bluuebunny/crossref_metadata_2025_split huggingface dataset:
Does insulin resistance alter pulse transit time in the cerebral circulation?
Authors: Muer Jessica D. , Ward Aaron T. , Carter Katrina J. , Kellawan J. Mikhail , Harrell John W. , Roldan Alejandro , Wieben Oliver , Eldridge Marlowe W. , Schrage William G. 
Published: April 2020

Approximately 80 million adults in the United States suffer from an insulin resistant state known as metabolic syndrome (MetSyn), associated with increased risk for strokes, cerebral infarcts and neurologic disease. Adults with MetSyn also exhibit increased peripheral arterial stiffness, but whether arterial stiffness occurs in the cerebral circulation remains unclear. We hypothesized adults with MetSyn would exhibit increased cerebrovascular stiffness, demonstrated as faster pulse transit time (PTT) compared to healthy adults. Participants were 32±7 years old, sedentary, non‐smoking, free of cardiovascular disease and unmedicated. Healthy normal‐weight adults (Control; n=11; weight=70±8 kg; BMI = 23±2 kg/m2) were normotensive and normolipidemic. Adults with MetSyn (n=6; BMI=38±8 kg/m2) met three or more of the five criteria according to American Diabetes Association (MAP=107±5 mmHg; glucose=82±17 mg/dL; triglycerides 167±123 mg/dL; HDL 39±9 mg/dL; waist circumference 119±16 cm). Subjects were instrumented to continuously monitor heart rate, blood pressure, and end‐tidal CO2. A 3T MRI using 4‐dimensional flow phase contrast vastly under‐sampled isotropic projection reconstruction (PC VIPR; ~6 minutes) was used to capture cerebral blood flow (CBF = blood velocity • vessel cross sectional area) in the internal carotid arteries (ICA) and superior sagittal sinus (SSS). Cerebral blood flow across 20 equal segments of the average cardiac cycle was used to determine the time to peak flow in the ICAs and SSS. Pulse transit time (PTT) was calculated as the difference in time to peak in the SSS from the time to peak in the ICAs. Data are presented as mean ± SD. Significance was determined using independent t‐tests and was set at p≤0.05. MAP, HR, SpO2, and end tidal CO2 were not different in magnet bore (p>0.05). ICA to SSS PTT was not different between Control and MetSyn (141±109 vs. 144±91 ms; p=0.743).ConclusionPulse transit time was similar between healthy adults and those with MetSyn. Perhaps greater disease severity, aging, or their combination are required to negatively change PTT in MetSyn. Alternatively, the cerebral circulation may be resistant to vascular stiffening.Support or Funding InformationADA 1‐12‐IN‐39
Store‐Operated Calcium Entry Regulates Macrophage Chemotaxis
Authors: White Carl , Fresquez Adriana M. 
Published: April 2020

The store operated calcium entry (SOCE) pathway is known to be important for cell migration; however, its involvement in regulating directed movement toward a chemotactic signal is less well understood. The aim of current study was to define the involvement of SOCE in macrophage chemotaxis. To achieve this aim, we used phase contrast microscopy to visualize primary mouse bone marrow derived macrophages migrating toward the source of the potent chemoattractant C5a (complement component 5a). A concentration gradient was established by pulsing C5a from a micropipette connected to a pressure regulating device. Chemotaxis was monitored for up to 90 minutes in the presence and absence of the SOCE blocker BTP2 (1 μM). We found that the chemotactic speed and total distance traveled was decreased in the presence BTP2. In addition, we quantified the morphological changes at the leading edge of the migrating cells and found that frequency and speed of protrusions was also reduced by BTP2. These studies establish the requirement for SOCE in macrophage chemotaxis and leading‐edge dynamics and suggest a role for SOCE in regulating the recruitment of macrophages to sites of inflammation in vivo.Support or Funding InformationThis work was supported by NIH Grant HL142906 to C.W.
Fusion protein EWS‐FLI1 partners with EWSR1 to regulate transcription in Ewing sarcoma
Authors: Ahmed Nasiha , Schwartz Jacob 
Published: April 2020

