bluuebunny/crossref_metadata_2025 huggingface dataset:
Protracted Effects of Ketamine Require Immediate Kappa Opioid Receptor Activation and Long‐Lasting Desensitization
Authors: Jacobson Moriah L. , Simmons Sarah C. , Wulf Hildegard A. , Cheng Huaiyan , Feng Ying-Hong , Nugent Fereshteh S. , Browne Caroline A. , Lucki Irwin
Published: April 2020

Ketamine produces rapid and sustained antidepressant activity, but its mechanism of action remains uncertain. Williams & colleagues (2018) reported that naltrexone pretreatment attenuated ketamine’s effects in treatment resistant depressed patients. Naltrexone antagonizes both kappa (KOR) and mu opioid receptors (MORs). We hypothesized that ketamine requires acute KOR activation, leading to long‐lasting suppression of KOR signaling to exert its antidepressant effects. The following series of experiments demonstrated: 1) exposure to ketamine produces internalization of KORs; 2) the behavioral effects of ketamine in mice were prevented by prior KOR blockade; and 3) ketamine pretreatment prevented behavioral and physiological responses caused by subsequent KOR activation.

KOR internalization was tested in HEK293 cells transfected with yPET‐OPRK1 plasmids following ketamine stimulation (2 μM). Approximately 30% of KORs were internalized, which was comparable to the internalization achieved with the endogenous KOR agonist dynorphin (40 μM). To demonstrate that activation of KORs by ketamine contributes to its protracted behavioral profile, C57BL/6J mice (8–12 weeks) were pretreated with naltrexone (1 mg/kg) or the selective KOR antagonist LY2444296 (3 mg/kg) 30 minutes prior to ketamine (10 mg/kg) and were tested in the forced swim test 24 h later. Reductions in immobility scores by ketamine were blocked by naltrexone and LY2444296 pretreatment, confirming the importance of KORs in initiating ketamine’s long‐lasting effects.

Next, the physiological impact of ketamine on KOR activity was examined ex vivo in the lateral habenula (LHb), a reward‐related brain region. Bath application of the KOR agonist U50,488 (10 μM) robustly increased the number of action potentials in response to depolarization. The increase in LHb neuronal excitability was absent when ketamine (10 mg/kg) was administered to mice 24 h previously. In parallel, U50,488 (10 or 20 mg/kg) induced behavioral deficits on nest building, prepulse inhibition and antinociception on the hot plate were effectively prevented by ketamine (10 or 20 mg/kg) given 24 h earlier. Finally, LY2444296 (3 mg/kg) administered 30 min prior to ketamine (10 mg/kg) prevented ketamine‐induced suppression of U50,488‐induced alterations on nesting behavior 24 h later. This established that KOR occupation prior to ketamine prevented the initiation of ketamine’s long‐lasting behavioral effects.

Collectively, these studies provide evidence supporting the hypothesis that activation of KORs by acute ketamine administration leads to enduring reductions of KOR signaling, which may contribute to the protracted clinical antidepressant effects of ketamine.

Support or Funding Information

This work was funded by US Public Health Service (USPHS) grant R01 MH105623.
The General Relationships of Giraffidae
Authors: Abdel-Karim Sarah , Monkash Nina , Dao Brandon , Solounias Nikos
Published: April 2020

Giraffidae is a small family of ruminants with approximately 53 species, all of which are extinct except for four extant giraffe species and the okapi. The Giraffa species are morphologically very similar. During the Miocene (18–6 million years ago), various diverse species of Giraffidae were found in numerous faunas in Eurasia and in Africa. Giraffidae are united by a set of major characteristics, including: (1) large body size (the smallest were the size of an okapi), (2) long and large metacarpals and metatarsals (when compared to antelopes and deer), (3) ossicones attached to the frontal bones and (4) presence of bi‐lobed lower canines. The family appears about 18 million years ago with the earliest fossils sampled in the Siwaliks of Pakistan, in Libya’s Gebel Zelten and in Kenya’s Moruorot Hill. From ancestral species of Gelocidae (Oligocene taxa), adaptive radiation resulted in numerous families, including Giraffidae, Palaeomerycidae and Climacoceridae (early to Middle Miocene 18–10 Ma). The basal Giraffidae (18–16 Ma) possess laterally directed ossicones, long necks and large elongated metapodials. One way to subdivide the family is into genera possessing simple premolars or complex premolars. Four groups of higher Giraffidae emerge separately from these basal taxa. The first branch, as represented by Sivatherium, is characterized by the presence of frontal sinuses, four ossicones, short necks and short metapodials. The second branch, as represented by Samotherium, is characterized by the lack of frontal sinuses, and the presence of two long slender ossicones, medium metapodials and longer necks. The third branch, as represented by Bohlinia, is characterized by the lack of a frontal sinus, and the presence of two short robust ossicones, long metapodials and a long neck. The fourth branch, as represented by Giraffa, is characterized by the presence of a frontal sinus, two robust and short ossicones, long metapodials and a long neck. The establishment of these four groups allows us to properly identify and classify a plethora of fossils and aid in the future classification of ruminants.
Abrogation of Ozone‐induced Oxidative Stress, Inflammation, and Aberrant Pulmonary Mechanics by Valproic Acid
Authors: Lee Jordan , Sunil Vasanthi R. , Vayas Kinal , Abramova Elena , Cervelli Jessica , Laskin Jeffrey D. , Laskin Debra L.
Published: April 2020

