bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Bioluminescent Tools to Study GPCR Pharmacology in Living Cells
Authors: Edenson Steven
Published: April 2020

G‐protein coupled receptors (GPCRs) continue to be prominent targets for new therapeutics. To support these efforts, we have developed a suite of bioluminescent assays to study GPCR pharmacology in real time in living cells. Quantification of ligand binding and receptor internalization relies on N‐terminal fusion to High BiT (HiBiT), an 11 a.a. peptide. HiBiT binds with high affinity to Large BiT (LgBiT) to form NanoBiT Luciferase. For both applications, a non‐lytic detection reagent is added containing cell‐impermeable LgBiT and furimazine substrate. Ligand binding dynamics at the cell surface can be measured in real time through bioluminescent resonance energy transfer (BRET) between NanoBiT Luciferase and a fluorescent tracer. Receptor internalization and recycling are monitored in real time by measuring changes in the cell surface density of HiBiT‐tagged GPCRs. Notably, the bright signal of NanoBiT Luciferase allows both assays to be run at endogenous levels of expression using CRISPR/Cas9 to introduce the HiBiT tag. Similarly, fusion proteins to LgBiT and Small BiT (SmBiT), an 11 a.a. peptide with low affinity binding to LgBiT, can be used to measure β‐arrestin‐1/2 recruitment to activated GPCRs in real time. Furthermore, changes in intracellular cAMP concentration can be sensitively measured using endpoint (cAMP‐Glo) or real time formats (GloSensor cAMP) using thermal stable or circularly permuted forms of luciferase, respectively. These homogenous, cell‐based assays, which are readily adaptable to laboratory automation, should be immensely useful in the study of GPCR pharmacology.

Support or Funding Information

Promega Corporation
How SFR2 is regulated to confer freezing tolerance of the chloroplast envelope membrane
Authors: Roston Rebecca L. , Barnes Allison C. , Shomo Zachary , Surber Samantha , Myers Jennifer
Published: April 2020

Internal cellular membranes must have their lipid composition remodeled for plants to survive low temperatures. One mechanism necessary for freezing tolerance of the chloroplast envelope membranes is well defined. An enzyme named “Sensitive to Freezing 2” (SFR2) changes monogalactolipid into oligogalactolipids at temperatures below freezing. Interestingly, SFR2 activity does not respond to initial cool temperatures, it only responds to barely tolerable freezing temperatures. Here, we show that SFR2 is post‐translationally regulated by modifications and changes to cytosolic acidification. We show that freezing increases cytosolic acidification and that proton pumps at both the plasma and vacuolar membranes participate in maintaining the acidification during low temperatures. Finally, quantitative measurements of SFR2’s product in a large number of plant species with diverse phylogenetic backgrounds shows that SFR2 is likely responding to membrane damage in some, if not all species. We conclude that plant low temperature sensing and response is likely a continuum rather than a switch, and that internal cellular membranes have systems set up to respond to damage in a diverse set of abiotic stresses.
Semaphorin 3F: A Novel Biologic Treatment for Oral Squamous Cell Carcinoma
Authors: Almazyad Asma , Gao Yao , Shahrabi-Farahani Shokoufeh , Sui Lufei , Gartung Allison , Watnick Randy , Panigrahy Dipak , Adam Rosalyn M. , Bielenberg Diane R.
Published: April 2020

