bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and Functional Insights into TAZ2‐mediated CBP/p300 Recruitment by the Melanogenic Transcription Factor MITF
Authors: Brown Alexandra , Vergunst Kathleen , Branch Makenzie , Baillie George , Dupre Denis , Langelaan David 
Published: April 2020

The microphthalmia‐associated transcription factor (MITF) is a master regulator of development and differentiation within the melanocyte lineage. However, aberrant MITF activity can lead to multiple malignancies such as skin cancer, where it plays a key role in modulating the proliferation and invasiveness of melanoma. MITF is a basic helix‐loop‐helix leucine zipper transcription factor that binds to specific gene promoters via a central DNA‐binding domain. MITF also recruits transcriptional co‐activators, such as the histone acetyltransferase CREB‐binding protein and its homologue p300 (CBP/p300) through an N‐terminal acidic transactivation domain (TAD), however the details of these interactions are not yet fully understood. In order to gain insight into the mechanisms of gene regulation by MITF, we investigated the structure and functional interaction between MITF‐TAD and the transcription adapter putative zinc finger (TAZ2) domain of CBP/p300. In mammalian‐one‐hybrid assays MITF transcriptional activity was enhanced in the presence of co‐transfected CBP/p300 and abolished upon deletion of residues within the MITF‐TAD. Peptide microarrays indicated that no one residue of MITF is essential for TAZ2 binding, however, deletion of multiple residues in MITF‐TAD ablated its ability to bind TAZ2. NMR‐based chemical shift mapping experiments determined that MITF‐TAD interacts with the same surface of TAZ2 as the adenoviral protein E1A, which has been shown to inhibit MITF function. We determined that E1A and MITF‐TAD directly compete for CBP/p300 through the TAZ2 domain using NMR‐based titrations, pulldown, and mammalian‐hybrid assays. Furthermore, we used qPCR to measure the effect of disrupting these interactions on the transcription of MITF‐specific target genes in melanoma‐derived cell lines. Understanding mechanistic details regarding the interaction between MITF and its co‐activators is fundamental to our understanding of gene regulation by MITF and may outline a potential new strategy to inhibit MITF function.Support or Funding InformationThis research was funded by the Beatrice Hunter Cancer Research Institute, Nova Scotia Heath Research Foundation Scotia Scholars Award, a Canadian Institutes of Health Research studentship, Dalhousie Nova Scotia Graduate Scholarship, and Izaak Walton Killam Predoctoral Scholarship.
"Effects of the Human SULT1A1 Polymorphisms on the Sulfation of 4‐hydroxytamoxifen and Endoxifen"
Authors: Rasool Mohammed Ibrahim , Liu Ming-Cheh 
Published: April 2020

BackgroundHuman sulfotransferase 1A1 (SULT1A1) catalyzes the sulfation of a wide variety of substances including 4‐hydroxytamoxifen (4‐HT) and endoxifen, the active metabolites of tamoxifen. Polymorphisms were reported in SULT1A1 gene. A functional single nucleotide polymorphism (SNP) in the SULT1A1 gene (R213H) showed lower catalytic activity and thermal enzyme stability. Previous data have suggested that this particular genotype may account for a portion of individual variation in responding to tamoxifen therapy. The current study was performed to investigate the effects of SNPs of human SULT1A1 genes on the enzymatic characteristics of the sulfation of 4‐HT and endoxifen by SULT1A1 allozymes.MethodsOnline databases were searched for SULT1A1 nonsynonymous coding SNPs. Nine SULT1A1 allozymes coded by missense SNPs were selected based on the predicted importance of the amino acid variations, were generated, bacterially expressed and purified by glutathione‐Sepharose affinity chromatography. Purified SULT1A1 allozymes were analyzed for sulfating activity using an established assay procedure.ResultsSpecific activity obtained showed the differential sulfating activities of SULT1A1 allozymes toward both antiestrogenic compounds.ConclusionThe activity data obtained for the SULT1A1 allozymes analyzed indicated the importance of the amino acid residues in the proper functioning of the SULT1A1 enzyme, which may affirm the variability in the metabolism of tamoxifen that could affect its efficacy in individuals with different SULT1A1 genotypes.Support or Funding InformationThis work was supported in part by a grant from National Institutes of Health (Grant #R03HD071146).
Kv7.1 Channels Contribute to Neonatal Renal Blood Flow Autoregulation Induced by a Step Decrease in Arterial Pressure
Authors: Peixoto-Neves Dieniffer , Soni Hitesh , Adebiyi Adebowale 
Published: April 2020