Acute exposure to inhaled ozone causes oxidative stress, inflammation, and impaired pulmonary mechanics. This is associated with an accumulation of inflammatory macrophages in the lung that contribute to toxicity by releasing reactive intermediates and pro‐inflammatory mediators. In the present studies we assessed the effects of valproic acid (VPA), a histone deacetylase inhibitor with antioxidant and anti‐inflammatory activity, on ozone‐induced pulmonary toxicity. Female C57B16/J mice (18–22 g) were exposed to air or ozone (0.8 ppm, 3 h) in whole body chambers. This was followed 30 min and 24 h later by i.p. administration of PBS control or VPA (300 mg/kg). Mice were euthanized 48 h later and bronchoalveolar lavage (BAL) and tissue collected. Ozone‐exposure resulted in increased levels of protein, IgM and cells in BAL, indicative of lung injury and inflammation. Ozone also caused oxidative stress as measured by increases in lung hemeoxygenase (HO)‐1 and 4‐hydroxynonenal (4HNE) modified proteins. Treatment of mice with VPA significantly reduced ozone‐induced increases in expression of HO‐1 and 4HNE modified proteins. Flow cytometric analysis of BAL lung cells showed that ozone‐induced injury and oxidative stress were associated with increases in pro‐inflammatory macrophages in the lung. These cells expressed ARL11 and TNFα, demonstrating that they are activated. VPA treatment reduced the number of activated macrophages accumulating in the lung in response to ozone; VPA also suppressed the accumulation of monocytic and granulocytic myeloid‐derived suppressor cells (MDSC). Ozone‐exposure caused alterations in pulmonary function, including increases in resistance and decreases in tissue elastance; these changes were blunted by VPA. Taken together, our data demonstrate that VPA is effective in reducing ozone‐induced inflammation, oxidative stress, and mechanical dysfunction. These findings may be useful in the development of therapeutics to treat oxidant induced lung injury.

Support or Funding Information

Supported by Rutgers SURF/ASPET, NIH ES004738, AR055073, and ES005022.
Effects of Different Cerium Oxide Nanoparticle Formulations on Oxidative Stress Survival in the Nematode C. elegans
Authors: Farber-Krug Acacia E. , Estevez Ana Yesenia
Published: April 2020

Cerium oxide (CeO2) nanoparticles (CeNPs or nanoceria) are potent antioxidants that are being explored as a potential therapeutic for diseases in which oxidative stress plays an important pathological role. Nanoceria catalytically neutralize multiple types of reactive oxygen species. However, both therapeutic and toxic effects of CeNPs have been reported. We hypothesize that these disparate reports are due to small differences in each type of nanoparticle arising from the synthetic process. These small differences could lead to particles that aggregate and induce toxicity versus particles that are monodispersed and display antioxidant properties. Due to its short lifespan and easily observable behaviors, we used the nematode worm C. elegans to examine, in parallel, the effects of three different types of cerium oxide nanoparticle formulations on survival after exposure to oxidative stress. To induce oxidative stress, we used juglone (5‐hydroxy‐1,4‐naphthoquinone), a compound derived from many plant species including the black walnut tree, Juglans nigra. We first conducted a dose‐response analysis to find a concentration of juglone that induced significant mortality in our model system. This dose of juglone, 250 μM, was then used in subsequent experiments to test the impact of nanoparticle pre‐treatment on worm viability. For these studies, animals were fed, starting at the L1 larval stage, a range of doses (0 to 580 μM) of three formulations of nanoceria that differed in size as well as the stabilizers used during synthesis. When the worms reached young adulthood, they were exposed to 250 μM juglone for 1 hour. Worms were then placed back onto fresh growth plates containing nanoceria and animal viability was determined 24 hr later via a nose‐touch assay conducted by an experimenter blind to treatment. Preliminary analysis shows that the three different formulations tested showed varying potency in protecting C. elegans from juglone‐induced oxidative stress, supporting our hypothesis that differences in synthetic parameters can impact the ultimate biological effect of the nanoparticles.

Support or Funding Information

Funding provided by St. Lawrence University.
Methcathinone Rapidly Decreases Dopamine Transporter Function: Role of Calcium and Protein Kinase C
Authors: Magee Charlotte P. , Siripathane Yasmeen H. , Le BaoMinh D. , Hanson Glen R. , Fleckenstein Annette E.
Published: April 2020

Methcathinone (MCAT) is a synthetic cathinone that causes both persistent striatal dopaminergic as well as striatal and hippocampal serotonergic deficits. It has been demonstrated that the rapid effects of similar stimulants on dopamine (DA) transporter (DAT) function contribute to persistent monoaminergic deficits. Thus, the effect of MCAT on DAT was assessed. Results revealed that MCAT self‐administration (SA) decreased striatal [3H]DA uptake, as assessed ex vivo in synaptosomes prepared from male rats 1 h after the final MCAT SA session. In vitro co‐incubation of synaptosomes with MCAT (1, 10, 100 μM) at 37°C likewise decreased striatal [3H]DA uptake. There was no effect of in vitro co‐incubation with MCAT at 4°C, suggesting that decreases in [3H]DA uptake are not due to residual drug. These effects were not limited to DAT as MCAT SA decreased hippocampal [3H]serotonin uptake. Further, in vitro co‐incubation of synaptosomes with MCAT decreased both striatal and hippocampal [3H]serotonin uptake. The in vitro effect on DAT function was attenuated by co‐incubation with the cell‐permeable calcium chelator, 1,2‐bis (2‐aminophenoxy) ethane‐N, N, N, N‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM), as well as with the structurally related non‐specific protein kinase C (PKC) inhibitors bisindolylmaleimide I and Ro‐31‐7549. In contrast, this in vitro decrease in DAT function was not attenuated by co‐incubation with other non‐specific PKC inhibitors (NPC‐15437 and Go6976), nor with the PKC‐β inhibitor ruboxistaurin. Neither co‐incubation with the Ca2+/calmodulin‐dependent protein kinase II inhibitor, KN‐93, nor the glycogen synthase kinase 3β inhibitor, SB‐216763, attenuated the MCAT‐induced decrease in [3H]DA uptake. Taken together, these results suggest that MCAT self‐administration and in vitro MCAT exposure have comparable effects to investigator administered MCAT on DAT and SERT function in the striatum and hippocampus; further, in vitro data suggest that the MCAT‐induced effect on DAT is dependent upon calcium and some, but not all, PKC isoforms.

