bluuebunny/crossref_metadata_2025 huggingface dataset:
The migration of radionuclide’s for the sanitary protection zone of uranium tailings enterprises (LB488)
Authors: Kazymbet Polat , Bakhtin Meirat , Kashkinbaev Erlan , Dzhumadilov Kasym , Eltokova Manshuk , Ribalkina Dina
Published: June 2021

Mining companies are potential sources of contamination of the environment by various pollutants, including technogenic radionuclides. Stepnogorsk Mining‐Chemical Combine (SMCC) functions over 50 years and is one of the largest complexes for production of uranium oxide and other rare metals. Radioactive waste of SMCC is accumulated in the tailing dumps with an area of 7.82 km2, located in 4 km to the North from Aksu village and Zavodskoy, where concentrated waste is with a mass of 44.17 million tons, and activity of 146.4 thousand Ku. The aim of this work was to evaluate the radiological situation of territories for sanitary protection zone of the tailings dumps (SZZH), including those in close vicinity to workers of the villages Aksu and Zavodskoy.

As a result of the pedestrian gamma survey there were identified local, contaminated sites in the North‐East of SZZH SMCC with a length of 5 km. The most contaminated with radionuclides is the top layer of the soil 0‐5 cm. in the North‐East and South‐East direction from SZZH specific activity is 238U, 226Ra in the soil exceed the reference value in 16 times, 210Pb exceeds up to 7 times. In the samples of plants selected from the vicinity of villages in the South‐East and North‐East directions from SZZH it is noted high concentrations of natural radionuclides. In reservoirs of Sulukamys and Manybay thalwegs, located at a distance of 1.5 to 2 km in the Eastern and North‐Eastern part of the tailings dumps, the specific activity of technogenic radionuclides in the tens times higher than control values.

On the territory of the village Aksu there were identified local, contaminated sites with a capacity of an equivalent dose of gamma radiation up to 0.86 mSv / h Possible additional radiation dose from external exposure for separate groups of the population of Aksu is from 0,86 mSv to 1,53 mSv per year.
Cellular responses to inflammation in the central nervous system: nitric oxide synthase isoform expression in cell culture and animal models (LB501)
Authors: Bick Diane , Doursout Marie‐Francois , Schiess Mya , Bick Roger
Published: June 2021

Inflammation plays a significant role in dopaminergic neuron damage associated with the development and progression of Parkinson’s disease (PD). Microglia within the central nervous system play are known to be activated during the inflammatory process and activated microglia can induce pleiotropic downstream sequellae. Activated microglia can serve a neuronal protective role, including their synthesis of glial cell line‐derived neurotrophic factor (GDNF). However, chronic or uncontrolled activation of microglia can lead to the production of pro‐inflammatory cytokines and other neurotoxic factors. A rat model of inflammation involving exposure to lipopolysaccharide (LPS) demonstrates many of the patho‐physiologic markers of PD. Furthermore our data has shown that there is a close temporal relationship between GDNF appearance and freely diffusible nitric oxide (NO) production. In the cardiovascular system an inflammatory response leads to a multiphasic production of NO and expression of different isoforms of nitric oxide synthase (NOS) which in turn activate protective pathways. We hypothesized that in the CNS, NO production can also regulate the activation of protective pathways. Our studies employed LPS exposure in both cell cultures and animal models. Cells and tissues were probed for the presence of GDNF and inducible, neuronal and endothelial nitric oxide synthase (iNOS, nNOS, eNOS), by fluorescence deconvolution microscopy in a time dependent manner after exposure to LPS. Both microglial cells and astrocytes in culture synthesized GDNF and nitric oxide synthase isoforms following LPS treatment. A similar time dependent production of these markers was observed in our animal model. Furthermore, we observed these differential changes were not generalized within the brain sections but occurred in specific areas.
Improvement of HDL and atherosclerosis by a mimetic peptide (18A) complexed with niclosamide and administered orally to mice (LB532)
Authors: Madahian Sepideh , Pourtabatabaei Nasim , Navab Kaveh Daniel , Safar Sheila , Vazirian Samra , Seyedali Seyedehsara , Hough Greg
Published: June 2021

18A was incubated together with niclosamide and within 6h of oral administration there was significant improvement (p<0.001) in the HDL‐inflammatory index (HII) in ApoE null mice. Oral L‐4F alone or niclosamide alone were ineffective. Subcutaneous injection of L‐4F alone but not niclosamide alone significantly improved the HII. Analysis of the L‐4F‐niclosamide complex by gradient gel electrophoresis or Fourier Transform Infrared Spectroscopy indicated that the self‐association of L‐4F was altered resulting in smaller micelles. Oral administration of L‐4F and niclosamide to old apoE null mice together with pravastatin resulted in regression of lesions. In another experiment ApoE null mice 3‐4 months of age were treated for 7 months with pravastatin in the drinking water and oral L‐4F together with niclosamide in doses which produced maximal L‐4F plasma concentrations of ~100 ng/mL. There was a significant reduction in lesion area compared to controls (p<0.001). The reduction in lesion area was not improved by administering L‐4F subcutaneously for the first 3 months to achieve maximal L‐4F plasma concentrations ranging from 5 ‐ >45 μg/mL. These results suggest that regimens achieving initial high L‐4F plasma concentrations followed by oral administration may be no more effective than oral treatment alone which produces maximal L‐4F plasma concentration.
HDL from Patients with Inflammatory Disorders has High levels of Oxidized Lipids and is Dysfunctional (LB536)
Authors: Bakhtiari Persiah
Published: June 2021