Chromosomal translocation events drive many types of pediatric cancers. Ewing sarcoma is a pediatric bone cancer driven by a chromosomal translocation where the N‐terminal low‐complexity domain of EWSR1 fuses to the DNA‐binding domain of the ETS transcription factor FLI1. This fusion creates the aberrant transcription factor EWS‐FLI1, which drives tumor formation by altering the transcriptional landscape. Interestingly, constitutive expression of EWSR1 is retained in Ewing sarcoma cells. The low‐complexity domain found in EWSR1 and EWS‐FLI1 is intrinsically‐disordered and can mediate protein‐protein interactions with itself and other protein partners. Moreover, this domain has the propensity to phase separate. Despite the common domain between the two proteins, very little is known about the effect of EWSR1 on EWS‐FLI1 activity. We performed RNA‐sequencing to reveal that EWSR1 and EWS‐FLI1 coregulate a large pool of genes in Ewing sarcoma cells. Loss of EWSR1 in inhibits anchorage‐independent growth, suggesting that EWSR1 plays a role in cancerous properties of Ewing sarcoma cells. We have shown EWSR1 and EWS‐FLI1 bind each other directly in vitro and in vivo. Finally, we have found the existence of EWSR1, EWS‐FLI1, and RNA Pol II in a novel granular body in Ewing sarcoma cell lines. This direct interaction of EWSR1 and EWS‐FLI1 presents a straight‐forward model that merges wild‐type and fusion properties to alter transcriptional output and drive Ewing sarcoma biology.Support or Funding InformationNational Institutes of Health, American Cancer Society
Development and Characterization of Spontaneously Immortalized Cell Lines Derived from Mouse Endometriotic Lesions
Authors: Chawla Shivane , Lang Stephanie A. , Burns Katherine A. 
Published: April 2020

Endometriosis affects approximately 1 in 10 women of reproductive age, where the tissue of the inner lining of the uterus, the endometrium, proliferates in and around the peritoneal cavity. Common symptoms of endometriosis include chronic pelvic pain, intense pain with menstruation, pain with urination and defecation, and infertility. Recently more attention is being brought to the links between endometriosis and other co‐morbidities such as heart disease, cancer, and autoimmune disorders. Other factors known to play a role in endometriosis are endocrine disrupting chemicals that are found in common household products that contain plasticizers. One of these environmental toxicants is a group of chemicals called phthalates. A challenge to the field of endometriosis is the availability of cell lines to study mechanism(s); therefore, we set forth to create cell lines from endometriosis lesions. In our model of endometriosis, uterine endometrial tissue expressing green fluorescent protein (GFP) is used to develop endometriosis lesions. From these lesions, multiple cell lines were generated, but for this study, the focus was on GFP positive (EmLe+) and GFP negative (EmLe‐) cell populations that emerged from the lesions. The EmLe+ line is phenotypically uterine derived exhibiting epithelial to mesenchymal transition (EMT) markers. The EmLe‐line phenotypically exhibits epithelial derived muscle markers. To characterize these lines and to determine their response to environmental toxicants, the cells were studied under normal conditions and exposure to a commonly found phthalate, bis (2‐ethylhexyl), also known as DEHP, to better understand the cellular properties in the development and progression of endometriosis. Cell proliferation, migration, and invasion assays were used to examine how endometrial cells cultured from a mouse model are able to proliferate, migrate, and invade under normal and DEHP exposure conditions. For the proliferation assay, each of the cell lines was plated at 5,000 cells per well in 24 well plates and counted each day under various conditions. To study cell migration and invasion, a 48‐well chemotaxis chamber was used with or without matrigel, respectively. From these studies, we find that the different cell types isolated from mouse endometriotic lesions differ in composition and behavior, and find that these cell lines may be a good model to study more in depth mechanisms of endometriosis development.Support or Funding InformationR00 ES021737P30 ES006096
Knockout of VDAC1 in H9c2 Cells Promotes tBHP‐induced Cell Apoptosis Through Decreased Mitochondrial HK II Binding and Enhanced Glycolytic Stress
Authors: Yang Meiying , Li Keguo , Sun Jie , Stowe David F. , Tajkhorshid Emad , Kwok Wai-Meng , Camara Amadou K.S. 
Published: April 2020