Oral squamous cell carcinoma (OSCC) accounts for the majority of all oral malignancies. The overall survival rate for patients diagnosed with advanced‐stage OSCC (with metastasis) is only 39%, and the prognosis for these patients has not improved dramatically despite advances in chemotherapeutic, radiotherapeutic, and surgical protocols. As such, there remains an urgent need to develop new therapies for OSCC to improve outcome. Recently, our laboratory identified the Neuropilin‐2 (NRP2) transmembrane receptor as a novel target in OSCC and its associated vasculature in human patient biopsies, human OSCC xenografts, and mouse carcinogenesis models of OSCC. NRP2 is an ideal drug target as it is not expressed in the oral epithelium nor the normal vasculature after birth, hence systemic toxicities are not expected. However, NRP2 is dramatically upregulated on the cell surface in OSCC and its associated tumor endothelium. In embryonic endothelium, NRP2 acts as a co‐receptor with VEGFR2 for the angiogenic factor VEGF‐A and as a co‐receptor with VEGFR3 for the lymphangiogenic factors VEGF‐C/D to regulate the developmental processes of angiogenesis and lymphangiogenesis, respectively. Alternately, NRP2 can bind Semaphorin‐3F (SEMA3F), a secreted chemorepulsive and inhibitory protein, which mediates cellular guidance and vascular patterning. SEMA3F is an inhibitor of the VEGFA/C/D signaling axes as it competes with binding to NRP2 as well as directly signals through the NRP2/PlexinA1 complex to collapse the cellular cytoskeleton and inhibit motility. Our hypothesis is that SEMA3F may potently inhibit OSCC progression by targeting NRP2 in tumor cells and neovessels in the tumor microenvironment. Our in vitro results demonstrate that SEMA3F exogenous protein inhibits the migration, invasion, and proliferation of Nrp2‐expressing mouse OSCC cells isolated from 4NQO‐induced tumors. In vivo, systemic SEMA3F protein delivered continuously via slow‐release mini‐osmotic pumps induces regression, as confirmed by bioluminescence imaging and histologic examination, in established syngeneic luciferase‐labeled OSCC implanted in the tongue of C57BL/6 mice. In human OSCC xenografts implanted orthotopically in immunodeficient mice, systemic SEMA3F treatment inhibits tumorigenicity and reduces tumor‐associated lymphangiogenesis compared to control (vehicle) treatment. Taken together, these preclinical data suggest that SEMA3F may be a powerful anti‐angiogenic and anti‐tumor therapy specifically targeting the NRP2 axis in carcinoma cells and stromal cells in the tumor microenvironment.

Support or Funding Information

None
Getting to the (Turning) Point: A Systematic Interactive Approach for Teaching Identification of Cross‐Sectional Cadaveric Structures
Authors: Nist Kayla M. , Afifi Linda M. , Zumwalt Ann C.
Published: April 2020

Introduction

The ability to interpret cross‐sectional anatomy in radiological scans is a necessary diagnostic skill for clinicians. The skills to spatially translate three‐dimensional (3‐D) anatomy into a two‐dimensional (2‐D) plane of section is challenging for beginner anatomy students. However, the capability to build these skills is critical for their medical and research training.

Aim

The following lesson was employed with the objective to aid first year medical and graduate students at Boston University School of Medicine in applying their understanding of 3‐D anatomy to interpret 2‐D transverse sections of the thorax, abdomen, and pelvis of human cadavers. Turning Technologies “Turning Point” application was used to measure students’ change in understanding of the material before and after.

Methods

The lesson began with an assessment of cross‐sectional knowledge using the Turning Point application on students’ phones. The students then participated in a lecture designed to systematically guide them through a general screening of osteological landmarks, muscles, blood vessels, organs, and other visceral structures at each vertebral level on cross‐sectional images of the thorax, abdomen, and pelvis. Students were guided on how to recognize the general topography and obviously identifiable organs in each section. They were then taught more specifically how to identify increasingly challenging anatomical features, such as organ‐specific structures, based on their previous 3‐D and 2‐D knowledge of the larger structures in the section. This lecture portion of the lesson gave students a system for how to apply and interpret 3‐D anatomy and transpose it into a 2‐D cross‐section. After the lecture portion of the lesson, students were asked to answer a series of practical‐style questions using the Turning Point app. Question types included multiple choice, short answer, and “hot
spot” style.

Results

In total, 42 students participated in the lesson. The overall class pre‐test average was 60.31 ± 26.79% while the post‐test average was 72.40 ± 13.28%. A comparison of individual pre‐test vs. post‐test scores using Wilcoxon Signed‐Ranks test revealed a significant improvement in student’s performance (p=0.0098, two‐tailed). Average scores of questions in each anatomical region were assessed (thorax: 67.21 ± 32.46%, abdomen: 71.71 ± 18.82%, pelvis: 70.76 ± 34.43%).