HDL from Patients with Inflammatory Disorders has High levels of Oxidized Lipids and is Dysfunctional. Persiah Bakhtiari, Omeed El Boudvarej MD, Susan Hama, Nasim Pourtabatabaei MD, Sheila Safar MD, Samra Vazirian MD, Seyedehsara Seyedali MD, Sepideh Madahian MD, Greg Hough. David Geffen School of Medicine at UCLA, Los Angeles California, 90095 Population studies have shown that plasma levels of HDL inversely correlate with the prevalence of coronary heart disease. HDL levels in an individual does not necessarily correlate with the risk for coronary heart disease. Many patients with high HDL, present with coronary events and many with low HDL do not. We demonstrated that HDL composition and function is important than HDL levels. Under excessive inflammatory pressure unsaturated fatty acids can undergo oxidative modification. Protective enzymes including paraoxonase, LCAT and PLTP are inactivated by oxidative modification reducing HDL anti inflammatory properties. This results in HDL not being able to protect LDL from oxidative modification. Methods: Fasting plasma from patients with coronary disease were analyzed for their ability to prevent LDL modification. Additionally the content of oxidized lipids in HDL was determined using LC‐ESI‐MS/MS. Results: Among coronary patients over 69 percent had non protective HDL while among healthy controls 4.5 percent had dysfunctional HDL. The content of a variety of oxidized lipids in the HDL from patients was orders of magnitude higher than that found in HDL from normal Controls. Conclusion: Until there are effective medications for raising HDL, it should be clear to the public that the best way of dealing with low HDL cholesterol is lowering LDL cholesterol.
Investigation of Angiostensin‐converting enzyme inhibitory potential and allergenicity of Sesamum indicum Linn seed proteins by an in silico approach (LB541)
Authors: Rotimi Oluwakemi , Oluwagbemi Olugbenga , Iyanda‐Joel Wisdom , Omotosho Elizabeth , Adebiyi Marion , Adelani Isaacson , Rotimi Solomon
Published: June 2021

The nutraceutical role of dietary proteins and peptides is gaining considerable interest in the management of cardiovascular disease like hypertension. In order to investigate the contribution of Sesamum indicum seed proteins to its antihypertensive properties was investigated, there seed protein sequences: 7S globulin (NCBI accession number, giǀ13183177), 2S albumin (NCBI accession number, giǀ13183174) and 11S globulin (NCBI accession number, giǀ13183172) were selected for BLAST, BIOPEP and allergenicity analysis. BLAST gave the following similarities: 7S globulin vs rice glutelin precursor (35%), 7S globulin vs oat 12S seed storage globulin 1 (29%), 11S globulin vs rice glutelin precursor (41%), 11S globulin vs oat 12S seed storage globulin 1 (39%) and 2S albumin vs oat 12S seed storage globulin 1 (60%). BIOPEP analysis showed that the sesame proteins demonstrated either di‐ or tri‐peptide with a total of 92, 91 and 34 potential ACE inhibitory peptides from 7S globulin, 2S albumin and 11S globulin, respectively. Papain hydrolysis theoretically released the highest numbers of predicted ACE inhibitory peptides (23, 14 and 5) from 7S globulin, 2S albumin and 11S globulin, respectively. The 8‐mer and 80‐mer allergenicity analysis of these proteins showed significant matches with allergenic proteins from Lens culinaris, Anacardium occidentale, Carya illinoinensis. Although, the ccombined digestion with pepsin, trypsin and chymotrypsin A, a simulation of human gastrointestinal digestion, gave a sum of 32 predicted ACE inhibitory peptides from these proteins, their allergenic property may be a limitation.
Akebiae caulis extract inhibits oxidative stress through AMPK/GSK3‐beta pathway (LB551)
Authors: Kim Young Woo , Jung Eun Hye , Byun Sung Hui , Kim Sang Chan , Cho Il Je
Published: June 2021

Akebiae caulis is commonly used for the oposiuria, inflammation, nociceptive and fever. We investigated the effect of Akebiae Caulis extract (ACE) to protect cells against the mitochondrial dysfunction and apoptosis. Arachidonic acid (AA)+iron promotes excessive reactive oxygen species production and dysfunction of mitochondria. ACE protected hepatocytes from AA+iron‐induced cytotoxicity. As a potential molecular mechanism for the ACE‐mediated cytoprotection, phosphorylation of AMPK, a key regulator in determining cell survival or death, was increased by ACE. Moreover, ACE treatment enhanced inactive phosphorylation of GSK3β, downstream substrate kinase of AMPK. ACE also prevented a decrease in the GSK3β phosphorylation derived by AA+iron, which might contribute to mitochondrial protection and cell survival. Moreover, we found that betulin in combination with hederagenin protected from AA+iron‐induced mitochondrial dysfunction. These results show that ACE has the ability to protect cells against AA+iron‐induced H2O2 production and mitochondrial impairment, which is mediated with inactive GSK3β phosphorylation downstream of AMPK.