VDAC1, the most abundant mitochondrial outer membrane protein, transports ions and metabolites between the cytoplasm and mitochondria. It is also involved in cellular energy production and apoptosis, but the role of VDAC1 in cell death remains controversial. Silencing or upregulation of VDAC1 may stimulate or inhibit cell apoptosis, growth and survival depending on cell types and stimuli. Using VDAC1 knockout (Vdac1−/−) embryonic stem cells (ES) and mouse embryonic fibroblast (MEF), it was revealed that VDAC1 was dispensable for both mitochondrial membrane permeability transition pore (mPTP) opening and Bax and Bid‐driven cell death. The effect of Vdac1−/− on oxidative stress‐induced cell death in a cardiac cell line has not been reported. We hypothesized that although VDAC1 is dispensable for mPTP and is not involved in mPTP‐mediated cell death, VDAC1 is an essential regulator of oxidative stress‐induced cell apoptosis in cardiac cells. To this end, we knocked out VDAC1 in the cardiac fibroblast cell line, H9c2 (Vdac1−/− H9c2) by the CRISPR‐cas9 technique and examined whether VDAC1 plays a role in cell death via oxidative stress induced by tert‐butylhydroperoxide (tBHP), an organic peroxide. We found that under physiologic conditions, Vdac1−/− did not affect H9c2 cell proliferation, and mitochondrial respiration. However, tBHP treatment enhanced the extracellular acidification rate (ECAR) and proton production rate (PPR) produced from glycolysis and promoted cell apoptosis in Vdac1−/− H9c2 cells compared to the wildtype. Vdac1−/− H9c2 cells also showed markedly reduced binding of mitochondria hexokinase II (HK II), a key glycolytic enzyme. Rescue of VDAC1 in Vdac1−/− H9c2 cells reinstated mitochondria HK II binding and concomitantly decreased tBHP‐induced cell death. Mitochondrial respiration remained the same after tBHP treatment in Vdac1−/− and wild type H9c2 cells. Our results suggest that Vdac1−/− in H9c2 cells enhances tBHP‐induced oxidative stress‐mediated cell apoptosis by reducing mitochondria HK II binding and by increasing ECAR and PPR produced from glycolytic stress.Support or Funding InformationNational Institutes of Health (R01 HL‐131673‐01A1), Veterans Administration (BX‐002539‐01), United States.
The exercise pressor reflex is inhibited by Brilliant Blue G in freely perfused and femoral artery ligated rats following intrathecal, but not hindlimb, treatment.
Authors: Estrada Juan , Ducrocq Guillaume , Kim Joyce , Kaufman Marc 
Published: April 2020

The exercise pressor reflex (EPR) is mediated in part by purinergic(P)2X receptors which are compromised of seven subtypes. Blockade of the P2X3 subtype on the peripheral endings of group III and IV muscle afferents attenuates the EPR. We investigated whether other P2X receptors were involved in the EPR arc in decerebrate Sprague Dawley rats whose femoral arteries were either patent or were ligated. We antagonized P2X receptors with Brilliant Blue G (BBG), which has highest affinity for P2X7 receptors. We injected BBG in both group of rats either intrathecally (50mg/mL; 25–50uL) or into the superficial epigastric artery, which is a side branch of the femoral artery. Intrathecal injection of BBG attenuated the magnitude of the EPR in both rats with patent femoral arteries and in rats with ligated femoral arteries (P<0.05). Intrathecal injection of BBG had no effect on the pressor response to intra‐carotid injection of sodium cyanide (2ug), which is a potent and selective stimulant to carotid chemoreceptors. This latter finding indicates that intrathecal injection of BBG did not ascend to the medulla and did not impact the spinal sympathetic outflow. BBG injected either into the superficial epigastric artery or intravenously (5mg) had no appreciable effect on the EPR. In addition the effect of intrathecal BBG could not be explained by “neural fatigue” caused by repetitive contractions in rats with ligated femoral arteries. We conclude that intrathecal BBG treatment inhibits the EPR by antagonizing P2X4 or P2X7 receptors within the dorsal horn of the spinal cord.Support or Funding InformationThis study was funded by NIH grants R01 AR 059397 and P01 HL 134609.
A Comparative Analysis of the Vascular Effects of Clopidogrel Metabolites
Authors: Kuszynski Dawn S. , Christian Barbara D. , Dorrance Anne M. , Lauver D. Adam 
Published: April 2020

