bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimizing Protein Function for Screening of Novel Inhibitors that Target Multi‐Drug Resistance in Cancers
Authors: Okwuone Dakota D. , de Oliveira Maisa C. , Jensen Madeline K. , Vogel Pia D. , Wise John G. 
Published: April 2020

P‐glycoprotein (P‐gp) belongs to the ATP‐Binding Cassette (ABC) transporter family of proteins and is strongly linked with multidrug resistance (MDR) in tumors where overexpression often abolishes the effects of chemotherapeutics by removing the therapeutics from the cells. In hopes of creating co‐therapeutics to re‐sensitize tumors to chemotherapeutics, we previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. To evaluate the mechanism of inhibition in human P‐gp, we have isolated human P‐gp from the yeast Pichia pastoris using mixed detergent micelles. In order to effectively assess the potential inhibitors, we must be able to sufficiently stimulate our protein and have a high enough activity to see the inhibitory effects of the potential inhibitors. In previous work, we found that the activity of P‐gp assembled into membrane nanodiscs had significantly higher basal activity in the absence of transport substrate than P‐gp in detergent micelles. However, significant increase in activity in the presence of the substrate verapamil was not observed in micelles or nanodiscs. Here, we report methods of protein purification and nanodisc assembly modified to have optimally functional human P‐gp. The resulting purified P‐gp will be used to evaluate the inhibition mechanism of lead compounds that were shown to reverse MDR in cancer cell culture.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, 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, Florida. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.
Modulation of Airway Remodeling by PF543, a Sphingosine Kinase 1 Inhibitor, in a Mouse Model of Bronchopulmonary Dysplasia
Authors: Sudhadevi Tara , Ha Alison W. , Ebenezer David L. , Fu Panfeng , Putherickal Vijay , Ackerman Steven J. , Kanteti Prasad , Berdyshev Evgeny , Natarajan Viswanathan , Harijith Anantha 
Published: April 2020

Bronchopulmonary dysplasia (BPD) is a chronic lung disease affecting extreme preterm newborn survivors with serious long‐term sequela of wheezing. Oxygen therapy, which is inevitable for the survival of such extremely premature infants, is partly responsible for the development of BPD and associated airway remodeling (AWRM). Altered Sphingosine‐1‐phosphate (S1P)/Sphingosine kinase 1 (SPHK1) signaling have been implicated in the pathophysiology of a number of lung diseases; however, the role of SPHK1/S1P signaling in BPD and AWRM is not well understood. The neonatal mice were subjected to hyperoxia (HO) at 95% oxygen (postnatal day 3 to 10) and allowed to grow as adults for 8 weeks. During exposure to room air (RA) or HO, the mice received 5mg/kg of PF543, a specific inhibitor of SPHK1, intraperitoneally on alternate days. Lungs were then collected and prepared for histology as well as morphometry of alveoli/airways. Lung function studies were performed using a ventilator and methacholine challenge was used to measure airway hyper reactivity (AHR). Levels of S1P and sphingoid bases in bronchoalveolar lavage (BAL) fluids and lung tissues were measured by LC‐MS/MS. In vitro mechanistic studies on HO‐induced AWRM and the protective effect of PF543 were performed using small airway epithelial cells (SAEC).The exposure of neonatal mice to hyperoxia resulted in AWRM in adults, as evidenced by increased peribronchial fibrosis and smooth muscle hypertrophy. These changes were significantly less in Sphk1 knock out (Sphk1−/−) mice exposed to HO indicating a potential role of SPHK1 in the pathogenesis of AWRM. Subsequent experiments using a specific SPHK1 inhibitor, PF543, during neonatal HO prevented alveolar simplification and, more significantly, ameliorated AWRM in adult mice. Lung function studies revealed AHR in adult mice that were exposed to neonatal HO. Interestingly, PF543 therapy reduced methacholine‐induced AHR and airway constriction along with a reduction in AWRM. Exposure of neonatal mice to HO showed epithelial mesenchymal transition (EMT) ‐like changes, which was reduced by PF543 treatment. Inhibition of SPHK1 with PF543 in SAECs ameliorated HO‐induced loss of E‐cadherin and gain of N‐cadherin and vimentin, markers of EMT.In conclusion, our data indicate a novel role for SPHK1/S1P signaling in HO‐induced BPD and AWRM, and amelioration by PF543.Support or Funding InformationThis work is supported by NIH/NICHD R01 HD090887‐01 A1 and AHA 18TPA34230095 to AH.
Beyond OIRD: The Multiple Negative Effects of Systemic Fentanyl in Adult Goats
Authors: Buchholz Kirstyn , Neumueller Suzanne , Burgraff Nicholas , Pan Lawrence , Hodges Matthew , Forster Hubert 
Published: April 2020