Conclusion

Based on student performance and feedback, this teaching method is an effective tool for improving students’ confidence and knowledge in interpretation of cross‐sectional anatomy. By using a systematic teaching approach with robust identification markers, Turning Point was an effective addition to help build students understanding and confidence in the material.

Support or Funding Information

No funding was received
NHE1 activity is required for profibrotic agonist activation of cytoskeleton remodeling in lung fibrosis
Authors: Mishika Manchanda , Nguyentu Trina T. , Provost Joseph John
Published: April 2020

Idiopathic pulmonary fibrosis (IPF) is an untreatable chronic lung disorder that is characterized by an accumulation of fibroblasts and scarring tissue due to aberrant tissue remodeling. The lung tissue loses flexibility as the disease upsurges and the affected patient’s alveolar architecture is destroyed. This leads to a disruption of gas exchange, eventual respiratory failure, and death. Sodium hydrogen exchanger isoform 1 (NHE1) is a membrane bound protein that moderates cellular pH, supports cell migration, and regulates cell behavior. NHE1 has also been found to impact the pathophysiology of cancer. Considering that a myriad of signaling pathways that activate tumor cells are also involved in profibrotic activity of fibroblasts and myofibroblasts, the purpose of this study was to investigate if NHE1 exacerbates IPF and if inhibiting NHE1 would decrease the advancing fibrosis. We measured one of the indicators of progressing fibrosis, the remodeling of skeletal proteins, through the formation of actin stress fibers. Actin stress fibers have an important role in cell migration and contractility in fibroblasts. To stimulate this formation, cells were treated with three agonists that increase fibrotic activity: transforming growth factor ‐ β(TGF‐β), serotonin (5HT), and lysophosphatidic acid (LPA) in the presence and absence of ethyl‐isopropyl amiloride (EIPA). LPA induced stress fibers 43.94+/−4.4 percent over the control, and both TGF‐βand 5HT also stimulated the formation of stress fibers by 32.08+/−2.1 and 39.1+/−4.2 percent over the control, non‐treated cells. In each case, addition of EIPA blocked agonist‐induced stress fiber formation, indicating that all three profibrotic factors require NHE1 activity for cytoskeletal remodeling. Interestingly, we also observed that stimulation in any combination of agonists had seemed to block stress fiber formation in comparison to the control. An initial interpretation indicates that the agonists negatively regulate the others in stimulating stress fiber formation. The role of each agonist in fibroblasts isolated from fibrotic patients and control, non‐diseased tissue are presented. Myofibroblasts are a specialized cell type that contribute to fibrosis. The role of NHE1 in TGF‐βinduced fibroblast‐myofibroblast differentiation is also determined. The marker for myofibroblast differentiation, α‐smooth muscle actin (α‐SMA) was measured with and without EIPA to determine the role of NHE1 in advancing disease. Finally, the role of NHE1 in myofibroblasts was investigated with EIPA and stress fiber changes with agonists. This work highlights for the first time that NHE1 has a role supporting profibrotic factors and may be a novel target or adjuvant to fight IPF.
Structural Basis of 7‐Deazaguanine Modification of DNA in Bacteria and Phage
Authors: Hellie Eric , Alkuzweny Sana , Bayooz Susan , Phan Phuc , Savage Joshua , Luthra Amit , Iwata-Reuyl Dirk , Swairjo Manal
Published: April 2020