Support or Funding Information

R01‐DA039145
RAGE and phospho‐ATM correlation during DNA Double Strand Breaks in trophoblast cells
Authors: Tsai K.Y.F. , Hirschi-Budge K. M. , Davis T. , Llavina S. , Tullis B. , Jones N. , Fowers R. , Graff T. , Reynolds Paul R. , Arroyo J.
Published: April 2020

Trophoblasts are critical for successful pregnancies and they mediate important steps including implantation, immune protection of the fetus, maternal blood flow to the placenta, and delivery. Increased DNA double strand breaks (DNA‐DSBs) in trophoblasts has been implicated in complicated pregnancies such as intrauterine growth restriction. Previous studies in our laboratory showed increased expression of the receptor for advanced glycation end‐products (RAGE) following cigarette smoke extract (CSE) treatment of trophoblast cells. More recently, a role for nuclear RAGE (nRAGE) has been implicated in DNA‐DSB repair in these cells. ATM is a regulator known to be involved in the repair of DNA‐DSBs through interaction with molecules associated with DNA repair. We tested the hypothesis that RAGE associates with ATM in facilitating DNA‐DSB repair in trophoblast cells. DSBs were induced in human trophoblasts by exposure to cigarette smoke extract (CSE) or bleomycin (BLM). Assessment of nRAGE and g‐H2AX (involved in DNA‐DSB repair) was done by immunoblotting. Immunoprecipitation was used to quantify RAGE and p‐ATM complexing in cells. Invasion was measured after CSE and BLM treatment in the presence or absence of a RAGE neutralizing antibody (nAb).

Increased expression of RAGE and g‐H2AX was detected (1.4‐fold and 1.6‐fold; p<0.05) in treated trophoblast cells compared to controls. Trophoblast invasion was decreased by 65% with BLM treatment compared to controls (p<0.0005) and diminished invasion was further enhanced (73%, p<0.0004) when RAGE nAb was added. Similarly, trophoblast invasion was decreased by 58% (p<0.002) when treated with 1% CSE and further decreased (79%, p<0.0006) in the presence of RAGE nAb. There was also a significant increase (56%, p<0.002) in RAGE expression when p‐ATM was immunoprecipitated from trophoblasts treated with CSE.

We conclude that CSE and BLM cause DNA‐DSBs in trophoblast cells and that DNA‐DSB repair may plausibly involve nRAGE association with ATM. We further conclude that DNA‐DSBs could be a factor that inhibits trophoblast invasion. Nuclear RAGE may be a potential target that maintains trophoblastic invasion by assisting in the repair of DNA‐DSBs. These studies provide a critical insight into dissecting tobacco‐mediated placental deficiency.

Support or Funding Information

This work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).
Regulation of GPCR activation of the Hippo pathway in metastatic breast cancer
Authors: Arakaki Aleena K. S. , Pan Wen-An , Trejo JoAnn
Published: April 2020

G protein‐coupled receptors (GPCRs) regulate the Hippo signaling pathway, a developmental pathway involved in tumorigenesis and metastasis. Although GPCRs promote cancer progression and are highly druggable, there are currently no FDA‐approved drugs targeting GPCRs for cancer treatment. Protease‐activated receptor‐1 (PAR1) is a GPCR that both activates the Hippo pathway and promotes breast cancer progression. PAR1 is overexpressed in breast cancer patient tumor biopsies and in invasive breast carcinoma cell lines, and correlates with increased metastasis and poor prognosis. We found that PAR1 overexpression is due to defective lysosomal trafficking and results in persistent signaling observed in multiple triple‐negative breast carcinoma cell lines including the model invasive MDA‐MB‐231 cells. We recently showed that the α‐arrestin domain containing protein‐3 (ARRDC3), a tumor suppressor, is reduced or lost in highly aggressive triple‐negative breast carcinoma and this is responsible for aberrant PAR1 lysosomal trafficking and persistent signaling, leading to increased invasion. However, the link between ARRDC3 expression and PAR1‐stimulated Hippo pathway regulation in invasive breast cancer is not known and was examined. Here, we show that ARRDC3 regulates GPCR activation of the Hippo pathway in triple‐negative breast cancer, where the transcriptional co‐activators YAP and TAZ display distinct functions. ARRDC3 re‐expression in invasive breast carcinoma cells attenuates GPCR‐stimulated Hippo signaling and invasion that is mediated by activation of TAZ but not YAP. Furthermore, siRNA‐targeted depletion of TAZ, but not YAP inhibits GPCR‐induced Hippo signaling and invasion. An understanding of the mechanisms by which the Hippo pathway is regulated by GPCRs may lead to new potential therapeutic targets for the treatment or prevention of metastatic breast cancer.