Grant Funding Source: This work was supported by the National Research Foundation of Korea grant funded by the Korea government [MEST](No.2012‐0009400).
The G Protein‐Coupled Estrogen Receptor 1 Regulates Endothelial Ca2+ efflux via the plasma membrane Ca2+‐ATPase. (LB568)
Authors: Tran Quang‐Kim , Burgard Michelle , VerMeer Mark , Giles Jennifer
Published: June 2021

The G protein‐coupled estrogen receptor 1 (GPER) has been demonstrated to have a vast array of cardiovascular effects. Ca2+ extrusion via the plasma membrane Ca2+‐ATPase (PMCA) plays important roles in Ca2+ homeostasis. We tested the hypothesis that GPER is involved in the regulation of calcium efflux in the vasculature. In primary vascular endothelial cells, the specific GPER agonist G‐1 dose‐dependently inhibits PMCA activity. Knockdown of GPER in endothelial cells using antisense directed against GPER is associated with a ~ 40% enhancement of PMCA activity. Heterologous expression of human GPER in fusion with the fluorescent protein DsRed2 in HEK 293 cells results in a 38% reduction of PMCA activity. Co‐IP experiments show that PMCA and GPER form a complex during Ca2+ signals triggered by the SERCA pump inhibitor thapsigargin in endothelial cells. In addition, the fusion proteins PMCA‐DsRed2 colocalizes with GPER‐ECFP heterologously expressed in HEK 293 cells. These data suggest that GPER regulates Ca2+ efflux in endothelial cells by interacting with PMCA.

Grant Funding Source: National Institutes of Health
Regulation of Store‐Operated Ca2+ Entry in the Vascular Endothelium by the G Protein‐coupled Estrogen Receptor 1 (LB570)
Authors: Terry Lara , VerMeer Mark , TRAN Quang‐Kim
Published: June 2021

The novel G protein‐coupled estrogen receptor 1 (GPER/GPR30) has been found to participate in numerous cardiovascular functions. Store‐operated Ca2+ entry (SOCE) is an essential mechanism that is required for many endothelial cell functions. We found that activation of GPER using the GPER specific agonist G1 causes a dose‐dependent inhibition of SOCE in primary vascular endothelial cells. Heterologous of GPER in HEK 293 cells is associated with substantial decreases in the total Ca2+ signal triggered by thapsigargin, and a 40% decrease in the rate of SOCE. Knockdown of GPER in endothelial cells using antisense directed against GPER increases SOCE by approximately 50% compared to cells transfected scrambled oligo. Interestingly, GPER specific antagonist G15 triggers total Ca2+ signals via SOCE by releasing the same internal Ca2+ stores as does thapsigargin. In primary endothelial cells, GPER forms a complex with the stromal interaction molecule 1 (Stim1), an essential molecular switcher of SOCE. In addition, Stim1‐ECFP colocalizes with GPER‐tetracysteine when heterologously expressed in HEK 293 cells. Heterologous expression of GPER‐DsRed2 fusion substantially decreases total Ca2+ signal stimulated by thapsigargin. Interestingly, SOCE measured in cells stably expressing fusions between EYFP and individual sub‐membrane domains (SMDs) 1 ‐ 4 of GPER does not differ from internal controls. These data indicate that GPER may be an important regulatory input of store‐operated Ca2+ entry via its interaction with Stim1. Since the EYFP‐SMD fusions are all cytoplasmically expressed, the data may also suggest that proper membrane docking (PM or ER) of full‐length GPER is required for it to exert its effect on SOCE.

Grant Funding Source: National Institutes of Health
Gravin‐mediated PKC signaling in heart (LB571)
Authors: Suryavanshi Santosh , Singh Sonal , McConnell Bradley
Published: June 2021

Gravin is an A‐Kinase Anchoring Protein (AKAP) that scaffolds protein kinase A (PKA), protein kinase C (PKC), phosphatase 2B, β2‐adrenergic receptors to specific subcellular compartments. Studies show that scaffolding of PKC by gravin decreases PKC activity due to sequestration. Also, PKC is involved in the translocation and distribution of gravin. Our goal is to study the role of gravin in PKC signaling in the heart. Basal PKC activity in the cytosolic fractions of the homogenized hearts was found to be greater in gravin truncated (t/t) mice as compared to wild type (WT). This correlation was preserved following stimulation of the samples with a PKC activator, PMA (Phorbol 12‐myristate 13‐acetate). However, no change was observed in the extent of PKC substrate phosphorylation between these groups. It supports previous studies that PKC scaffolding by gravin decreases PKC activity. The next step is to study the effect of PKC on gravin translocation in cardiomyocytes using PKC activator PMA and inhibitor chelerythrine. We expect to see increased translocation of gravin to cytosol after PKC activation. These studies will help us better understand the functional significance of the role of gravin in PKC signaling.