Two important modifications of tRNA, the 7‐deazaguanine nucleosides queuosine (Q) and archaeosine (G+), are biosynthesized from GTP in bacteria and archaea, respectively, in a well characterized multi‐enzyme pathway leading to the shared advanced intermediate, 7‐cyano‐7‐deazaguanine (preQ0). In bacteria, preQ0 is converted to an aminomethyl derivative that is then inserted in tRNA by the bacterial tRNA‐guanine transglycosylase (TGT) enzyme. In archaea, preQ0 is inserted directly in tRNA by the archaeal TGT before conversion to G+. Recently, in 230 bacterial and phage genomes, a genomic island that contains a paralog of TGT was identified, which led to the discovery of Q, G+ and preQ0 deoxy derivatives in the DNA of some of these organisms. This gene cluster was renamed DpdA‐K, for “7‐deazapurine in DNA.” The G+‐modified DNA of E. coli bacteriophage 9g has been shown to resist restriction by >140 Type II restriction endonucleases (REases), consistent with a role of the modification as a defense mechanism. Recent experiments have shown that DpdA is a DNA‐guanine transglycosylase that catalyzes the insertion of preQ0 at a specific palindromic sequence in DNA. Here we report overexpression, purification, and crystallographic analysis of S. Montevideo DpdA (SmDpdA, ~47 kDa). The crystal structure, determined at 2.25‐Å resolution by multi‐wavelength anomalous diffraction methods, reveals a TIM‐barrel enzyme similar to the bacterial TGT in overall fold, active site and structural Zn2+ site. However, a large DpdA‐specific insertion in the TIM barrel provides an extended positively charged 26‐Å wide surface groove, consistent with a DNA binding surface. Free docking of a DNA duplex using the HADDOCK server places DNA to this surface in a fashion reminiscent of DNA binding to the ubiquitous Zn2+‐dependent DNA repair enzyme apurinic/apyrimidinic endonuclease IV. The structure and docking model suggest that DpdA bends its substrate DNA by 60° and flips the modification‐site guanine base out of the helix for transglycosylation. The results also inform the design of a substrate DNA duplex for future crystallization of the nucleoprotein complex.

Support or Funding Information

NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).
PERINEURAL IMPLANTATION OF STEM CELLS FOR CORRECTION OF PATHOLOGICAL PROCESSES IN HEART AND LIVER
Authors: Zamaro Aleksandra , Kulchitsky Vladimir
Published: April 2020

Pathological processes in cardiovascular system and liver are fatal for citizens of all over the world. New preventive and therapeutic approaches are needed in this field of medicine. Using of cell technologies for the treatment of patients with heart attack and/or cirrhosis is the one. Systemic or intraorganic stem cells (SCs) administration has shown poor therapy results. New technology was substantiated in our experiments on 27 male Wistar rats weighing 240–270 g. Vagus was cut off in 9 animals after cervical midsection on the left side under ketamine‐xylazine‐acepromazine (55.6, 5.5, and 1.1 mg/kg, respectively) anesthesia (I) in order to initiate destructive processes in heart. 9 rats were subjected to laparotomy followed by 2×2 mm injury of liver right lobe (II). Both laparotomy and cervical midsection but without liver and vagus damage was performed in the last 9 rats. All the rats received 10 thousands SCs in 10 μl culture medium into perineural space of vagus on the right side of the neck during anesthesia. SCs were marked with monoclonal antibodies to CD90 and PKH67 Green Fluorescent Cell Linker. Two animals from each group were decapitated in one day, one week and two weeks and slices from heart and liver 8 μm in thickness were prepared. SCs distribution was visualized using Zeiss AxioVert 200M fluorescence microscope with Zeiss AxioCam HRm CCD camera. Fluorescent cells were revealed in heart and liver areas in groups I and II, respectively. Zero fluorescent cells have been found in heart and liver of group III. Therefore, perineural way of SCs administration is an alternate path of SCs delivery to damaged inner organs only.
ANTITUMOR EFFECTS OF MESENCHYMAL STEM CELLS
Authors: Kulchitsky Vladimir , Zamaro Aleksandra
Published: April 2020

