bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of functional partners of translation Elongation Factor 3 in Saccharomyces cerevisiae
Authors: Manzon Emily R. , Kinzy Terri Goss 
Published: April 2020

Fungal infections are responsible for 1.6 million human deaths each year. Additionally, fungal pathogens damage a significant portion of food crops. The number of effective anti‐fungal drugs available is small and many have serious side effects. Furthermore, most of these drugs target aspects of the cell membrane, offering a limited selection as cases of resistance increase. While protein synthesis is not a common focus for drug discovery because of similarities between fungi and host cells, eukaryotic Elongation Factor 3 (eEF3) is highly conserved in fungi but not found in higher eukaryotes. This makes it an excellent target for novel anti‐fungal drugs. eEF3 interacts with the ribosome during the translation elongation step of protein synthesis and is essential for yeast survival. However, little is known about the functional partners of eEF3. We are identifying genes that influence this factor with a screen for suppressors of a conditional eEF3 mutant. The mutant contains two missense mutations F803A R806A in a unique chromodomain insertion in the second ATP binding domain of eEF3 that shows significant interactions with multiple ribosome components by cryo‐EM. Through UV mutagenesis, we randomly generated putative suppressor mutations that restore growth at 37°C to the eEF3 temperature‐sensitive phenotype. Eleven strains showed improved growth rate, reduced temperature sensitivity, and many have altered susceptibility to translation‐inhibiting drugs. Whole genome sequencing of the putative suppressors showed multiple genes potentially involved in eEF3 function. Further analysis of the mutations in these proposed suppressor genes will determine their impact on cell function. These experiments will lay the foundation for the development of a novel class of anti‐fungal therapeutics that target eEF3.Support or Funding InformationNational Institutes of Health RO1 GM57483
Discordance between the neutralization profile of apixaban, betrixaban, edoxaban and rivaroxaban in the clotting assays and anti‐Xa measurements
Authors: Siddiqui Fakiha , Tafur Alfonso , Hoppensteadt Debra , Jeske Walter , Lewis Bruce , Fareed Jawed 
Published: April 2020

IntroductionOral factor Xa inhibitors such as Apixaban, betrixaban, edoxaban and rivaroxaban are widely used for the management of thrombotic and cardiovascular indications. A specific antidote namely andexanet alfa (AA) is approved for the management of bleeding complications with apixaban and rivaroxaban. The dosing regimen of andexanet alfa ranges from 400 – 800 mg bolus followed by 4 – 8 mg/min for up to two hours. This dosing regimen is projected to result in a circulating concentration of 75 – 150 ug/ml. This study is designed to compare the neutralization efficacy of andexanet alfa for various factor Xa inhibitors in clot‐based and anti‐Xa assays.Materials and MethodsCitrated pooled plasma (CPP) was prepared from individual donors. Active pharmaceutical ingredients (API) version of various factor Xa inhibitors were commercially obtained. Stock solutions of each of the agents were prepared at 1mg/ml and working concentrations at 100 and 10 ug/ml. Each of the factor Xa inhibitor was supplemented in plasma in the concentration range of 0 – 1 ug/ml. The neutralization profile of AA was studied at 100ug/ml and 200ug/ml and saline was used as a control. Clot‐based PT, aPTT and amidolytic anti‐Xa assays. The IC50 for the Xa inhibitory activity were calculated for the control drugs and after supplementation with AA. The neutralization profile in the clot‐based assays was measured in terms of decrease in clotting times.ResultsApixaban and edoxaban exhibited similar inhibitory profiles in the anti‐Xa assays, where as rivaroxaban and betrixaban exhibited much higher IC50s. AA effectively neutralized the anticoagulant effects of all agents at both the 100 and 200 ug/ml, however the neutralization profile in the clot based was not proportional to the anti‐Xa effects. At 200ug/ml only betrixaban was completely neutralized whereas all the other agents were partially neutralized. The order of neutralization at 200ug/ml was betrixaban > rivaroxaban > apixaban > edoxaban.ConclusionsThese studies show that the neutralization profiles of various Xa agents by AA exhibit differential characteristic response for each of the individual agent. These results indicate that individual dosing based on clinical endpoints for each agent rather than the neutralization of anti‐Xa activity measurement may be more relevant to the clinical outcome.
Effects of aging on substantia nigra dopamine neuron excitability and inhibitory synaptic transmission in mice
Authors: Howell Rebecca Dean , Beckstead Michael 
Published: April 2020