Grant Funding Source: R01HL085487
Determination of the IC50 and LD50 of Calcium Sulfide (CaS) Clusters on Malignant Carcinoma and Normal Fibroblasts Cell Lines (LB580)
Authors: Manoharan‐Valerio Michael , Ortiz Carlos , Quiñones Jaileen , Feliciano Geishamarie , Diaz Angel , Rivera Daniel , Castro Miguel , Suarez Edu
Published: June 2021

Preliminary results in our lab showed that naked Calcium Sulfide (CaS) nanoparticles (5.0nm) decreased cell proliferation in the carcinoma cell lines CRL‐2124. Objective: The objective of this study was to determine the IC50 and LD50 for CaS clusters (measuring < 3.0 nm) in the carcinoma cell lines CRL‐2124 and the normal fibroblasts CRL‐2522 by using the n3D BiOAssay (Biosciences, Inc.). Methods: A total of 100,000 cells per cell line were seeded in T‐75 flasks. When reaching 80% confluence, they were incubated with the n3D nano‐shuttles overnight (ON) at 37oC, 5% CO2, and 98% relative humidity. Next day, cells were trypsinized, counted, and seeded (1.4 ‐ 1.6 M/per well) in 6‐well plates using the n3D BiOAssay to levitate the cells. A total of 150,000 cells/well were seeded in a 96‐well plate in triplicates with a total volume of 300ul (150ul of cells, 120ul of media, and 30ul of the treatments). The treatments consist of doses ranging 10‐100 pmoles of CaS, 2% DMSO, or growth media. A 24‐hours recording consisting of four (4) photos per minute was collected with an iPod. The analyses were performed using the ImageJ software by measuring the “O” ring outer and inner diameters. Results: We observed changes in the diameters for the wells treated with CaS at different concentrations when compared to controls. Conclusions: We determined the IC50 and LD50 for adenocarcinoma and normal fibroblast cell lines. This step is important for the translation of the use of CaS as an anti‐cancer therapy when translated into in vivo models.

Grant Funding Source: NIH‐NIGMSR25GM096955, NIH‐R25GM088023
In vivo Anti‐ulcer and Antioxidant Enzyme Effect of Partially Purified Leaf Extract from Labisia Pumila in Indomethacin‐ Induced Ulcer Animal Model (LB585)
Authors: Okechukwu Patrick , Siyumbwa Stepfanie , Nomishan Ngizan
Published: June 2021

This study is aimed at investigating the effect of partially purified leaf extract of Labisia Pumila (LP) on Indomethacin (IND) induced ulcer and on selected antioxidant enzymes: ‐ catalase (CAT), glutathione (GSH), superoxide dismutase (SOD) and lipid peroxidation(LPO). Column chromatography was used to partially purify the DCM crude extract into 4 fractions (A, B, C and D). 10mg/kg Indomethacin was administered for 5 days to induce ulcer. The oral administration of the LP fractions (A, B, C and D) at a concentration of 100mg/kg were found to inhibit IND induced gastric lesions by 67.5%, 27.2%, 47.82% and 66.2%, respectively, the effect was highest in fraction A and D. The level of antioxidant activity was enhanced by the oral administration of fractions A and D, the other fractions did not show any significant antioxidant activity. In conclusion, the oral administration of LP fractions A to D showed antiulcer activities in IND induced ulcer and fractions A and D enhanced the antioxidant activities in ulcer models.

Grant Funding Source: UCSI University
Validation of new venom protease assays for translational research (LB589)
Authors: Price Joseph
Published: June 2021

Snake envenomation is an understudied significant world health problem, designated an orphan disease by the WHO. In the U.S. lives are usually saved, but significant tissue is often lost. Overseas without hospitalization and antivenin, lives are forfeit. For most American snake venoms the major pathologies of tissue loss and coagulopathies are due to proteolytic degradation of host tissues and plasma cascades. For HTS leading to therapeutics including drug repurposing, sensitive new substrates may finally allow direct comparisons of venom activities, but only if the reptile enzymes are active upon them, which has been not previously been published. Here I show direct comparisons of representative American venoms strong protease and collagenase, but weak elastase activities using the new technology. Assay CV range 1‐5%. Caseinolytic activity is inhibited by the nonspecific protease inhibitor 1,10‐phenanthroline and by EDTA. So is collagenase activity, consistent with the action of matrix metalloproteases. The Km measured shows that high throughput assays could be done at substrate levels well below the Km for the reaction. Such a system will be invaluable for the testing leading to the repurposing of drugs to treat envenomations.

Grant Funding Source: Supported by CHS‐OSU
Formulation Development And Evaluation Of Omeprazole Microspheres by Using the pH sensitive enteric polymers (LB590)
Authors: Ramana M , Babu Ancha , Thadanki madhurilatha
Published: June 2021

Mostly in infections like peptic ulcers, gastro‐esophageal‐reflux disorder, Zollinger‐Ellison syndrome and H.pylori we use Omeprazole, which is a proton‐pump inhibitor.. The omeprazole is unstable at acidic pH, undergoes degradation in stomach. Recently, pH‐sensitive enteric polymers are utilized to deliver drug to intestine and this polymer swells only in alkaline pH and releases the drug as it enters the intestine. In this research, the formulations were developed to deliver the omeprazole effectively by Using the pH sensitive enteric polymers (Ethyl Cellulose, Cellulose acetate, Eudragit L‐100, Hydroxy propyl methyl cellulose pthalate‐55, Cellulose acetate phthalate), by solvent evaporation microspheres are prepared, Omeprazole drug were loaded into those microspheres. All microspheres were evaluated for surface & morphology, physico chemical characterization, invitro dissolution profile, release mechanisms and stability studies. Results and conclusion indicated that, there is an increase in the ratio of polymer with a decrease in the release rate of drug in respective formulations. When the R2 values of First order and Zero order were considered, according to the regression plots it was evident that the drug release from all omeprazole microspheres formulations follow Zero order release kinetics
Methamphetamine‐induce neurotoxic effects in rat c6 glioma cell (LB620)
Authors: Wiley Chantel
Published: June 2021