Support or Funding Information

Funding for this project was provided by the HHMI Gilliam Fellowship for Advanced Study, the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through NIH General Medical Sciences, T32 GM007752 and the UCSD Tribal Membership Initiative (Aleena Arakaki) and by the National Institute of Health NIGMS R01 GM090689 and R35 GM127121 (JoAnn Trejo).
IH‐dependent HIF1a signaling upregulates reactive oxygen species signaling to suppress expression of the hippocampal NMDA receptor by downregulating the NR1 subunit.
Authors: Arias-Cavieres Alejandra , Nwakudu Chin , Fonteh Ateh , Garcia Alfredo J.
Published: April 2020

When left untreated, the periodic cessation in breathing occurring with sleep apnea can cause oscillations in blood oxygenation leading to intermittent hypoxia (IH) and disrupted cognitive function. IH causes oxidative stress in the nervous system and impair spatial learning and memory. This study examines how IH influences synaptic physiology of principal neurons in the hippocampus. We hypothesize that IH‐dependent HIF1a signaling remodels the glutamatergic synapse by upregulating reactive oxygen species (ROS), weakening N‐methyl‐D‐aspartate receptor (NMDAR)‐dependent long‐term potentiation (LTP), and impairing cognitive performance on hippocampal based behavior. Wildtype mice (WT) and mice hemizygous for HIF1a (HIF1a+/−) were exposed to ten days of IH. While spatial memory in the Barnes maze was impaired in WT following IH (n=6), performance in the Barnes maze was unaffected by IH among HIF1a+/− (n=6). Electrophysiological recordings in brain slices from WT (n=6) and HIF1a+/−(n=6) exposed to IH also revealed that NMDAR‐dependent LTP was suppressed only in WT slices. While IH caused a down regulation of the NR1 subunit from WT, protein carbonylation and HIF1a regulated oxidase, NOX4, was increased by IH in the WT exposed to IH. No changes in NR1, protein carbonylation, or NOX4 were observed in HIF1a+/− exposed to IH. Furthermore, the administration of the superoxide anion scavenger MnTMPyP (5 mg/Kg i.p) during IH mitigated the downregulation of NR1 and suppression of NMDAR‐dependent LTP in WT. These results show that IH‐dependent HIF1a signaling causes targeted disruption to NMDAR‐dependent LTP through a ROS‐based mechanism that involves the downregulation of NR1 subunit. We provide new insight into the mechanistic basis by which sleep apnea may disrupt cognitive performance.

Support or Funding Information

Supported by NIH Grant R01‐NS‐1074210100
Tailoring HDL mimetics for in vivo delivery of mRNA
Authors: He Wei , Rasley Amy , Gilmore Sean F. , Peters Sandra , Coleman Matthew A. , Fischer Nicholas O.
Published: April 2020

In vivo delivery of nucleic acid‐based therapeutics utilizing messenger RNA (mRNA) has significant implications for gene therapy, counteragent delivery, and vaccine applications. The mRNA therapeutics harness the body’s own cells to produce and deliver therapeutic protein molecules. However, large RNA molecules are prone to RNase degradation and exhibit marked difficulty in in vivo delivery compared with smaller RNA molecules. We have successfully engineered reconstituted high‐density lipoproteins (termed nanolipoprotein particles, NLPs) to deliver self‐amplifying mRNA (aka. Replicon) constructs in vivo. NLPs are nanometer‐scale, discoidal particles. They are safe, non‐immunogenic, and amenable to multiple routes of delivery. Also, the size and surface chemistry of NLPs allow for facile optimization of the delivery of compounds with wide range of physicochemical properties. To tailor NLPs for in vivo delivery of large nucleic acids molecules, a panel of NLPs formulated with a wide range of cationic lipids and assembly constituents were screened. Cationic lipids confer NLPs positive charge which successfully complexed with RNA Replicons encoding luciferase, provided protection from RNase degradation, and enhanced mRNA in vivo expression efficiency. These results represent the experimental foundation of utilizing the NLP technology to advance for RNA delivery. Future efforts focus on elucidating the correlation of NLP composition, NLP‐Replicon structure, and in vivo efficacy that will eventually allow for the rational design of a highly potent in vivo delivery platform that can be used as a universal mRNA delivery technology.

Support or Funding Information

This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE‐AC52‐07NA27344, through LDRD award 20‐ERD‐004. This work was also supported by U19 AI144184 from the National Institute of Allergy and Infectious Diseases
NAD Redox Imbalance Drives Diabetic Cardiomyopathy: Roles of oxidative stress and post‐translational modifications
Authors: Lee Chi Fung
Published: April 2020

Diabetes is a major risk factor of heart failure. Diabetes and heart failure both have been linked to NAD redox imbalance. However, the causal role of NAD redox state in the progression of diabetic cardiomyopathy (DC) has not been directly tested. We induced diabetes in wild type mice (WT) by streptozotocin (STZ) injections. Chronic diabetic stress to WT mice (16 weeks) led to gradual declines in cardiac function and lowered NAD/NADH ratio, suggesting the association of NAD redox imbalance in linking diabetic stress with cardiac dysfunction. To determine the causal role of NAD redox imbalance to drive DC, we used cardiac‐specific Ndufs4‐KO mice (cKO), which exhibit lowered cardiac NAD/NADH ratio without overt dysfunction. While insulin depletion and hyperglycemia were similar in diabetic control and cKO mice, cardiac dysfunctions were exacerbated in diabetic cKO mice. Cardiac fibrosis levels of diabetic control and cKO hearts were measured by trichrome staining. Although cardiac fibrosis was slightly elevated in diabetic hearts compared to non‐diabetic controls, but there was no difference between diabetic control and diabetic cKO hearts. Similar in vivo and histological observations were made in both diabetic male and female cohorts. The results suggest that the accelerated decline of cardiac function associated with the pre‐existed NAD redox imbalance is not be due to enhanced fibrosis and is independent of sex. We next analyzed acetylation‐dependent pathways to account for the accelerated decline of function in diabetic cKO hearts. NAD redox imbalance in diabetic cKO hearts promoted protein acetylation, including SOD2 acetylation (SOD2‐K68Ac). SOD2 acetylation inhibits its antioxidant function. We observed elevated protein oxidation levels in diabetic cKO hearts, while expression of genes regulating oxidative stress (Nox1, 2, 4) remained unchanged. The results suggest that NAD redox imbalance promotes oxidative stress by suppressing antioxidant pathway via protein acetylation. To gain further insights how NAD redox imbalance may regulate cardiomyocyte function, phosphorylation levels of myosin binding protein C (MyBPC) and troponin I (TnI) were examined. In diabetic cKO hearts, TnI phosphorylation was increased while MyBPC phosphorylation remained unchanged, suggesting that hyperacetylation in cKO hearts regulates cardiomyocyte contraction‐relaxation to promote cardiac dysfunction. To normalize NAD redox imbalance, we elevated NAD levels in diabetic cKO mice with cardiac‐specific NAMPT overexpression. NAMPT is the rate‐limiting enzyme in salvage pathway. Cardiac NAMPT expression slowed the accelerated decline of cardiac function in diabetic cKO hearts. Our findings collectively suggested that NAD redox imbalance drives DC and the NAD‐dependent pathways regulating DC progression warrant further investigations.