The degeneration of substantia nigra dopamine neurons is involved in the etiology for Parkinson’s disease, which is characterized by progressive loss of motor and cognitive functions. The biggest risk factors for Parkinson’s disease are age and sex; most cases occur after age 60 and prevalence is almost two times greater in men than women. While much research in Parkinson’s has focused on the molecular mechanisms underlying dopamine neuron degeneration, very little work has considered the influence of sex and normal aging on the functioning of dopamine neurons, which may contribute to disease risk and presentation. In this work, we studied intrinsic firing properties and synaptic transmission to substantia nigra dopamine neurons with in vivo single unit recordings and whole cell patch clamp recordings in brain slices in male and female C57Bl/6 mice at ages 4, 18 and 24 months. Dopamine neurons recorded from male mice ages 18 months and older showed disruptions in firing activity and an increased propensity to depolarization block compared with younger males, while neurons from females remained largely unchanged across ages. Also, preliminary data suggest enhanced GABAergic transmission in dopamine neurons with age in males; likely a compensatory mechanism to balance the increased dopamine neuron excitability in order to maintain normal motor functioning. Understanding how aging affects dopamine neuron functioning, as well as the balance of excitatory and inhibitory input can help determine areas in the nigrostriatal circuity that are vulnerable to impaired functioning, such as in Parkinson’s disease, as well as therapeutic targets.Support or Funding InformationNIA grant ‐ R01 AG052606, Presbyterian Health Foundation, Oklahoma Center for Adult Stem Cell Research (OCASCR)
A Comparison of Mass and Potency Equated Bovine and Porcine Heparins
Authors: Krishnappan Sharan , Siddiqui Fakiha , Farooqui Ambar , Iqbal Omer , Kouta Ahmed , Hoppensteadt Debra , Jeske Walter , Walenga Jeanine , Fareed Jawed 
Published: April 2020

IntroductionHeparins are extracted from the mast cell rich mammalian tissues such as lung and intestine. While the current source of pharmaceutical grade heparin nationwide is primarily derived from porcine mucosa, bovine mucosal heparins (BMH) are currently being considered by the FDA for re‐introduction in medical and surgical procedures as an alternative to porcine mucosal heparins (PMH). We hypothesized that at USP potency equated levels, bovine and porcine mucosal heparins should be comparable, however at gravimetric concentrations, mass adjusted porcine heparins should exhibit stronger anticoagulant effects in comparison to bovine heparins. The purpose of this study is to compare the clotting profiles and biological properties of mass and USP potency equated BMH and PMH.Materials and MethodPowdered form of pharmaceutical grade heparins of BMH (140 U/mg) and PMH (190 U/mg) origin were obtained from commercial vendors and stock solution was prepared in saline. Powdered protamine sulfate (USP grade) was reconstituted at 1.0 mg/ml in saline. Whole blood thromboelastographic studies were carried out in TEG 5000 Hemostasis system. Clotting (thrombin time; TT and activated partial thromboplastin time; aPTT) and anti‐protease (anti‐Xa and anti‐IIa) assay were measured by using ACL instrument. Relative neutralization by protamine sulfate was carried out at a fixed concentration of 10 ug/ml.ResultsIn whole blood TEG, clotting profile (TT and aPTT) and anti‐protease (anti‐Xa and anti‐IIa), USP potency equated BMH and PMH at concentration range of 0 – 1.0 U/ml showed comparable effects. Protamine sulfate at 10 ug/ml completely neutralized BMH and PMH in in a comparable fashion in clotting profile (TT and aPTT) and anti‐protease (anti‐Xa) while drugs were only partially neutralized in anti‐IIa assay. In mass ratio at 0 – 10 ug/ml, PMH showed strong effects in TEG, anti‐protease (anti‐Xa and anti‐IIa) and clotting profile (TT and aPTT) compare to BMH. BMH and PMH were completely neutralized by protamine sulfate in clotting assay, however in anti‐protease assay BMH was completely neutralized while PMH was only partially neutralized at higher concentrations.ConclusionThese studies suggest that mass porcine heparin exhibits strong effects compared to mass bovine heparin in gravimetric ratios. However, at potency adjusted levels, bovine and porcine heparins exhibit comparable effects sand can be considered biosimilar. These studies conclude that bovine heparin can be an alternative source of heparin to the prominent porcine heparin.
Development of Phenylazo Piperidine Triazenes for Initiated Release of Aryl Diazoniums in Physiologically Relevant Conditions
Authors: Morrow Madeline Gladys , Davis Garrett J. , Jewett John 
Published: April 2020