PURPOSE: Methamphetamine (MA) is a potent, highly addictive central nervous system stimulant. It has been classified as a Schedule II drug, which makes it legally available, but only through a no refillable prescription. MA addiction has become a major public concern because its chronic abuse is associated with serious, yet fatal side effects such as memory loss, stroke, hyperthermia, and convulsions. These complex complications of the central nervous system may be related to a MA‐induced neurotoxic effect, which include damage to the dopamine system, activation of microglial and astrogial cells, and ultimately destruction of specific brain regions in humans. The present study seeks to investigate the cellular events associated with cytotoxic effects of MA in the Rat C6 glial cells. METHOD: Using the crystal violet assay to quantitate cell viability, methamphetamine at various concentrations (0.1 mM, 0.5 mM, 1 mM, 3 mM, and 5 mM) was exposed to the glial cells for 1, 24 and 48 hrs. To measure the morphological alterations after MA exposure, FITC Annexin V flow cytometry was used to distinguish cells that were in early and late stage of apoptosis. RESULTS: MA was found to have a dose and time dependent effect on cell viability. In addiction, the FITC Annein V Flow cytometry data revealed that as the concentrations increased more cells underwent apoptosis. These results revealed that MA not only cause a decrease in viability among C6 glioma cells, but is also the causes of the cellular demise was through the action of apoptosis. CONCLUSION: Further studies are underway to elucidate the mechanism by which MA induces apoptosis. The data generated from this study has provided some significant findings regarding the cellular mechanism of MA‐induced cytotoxicity in the Rat C6 glial cells. Moreover, our results can lead to potentially safe therapeutic treatments for neurodegenerative disorders that are associated with chronic drug use.

Grant Funding Source: Research supported by NIMHD of the National Institutes of Health NIH under Award Number G12MD007582
Effects of Systemic [Pyr] Apelin‐13 Administration on Coronary and Peripheral Hemodynamics (LB643)
Authors: Goodwill Adam , Casalini Eli , Noblet Jillian , Christe Mike , Kowala Mark , Zhen Eugene , Berwick Zachary , Moberly Steven , Tune Johnathan
Published: June 2021

This study was designed to assess effects of acute apelin administration on cardiac and coronary performance in anesthetized, open‐chest swine. Systemic intravenous administration of pyr‐apelin 13 (3nM to 1μM) did not significantly affect coronary blood flow (0.37 + 0.07 to 0.29 + 0.06 mL/min/g; p=0.93) or myocardial oxygen consumption (46.47 + 8.64 to 36.84 + 5.93 μL O2/min/g; p=0.91). However, pyr‐apelin 13 elicited a dose‐dependent peripheral dilator response as mean aortic pressure was decreased by 12 + 4 mmHg at 1μM pyr‐apelin 13 (p=0.001). Left ventricular pressure volume relationships were assessed to establish the potential inotropic effects of pyr‐apelin 13. No changes in cardiac output (2074 + 347 to 2052 + 185 mL/min; p= 0.96), stroke volume (35 + 2 to 33 + 1 mL; p=0.34) or end systolic pressure volume relationship (Ees 10.55 + 3.78 to 18.23 + 3.84; p = 0.20) were noted in response to pyr‐apelin 13 (3nm to 1μM). Mass spectroscopic analyses of plasma samples at each dose confirmed a ~700 fold increase in plasma apelin concentrations (0.39 + 0.14 to 287.55 + 43.58 ng/mL; p<0.001) with treatment. Taken together, these data support that acute systemic administration of pyr‐apelin 13 dose‐dependently decreases arterial pressure via reductions in systemic vascular resistance, independent of alterations in cardiac inotropic function.
Role of C‐jun N‐terminal Kinase in the Mitochondria of Ischemia‐Reperfusion Injury of Isolated Heart (LB648)
Authors: Jang Sehwan , Soto Jessica , Agostini Bryan , Javadov Sabzali
Published: June 2021

Background: Previous studies indicate that inhibition of JNK activation during heart reperfusion reduces cell death and infarct size. However, the studies could not eliminate off‐target effects due to the use of non‐specific JNK inhibitors. In the present study, we observed whether two mechanistically unrelated, specific JNK inhibitors, SU3327 and SR3306, would reduce damage from ischemia/reperfusion (IR) and improve cardiac function.

Methods: Using the Langendorff model of ischemia‐reperfusion, isolated rat hearts underwent 25 min of global ischemia followed by concurrent JNK inhibitor administration (2 and 10 µM for SU3327, 0.5 and 2 µM for SR3306) and 30 or 60 min of reperfusion. Throughout perfusion, we monitored cardiac function based on the percent recovery of left ventricular developed pressure, minimum and maximum rates of pressure change in the ventricle, and the rate pressure product. Myocardial damage was assessed by measuring lactate dehydrogenase activity in heart effluent. At the end of reperfusion, we used isolated mitochondria to determine respiration rates and mitochondria permeability transition pore opening. Proteins from the heart homogenate were analyzed using Western blot.

