stehlik Josef , Selzman Craig H. , Drakos Stavros G. , Richardson Russell S. 
Published: April 2020

Vascular function is further diminished in chronic heart failure patients implanted with a continuous‐flow left ventricular assist device (LVAD), which contributes to vascular‐related adverse events. A single pulsatility treatment has been documented to recover vascular function, however, the impact of a chronic treatment regimen is unknown.MethodsEleven LVAD recipients and ten well‐matched controls, received unilateral, 45‐minute, arm pulsatility treatment, created by intermittent cuff inflation and deflation (2‐second duty cycle), distal to the elbow, three days a week for 6 weeks. Vascular function was assessed by percent brachial artery flow mediated dilation (%FMD) (Doppler ultrasound).ResultsAt baseline, %FMD was not different between LVAD recipients (4.2±0.3%) and controls (4.4±0.4 %), while LVAD recipients exhibited an attenuated %FMD/Shear (LVAD: 0.09±0.01; Controls: 0.14±0.01 %/s−1, p<0.05) and a longer time‐to‐peak dilation (LVAD: 85±6; Controls: 51±5 s, p<0.05). Pulsatility index (PI) was lower in the LVAD recipients compared to controls (LVAD: 2±1; Controls: 13±2 AU, p<0.05). Importantly, during the pulsatility treatment, the PI level was not different between groups (LVAD: 28±5; Controls: 34±5 AU). Although time‐to‐peak dilation was not altered by 6‐weeks of pulsatility treatment, %FMD (LVAD: 7.9±0.5; Controls: 7.3±0.7 %) and %FMD/Shear (LVAD: 0.16±0.02; Controls: 0.26±0.03 %/s−1) improved significantly. Notably, %FMD/Shear in the LVAD recipients recovered to the baseline level of controls. All measures returned to baseline levels after a 6‐week washout period.ConclusionsA 6‐week pulsatility treatment regimen restored vascular function in LVAD recipients, demonstrating the efficacy of a pulsatility targeted intervention to potentially mitigate vascular related adverse events in this population.Support or Funding InformationCOI: Dr. Stavros G. Drakos serves on the Steering Committee of the Abbott‐sponsored INTELLECT‐2 LVAD. Funding: This research was supported by the National Institutes of Health (1T322HL13945, R01HL118313) and the U.S. Veteran’s Administration (E6910‐R, E1697‐R, E1433‐P, E9275‐L, E1572‐P, and I01RX001311).
Analysis of A. thaliana Hsp40s further supports prion‐specific domain‐based functions and primacy of the glycine‐rich region in [RNQ+]/[PIN+] propagation
Authors: Hayward Rachel E. , Cole Sierra J. , Singh Atul K. 
Published: April 2020

Amyloid‐based prions of the baker’s yeast, Saccharomyces cerevisiae rely upon fragmentation by the chaperone machinery for propagation in cell populations. In addition to Hsp104 and Hsp70, the Hsp40 Sis1 is specifically essential for prion propagation, yet different prions rely upon distinct parts of Sis1. We recently identified six functional orthologs of Sis1 in the model plant Arabidopsis thaliana which rescue both cell viability and strong [PSI+] propagation in a Δsis1 strain, however these orthologs vary in their ability to maintain [RNQ+]/[PIN+] and weak variants of [PSI+]. Despite high sequence homology among the six A. thaliana paralogs, significant differences in the glycine‐rich regions exist. This region of Sis1 is universally required for the propagation of all known variants of [RNQ+], though its role in weak [PSI+] propagation in less clear. Here we report the results of a domain‐based analysis utilizing chimeric forms of two orthologs and validated in two distinct yeast strain backgrounds. Our data indicate that the glycine‐rich region specifies the propagation of [RNQ+] but not weak [PSI+], as no change in weak [PSI+] propagation was observed.Support or Funding InformationThis work was supported by the Lafayette College Chemistry Department, the EXCEL research scholarship program (
www.lafayette.edu), the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606, and the Henry Dreyfus Teacher Scholar Award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Dreyfus Foundation.
Rapamycin and chloroquine insensitivity reveal mTORC1‐independent regulation of epithelial permeability in differentiated intestinal Caco‐2 cells
Authors: Kaur Harleen , Moreau Regis 
Published: April 2020

Intestinal barrier integrity dictates the intestinal homeostasis by stringently regulating the permeability of the luminal contents into the intestinal tissue of the human body. The protective role of a well‐formed epithelial barrier is substantiated by incidences of increased mucosal inflammation upon epithelial barrier disruption. The pathophysiology of many serious intestinal disorders such as inflammatory bowel disease, necrotizing enterocolitis, and celiac disease is associated with increased permeability of the intestinal wall to luminal contents. In order to identify new therapeutic strategies against these disorders, it is critical to better understand the underpinning molecular mechanisms regulating epithelial barrier permeability. The role of the mTORC1 (mechanistic target of rapamycin complex 1) signaling in the regulation of intestinal barrier function and permeability remains to be fully elucidated. To that end, we genetically manipulated human epithelial intestinal Caco‐2 cells to generate three stable cell lines with baseline (shScramble), low (shRaptor) or high (shTSC2) mTORC1 activity. The three cell lines maintained their intended mTORC1 activity upon differentiation on Transwell® inserts, i.e., shRaptor cells exhibited 50% lower mTORC1 activity vs. shScramble cells, while shTSC2 cells exhibited 80% higher mTORC1 activity vs. shScramble cells. Transepithelial electrical resistance (TEER), a measure of epithelial permeability was inversely proportional to mTORC1 activity (r2= 0.98, P<0.05). shRaptor cells had higher expression of p‐(Thr172)AMPK (+46%, P<0.05), lower abundance of p‐(Ser757)ULK‐1 (−50%, P>0.05), higher autophagic flux (+180%, P<0.05), and lower expression of pore‐forming tight junction protein Claudin‐2 (−65%, P<0.05) compared with shScramble cells. In contrast, shTSC2 cells had higher levels of p‐(Ser757)ULK‐1 (+100%, P<0.05) and Claudin‐2 (+250%, P<0.05) compared with shScramble cells. We treated shScramble cells with rapamycin in an attempt to pharmacologically reproduce the effects of Raptor knockdown on epithelial permeability and found that rapamycin modestly but statistically increased TEER (+34%, P<0.05) and decreased Claudin2 (−36%, P<0.05). The role of autophagy in rapamycin‐mediated increase of TEER was dismissed on the basis that chloroquine (inhibitor of autophagosome‐lysosome fusion) did not significantly reduce TEER in shScramble cells. Our results suggest that although mTORC1 regulates epithelial permeability during the early stages of Caco‐2 cell differentiation, TEER is not substantially affected by rapamycin and chloroquine in fully differentiated cells.Support or Funding InformationSupported by USDA‐NIFA