Triazenes can easily be synthesized from various anilines via diazotization and conjugation with secondary amines, including piperidine. Previous literature has noted that such triazene formation can act as a protecting mechanism for aryl diazoniums that can be subsequently released in highly acidic conditions. Previous work in our own lab pertaining to triazabutadienes (TBDs) has shown analogous reactivity, whereas aryl diazoniums can be released under various conditions including low pH, or by irradiation with UV light. Such initiated release shows potential advantages for studying various biological systems. In this work we show that a class of phenylazo‐piperidine triazenes have similar potential utility to be used as protected diazoniums for biological study. We were able to easily synthesize a small library of triazenes with various electronic properties including those with benzene, p‐nitrobenzene, p‐benzamide, and m‐methoxy substituents, among others. Subsequent studies on these compounds showed that the electronics of the functional groups alters the stability and subsequent reactivity of the respective triazenes allowing for different rates of aryl diazonium release. We have seen that decreasing electron density within the aryl ring leads to more stable, and less reactive triazenes, whereas increasing the electron donating potential of these substituents increases reactivity. Furthermore, we have shown that these triazenes are capable of releasing their diazonium species in neutral pH via irradiation with 350 nm UV light, a phenomenon that has not been previously reported to our knowledge. Lastly, we report the ability for these triazenes to label proteins in situ using UV irradiation and display the utility of a novel coumarin triazine that has analogous reactivity to previously reported coumarin triazabutadienes developed in our lab.Support or Funding InformationNSF CAREER Award
Protein Tyrosine Phosphatase Type 1B (PTP1B) Contributes To Atherosclerotic Processes By Mechanisms That Involve NADPH‐Oxidase And Calcium Influx
Authors: da Silva Josiane Fernandes , Alves Juliano Vilela , Bolsonni Juliana A. , Costa Rafael M. , Rios Francisco J. , Camargo Livia L. , Montezano Augusto C. , Touyz Rhian M. , Tostes Rita C. 
Published: April 2020

PTP1B is a protein tyrosine phosphatase (PTP) widely expressed in different cell type including vascular smooth muscle and endothelial cells. PTP1B activation is linked to downregulation of insulin signalling, impairment of nitric oxide production, induction of endoplasmic reticulum stress and angiogenesis. While inhibition of PTP1B decreases atherosclerotic plaque size and increases systemic levels of interleukin 10 (IL‐10), effects on vascular function are unclear. This study tested the hypothesis that PTP1B inhibition attenuates atherosclerosis‐associated vascular dysfunction by reducing endothelial ROS production.MethodsMale 12 week‐old apolipoprotein E knockout (apoEKO) and wild type (WT) mice were fed a control diet or a high fat (HF) diet (15% cocoa butter; 0.5% cholesterol; 10% protein; 25% carbohydrate; 65% fat; 9,6 kcal/g) for 8 weeks. Animals were treated with the PTP1B inhibitor claramine, 2 mg/Kg/week, i.p., for 3 weeks. Glucose, cholesterol, low density lipoprotein (LDL) and triglycerides (TG) were measured. In vitro studies were performed in human aortic endothelial cells (HAEC) stimulated with lysophosphatidicholine (10−5 M). ROS production was evaluated by lucigenin and DHE fluorescence. Aortic reactivity was evaluated using isometric transducer myograph. Global PTP irreversible oxidation was determined by immunoprecipitation and western blot. PTP, VCAM, NOX4, NOX5 mRNA expression was determined by qPCR. Phosphorylated protein levels, PTP activity, ROS production and calcium influx were evaluated in stimulated HAECs ± PTP1b inhibitor or vehicle. Experiments were approved by the Ethics Committee on Animal Research of the Ribeirao Preto Medical School, University of Sao Paulo (protocol n°106/2019).ResultsApoEKO mice showed dyslipidaemia, characterized by increased serum levels of cholesterol (WT = 154.7 ± 2.8 vs. apoEKO = 1668.0 ± 26.7 mg/dL) and LDL (WT = 86.5 ± 1.4 vs. apoEKO = 976.2 ± 17.8 mg/dL). Aortic arch and carotid artery atherosclerotic plaque areas showed higher PTP1B, NOX4 and VCAM gene expression than control areas (p < 0.05). Vasorelaxation to acetylcholine was decreased in apoEKO mice vs. WT mice. Claramine restored this response. In HAECs, LPC stimulated ROS generation by NOX5‐dependent mechanisms. LPC also induced PTP1B expression and activation. PTP1B inhibition in HAECs reduced ROS production and normalized NO production and intracellular calcium responses.ConclusionPTP1B contributes to endothelial dysfunction by mechanisms that involve ROS production. PTP1B inhibition may be targeted to reduce vascular injury in atherosclerosis.Support or Funding InformationFAPESP
In Female Rats, the Sympathoexcitatory and Pressor Actions of Arcuate Nucleus Angiotensin II Vary During the Estrus Cycle and are Mediated by Inhibition of Neuropeptide Y, and Excitation of α‐Melanocyte Stimulating Hormone, Projections to the Hypothalamic Paraventricular Nucleus
Authors: Shi Zhigang , Wong Jennifer , Brooks Virginia L. 
Published: April 2020