Results: Our results indicate that the specific JNK inhibitors did not improve cardiac function. As expected, mitochondrial respiration control index was correlated to recovery of heart function. We observed marginal reduction of myocardial damage by JNK inhibitor SU3327 at the 10 min time point after reperfusion. Western blot analysis demonstrated inhibition of phospho‐JNK and total‐JNK at high doses (10 uM) of SU3327, but it coincided with activation of p38.

Conclusion: JNK inhibition may provide cardioprotection against IR. However, in practice, it may be ineffective due to compensatory activation of p38 by an unknown mechanism.

Grant Funding Source: Supported by SC1HL118669 (to S.J.), and in part, G12RR‐03051 NIH grants.
Pannexin‐1 regulates eNOS activation through a superoxide‐dependent pathway (LB652)
Authors: Lillo Mauricio , Gaete Pablo , Figueroa Xavier
Published: June 2021

Pannexin‐1 (Panx1) is expressed in smooth muscle and endothelial cells of resistance arteries. In smooth muscle, Panx1‐formed channels mediate the α1‐adrenergic receptor‐initiated constriction, but the function of Panx1 in endothelial cells has not been determined. We analyzed the participation of Panx1 in the NO‐mediated dilation induced by the endothelium‐dependent vasodilator acetylcholine (ACh) in rat mesenteric resistance arteries contracted with 70 mM KCl. The activating phosphorylation of eNOS at serine 1177 (P‐eNOSS1177) and Akt at serine 473 (P‐AktS473) was evaluated by Western blot and superoxide production was detected using dihydroethidine. Blockade of Panx1 channels with probenecid, but not connexin‐formed hemichannel inhibition with La+3, enhanced the ACh‐induced vasodilation. Consistent with this, treatment with Panx1 channel blockers, probenecid or the mimetic peptide 10Panx, elicited an increase in P‐eNOSS1177, which was associated with a rise in P‐AktS473 and superoxide production. This effect of Panx1 blockers was not altered by ACh stimulation and was abolished by TEMPOL, a superoxide dismutase mimetic. It has been shown that superoxide production can lead to Akt activation by P‐AktS473. Therefore, these results indicate that Panx1 controls eNOS activity. Panx1 channel blockade initiates a superoxide‐dependent pathway that elicits Akt‐mediated eNOS activation via P‐eNOSS1177.

FONDECYT 1100850
Improved signal quality using solid tip vascular placement of ECG leads in rats (LB667)
Authors: Simenson Heather , Bogie Heather , Lindquist Ryan , Doyle Jennifer , Fine Megan , Sarazan R , Main Bradley
Published: June 2021

Continuous data recording, using telemetry systems, has proven to be the preferred way of collecting electrocardiogram (ECG) data in conscious freely moving animals. In small animals, however; the resulting recordings can have indistinguishable P, Q, R, S and T segments due to low signal to noise ratios. In large animals, similar challenges have been addressed by equipping wireless telemetry devices with an intravascular negative electrode, or solid tip lead (STL). The purpose of this study was to determine if STLs could be implanted in rats and result in improved ECG signal quality when compared to conventional subcutaneous leads. Two groups of Sprague Dawley rats, one with conventional lead placement (subcutaneous) and the other using the STL (vascular) were surgically implanted. ECG data were collected multiple times over a one year period. Signal quality was qualitatively and quantitatively evaluated between the STL and conventional lead groups, using a side by side visual comparison, and automated Q, R, S and T match percentage, by an experienced ECG analyst. ECG data collected with a STL was observably clearer when compared to the conventional lead group. At the completion of the study, the rats implanted with STLs were subjected to thorough necropsies. A histological examination of the tissue surrounding the STL was completed. In conclusion, STLs were successfully implanted in rats, resulting in improved signal quality with no detrimental evidence of inflammation noted at necropsy. The two ECG signals were compared for ease of automated analysis through comparison of Q, R, S and T match. This application could be especially useful in other small animal models such as the ferret or guinea pig, to increase ECG quality and improve automated ECG analysis.
Extracellular Matrix Controls Notch Signaling via Integrin Activation and NICD Ubiquitination. (LB67)
Authors: Albig Allan , Malik Arpita , DeFord Peter , Westover Katherine
Published: June 2021

The extracellular Matrix (ECM) has recently been implicated with the regulation of angiogenesis via manipulation of the Notch signaling system however the molecular basis for this has not been explored. We previously showed that the ECM protein microfibril‐associated glycoprotein 2 (MAGP2), suppresses Notch signaling in endothelial cells but promotes Notch signaling in non‐endothelial cells. The goal of this work was to dissect the molecular mechanism by which MAGP2 manipulates Notch signaling. We found that control of Notch by MAGP2 was not dependent on specific Notch receptor‐ligand expression patterns. Interestingly however, we found that mutation of the MAGP2 RGD to a non‐integrin binding RGE caused MAGP2 to promote rather than suppress Notch signaling in endothelial cells. Similarly, soluble integrin blocking RGD peptides and αVβ3 blocking antibodies also increased Notch signaling even in the absence of MAGP2. Further mechanistic dissection revealed that inhibitors of SRC and FAK kinases but not other kinases also increased Notch signaling. Interestingly, in addition to intensifying Notch signaling, RGD peptides and SRC inhibitors also triggered an increase in higher mobility NICD western blot bands and extended NICD half‐life. Our results suggest that these high mobility NICD bands are non‐ubiquitinated forms of NICD fragments. Collectively, we believe these results have revealed a previously undiscovered interaction between integrins and the Notch signaling system that acts to limit Notch signaling via ubiquitination and degradation of active NICD fragments. This novel mechanism may be responsible for ECM control of Notch signaling and angiogenesis.