Opioids are effective analgesics but are addictive and negatively impact multiple physiologic functions. Opioid abuse has reached epidemic levels and led to an exponential increase in deaths and societal costs, with a disproportionate impact on women. A majority of studies have focused on the depressant effect of opioids on breathing frequency. However, there are data suggesting that opioids cause multiple life‐threatening physiologic effects, but there is a paucity of data assessing the multiple deleterious effects simultaneously in freely behaving mammals. Here we tested the dose‐dependent effects of systemic fentanyl (a major cause of the current opioid crisis) in adult female goats. Systemic fentanyl acutely and dose‐dependently depressed breathing frequency through prolongation of expiratory time while increasing tidal volume similar to effects of fentanyl in humans (J Clin Monit Comput 18: 2004; Eur J. Anest 20: 2003; Br J. Anest 96: 2006). Fentanyl also increased the difference between alveolar and arterial PO2 (A‐a oxygen gradient,) and at high doses increased tonic activity of intercostal and abdominal muscles (consistent with “wooden chest”) while also prolonging post‐inspiratory muscle activation and in rare instances caused obstructive apnea. Beyond these acute (<15 min) effects were prolonged increases in body temperature, metabolic rate, and arterial blood pressure and decreases in heart rate. Furthermore, high dose opioids had long‐lasting effects, including induction of acute withdrawal symptoms, and led to decreased fecal output for >24 h. We conclude that a triad of deleterious effects on cardiorespiratory responses (respiratory frequency depression, A‐a oxygen gradient, and sustained contraction of respiratory muscles) conceivably contribute to hypoxemia induced opioids fatalities. Furthermore, opioids have profound, deleterious effects (withdrawal and constipation) beyond cardiorespiratory control.Support or Funding InformationDepartment of PhysiologyZablocki VA Medical Center
The protease inhibition with Ritonavir impairs endothelial function and promotes vascular smooth cell proliferation via RANTES/C‐C chemokine receptor type 5 and Nox1‐derived reactive oxygen species pathway.
Authors: do Nascimento Thiago Bruder , Belin de Chantemele Eric J. 
Published: April 2020