The problem of treatment of oncologic patients is not resolved. Our experiments focused on the ability of autologous mesenchymal stem cells (MSCs) to inhibit glioma cell growth in vitro and in vivo. 28 thousand cells of C6 glioma in 20 μl of culture medium were stereotactically introduced into occipital region of brain (Oc2L) from the right side after craniotomy in experiments on 30 male Wistar rats weighing 250–270 g under ketamine‐xylazine‐acepromazine (55.6, 5.5, and 1.1 mg/kg, respectively) anesthesia. 20 rats received 400 thousand MSCs labeled with CD90s and FITC in 50 μl of culture medium under nasal mucosa during the operation. Brain slices 8 μm in thickness were prepared after decapitation of one rat from each group in one day after surgery. MSCs distribution was visualized using Zeiss AxioVert 200M fluorescence microscope with Zeiss AxioCam HRm CCD camera. Fluorescent MSCs were revealed only in damaged Oc2L area of the rat with previously injected MSCs. 10 rats from the same group (with preimplanted MSCs) received 400 thousand MSCs in 50 μl of culture medium via weekly spray injections into both nasal cavities. Rats with implanted glioma and without MSCs died in 15.0±4.3 days. Rats with implanted glioma and single intranasal MSCs injection died in 21.1±5.2 days. 8 of 10 rats that received both intranasal MSCs injection and subsequent weekly spray MSCs injections died in 33.6±3.4 days. Two rats are still alive (108 days from the date of operation). Morphological examination of brain slices of rat from the last group showed signs of apoptosis of tumor cells. It was assumed that MSCs trigger apoptosis in C6 glioma cells via paracrine way by excretion of exosomes and cytokines.
Role of Phosphorylation and Palmitoylation Post‐Translational Modification of NHE1 on Fibroblast Motility
Authors: Armand Aryna , Foster James D. , Provost Joseph John
Published: April 2020

The sodium hydrogen exchanger protein isoform 1 (NHE1) is a trans‐membrane protein expressed in nearly all cells and plays an essential role in regulating intracellular pH as well as cell motility. In actively motile cells, NHE1 localizes to the leading edge of migrating cells and regulates directional cell movement. These mechanisms include the formation of intra‐ and extracellular pH‐gradients through Na+/H+ ion exchange, and likely also direct protein‐protein interactions and local cell volume changes. The purpose of this study was to understand the impact of palmitoylation on the NHE1 driven migration of lung cells. Palmitoylation of NHE1 involves the reversible covalent attachment of a palmitic acid to an amino acid found in NHE (cysteine): proteins are modified by palmitate via enzyme palmitoyl acyl‐transferases (PATs). To study the impact of palmitoylation on NHE1 and cell motility, an inhibitor of palmitoylation known as 2‐bromo‐palmitate (2BP) was incubated during a wound‐healing cell migration assay. Treatment with 2‐BP resulted in a significant and dose‐responsive reduction in cell motility, indicating a positive correlation between palmitoylation and NHE1 driven migration. Previous research indicates that phosphorylation also plays an important role in NHE1 activity and potentially palmitoylation. Therefore we investigated the impact of inhibition of both posttranslational modifications on cell motility. Specifically, we focused on agonists and stimulation factors which lead to increases in both NHE1 phosphorylation and palmitoylation: LPA, insulin, serum and PMA. The interplay between kinases and PAT regulation of NHE1 will provide a novel and underappreciated level of regulation of the transporter that may be a model for other transport proteins.
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

Aims

Protein 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 Results

To 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.

Conclusions

Combined 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 Information

This 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.
Student Performance on Pharmacology Topics After Integrating Pre‐recorded Video Lectures as an Additional Resource
Authors: Kolodziejczyk Tesia , Presley Colby , Maguire Keaton , Waller Jacquelyn , Linger Rachel
Published: April 2020