Support or Funding Information

1. Scientist Development Grant (17SDG33330003), American Heart Association;

2. Seed Funding, Presbyterian Health Foundation of Oklahoma City
Acute Voluntary Wheel Running Attenuates Olanzapine‐Induced Hyperglycemia in Male Mice
Authors: Ashworth Kristen E. , Shamshoum Hesham , Medak Kyle D. , Wright David C.
Published: April 2020

Background

Olanzapine is a second‐generation antipsychotic often used in the treatment of schizophrenia and other “off‐label” conditions. Though effective in reducing psychoses, the use of olanzapine is associated with adverse metabolic side effects. Acutely, olanzapine increases liver glucose output and the development of insulin resistance. Chronic olanzapine treatment leads to weight gain and predisposes individuals to the development of cardiovascular disease and type 2 diabetes. Exercise is an intervention which helps to maintain glucose homeostasis, and with long‐term adherence, can decrease glucose intolerance and insulin resistance. We have previously demonstrated that a single bout of exhaustive exercise mitigates olanzapine‐induced hyperglycemia. While these findings are encouraging, the clinical relevance of exhaustive exercise in individuals taking olanzapine is limited.

Purpose & Hypotheses

The purpose of this study was to investigate if increases in short‐term, habitual physical activity in the form of voluntary wheel running (VWR) would have a protective effect against olanzapine‐induced hyperglycemia.

Methods

10‐week old male C57BL/6J mice remained sedentary or were provided running wheels at the start of the dark cycle (~20:00 h), until the following morning (~8:00 h; start of light cycle). Olanzapine (5 mg/kg body weight (BW)) was administered intraperitoneally (IP) immediately following overnight VWR, or 6 hours and 24 hours post wheel‐lock. Blood glucose was measured at baseline, 30, 60, 90, and 120 mins post‐olanzapine. In parallel experiments following the same overnight VWR protocol, an insulin tolerance test (ITT) (0.5 IU/kg BW; IP) was conducted 60 mins post‐olanzapine treatment to assess olanzapine‐induced insulin resistance among groups.

Results

A single overnight session of VWR protected against acute olanzapine‐induced hyperglycemia immediately post‐wheel lock. This protective effect was still present 6 hours post‐wheel lock, and had reversed by 24 hours after the cessation of exercise. Under insulin‐stimulated conditions, the sedentary olanzapine‐treated mice had significantly higher blood glucose area‐under‐the‐curve compared to the other three groups, providing evidence that a prior bout of VWR protects against olanzapine‐induced insulin resistance. Under insulin‐stimulated conditions, serum free fatty acid concentrations were increased with olanzapine and reduced with VWR.

Conclusions

Our findings demonstrate that with a single session of overnight habitual physical activity, olanzapine‐induced hyperglycemia is mitigated for at least 6 hours post‐exercise and insulin‐stimulated glucose disposal is recovered under olanzapine‐treated conditions.

Support or Funding Information

This study was supported by the Canadian Institutes of Health Research.
Assessing the Biochemical Mechanism of Reversers of Multidrug Resistance in Cancer
Authors: Jensen Madeline Kay , de Oliveira Maisa Correa , Okwuone Dakota D. D. , Wise John G. , Vogel Pia D.
Published: April 2020

The breast cancer resistance protein (BCRP) is an ABC transporter that uses energy derived from ATP hydrolysis to export toxins from the cell. In cancer cells, these transporters are overexpressed and remove chemotherapeutic drugs from the cell. This confers multidrug resistance. We have previously conducted high‐throughput in silico ligand docking studies to identify potential inhibitors of BCRP. These drug‐like molecules inhibit ATP hydrolysis by targeting the nucleotide binding domain. Biochemical and biophysical analyses of these inhibitors are necessary to study their inhibition mechanisms. Large amounts of purified protein are required to carry out these analyses. Our lab previously generated a gene for human BCRP that was codon optimized for protein expression in the yeast, PichiaPinkTM. Here we report our attempts to purify BCRP from this strain as well as from a second, BCRP over‐expressing Pichia Pastoris strain. Growth conditions were previously optimized using methanol to induce BCRP expression. We also report our efforts to purify BCRP into detergent mixed micelles using Ni‐NTA affinity and ion exchange chromatography. Attempts to assemble BCRP containing nativelike membrane nanodiscs to achieve maximum ATP hydrolysis are also reported. Obtaining purified BCRP in a native‐like environment with high activity is crucial for the assessment of BCRP inhibition mechanisms of our newly found drug‐like compounds.