Grant Funding Source: Supported by NIH (NIGMS)
Influence of Dietary Fat Intake on Endothelial Progenitor Cell Function (LB678)
Authors: Bammert Tyler , Beckstrom Collin , Gooding‐Lord Emma , Paswaters Katie , Dow Caitlin
Published: June 2021

High dietary fat intake has been linked to promoting cardiovascular disease and increasing the risk of heart attack. Bone‐marrow derived circulating endothelial progenitor cells (EPCs) play an important role in repairing and maintaining the health of the cardiovascular system. Circulating EPCs home to sites of vascular damage to initiate a repair response. Declines in circulating EPC function has been linked to greater risk of cardiovascular disease and vascular events. It is currently unknown whether habitual consumption of a diet high in fat is associated with impairments in EPC function. Cells with phenotypic EPC characteristics were isolated from peripheral blood samples collected from 26 middle‐aged and older adults: 13 who habitually consumed a diet high in fat (蠅35% of total calories); 13 who habitually consumed a diet lower in fat (<35% of total calories). All subjects were sedentary and free of cardiometabolic disease. Diet was assessed using 4‐day food records and analyzed using a nutritional software. EPC functional characteristics were assessed by the following: colony formation (in vitro colony forming assay); migration (modified Boyden chamber); release of angiogenic growth factors (ELISA); and apoptosis (active intracellular caspase‐3). There were no significant differences between the high and lower fat groups in EPC colony formation (11±3 vs 8±2) and migration (1297±114 vs 1035±124 RFUs). Basal and phytohemagglutinin (PHA; 10 µg/mL) stimulated release of vascular endothelial growth factor (21.5±2.1 to 84.1±14.3 vs 22.9±1.4 to 95.6±14.1 ng/mL) and granulocyte colony stimulating factor (34.0±6.3 to 1246.9±283.6 vs 38.0±6.6 to 873.7±143.1 ng/mL). Moreover, there was no difference in unstimulated (0.3±0.1 vs 0.3±0.1 ng/mL) and stimulated (2.6±0.3 and 2.5±0.3 ng/mL) active caspase‐3 concentrations between the high and lower fat groups. In conclusion, regular consumption of a diet high in fat (蠅35% of calories) does not appear to adversely influence circulating EPC function in middle‐aged and older adults. Impaired EPC function may not contribute to the increased cardiovascular risk associated with a high fat diet.
Connexin 37‐mediated growth regulation in primary endothelial cells (LB680)
Authors: Jacobsen Nicole , Burt Janis
Published: June 2021

We have previously shown that connexin 37 (Cx37) is growth suppressive in vivo, as suggested by increased in ischemia‐induced angiogenesis and collateral vasculogenesis in Cx37‐deficient mice (Am.J.Physiol. 301: H1872‐H1881, 2011). To provide an in vitro system for studies of microvascular capillary growth control mediated by Cx37, we isolated primary skeletal muscle endothelial cells (ECs) from the hindlimb of both wild type (WT) and Cx37‐/‐ mice. Using VE‐cadherin as the positive selection marker and a live cell sorting strategy, we are able to culture primary ECs for 14‐21 days before they lose endothelial character. Evidence for endothelial nature was provided by cell morphology and the continued presence of VE‐cadherin. The growth of ECs was monitored and revealed that proliferation of ECs isolated from WT mice is slowed compared to ECs originating from Cx37‐/‐ mice. Additionally, there is a 4‐day difference between the time it takes to reach confluence of Cx37‐/‐ EC cultures and WT ECs. These data implicate Cx37 in the growth regulation of primary ECs and suggest Cx37 is growth suppressive in microvascular ECs. Future studies will provide further insight into the growth suppression mechanism by Cx37 in primary ECs.

Grant Funding Source: Supported by R01HL058732 and T32HL007249
Expression of enzymes of sphingolipid metabolism in the blood vessel. (LB697)
Authors: Bautista Rocio , Del Valle‐Mondragón Leonardo , Cano‐Mártinez Agustina , Mercado Adriana , Franco Martha , Pérez‐Méndez Oscar
Published: June 2021

Background: Sphingolipids represent a class of diverse bioactive lipid molecules that are increasingly appreciated as key modulators of diverse physiologic and pathophysiologic processes that include cell growth, cell death, autophagy, angiogenesis, and stress and inflammatory responses. The cellular contents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways: sphingomyelinase (neutral‐SMase), Ceramidase (neutral‐CDase), Sphingosine kinase 1 (SPHK1) and Sphingosine kinase 2 (SPHK2). Therefore, the purpose of this study was to determine the expression of enzymes of sphingolipid metabolism in the blood vessel of kidney and heart as well as in aorta.

Methods: The expression of neutral‐SMase, neutral‐CDase, SPHK1, SPHK2, α‐actin (Smooth muscle cells marker) and PECAM (endothelial cell marker) were measured by Immunofluorescence and Confocal Microscopy.

Results: neutral‐SMase, neutral‐CDase, SPHK1 and SPHK2 immunoreactivity were found to have a widespread distribution in the blood vessel of kidney and heart as well as in aorta.