Kv7 channels, the voltage‐gated potassium channels encoded by KCNQ genes, are expressed in a wide variety of blood vessels, including coronary, mesenteric, cerebral, and renal arteries. In adult rodents, Kv7 channels mediate heterogeneous basal and agonist‐induced vascular reactivity. Although postnatal changes in functional expression of mammalian Kv7 channels have been reported, the physiological significance of these channels in neonatal renal microvessels is unknown. The objective of this study was to determine whether Kv7 channels regulate neonatal renal microcirculation. Polymerase chain reaction indicated that out of the five members of KCNQ genes, only KCNQ1 (Kv7.1) is expressed in smooth muscle cells (SMCs) isolated from preglomerular microvessels of neonatal pigs. Confocal microscopy demonstrated that Kv7.1 is expressed in the plasma membrane of renal vascular SMCs. Kv7.1 channel activators induced whole‐cell currents in neonatal pig renal vascular SMCs, which were inhibited by HMR1556 (HMR), a selective Kv7.1 inhibitor. However, HMR did not cause a significant change in steady‐state diameters of neonatal pig distal interlobular arteries that were pressurized to physiological renal arterial pressure. Also, intrarenal artery infusion of HMR did not alter the mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance in the pigs. An approximately 20 mmHg reduction in the MAP evoked effective autoregulation of the RBF in neonatal pigs, which was inhibited by HMR. Together, these findings suggest that renal vascular SMC Kv7.1 does not control basal renal vascular tone in neonatal pigs, but contribute to RBF autoregulation induced by a step decrease in arterial pressure.Support or Funding InformationAmerican Heart Association (AHA), National Institutes of Health (NIH).
19‐(S/R)Hydroxyeicosatetraenoic Acid is a Novel Endogenous Inhibitor of Cytochrome P450 1B1 in Enantioselective Manner
Authors: Shoieb Sherif , El-Sherbeni Ahmed A. , El-Kadi Ayman O. S. 
Published: April 2020

PurposeCYP1B1 is known to contribute to the pathogenesis of several diseases such as cancer and cardiac hypertrophy by forming the toxic metabolites. We recently reported that the arachidonic acid metabolite, 19(S/R)hydroxyeicosatetraenoic (HETE) acid, confers cardioprotection against cardiac hypertrophy by inhibiting the formation of cardiotoxic metabolites, midchain HETEs, known to be formed by CYP1B1. This raised the question whether 19(S/R)‐HETE can directly inhibit the CYP1B1.MethodsHuman ventricular cardiomyocytes, RL‐14 cells were treated with vehicle or 10 μM Ang II in the absence and presence of 20 μM 19(R)‐HETE or 19(S)‐HETE for 24 h. Gene and protein expression were measured using real‐time PCR and Western blot analysis, respectively. The level of mid‐chain HETEs was determined using liquid chromatography–mass spectrometry (LC/MS). The O‐dealkylation rate of 7‐ethoxyresorufin (EROD) by recombinant human CYP1B1 was measured in the absence and presence of 19(R)‐HETE or 19(S)‐HETE.ResultsThe results showed that both 19(R)‐HETE and 19(S)‐HETE significantly decreased the metabolite formation rate of midchain HETEs, namely 8‐, 9‐, 12‐ and 15‐HETE compared to control group. Nonlinear regression analysis and comparisons showed that the mode of inhibition for 19(R)‐HETE and 19(S)‐HETE is non‐competitive inhibition of CYP1B1 enzyme. Dixon plots showed that 19(R)‐HETE and 19(S)‐HETE have Ki values of 89.1 and 37.3 nM, respectively. The Ki values of both enantiomers showed that the S‐enantiomer is more potent than the R‐enantiomer by approximately 2.4 fold.ConclusionThe current study suggests that 19(R)‐HETE and 19(S)‐HETE could be considered a novel therapeutic modality in the treatment of cardiac hypertrophy, and cancer. Moreover, given that a non‐competitive inhibitor might bind to the enzyme regulatory region, 19(R)‐HETE and 19(S)‐HETE could be the focus for unraveling the obscure mechanisms of CYP1B1 enzymatic reaction.Support or Funding InformationThis work was supported by a grant from CIHR to A.O.S.E. S.M.S. is the recipient of Alberta Innovates Graduate Student Scholarship.
Effects of Wastewater Treatment Plant Effluents on the Microbial Biomass and Community Structure of the Big Walnut Creek
Authors: Atkinson Sophia , Luchmun Hashleyna , Akinwole Philips 
Published: April 2020