Thanks to the advent of highly active antiretroviral therapy (HAART), the life of people living with HIV has been extended to close to that of healthy people. However, patients with HIV are now presenting with accelerated development of cardiovascular disease (CVD) and CVD is currently the leading cause of death in patients with HIV on HAART. Herein, we tested the hypothesis that the protease inhibitor ritonavir contributes to HAART‐associated CVD by impairing vascular function via C‐C chemokine receptor type 5 (CCR5)‐ and reactive oxygen species‐mediated mechanisms. We treated 8–10‐week‐old male C57BL6/J with ritonavir for 4 consecutive weeks (5mg/kg/day, i.p). At the end of the treatment, blood was collected and aortas excised to measure H2O2 via Amplex Red and vascular function via wire myography. Aorta from ritonavir treated animals exhibited higher H2O2 levels, impaired relaxation to acetylcholine (Ach) and increased contractility to phenylephrine (Phe). Nox1 inhibition with GKT771, (10μM) fully restored endothelial function in rings from ritonavir treated animals. Ritonavir treatment also elevated RANTES (CCR5 ligand) plasma levels, as well as CCR5, NADPH oxidase 1 (Nox1) and NoxA1 transcript levels in the aorta. No difference was observed for others oxidases, such as Nox2 and Nox4. Repetition of the ritonavir treatment in CCR5 deficient mice revealed that CCR5 deficiency protects vessels from ritonavir induced‐endothelial dysfunction and upregulation of Nox1 and NoxA1 enzymes. To analyze the direct effects of RANTES on the vasculature, thoracic aorta, endothelial, and vascular smooth muscle cells were exposed to RANTES in different times (20ng/mL). RANTES elevated endothelial NOX1 expression and impaired ACh‐mediated relaxation. Concomitantly, Nox1 inhibition prevented RANTES‐mediated endothelial dysfunction. In addition, RANTES time‐dependently stimulated rat aortic smooth muscle cell proliferation, as measured by Ki67 expression, which was partially reverted by Nox1 inhibition. Our data suggest that the protease inhibitor ritonavir contributes to vascular dysfunction and cardiovascular disease by increasing RANTES‐mediated CCR5 activation ultimately leading to increases in Nox1 expression, ROS production, endothelial dysfunction and vascular smooth muscle cell proliferation. Taken together, these findings provide potential explanation for the increased risk of cardiovascular disease developed by patients living with HIV and present CCR5 as a potential target for treatment.Support or Funding Information1R01HL130301‐01 to EBC; K99: 1K99HL140139‐01A1; R00: 4R00HL140139‐03 to TBN
Trimethylamine N‐Oxide (TMAO) Impairs Endothelium‐Dependent Relaxation in Mouse Mesenteric Resistance Artery and Aorta
Authors: Priviero Fernanda , Justina Vanessa Dela , Arishe Olufunke , Webb R. Clinton 
Published: April 2020

Trimethylamine N‐oxide (TMAO) is a gut microbiota‐dependent metabolite produced from choline and phosphatidylcholine and it has been related to risk for cardiovascular diseases. Studies have shown that increased levels of TMAO are associated with heart attack, stroke and heart failure. This study tested the hypothesis that TMAO has a deleterious effect on the vascular beds leading to vascular dysfunction. Sixteen‐week old male lean mice were euthanized and the aorta and mesenteric resistance arteries (MRA) were mounted in a wire myograph. Concentration response curves to acetylcholine (ACh; 0.001 – 30 μM), A23187 (0.1 – 10 μM) and sodium nitroprusside (SNP; 0.001 – 1 μM) were performed in the absence or in the presence of TMAO (100 μM – incubated for 30 min) in order to evaluate endothelium dependent and independent‐relaxation of the aorta and MRA. In the aorta, TMAO decreased the potency of ACh (pEC50: 7.96 ± 0.22 versus 7.25 ± 0.16) and EMAX to ACh (89 ± 06% versus 63 ± 4%). Similarly, potency to ACh was decreased by TMAO in the MRA (pEC50: 7.52 ± 0.43 versus
6.01 ± 0.15, with vs. without TMAO, respectively). TMAO did not alter endothelium‐dependent relaxation induced by the calcium ionophore A23817 in the MRA, but completely abolished the relaxation in the aorta. Endothelium‐independent relaxation elicited by SNP was not affected by incubation with TMAO in aortic rings, whereas it caused a significant decrease in the maximal response to SNP in the MRA. In conclusion, TMAO leads to endothelial dysfunction in aorta and MRA and impaired smooth muscle relaxation of the MRA which might contribute to the development of cardiovascular diseases.Support or Funding InformationNIH: NL13604
Small Changes of Active Site Residues of an Acinetobacter Aminoglycoside Acetyltransferase Affect Antibiotic Susceptibility
Authors: Pradhananga Anjani , Barol Irina , Stogios Peter , Pennings Pleuni , Kuhn Misty L. 
Published: April 2020