Support or Funding Information

This work is supported by the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D., of Naples, FL.
‐The Function of CASK in Transcriptional Regulation in Neurons
Authors: Meng Yinuo , Montilla-Perez Patricia , Hillary Ryan , Wen Junneng , Benner Chris , Telese Francesca
Published: April 2020

CASK is a scaffold protein that functions in trafficking and targeting of synaptic proteins in the central nervous system. Emerging literature suggests that CASK localizes inside nucleus and might regulate transcription by interacting with TBR1, a transcription factor essential for brain development. However, these regulatory mechanisms are not well established. We hypothesize that CASK is involved in neuronal activity‐regulated transcription. To test our hypothesis, we used primary cultures of mouse cortical neurons stimulated with KCL depolarization buffer, which is known to activate a robust CREB‐dependent gene expression program and to induce the activation of distal enhancer elements. To measure gene expression genome‐wide, we performed RNAseq in unstimulated or KCL‐treated neurons after knock‐down of CASK expression using specific shRNA. Our results show that CASK knock‐down strongly reduces the number of genes that are regulated by depolarization. These results indicate that CASK is required for gene expression regulation in neuronal cells. To gain further insights in the mechanisms by which CASK regulate transcription, we used RNA in situ hybridization (RNAscope) to test the hypothesis that CASK is not only required for the transcription of coding genes, but also of non‐coding RNAs, termed ‘enhancer RNAs (eRNAs), in proximity of KCL‐regulated genes. We showed that RNAscope is a valid method to measure eRNAs induction after KCl treatment. Ongoing experiments in the lab are assessing the effect of CASK knock‐down on eRNA transcription. Interestingly, we observed by immunoprecipitation that nuclear CASK dimerizes, and that the stability of this homodimer is dependent on the presence of RNA. Finally, we used biochemical fractionation to study CASK localization in the nucleus in both mouse neuronal cultures and brain tissues. These results shed light on how CASK functions in neurons. This study is relevant to human disease because mutations in CASK gene are linked to microcephaly and X‐linked intellectual disability.

Support or Funding Information

This project has been supported by CABRI Undergraduate Research Fellowship
Diabetes Attenuates the Increase in Estrogen‐Mediated Endothelial Function
Authors: Derella Cassandra C. , Bieck Maire Rose , Blackburn Marsha , Harris Ryan A.
Published: April 2020

Background

Apparently healthy premenopausal women are protected against cardiovascular disease (CVD) compared to men; and estrogen has been thought to play a key role. In the presence of diabetes; however, this overall sex‐dependent cardiovascular protection is lost. The increase in vascular function throughout the menstrual cycle in healthy premenopausal women has been established. The present investigation sought to test the hypothesis that the increase in vascular function from menses to the late follicular phase of the menstrual cycle is lost in premenopausal women with type 1 diabetes (T1D).

Methods

Premenopausal women with a clinical diagnosis of T1D (n=22, HbA1c=8.2±1.7%, age=26±8 years, BMI=26.9±5.4 kg/m2) and seemingly healthy, age‐ and sex‐matched controls (n=10, HbA1c=5.2±0.3%, age=26±9 years, BMI=23.3±4.1 kg/m2) were recruited to participate. Flow mediated dilation (FMD), a measure of endothelial function, was assessed during the menses (cycle day=4±1) and late follicular phases (cycle day=13±1) of the menstrual cycle to represent periods of low and high concentrations of estrogen, respectively. Visit 1 (menses) was confirmed by a positive hemolytic urological multistix test and visit 2 (late follicular) was scheduled based on indications from an ovulation prediction kit. A small venous blood sample was collected at each visit to measure concentrations of estrogen. Endothelial independent vasodilation was determined by administration of sublingual nitroglycerin after each FMD test.

Results

The increase in estrogen from menses to the follicular phase was similar (p=0.73) in both groups (Δ estrogen: T1D=78.3±92.6 vs Controls=85.6±30.0 pg/mL). Baseline brachial artery diameter was similar between T1D and controls (p=0.25) and was not different between visit 1 and visit 2 (p=0.76). Fasting blood glucose was higher (p<0.01) in women with T1D compared to controls at both visits; however, the change within groups was similar (p=0.49) across time. A near significant interaction (p=0.07) for endothelial function was observed such that FMD increased from the menses to follicular phase in controls (ΔFMD=1.151±1.603%), whereas no change was observed in patients with T1D (ΔFMD=−0.633±2.126%). Endothelial independent vasodilation was not different between groups (p=0.82). Additionally, the endothelial independent response was similar across the menstrual cycle (p=0.97).

Conclusion

These data suggest that the presence of diabetes may attenuate the beneficial effects of estrogen on endothelial function in premenopausal women. The loss of estrogen’s cardiovascular protective effect on the vasculature in diabetes may explain the increased risk of CVD observed in women with T1D.

Support or Funding Information

1RO1HL137087‐01A1.
Effects of TNF‐Alpha on TGF‐Beta Expression in Human Skeletal Muscle Cells: A Cell Model for Inflammation
Authors: Kennedy Christina , Gilkeson Josephine
Published: April 2020

Approximately 25.3 million adults suffer from chronic pain, often caused by chronic inflammatory conditions. We hypothesize that chronic inflammation negatively alters myofascial tissue structure and function. To better understand this process and test our hypothesis, we are developing a cell model to examine chronic inflammation in myofascial tissue. The goal of this investigation is to determine if chronic inflammation changes cell signaling of the myofascial tissue and lead to fibrosis. Human skeletal muscle (HSkM) cells and human fibroblasts make up the myofascial tissue. Therefore, we will first examine the relationship between the chronic inflammatory mediator, TNF‐alpha, and HSkM cell expression of TGF‐beta. We find that TNF‐alpha increases expression of TGF‐beta in HSkM cells changing the cell phenotype to a fibrotic state.
Inhibition of PDE4 Induces Hypothermia in Mice
Authors: McDonough Will , Aragon Ileana , Rich Justin , Saleh Lina Abou , Richter Aris , Boyd Abigail , Koloteva Anna , Richter Wito
Published: April 2020