Excessive organic matter input and nutrient loading are considered a major ongoing threat to water quality. Communal wastewater treatment is a process whereby sewage water is treated to remove contaminants before being discharged back to the environment. As most Wastewater Treatment Plants (WWTPs) do not remove all contaminants from sewage waters, we examined the impact of WWTPs effluents on microbial biomass and community structure of the Big Walnut Creek, Greencastle, IN. We took triplicate sediment samples along a 6,000 meter transect at five different locations, two upstream and three downstream of the outfall in June, September, and November of 2019. We quantified microbial community structure as community level physiological profiling (CLPP) using Biolog EcoPlates as well as microbial biomass using phospholipid phosphate analysis. The samples in June right above and below the outfall had comparably lower carbon to nitrogen (C/N) ratios (39.63 and 32.44), and therefore more available nutrients for microbial uptake. The samples further downstream from the outfall (1,452 m and 4,677 m) had high C/N ratios of 65.8 and 88.09 respectively, while samples farther upstream from the outfall (1,600 m) had a C/N ratio of 93.53. Microbial biomass increased from June to November for all five sites due to seasonal allochthonous material inputs. We noticed that the C/N ratio correlated to the increase in microbial biomass observed at the outfall for the June samples. Interestingly, the further away from the outfall, the lower the biomass measured and the higher the C/N ratios. We did not observe any significant difference in microbial biomass across sites during the months of September and November. Increased inputs of allochthonous materials during these months (falling leaves and algae) might have obscured the effects of the WWTP effluent. Principal component analysis (PCA) of the CLPP showed two distinct microbial communities for samples collected at the outfall across seasons and separately grouped together microbial communities from non‐outfall samples. PC 1 explained 28.04% variation observed in microbial community and the most dominant microbial groups on the positive component of PC 1 includes Corynebacteriaceae, Acetobacteraceae, and Lactobacillaceae. Overall, this study showed that WWTP effluent impacts the immediate surroundings of its input, but can be obscured by allochthonous materials or biological transformation of nitrogen downstream. Further research should look into antibiotic resistant genes and resistant bacteria at these separate sites to see if WWTP effluents might have further health impacts on the stream ecology of the Big Walnut Creek.Support or Funding InformationSupport from the Biology Department and the Science Research Fellows program at DePauw University.
Capillary Oxygen Triggers Conducted Hyperpolarization and Dilation Independent of Extracellular K+ and Endothelial KIR2.1
Authors: Kowalewska Paulina M. , Milkovich Stephanie , Ellis Christopher G. , Welsh Donald G. 
Published: April 2020

The matching of red blood cell (RBC) supply to oxygen demand is an intricate process which requires generation of a capillary stimulus and triggering of a transduction process that dilates upstream arterioles. The sensor’s identity, the stimulus it generates and the mechanisms that enables it are openly debated and a source of active inquiry. This study tested whether extracellular K+ is the stimulus and if capillary KIR2.1 channels enable upstream arteriolar dilation via conduction. Experiments were performed in vivo, using a live microcirculatory preparation (extensor digitorum longus); the local oxygen environment was tightly controlled (custom stage insert) and capillary RBC kinetics monitored in mice lacking endothelial KIR2.1 or connexin 40. Dropping PO2 on the muscle surface (53 mmHg to 15 mmHg or 0 mmHg (3 min)) induced a stark microvascular response in control animals (RBC supply rate, velocity and hematocrit increased by 56%, 28% an 18%, respectively) without diminishing mitochondrial respiration. This blood flow response failed to materialize in connexin 40−/− mice. As this response was absent, capillary RBC O2 saturation was lower under normal conditions in the connexin 40−/− animals. In contrast, endothelial KIR2.1−/− mice reacted normally to O2 challenges, with RBC supply rate, velocity and hematocrit rising by 43%, 28%, and 11%, respectively. These findings show that coupling O2 demand‐to‐O2 supply requires coordinated electrical signaling among cells connected by gap junctions comprised of connexin 40. They also show that endothelial KIR2.1 channels are not responsible for initiating the hyperpolarization needed to drive conduction. These findings begin the process of restructuring our understanding of blood flow regulation and how O2 initiates this process independent of metabolite production.Support or Funding InformationThis work was funded by NSERC Discovery grants to CGE and to DGW.
Vascular Dysfunction and Posttraumatic Stress Disorder: Examining the Role of Oxidative Stress and Sympathetic Activity
Authors: Weggen Jennifer B. , Autler Aaron S. , Hogwood Austin C. , Darling Ashley M. , Decker Kevin P. , Garten Ryan S. 
Published: April 2020