In male rats, nanoinjections of Angiotensin II (AngII) into the arcuate nucleus (ArcN) increases sympathetic nerve activity (SNA) by suppressing tonic sympathoinhibitory Neuropeptide Y (NPY) inputs, and activating excitatory pro‐opiomelanocortin (POMC) inputs that release alpha melanocyte stimulating hormone (α‐MSH), into the hypothalamic paraventricular nucleus (PVN). In female rats, ArcN AngII AT1a receptor (AT1aR) expression is generally higher than in males, but varies with the estrus cycle (highest during estrus; lowest during proestrus). However, whether ArcN AngII increases SNA in females, or if the response varies during the cycle, is unknown. Therefore, we tested bilateral ArcN AngII injections (30 pmol in 30 nL) in α‐chloralose anesthetized female rats throughout the reproductive cycle, and found that 90 min after ArcN AngII injections, LSNA increased (P<0.05) in rats during estrus (by 72±12 %; n=6) and diestrus (by 40±9 %; n=4), but not proestrus (−1±8 %; n=5). ArcN AngII also induced an initial rapid rise (P<0.05) in mean arterial pressure (MAP) in all groups (estrus: 13±2 mmHg; diestrus: 11±3 mmHg; proestrus: 13±3 mmHg), but the increase was sustained only in estrus rats (by 12±2 mmHg; P<0.05). We next tested if ArcN AngII suppresses inhibitory NPY inputs by determining if blockade of PVN NPY Y1 receptors (Y1R) with bilateral nanoinjections of BIBO3304 (60 pmol/60nL) elicits smaller increases in MAP and LSNA 60 min after nanoinjection of AngII into the ArcN. As previously reported, PVN injections of BIBO3304 (without ArcN AngII; n=3) increased (P<0.05) LSNA (by 34±6 %) and MAP (by 10±1 mmHg). However, the increases (P<0.05) in LSNA (to 93±16 %) and MAP (by 19±1 mmHg) (n=4) following ArcN AngII and PVN BIBO3304 were the same as ArcN AngII followed by PVN artificial cerebrospinal fluid (n=3; LSNA by 107±30 % and MAP by 17±4 mmHg). Thus, ArcN AngII markedly attenuated the pressor and sympathoexcitatory effects of blockade of PVN Y1R, indicating that AngII increases LSNA and MAP in part by inhibition of NPY projections to the PVN. Finally, because suppression of tonic NPY inhibition of PVN presympathetic neurons permits increased activation of PVN α‐MSH receptors (MC4R), we tested if blockade of PVN MC4R with SHU9119 decreases LSNA and MAP 90 min after ArcN AngII (n=4). Indeed, bilateral PVN SHU9119 decreased (P<0.05) LSNA (by 30±10 %) and MAP (by 10±1 mmHg). In conclusion, in females as in males, ArcN AngII increases SNA and MAP by inhibiting NPY inputs and increasing α‐MSH inputs into the PVN. However, these effects vary during the estrus cycle in parallel to AngII AT1aR expression: highest during estrus and undetectable during proestrus.Support or Funding InformationHL128181
Prior Ablation of the Splanchnic Sympathetic Nerves Increases Plasma Inflammatory Cytokine Levels and Suppresses Bacteremia in Response to Systemically Administered E. coli in Sheep
Authors: McKinley Michael J. , Almeida Kaylani , Bedoui Sammy , Farmer David G.S. , Hocking Dianna , Lankadeva Yugeesh , Martelli Davide , May Clive N. , Robins-Browne Roy M. , McAllen Robin M. 
Published: April 2020