Type 4 cyclic nucleotide phosphodiesterases (PDE4s), a group of isoenzymes that hydrolyze and inactivate the second messenger cAMP, are established targets for development of enzyme inhibitors with anti‐inflammatory properties. While testing the anti‐inflammatory effects of a nonselective PDE4 inhibitor in a murine model of bacterial lung infection, we noticed that PDE4 inhibition per se induced significant hypothermia in the animals in the absence of bacterial infection. Because drug‐induced hypothermia may critically impact our animal model, and the role cAMP‐phosphodiesterases play in the regulation of body temperature is poorly understood, we further explored this observation. We found that treatment with a number of structurally distinct PAN‐PDE4 inhibitors, including Rolipram, Roflumilast and RS25344 (all 1 mg/kg, i.p.), but not the PDE3‐selective inhibitor Cilostamide, induced a rapid decrease in core body temperature of C57Bl6 mice as measured with a rectal thermometer at 30 min after drug application, suggesting that hypothermia in mice is a class‐effect of PDE4 inhibitors. PDE4 inhibitor‐induced hypothermia was considerable (~4°C drop), rapid in onset (max effect is reached within 20–30 min), and long lasting (core body temperature remains below controls for up to 5 h). As little as 40 μg/kg of the archetypal PDE4 inhibitor Rolipram induced a hypothermic effect. Similar or higher doses of Rolipram were used in most published animal studies. Thus, the reported findings are likely associated with, or at least paralleled by, drug‐induced hypothermia. The initial decrease in body temperature after PDE4 inhibitor injection is so rapid that it is no different from heat dissipation after euthanasia, suggesting that PDE4 inhibition affects central body temperature regulation so that both heat‐generating (e.g. shivering/non‐shivering thermogenesis) as well as heat‐preserving (e.g. skin vasoconstriction) mechanisms are simultaneously suspended, either by lowering the cold defense set point or by impairing its enforcement. Indeed, PDE4 inhibitor treatment induced a temporary heat‐loss via tail vasodilation as assessed by thermography. Consistent with the idea of an effect on central body temperature regulation, hypothermia was induced by moderate doses (≤1 mg/kg) of various brain‐penetrant PDE4 inhibitors. Conversely, YM976, a PDE4 inhibitor that does not cross the blood‐brain barrier, had no effect. Finally, to begin delineating the mechanism of drug‐induced hypothermia, we show that a blockade of serotonergic‐ and dopaminergic‐, but not β‐adrenergic‐, histaminergic‐ or opiate receptors, can alleviate PDE4 inhibitor‐induced hypothermia.

Support or Funding Information

Supported by grants from the Cystic Fibrosis Foundation (SALEH18H0,RICHTE16GO) and the NIH (HL76125, HL141473, HL066299).
The Role of Endothelin‐1 Receptor Subtypes in Limiting Insulin‐Stimulated Blood Flow in Middle‐Aged Adults
Authors: Young Ben E. , Padilla Jaume , Finsen Stine H. , Mortensen Stefan P. , Fadel Paul J.
Published: April 2020

Beyond its metabolic actions, insulin stimulates peripheral vasodilation, which serves to increase glucose delivery to skeletal muscle. Insulin activates two distinct signaling pathways leading to the production of nitric oxide versus endothelin‐1 (ET‐1). Several lines of research suggest that insulin‐stimulated ET‐1 production may limit insulin‐stimulated nitric oxide‐mediated vasodilation. Recently, it was demonstrated that basal ET‐1‐mediated vasoconstricton is elevated in middle‐aged adults. However, no studies have examined the role of ET‐1 during hyperinsulinemia in middle‐aged individuals. Therefore, we tested the hypothesis that ET‐1 receptor antagonism would improve insulin‐stimulated blood flow (BF) in a cohort of middle‐aged adults. In 14 subjects (age: 54±2 years, BMI: 26±1 kg/m2) with a range of insulin resistance (glucose infusion rate range: 2.0 to 7.2 mg/kg/min), two femoral arterial catheters were placed. One catheter in the left leg for measurement of arterial blood pressure (BP) and one in the right leg for determination of arterial glucose concentrations and intra‐arterial administration (25nmol/min) of BQ‐123 (ETA antagonist) and BQ‐788 (ETB antagonist). In addition, a femoral venous catheter (right leg) was placed for determination of venous glucose concentrations, and one antecubital venous catheter was placed for infusion of insulin and glucose. Femoral artery BF (right leg: Doppler Ultrasound), BP, and glucose uptake (arterial‐venous glucose concentration x BF) were measured during a hyperinsulinemic‐euglycemic clamp alone (insulin: 40 mU/m2/min), followed by hyperinsulinemia with intra‐arterial BQ‐123 administered for one hour (Insulin+BQ‐123). BQ‐788 was then co‐infused with BQ‐123 for an additional hour (insulin+BQ‐123+BQ‐788; n=10). The infusion of BQ‐123 during hyperinsulinemia augmented leg BF (Insulin: 399±42 mL/min; Insulin+BQ‐123: 500±47 mL/min, P<0.001) and vascular conductance (Insulin: 4.3±0.5 mL/min/mmHg; Insulin+BQ‐123: 5.8±0.7 mL/min/mmHg, P<0.001), and increased leg glucose uptake (Insulin: 76±13 mg/min; Insulin+BQ‐123: 103±15 mg/min, P=0.02). Subsequently, co‐infusion of BQ‐788 provided no further increase in leg BF (Insulin+BQ‐123: 485±60 mL/min; Insulin+BQ‐123+BQ‐788: 511±70 mL/min, P=0.42), vascular conductance, or glucose uptake. Sub‐analysis of subjects with type 2 diabetes (T2D, n=7) versus non‐T2D (n=7) suggested no difference in responsiveness to either drug intervention between groups (both P>0.05). These data suggest ET‐1 signaling through ETA receptors plays an important role in limiting insulin‐stimulated BF in middle‐age, whereas ETB receptor signaling has little effect. These findings provide novel insight into the role of ET‐1 during hyperinsulinemia, favoring selective targeting of the ETA receptor for improving insulin‐stimulated BF and glucose uptake with aging.