PurposeThe physiological manifestations of posttraumatic stress disorder (PTSD) have been associated with an increase in risk of cardiovascular disease (CVD) independent of negative lifestyle factors. The goal of the study was to better elucidate the mechanisms behind the increased CVD risk by examining peripheral vascular function, a precursor to CVD. Moreover, this study sought to determine the role of oxidative stress and sympathetic nervous system (SNS) activity in PTSD‐induced vascular dysfunction.MethodsSixteen individuals with PTSD (10 women, 6 men; age 24 ± 4 years), and twenty‐four healthy controls (CTRL; 15 women, 9 men, 24 ± 4 years), participated in the study. The PTSD group participated in two visits, consuming either a placebo or antioxidant cocktail (AO ‐ vitamins C and E and alpha lipoic acid) prior to their visits, in a randomized order. Arm vascular function was assessed via the reactive hyperemia‐induced flow mediated dilation of the brachial artery (BAFMD) technique and evaluated with Doppler ultrasonography. Brachial artery and arm microvascular function were determined by percent change of diameter from baseline normalized for BA shear rate (BAD/Shear), and blood flow area under the curve (BF AUC), respectively. Heart rate variability (HRV) was used to assess autonomic nervous system activity.ResultsBF AUC was significantly lower (p = 0.02) and SNS activity was significantly higher (p = 0.003) in the PTSD group when compared to the CTRL group. BAD/Shear was not different between groups. Following the acute AO supplementation, BF AUC was augmented and SNS activity was reduced, to which it was no longer significantly different (BF AUC: p = 0.16, SNS activity: p=.41) when compared to the CTRL group.ConclusionYoung individuals with PTSD demonstrated lower arm microvascular, but not brachial artery, function as well as higher sympathetic activity when compared to healthy controls matched for age, sex, and physical activity level. Furthermore, this microvascular dysfunction and SNS activity was attenuated by an acute AO supplementation to the level of the healthy controls. Taken together, this study revealed that the modulation of oxidative stress, via an acute AO supplementation, improved vascular dysfunction in individuals with PTSD, potentially by reducing the substantial SNS activity associated with this disorder.
Characterizing the Effect of the Hypoxic Vasoconstriction Response Curve on Blood Flow Distribution and Oxygenation in the Lung
Authors: Roy Tuhin K. , Secomb Timothy W. 
Published: April 2020

The ability of the lung to take up oxygen can be characterized by its effective diffusing capacity, which is determined by the transport characteristics of the alveolar‐capillary membrane as well as heterogeneity of pulmonary blood flow. At rest, the healthy lung has a high degree of tolerance for pulmonary flow heterogeneity, but under conditions of physiological stress such as exercise, oxygen uptake may be inadequate to satisfy tissue demand if flow is very heterogeneous. We previously used a mathematical model of pulmonary oxygen uptake to examine the ability of hypoxic pulmonary vasoconstriction (HPV) to mitigate flow heterogeneity. In the model, flow heterogeneity is characterized by the coefficient of variation (CV) of pulmonary capillary blood flow. Assuming an initial lognormal distribution of blood flow with a normal level of heterogeneity (CV = 1.66), we simulate flow regulation over a range of oxygen consumption from rest to maximal exercise by adjusting pulmonary flow resistance in each vessel in accordance with an HPV response curve obtained from previously published experimental data. The effect of HPV on vessel flow resistance is assumed to be characterized by a sigmoidal response dependent on the capillary oxygen tension, which is approximated as the mean of the venous and arterial values in each pulmonary capillary; its maximal sensitivity occurs at a stimulus oxygen tension of approximately 30 torr. The purpose of the present study was to determine whether altering the shape of the HPV response curve would result in an improvement in CV reduction. Altering the HPV by shifting the sigmoidal curve to the right by 10 or 20 torr worsened CV, extraction and total pulmonary vascular resistance (PVR). Similar detrimental results were obtained when the range of oxygen tensions over which HPV varied was expanded by a factor of two, increasing the oxygen tension at which the sensitivity of HPV to oxygen tension was maximal. On the other hand, shrinking the range of variation of the HPV curve by a factor of two neither improved nor significantly worsened the CV or extraction, and resulted in a PVR distribution similar to the case of the normal HPV curve. These results demonstrate that the observed shape of the HPV response tends to minimize heterogeneity of pulmonary flow under conditions of exercise.Support or Funding InformationSupported by NIH U01 HL133362.
Mechanosensitive Mechanism of Lung Regeneration through Endothelial YAP1
Authors: Hendee Kathryn , Mammoto Tadanori , Mammoto Akiko 
Published: April 2020