Dual antiplatelet therapy using low‐dose aspirin with a purinergic receptor 2Y12 (P2Y12) antagonist is the most common preventative method for arterial thrombosis. Clopidogrel, a P2Y12 antagonist, effectively inhibits ADP‐induced platelet aggregation. However, it also increases adverse bleeding side effects which do not correlate with the antiplatelet effect. Of patients who have taken clopidogrel for at least one year, 31% have cerebral microbleeds and 47% of those patients also develop intracerebral hemorrhage. Through clopidogrel’s complex metabolism only 5% of the active metabolite responsible for the antiplatelet effect is generated. In order to avoid the complex metabolism associated with clopidogrel, DT678 was developed. DT678, a conjugate of the clopidogrel active metabolite, is completely converted into the active metabolite to inhibit platelet aggregation. We have previously reported that DT678 is equally as effective in preventing platelet aggregation however, it has a reduced bleeding risk compared to clopidogrel in a rabbit model. A more complete mechanistic understanding of the cellular pathways affected by clopidogrel and its metabolites are required in order to explain the difference in bleeding risk.The purpose of this study was to determine the effects of clopidogrel on spontaneous myogenic tone generation, vasoconstriction or vasodilation in middle cerebral arteries (MCA). Rabbits were anesthetized and the jugular vein was cannulated for blood collection and intravenous drug administration of various doses of DT678, clopidogrel or vehicle. Blood collections were taken at baseline and two‐hour post IV drug administration. The blood was then used in a platelet aggregometry assay to determine the inhibition of platelet aggregation. Two‐hours post treatment the brain was collected and placed in Ca2+ free physiological salt solution. The MCA was isolated and cannulated on an arteriography chamber. The cannulated artery, in Ca2+ physiological salt solution, was pressurized to 80mmHg with a flow rate of 0.25ml/min through the lumen at 37°C. Spontaneous myogenic tone was allowed to develop before assessing the vascular effects of increasing concentrations (10−9–10−5M) of ADP or specific purinergic receptor agonists (P2Y1: MRS 2365; P2Y2 MRS 2768; P2Y4 MRS 4062; P2Y6: MRS 2693; P2Y11: NF 546; or P2Y14: MRS 2905). If spontaneous myogenic tone was not generated the MCA was preconstricted with 10−6M serotonin. Spontaneous myogenic tone and ADP‐induced vasodilation was inhibited in clopidogrel‐treated, but not DT678‐treated middle cerebral arteries. These data suggest that clopidogrel has non‐platelet mediated effects on purinergic receptors expressed in the MCA and may explain the cerebral bleeding side effects observed.Support or Funding InformationThis work was supported in part by MSU PHM/TOX and CVM Startup Funds, Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation Research Starter Grant and NIH/NHLBI R43HL139380.
Clarifying Ded1 Function in Translational Control using an in vivo Fluorescence‐Based Reporter Assay
Authors: Schuessler Peter Joseph , Srinivasan Aravind , Barbi Joseph , Walker Sarah E. 
Published: April 2020

Eukaryotic translation initiation is a critical node at which gene expression is regulated. Among the factors that are known to regulate this process, DEAD‐box RNA helicases are believed to promote translation by resolving secondary structure in the 5′ UTR of an mRNA. These helicases include the protein Ded1 in budding yeast, which is required to remove cap‐distal structure in the 5′ UTR of an mRNA, thus enabling scanning of the preinitiation complex. Yet the role of Ded1 in translation is complex; a translationally repressive role has also been demonstrated by Ded1 overexpression. Further confounding investigations of Ded1 function are the large number of protein‐protein and protein‐RNA contacts made by Ded1 to regulate activity. To study the diverse roles that Ded1 plays in translation, we have developed a system that reports on Ded1‐specific changes in translation in vivo. This system relies on a fluorescence‐based reporter that simultaneously promotes the transcription of two mRNA transcripts: one encoding RFP with a 5′ UTR that is translated independent of Ded1 activity, and the other encoding GFP with a 5′ UTR that requires Ded1‐activity for translation. We are using this system in combination with a library of random Ded1 mutants to broadly interrogate Ded1 activities for effects on translation initiation and repression.Support or Funding InformationThis work was supported by NIH grant R00GM119173 and start‐up funds from SUNY at Buffalo, College of Arts and Sciences.
ATP‐sensitive K+ Channel Inhibition Diminishes Critical Speed in Male and Female Rats but V̇O2max in Females Only
Authors: Weber Ramona E. , Colburn Trenton D. , Schulze Kiana M. , Hageman K. Sue , Behnke Bradley J. , Musch Timothy I. , Poole David C. 
Published: April 2020