Antibiotics are drugs used to treat bacterial infections by inhibiting growth and proliferation of microorganisms. Commonly prescribed broad‐spectrum antibiotics include aminoglycosides, but many bacteria have developed resistance to these drugs by acquiring enzymes that acetylate them. We previously reported the structural and kinetic characterization of two of these enzymes from Acinetobacter. However, several outstanding questions regarding the role of specific residues in the active site of these proteins remain. In particular, we observed an active site residue that was different between these two enzymes and appeared to alter the aminoglycoside binding pocket. It has been previously shown that active site mutations within aminoglycoside acetyltransferase enzymes can adjust their ability to modify aminoglycosides and therefore their susceptibility. Since we cannot predict which point mutations will ultimately enhance their activity and therefore resistance to aminoglycosides, we performed saturation mutagenesis of this residue in the Acinetobacter haemolyticus AAC(6′)‐Ig enzyme and tested the antibiotic susceptibility of Escherichia coli to four different aminoglycosides when these mutants were overexpressed. We found the identity of the amino acid at this position was critical for altering the susceptibility of E. coli to these antibiotics. Additionally, these mutants exhibited different susceptibility patterns depending upon the identity of aminoglycoside tested. Our results show how small changes of active site residues of aminoglycoside acetyltransferase can drastically affect bacterial susceptibility to aminoglycosides, and may provide insight to which antibiotics could be effective for treating Acinetobacter infections.Support or Funding InformationResearch reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).
Diagnostic Conundrum of an Osteocartilaginous Exostosis of the Patella: An “Ancient” Osteochondroma?
Authors: Cook Ryan L. , Ohan Hovsep , Adelanwa Ayodele , Herrold Christina B. , Bacaj Patrick , Zdilla Matthew J. , Lambert H. Wayne 
Published: April 2020

The patella is an uncommon site for a tumor. This report details the incidental finding of an excrescence on the right patella identified in a cadaver during a gross anatomy course. Histological assessment revealed no periosteal surface, a very thin cartilage cap, minimal endochondral ossification, bone intermediate to more cartilage, and degenerative changes. The assessment remains unclear. What is clear, however, is that this incidental finding has served as an excellent interdisciplinary exercise for anatomists and pathologists and has benefited the education of undergraduate and graduate medical students. Accordingly, in addition to the gross pathology and histopathology, a workflow is presented.Support or Funding InformationT. Walley Williams Summer Anatomy Research Fellowship; WV Research Challenge Fund [HEPC.dsr.17.06]
Fiber‐Type Effects of KATP Channel Inhibition via Glibenclamide on the Recovery of Interstitial PO2 Following Muscle Contractions in Rats
Authors: Schulze Kiana M. , Colburn Trenton D. , Weber Ramona E. , Hageman K. Sue , Behnke Bradley J. , Poole David C. , Musch Timothy I. 
Published: April 2020

KATP channels allow influx of potassium (K+) which hyperpolarizes cell membranes. Glibenclamide (GLI) is a second‐order sulphonylurea often prescribed to Type II diabetic patients to increase insulin release via the inhibition of pancreatic KATP channels. Unfortunately, in vivo studies demonstrate that GLI impairs vasodilation and O2 delivery‐utilization matching (assessed via interstitial PO2, PO2is) in contracting skeletal muscle. Determination of GLI’s impact on PO2is kinetics (T63; time taken to reach 63% of the final response) across muscles comprised of different fiber types during post‐exercise recovery may provide novel insights into mechanisms of fatigue in patients prescribed this medication, particularly in repeated bouts of activity.PurposeTo test the hypothesis that KATP channel inhibition via GLI will slow PO2is kinetics, thus lengthening recovery time following contractions in rat fast‐(mixed gastrocnemius, MG) and slow‐(soleus, SOL) twitch muscles.MethodsIn twenty Sprague‐Dawley rats (male (n=5), female (n=8) and ovariectomized female (F‐OVX; n=7)), PO2is was determined, before and after GLI superfusion (5 mg kg−1), via phosphorescence quenching (G4). Measurements were taken for 3 min throughout the duration of, and 3 min following, the cessation of electrically‐induced contractions (180 s, 1 Hz, 7V). Blood flow was measured via fluorescent‐labeled microspheres in the last 30 seconds of contractions (15 μm).ResultsIn both muscles, PO2is recovered to its precontracting level in control and GLI conditions within the 3 min measurement window. However, GLI significantly slowed PO2is recovery following contractions in the MG (T63: 87.7 ± 4.6 vs 117.4 ± 7.9 s; p < 0.05), but not in the SOL (73.7 ± 8.8 vs 84.8 ± 11.2 s; p > 0.05). The PO2is value at which T63 occurred was not significantly different between the control and GLI conditions for MG or SOL. During contractions, blood flow was attenuated in the MG following GLI (53 ± 4 vs 39 ± 3 ml/100 g/min; p < 0.05), but not in the SOL (37 ± 4 vs 35 ± 5; p > 0.05).ConclusionThese data highlight the role of KATP channel function in supporting PO2is recovery of fast‐twitch oxidative fiber types due in part to elevated blood flow. These effects of sulphonylurea medication (i.e. GLI) may explain, in part, the early onset of exhaustion in patients performing high‐intensity exercise and/or activities of daily living that recruit fast‐twitch muscles.Support or Funding InformationSupported by NIH Grants: HL108328 (T.I.M and D.C.P) and F31HL145981 (T.D.C)
Probiotic Mitigates High Fat Diet‐Induced Mammary Gland Inflammation and Matrix Remodeling
Authors: Molnar John Christian , Homme Rubens Petit , Smolenkova Irina , Mallonee Carissa Jo , Stanisic Dragana , George Akash , Singh Mahavir , Tyagi Suresh C. 
Published: April 2020