During the 2018–2019 academic year, OMS‐II students at Rocky Vista University were introduced to pre‐recorded video lectures (PRVL) as a modality of learning and preparing for pharmacology clinical integration sessions. Traditionally, our university uses designated student assignments (DSA), traditional lectures, and clinical integration sessions (CIS) as methods of delivering pharmacology material. The purpose of adding PRVL was to introduce or “prime” students for future lectures and clinical integration sessions. In contrast to a flipped classroom, the PRVL simply highlight important concepts for lecture or introduce difficult subjects. They are meant to augment the other learning modalities, not to replace traditional lecture, which is typically seen in a true flipped classroom. We focused our research on assessing the efficacy of PRVL as measured by performance on exam questions. We hypothesized that access to PRVL would improve performance on pharmacology topics compared to previous classes at Rocky Vista University who did not have this additional resource. To determine whether students utilized the pre‐recorded video lectures, we administered a survey to all OMS II students, focusing on identifying which resources students used most often to learn pharmacology. Survey responses and student performance data were linked using participant numbers that were deidentified. Out of 271 total students, 97 students (a 35.8% response rate) responded to this voluntary survey. We found that 85% of survey respondents utilized the pre‐recorded video lectures >60% of the time. Comparing student performance on 30 pharmacology test items that assessed topics presented in PRVL, students who reported using PRVL <60% of the time scored slightly higher than students who used PRVL the majority of the time. In addition, 97% of students found PRVL to be a useful resource, but when asked whether it was the most useful resource of all modalities available, it was the least favored by students with only 8.2% of respondents preferring PRVL. These results are limited by small sample size, particularly in the group of students who used PRVL < 60% of the time (N=14). We hope to expand these findings by comparing cumulative performance on pharmacology test items before and after PRVL implementation.
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 Information

Brody 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 Information

Supported 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 Information

Nord 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 Information

Grant‐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 Information

NIH/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 Information

Supported 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.

Methods

Eleven 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).

Results

At 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.

Conclusions

A 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 Information

COI: 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 Information

This 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 Information

Supported by USDA‐NIFA
Neural control of respiratory musculature in the mdx mouse model of Duchenne muscular dystrophy
Authors: Burns David P. , O’Driscoll Eabha , Lucking Eric F. , O’Connor Karen M. , Clarke Gerard , Walter Glenn A. , Edge Deirdre , Fuller David D. , O’Halloran Ken D.
Published: April 2020

Duchenne muscular dystrophy (DMD) is an inherited fatal neuromuscular disease. Despite deficits in respiratory muscle performance being well recognised, there is a paucity of knowledge in respect of the neural control of breathing in dystrophinopathies. We sought to examine neural control of the diaphragm and upper airway musculature in dystrophin deficient mdx mice.

Wild‐type (WT) and mdx mice were studied to examine diaphragm and genioglossus (GG) EMG activity using fine concentric needle electrodes in urethane (1.7mg/kg) anaesthetized spontaneously breathing mice. Basal EMG and responsiveness to chemostimulation were determined. In separate studies, phrenic and hypoglossal (XII) motor output were assessed in anaesthetized, vagotomized, paralyzed and mechanically ventilated WT and mdx mice.

Diaphragm and upper airway (sternohyoid) muscle form and function were assessed. Monoamine concentrations, density of glial cells, and pro‐inflammatory gene expression were determined in the spinal cord (C3–C5) and brainstem. Diaphragm and GG EMG activities were depressed in mdx compared to WT during baseline and hypoxic‐hypercapnic challenge. Phrenic and XII nerve recordings revealed enhanced neural drive to diaphragm and GG in mdx mice during hypoxic‐hypercapnic challenge. Excitatory monoamine concentrations were elevated in mdx spinal cord (noradrenaline and serotonin) and brainstem (serotonin). Putative phrenic and hypoglossal motor nuclei in mdx mice had unaltered expression of microglia and astrocytes compared to WT. NFkB and GFAP mRNA expression were increased in mdx brainstem compared to WT, while GFAP mRNA was reduced in mdx spinal cord.

Our study revealed a potentiated neural drive in phrenic and XII motor pathways in mdx mice during chemoactivation, suggesting plasticity in motor pathways facilitating breathing. Depressed EMG activity despite potentiated neural outputs, suggests compromised neuromuscular transmission, which we have also observed in respiratory EMG recordings during protracted airway obstruction (Burns et al., 2019, J Physiol). Studies of the fundamental control of breathing in models of DMD should provide a comprehensive vista of the disease, with the potential to identify novel therapeutic targets.