bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationThis 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

BackgroundApparently 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).MethodsPremenopausal 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.ResultsThe 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).ConclusionThese 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 Information1RO1HL137087‐01A1.
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 InformationSupported 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 InformationSupported 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 InformationThis 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

IntroductionThe 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 MethodsUSP 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).ResultsIn 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.
The Ventilatory Response to Hypercapnia in vivo Requires Serotoninergic Afferents to the Retrotrapezoid Nucleus
Authors: Leirão Isabela P. , Colombari Débora S.A. , da Silva Glauber S.F. , Zoccal Daniel B. 
Published: April 2020

Chemosensory cells located in the brainstem are responsible to monitor the arterial levels of CO2/pH and provide excitatory inputs to the respiratory network to evoke a reflex increase in ventilation in conditions of hypercapnia. Among the candidates responsible for central chemoreception are the Raphe serotoninergic neurons. However, there is scarce functional evidence about the role of specific serotoninergic projections to the respiratory network and other chemosensitive sites. Herein, we explored the hypothesis that the hypercapnic ventilatory response in vivo requires, at least in part, the stimulation of the Raphe serotoninergic neurons that project to the retrotrapezoid nucleus (RTN) ‐ an important respiratory and CO2/pH chemosensitive region of the ventrolateral medulla. To reach this goal, pulmonary ventilation was evaluated under baseline conditions and during the exposure to hypercapnia (7% CO2) in unanesthetized juvenile Holtzman rats (60–90 g) that received bilateral microinjections of the toxin anti‐SERT‐SAP (0.1 mM) in the RTN to selectively lesion serotoninergic neurons that send projections to this region. After 15 days of anti‐SERT‐SAP microinjections in the RTN (n=7), baseline respiratory frequency (Rf) and tidal volume (VT) were similar to control rats that received vehicle microinjections (n= 9). On the other hand, the ablation of RTN‐projecting serotoninergic neurons markedly attenuated the CO2‐induced response of increase in the Rf (P=0.001), but not in the VT. Our preliminary analyses suggest that the impaired tachypneic response to hypercapnia induced by anti‐SERT‐SAP microinjections in the RTN was associated with reduced number of serotoninergic neurons in the Raphe Obscurus (Rob) and Magnus (RMg), but not in the Raphe Pallidus. Overall, our data support the idea that interactions between the serotoninergic neurons of the Rob/RMg and the RTN neurons are necessary for the processing of the hypercapnic ventilatory response in unanesthetized conditions.Support or Funding InformationSupported by FAPESP and CNPq
Glutamate strengthening of GABAergic inhibition in the hypothalamic PVN: Dependence on EAAT3‐mediated Glu uptake into local GABA terminals
Authors: Yamaguchi Junya , Andrade Mary Ann , Truong Tamara , Toney Glenn M. 
Published: April 2020

In unstressed animals, pre‐sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) are held in a state of near discharge quiescence by the dominance of GABAergic inhibition. In brain slice studies we found that the amplitude of PSN GABAergic inhibitory postsynaptic currents (IPSCs) is increased by elevated ECF L‐glutamate (Glu). This Glu‐GABA strengthening (GGS) phenomenon, originally described in the hippocampus, can be elicited in PVN PSNs both by exposure to bath Glu (100 μM) or high K+ (+10 mM) for 10 min. PVN GGS is (1) mediated by a presynaptic mechanism that increases unitary quantal size (more GABA molecules/vesicle), (2) is unaffected by inhibition of glial GLT1‐mediated Glu uptake, and (3) is prevented by global inhibition of Glu uptake. Collectively, these findings indicate that PVN GGS depends on Glu uptake by the neuronal Glu transporter EAAT3. Importantly, bath Glu elicits a delayed GABA‐AR‐mediated suppression of PSN firing, indicating that the Glu‐induced increase of IPSC amplitude (i.e., GGS) is sufficient to regulate PSN discharge quiescence. Moreover, in vivo studies indicate that PVN Glu enhances the increase of renal SNA caused by local blockade of GABA‐AR, consistent with elevated Glu increasing functional GABAergic inhibitory tone in the PVN. Here we performed PVN immunohistochemistry and found EAAT3 localized diffusely within somatodendritic compartments and densely in puncta resembling synaptic terminals. EAAT3+ puncta co‐localized with GAD65 punctate staining, consistent with EAAT3 expression on local GABAergic synaptic terminals. To test dependence of GGS on EAAT3 uptake of Glu specifically by PVN GABA terminals, we used a CRISPR/Cas9 gene editing strategy in which spCas9 was expressed in a Cre‐dependent manner amongst vGAT+ (GABAergic) neurons. To knockdown (KD) EAAT3 specifically amongst GABAergic inputs to PVN, AAV1‐CAG‐EAAT3:sgRNA‐GFP was nanoinjected (400 nl) into the surrounding peri‐nuclear zone (PNZ). Western blot analysis of tissue punches (Cntrl: n=10, CRISPR EAAT3: n=12) revealed a nearly 40% loss of EAAT3 in PVN. Given that EAAT3 amongst non‐GABAergic (i.e., glutamatergic) PVN neurons were not targeted by our CRISPR/Cas9 strategy, loss of EAAT3 specifically amongst PVN‐projecting GABAergic PNZ neurons would likely have been much greater than ~40%. Crucially, strengthening of PNZ evoked GABAergic IPSCs by bath Glu was effectively abolished (before: −238 ± 10 pA, after: −226 ± 12 pA; n=3) by CRISPR EAAT3 KD amongst PNZ GABAergic neurons. Results indicate that EAAT3‐mediated Glu uptake into PVN GABA terminals actively defends and restores PSN quiescence following acute glutamatergic excitation.Support or Funding InformationNIH HL088052 (GMT)