Glibenclamide (GLI) stimulates pancreatic insulin secretion in patients with Type II diabetes by inhibiting ATP‐sensitive K+ (KATP) channels. KATP channels also support vasodilation, increasing blood flow and O2‐delivery‐utilization matching in contracting muscle. Whether KATP channels support high‐intensity exercise tolerance and the sex dependency of any such effect remains unknown.PurposeTo determine the role of KATP channels in supporting maximal aerobic capacity (V̇O2max) and the determinants of the speed‐duration relationship (CS, critical speed, and D′, distance covered above CS). We hypothesized that KATP channel inhibition via GLI would impair V̇O2max and CS with females exhibiting the greatest reduction.MethodsMale (n=11), female (n=10, proestrus), and ovariectomized female (F+OVX; n=12) Sprague‐Dawley rats were tested with and without GLI (10 mg kg−1 in DMSO i.p.). V̇O2max and CS were assessed using a motorized treadmill with an incline of 5%. V̇O2max was measured via plexiglass metabolic chamber connected to an O2 analyzer with treadmill speed increasing 5–10 m min−1 every minute until V̇O2 plateaued. Multiple (i.e., ≥ 4) constant speed runs to exhaustion on separate days were used to determine CS and D′.ResultsGLI reduced V̇O2max in female (71.5 ± 1.0 vs 67.9± 1.5 mL O2 min−1 kg−1; p<0.05) and F+OVX (76.8 ± 1.4 vs 74.4 ± 1.5 mL O2 min−1 kg−1; p<0.05) without affecting males (81.5 ± 2.0 vs 80.8 ± 2.0 mL O2 min−1 kg−1; p>0.05). In contrast, GLI reduced CS equivalently across groups (8–11%; p<0.05). D′ was unchanged (p>0.05).ConclusionConsistent with our hypothesis, KATP channel function supports exercise tolerance. Interestingly, whilst CS between sexes was not impacted differently, V̇O2max was diminished only in females and not males. These data demonstrate that the exercise intolerance in Type II diabetes patients may be exacerbated by sulphonylurea medication (i.e. GLI) with women being affected more severely in terms of O2 transport (V̇O2max).Support or Funding InformationSupported by NIH Grants: HL108328 (D.C.P and T.I.M) and F31HL145981 (T.D.C)
Synthesis and secondary structure characterization of sialyl‐Tn bearing MUC1 glycopeptides
Authors: Ormaza David , Cudic Mare 
Published: April 2020

ObjectiveTo synthesize carbohydrate building blocks bearing the sialyl‐Tn antigen, and incorporate them into a MUC1 peptide backbone for structural and functional studies with endogenous lectins.Mucin 1 (MUC1), a transmembrane mucin protein, is a well‐known biomarker of different cancer types. The aberrantly glycosylated forms of MUC1 display specific truncated tumor‐associated carbohydrate antigens (TACAs) on the surface of the cell such as: Tn (GalNAc), T (Galβ1,3GalNAc), sTn (NeuAcα2,6GalNAc), and sT (2,3‐sT and 2,6‐sT). These truncated glycan structures, often capped by sialic acid, play a key role in tumor initiation, progression, and metastasis, and accumulating evidence suggests their involvement in tumor escape from immune defenses through interaction with endogenous carbohydrate‐binding proteins (lectins). Macrophage galactose binding lectin (MGL) is a C‐type lectin receptor found on antigen‐presenting cells (APCs) which facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. The main goal of this research is to evaluate the interactions between Tn and sTn antigen of tumor‐associated MUC1 with MGL and to characterize the molecular origin of the high affinity and selectivity of this endogenous lectin for its natural MUC1 ligands. This interaction plays a crucial role in tumor immune evasion. Nonetheless, MGL targeting is an attractive approach to enhance cancer vaccine efficacy. It is of crucial importance to understand the fine‐intricate nature of immune cells to either augment the anti‐tumorigenic effects or minimize the pro‐tumorigenic effects. The aim of this research is to synthesize structurally well‐defined chemical probes, mono‐ and multiple‐glycosylated MUC1 peptide models carrying the Tn or sTn O‐glycans, that allow for the control of the complexity of the chemical space of the multivalent ligands. For this purpose, a concise scheme was developed for the synthesis of the sTn antigen building blocks to be used in the solid‐phase synthesis of glycopeptide models. We employed the use of orthogonally protected phenyl thioglycoside glycosyl donors in the presence TfOH/NIS or TMSOTf/NIS as promoter system. The glycosylated threonine and sialylated glycosylated threonine were synthesized in a relatively high yield, 85% and 60%, but with moderate stereoselectivity, α/β ratio 3.5:1 and 2:1, respectively. The subsequent reduction and N‐acetylation of an azido group resulted in targeted glycosylated amino acid building block that was used in the solid‐phase peptide synthesis of MUC1 sTn bearing glycopeptides. The secondary structure of synthesized peptides was determined by circular dichroism (CD) spectroscopy. The synthetic efforts will be followed by mechanistic studies including isothermal titration calorimetry (ITC) and AFM.Support or Funding InformationThis work was supported by the National Institutes of Health Grant CA242351 to M. Cudic.