Support or Funding Information

Supported by UTA College of Nursing and Health Innovation
Introducing Substrate Features in Fmoc‐Amino Acid‐Based Compounds Increases the Potency of Butyrylcholinesterase Inhibitors: A Potential Therapeutic for Alzheimer’s Disease
Authors: Godinez Jonathan , Lee Catherine , Schwans Jason P.
Published: April 2020

Butyrylcholinesterase (BChE) and Acetylcholinesterase (AChE) are enzymes responsible for terminating neurotransmission by hydrolyzing acetylcholine. Low levels of acetylcholine in the brain is correlated to the reduced cognitive function in individuals suffering from neurodegenerative disorders such as Alzheimer’s Disease (AD). Relative to healthy individuals, those suffering from AD exhibit a significant increase in BChE activity, and little change in AChE activity. We previously found that 9‐fluorenylmethoxycarbonyl (Fmoc) amino acid‐based inhibitors have been effective to inhibit BChE. As enzymes are specific for their substrate, esterification of the carboxy‐terminus with a trimethylammonium moiety to mimic the cationic group of the substrate may lead to increased potency and specificity. To test this model, a series of inhibitors bearing a cationic trimethylammonium group were synthesized, purified by silica gel chromatography, and characterized by NMR. Enzyme assays monitored by UV‐Vis spectrometry suggested the compounds were potent inhibitors. However, high‐performance liquid chromatography (HPLC) experiments suggest that the Fmoc‐ester derivatives were acting as a substrate and being hydrolyzed by BChE, complicating analysis and minimizing their potential to act as inhibitors. To combat this limitation, a more stable amide was substituted for the ester. HPLC results indicate this substitution prevents enzymatic hydrolysis. Initial biochemical results for the Fmoc‐amide derivatives suggest the compounds inhibit BChE with KI values in the 0.06–10.0 μM range. Overall, the results suggest that introducing substrate‐like characteristics within the Fmoc‐amino acid‐based background increases their potency and may help guide the generation of future inhibitors.

Support or Funding Information

This research was supported by NIH award number R25GM071638
Resourcing of Porcine Unfractionated and Low‐Molecular Weight Heparins from Ovine, and Bovine Sources
Authors: Baig Nausheen , Kouta Ahmed , Siddiqui Fakiha , Bontekoe Emily , Hoppensteadt Debra , Jeske Walter , Iqbal Omer , Fareed Jawed
Published: April 2020

Introduction

The current shortage of porcine mucosal heparin supply warrants the need for exploring alternate sources to manufacture heparins. UFHs and LMWHs can also be derived from ovine and bovine sources, which could provide substitute for porcine derived anticoagulants. The purpose of this study is to compare the biologic properties of UFHs and LMWHs from porcine, ovine, and bovine sources at gravimetric concentrations.

Materials and Methods

USP standard (100 ug/ml), NIBSC (100 U/mL), APTT reagents (Triniclot), Spectrozymes Xa and IIa (Sekisui), Bovine Factor Xa (Enzyme Research, USA), Thrombin (Enzyme Research, USA), and blood bank plasma. The following agents were tested: porcine UFH (Medefil 13353), porcine LMWH (Enoxaparin 7L010), ovine UFH (lot# 1001), ovine LMWH (Enoxaparin lot# 404D), bovine UFH (GAG 25), and bovine LMWH (Enoxaparin lot# 003). All agents were diluted at a stock solution of 100 ug/mL in saline. Working dilutions were prepared at 100 ug/mL. Whole blood ACT was measured using a Hemochron instrument at a fixed concentration of 10 ug/mL and 25 ug/mL. Prothrombinase induced clotting time (PiCT), TT, aPTT, anti‐Xa, and anti‐IIa activities were measured in plasma supplemented with each of these agents at a concentration range of 0–10 ug/mL using the ACL Elite instrument. The USP potency assay was carried out using chromogenic antiprotease assays (Biophen Kits). The inhibitory concentrations at 50% (IC‐50) were measured using a purified antithrombin biochemical system employing a kinetic method. Thrombin generation inhibition assay was carried out using a CAT method (Diagnositca Stago, Paris, France).

Results

In the ACT method, ovine UFH showed the highest value followed by porcine UFH, bovine UFH, ovine LMWH, porcine LMWH, then bovine LMWH. The PiCT results showed ovine UFH with the strongest anticoagulant activity followed by porcine UFH and bovine UFH. All three LMWH demonstrated comparable PiCT results and were significantly lower than their UFH counterparts. In the plasma supplementation studies, a similar trend was seen in the aPTT, TT, factor Xa, and factor IIa studies: ovine UFH demonstrated the strongest anticoagulant and antiprotease effects followed by porcine UFH, bovine UFH, ovine LMWH, porcine LMWH, then bovine LMWH. The IC‐50 values for the anti‐Xa ranged from 0.71–2.12 ug/mL for the UFH and from 2.23–3.92 for the LMWH, whereas the anti‐IIa values ranged from 3.91–8.37 ug/mL for the UFH and were greater than 10 ug/mL for the LMWH. Ovine UFH and LMWH had the lowest IC‐50 values for anti‐Xa in their respective categories while bovine UFH and LMWH had the highest. In the thrombin generation assays, the ovine UFH showed slightly stronger activities followed by porcine UFH, bovine UFH, ovine LMWH, porcine LMWH, then bovine LMWH in all three parameters including peak thrombin, lag time, and percent inhibition of area under the curve.