Hidradenitis Suppurativa (HS) is a chronic inflammatory condition about which little is known. HS results from occlusion of the apocrine sweat glands of the skin. The blocked follicles will become inflamed, swell, and burst, leaving scar tissue after lesional skin heals. As the disease progresses, tracts develop beneath the skin, which connect lesional areas to new regions. MMP activity is elevated in lesional, perilesional, and tract biopsies. Obesity is known to exacerbate the condition, while the mechanism is unknown. A High Fat Diet (HFD) causes gut dysbiosis and we hypothesize that this shift in the microbiome leads to an increase in circulating inflammatory cytokines and inflammation in the mammary gland (MG), a modified apocrine gland. Mice fed a HFD showed elevated inflammatory cytokines in serum and MG tissue. MMP activity was increased in mice fed a HFD compared to normal diet (ND), but was mitigated when given probiotic (PB). MMP inhibitors such as TIMP‐3 were significantly over expressed in the HFD+PB group. Administration of probiotic and restoration of the gut microflora has a protective effect against inflammation caused by a HFD.Support or Funding InformationResearch supported by NIH grants HL74185, HL139047, and AR‐71789.
Adrenodoxin Modulates Substrate Binding in the Vitamin‐D Metabolizing Human Cytochrome P450 24A1
Authors: Jay Natalie , Estrada Fernando 
Published: April 2020

Vitamin‐D insufficiency is associated with rickets, osteomalacia, cardiovascular disease, and chronic kidney disease and affects nearly 50% of the population. In humans, vitamin D is metabolized by a series of cytochrome P450 enzymes, including inactivation by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 in humans hydroxylates either the carbon‐23 or carbon‐24 position of 1,25‐(OH)2D3 followed by a series of reactions to produce additional metabolites. The human isoform of CYP24A1 is not easily expressed and purified recombinantly, likely due to the labile nature of the enzyme. Therefore, most current structural studies are based on the rat isoform, which does not display carbon‐23 regioselective hydroxylation. As a way to study the human isoform, we have initiated use of a fusion construct of human CYP24A1 with its reduction‐oxidation protein partner adrenodoxin (Adx). The fusion product contains a short linker sequence connected to a cleavable tobacco etch virus protease recognition site, thus allowing for production and then separation of human CYP24A1 from Adx. This construct provides a platform for interrogating the structural effects of an Adx‐CYP24A1 complex on substrate, analog, and inhibitor binding in CYP24A1. Notably, we’ve observed that when Adx is present, titration with 1,25(OH)2D3 primarily populates a low‐spin state. This effect is removed upon cleavage and separation of Adx. Our interpretation is that the presence of adrenodoxin stabilizes a closed form of CYP24A1 that prevents water from departing the active site as the sixth ligand. This finding points toward close structural coupling between substrate binding and redox partner recognition in mitochondrial P450 enzymes.Support or Funding InformationR00GM112862