Angiogenesis, the formation of new blood capillaries, plays a key role in organ development, homeostasis and regeneration. In addition to chemical factors, mechanical forces control angiogenesis and organ regeneration. We have reported that the mechanosensitive transcriptional co‐activator Yes‐associated protein (YAP1) in endothelial cells (ECs) is necessary for post‐pneumonectomy (PNX) lung growth. Right pulmonary artery (PA) pressure and shear stress increase after left unilateral PNX. However, it remains unknown whether changes in PA pressure and shear stress after PNX control post‐PNX regenerative lung growth through endothelial YAP1 signaling. Here we found that ligation of the right cardiac lobe PA after left PNX further increases right PA pressure and shear stress, upregulates expression of YAP1 and angiogenic factor Tie2, and stimulates post‐PNX lung growth in the remaining non‐occluded lobes. These effects are attenuated in VE‐cadherin‐specific Yap1 knockout mice. Hydrostatic pressure or shear stress applied to cultured human pulmonary arterial ECs also increases the expression and activity of YAP1 and induces EC proliferation and migration in vitro. These results suggest that increases in the right PA pressure together with elevation in shear stress after left PNX stimulate angiogenesis and post‐PNX lung growth through endothelial YAP1. Modulation of the mechanical environment or endothelial YAP1 expression/activity could be an efficient strategy for regenerative lung growth.Support or Funding InformationNIH RO1HL142578
Optimal Cutting Temperature Medium‐Embedding Is a Valid Method for Storing and Preparing Myocardial Biopsies Preceding Myofilament Function‐Assessment
Authors: Isackson Henrik , Ng Henry , Agic Mediha Becirovic , Hultström Michael 
Published: April 2020

Background and objectiveOptimal Cutting Temperature (OCT) media is commonly used for histological characterisation. Myocardial tissues from clinical biopsies are precious and thus utilising them for both histological and functional assessment is an attractive prospect. This study was designed to assess whether OCT media stored samples can be used for preparing myocardial tissue for myofilament function‐assessment.MethodsLiquid nitrogen (LN) – and OCT‐stored myocardium from mouse was compared using a permeabilised fibre set‐up for functional characterisation of thin and thick filament‐interaction including Ca2+ sensitivity (pCa50), cooperativity along the thin filament (nH), cross‐bridge cycling rate (ktr), Ca2+ activated maximum tension (Fmax)/cross sectional area, cross‐bridge recruitment upon Ca2+ activation, as well as passive strain (kstrain) and the relative titin contribution to passive stiffness. LN samples were prepared in a conventional fashion and OCT‐stored tissue was first sectioned in parallel with papillary muscle longitudinal axis before chemical demembranisation and further trimming under the microscope.ResultsCa2+ sensitivity (pCa50): LN (n=8): 5.75 ±0.019, OCT (n=8): 5.69 ± 0.022 SEM (p>0.05). Cooperativity along the thin filament (nH): LN (n=8): 2.63 ± 0.24, OCT (n=8): 2.53 ± 0.25 SEM (p>0.05). Fmax: LN (n=8): 19.6 ± 1.9, OCT (n=8): 11.6 ± 1.7 mN/mm2 SEM (p<0.05). Ktr: LN (n=8): 37.5±1.6, OCT (n=8): 37.9 ± 1.6 s−1 SEM (p<0.05). Relative cross‐bridge recruitment upon full Ca2+ activation: LN (n=8): 0.32 ± 0.015, OCT (n=7): 0.31 ± 0.014 SEM (p<0.05). kstrain: LN (n=7): 13.9 ± 0.67, OCT (n=7): 11.4 ± 0.49 L−1, p<0.01). Relative titin contribution to stiffness at 20 % stretch from slack: LN (n=6): 0.52 ± 0.07, OCT (n=7): 0.80 ± 0.03 SEM, (p<0.01).ConclusionsOCT‐storage and cryostat sectioning before mounting fibre bundles on a force‐measuring rig can be used for investigating relative measures of function depending on active force development, ie pCa‐tension relationship, cooperativity along the thin filament and cross‐bridge cycling rate. There appears to be no negative effect on the amount of cross‐bridge recruitment upon Ca2+ activation compared to a conventionally LN‐stored and prepared sample. Absolute values such as Fmax, as well as passive strain, need to be interpreted with great caution as they clearly differ between mode of preparation and thus these parameters are not suited for investigation. We speculate this may be due to how cutting of fibre bundles using a cryostat, to an unknown degree, disrespects the natural continuity of the bundles and thus hampers force relay.Support or Funding InformationThis work was supported by: Swedish Stately Grant for Medical Research (ALF‐means) & Lars Hierta Memorial Fund