The splanchnic sympathetic nerves exert a powerful anti‐inflammatory action in response to systemic administration of lipopolysaccharide in rats (Martelli et al. J. Physiol. 592: 1677–1686, 2014). However, it is not known how this endogenous anti‐inflammatory influence of the splanchnic nerve affects the body’s ability to fight infection. The aim of this study was to determine if and how the endogenous reflex activation of the splanchnic anti‐inflammatory pathway affects blood cytokine levels and the disease course of bacteremia after intravenous infusion of live E. coli into a conscious large animal (sheep). One month prior to infection, the greater splanchnic nerve was cut bilaterally (SplancX, n = 8) or sham surgery (Sham, n = 7) performed in anesthetized sheep (isoflurane/O2). After 1 month, these conscious sheep were infused with live E. coli (2.8 × 109 CFU in 30 min) into a jugular vein. Blood samples were obtained at 1.5, 3, 6, 24 and 48 h after bacterial injection. As expected, i.v. infusion of E. coli caused systemic bacteremia with high blood counts of E. coli [3.02 ± 0.64 Log10(CFU/ml + 1), mean ± sem] in 5 of 7 sham sheep. By contrast, no significant bacteremia was observed in any of the 8 SplancX sheep (0.18 ± 0.18 Log10[CFU/ml + 1], p < 0.01, c.f. Sham sheep). Plasma levels of tumour necrosis factor‐α peaked after 1.5 h, and were significantly greater in SplancX vs Sham sheep (2.4 ± 0.6 vs 1.5 ± 0.2 ng/ml, p < 0.05) at this time. Plasma levels of another pro‐inflammatory cytokine, interleukin‐6, peaked at 6h and were higher in SplancX vs Sham sheep (7.4 ± 0.6 vs 2.4 ± 0.6 ng/ml, p < 0.01). Although plasma levels of the anti‐inflammatory cytokine interleukin‐10 increased in both groups, they were lower in SplancX vs Sham sheep from 1.5 h to 24 h (2.9 ± 0.4 vs 6.7 ± 1.1 ng/ml at 24 h, p < 0.01). All plasma cytokine levels measured had returned to baseline by 48 h. Plasma cortisol levels were higher (p<0.01) in SplancX sheep only at 6 h (298 ± 29 vs 138 ± 37 ng/ml), while the fever was less (p < 0.05) in SplancX at 6 h (40.3 ± 0.3 vs 40.8 ± 0.3°C). The greater pro‐inflammatory response in splanchnic denervated animals most likely contributes to their enhanced ability to clear bacteremia. The present findings demonstrate the powerful consequences of neural control of inflammation in the context of an acute systemic infection.Support or Funding InformationSupported by the National Heart Foundation of Australia
A reliable LC‐MS/MS method for targeted analysis of amino acid metabolome: Method validation and application to studies on amino acid dynamics during tumor progression
Authors: Liu Zhenzhen , Tu Mei-Juan , Zhang Chao , Jilek Joseph Lionel , Zhang Qian-Yu , Yu Ai-Ming 
Published: April 2020

A simple and fast LC‐MS/MS method was developed and validated for targeted quantification of 20 proteinogenic L‐amino acids (AAs) in mouse plasma. Chromatographic separation was achieved on an Intrada Amino Acid column within 13 min via gradient elution with an aqueous solution containing 100 mM ammonium formate and an organic mobile phase containing acetonitrile, water and formic acid (v: v: v = 95: 5: 0.3), at the flow rate of 0.6 mL/min. Individual AAs and corresponding stable‐isotope‐labeled AA internal standards were analyzed by multiple reaction monitoring (MRM) in positive ion mode under optimized conditions. Method validation consisted of linearity, sensitivity, accuracy and precision, recovery, matrix effect, and stability, and the results demonstrated this method as a specific, accurate, and reliable assay. This LC‐MS/MS method was thus utilized to examine the dynamics of plasma AA metabolome during tumor progression in an orthotropic hepatocellular carcinoma (HCC) xenograft mouse model. The results reveal a significantly lower Arg concentration as well as higher Ala and Thr levels during HCC progression. These findings support the utilities of this LC‐MS/MS method and the promise of specific AAs as possible biomarkers for HCC.Support or Funding InformationNational Cancer Institute (grant No. R01CA225958)National Institute of General Medical Sciences (R01GM113888)