Conclusions: The blood vessel express neutral‐SMase, neutral‐CDase, SPHK1 and SPHK2, enzymes involved in the sphingolipid metabolism and can therefore endogenously form sphingomyelin metabolites in the vasculature.
Effects of Neuromuscular Joint Facilitation Treatment on Electromechanical Reaction Times of Hip Flexion (LB73)
Authors: HUO MING , Murakami Shinyichiro
Published: June 2021

[Purpose] The aim of this study was to investigate the change in electromechanical reaction times (EMG‐RT) of hip flexion of younger persons after neuromuscular joint facilitation (NJF) treatment. [Subjects] The subjects were 39 healthy young people, who were divided into two groups: a NJF group and a proprioceptive neuromuscular facilitation (PNF) group. The NJF group consisted of 16 subjects (7 males, 9 females), and the PNF group consisted of 23 subjects (10 males, 13 females). [Methods] Participants in the NJF group received NJF treatment. We measured the EMG‐RT, the premotor time (PMT) and the motor time (MT) during hip flexion movement before and after the intervention in both groups. [Results] There were no significant differences among the results of the PNF group. For the NJF group, there were significant differences in PMT and EMG‐RT after NJF treatment. [Conclusion] These results suggest that there is an immediate effect of NJF intervention on electromechanical reaction times of hip flexion.
Skeletal muscle TLR4 expression is associated with body composition, but not physical activity level in older adults (LB74)
Authors: Connors Ian , Mott Rachael , Trivedi Rudra , Combs Larissa , Heilbronn Chase , Timmerman Kyle
Published: June 2021

Elevated skeletal muscle expression of toll‐like receptor 4 (TLR4) has been linked to increased inflammation in clinical populations. Higher physical activity level (PA) is associated with decreased disease risk and inflammation. The purpose of this study was to examine the relationships among PA, body composition, and skeletal muscle TLR4 in older adults. Methods: In 23 subjects (age=67±4y, BMI=26±3 kg•m‐2) self‐reported PA (kilocalories•week‐1), estimated maximal oxygen consumption (VO2max), and body composition (air plethysmography) were measured. TLR4 was measured in skeletal muscle biopsies (vastus lateralis) using western blot analyses. Pearson product‐moment correlations were run between variables. Significance was set to p<0.05. Results: There was no significant correlation between TLR4 and PA. There was a significant correlation between %body fat and skeletal muscle TLR4 (r=0.496, p<0.05). There was also a significant negative correlation between VO2max and BF% (r=‐0.45, p<0.05). Conclusion: These preliminary data do not show an association between PA and TLR4 in skeletal muscle from healthy older adults. However, a positive correlation was found between skeletal muscle TLR4 and % body fat. Thus, elevated skeletal muscle expression of TLR4 may underlie reports indicating that increased adiposity is associated with elevated inflammation and disease risk.
Quantitative analysis of transcriptional responses of the porcine small and large intestines to lipopolysaccharide challenge (LB745)
Authors: Hou Yongqing , Yi Dan , Zhao Di , Long Minhui , Ding Binying , Wang Lei , Wu Guoyao
Published: June 2021

BACKGROUNDS & AIMS: Lipopolysaccharide (LPS) causes intestinal dysfunction, but the underlying mechanisms remain largely unknown. This study determined transcriptional responses of four different porcine intestinal segments (the duodenum, jejunum, ileum, and colon) to LPS challenge. METHODS: Intestinal mucosal samples were collected from 47‐day‐old pigs at 24 h post LPS injection to quantify the mRNA levels for 28 genes involved in intestinal integrity and function using quantitative real‐time PCR assays. RESULTS: LPS administration affected expression of genes related to intestinal growth, absorption, defense, transport, and energy metabolism in the four different intestinal segments. Specifically, expression of the genes encoding for tight junction proteins, insulin‐like growth factor 1 (IGF‐1), aquaporin 8 (AQP‐8), regenerating islet‐derived protein 3 alpha (REG3A), and uncoupling protein 2 (UCP‐2) was decreased (P < 0.05), whereas expression of porcine β‐defensins 1 (pBD‐1), AMP‐activated protein kinase (AMPK), and HSP70 was enhanced (P < 0.05). Expression of the gene for cystic fibrosis transmembrane conductance regulator (CFTR) was reduced in the ileum, but increased in the colon, post LPS challenge. In addition, differences in expression of genes (EGF, AR, IGF‐1, Caspase‐3, Occludin, pBD‐1, pBD‐2, SGLT‐1, REG3A, ALP, HSP70, AMPK, Sirt1, and UCP2) observed among the four intestinal segments. CONCLUSIONS: There are regional differences in gene expression profiles along the porcine intestine after LPS challenge.

Changes in gene expression relative to unchallenged controls within each intestinal segment

Items
Duodenum fold‐change
Jejunum fold‐change
Ileum fold‐change
Colon fold‐change

IGF‐1
0.76
0.72*
0.65*
0.68*

Claudin‐1
0.64*
0.74*
0.69*
0.48*

AQP‐8
0.22**
0.34**
0.29**
0.48**

REG3A
0.41**
0.07**
0.33**
NA

UCP2
0.56*
0.39**
0.43**
0.66*

Sirt1
0.91
0.79*
0.79*
0.93

pBD‐1
2.43**
3.65**
2.19**
3.01**

AMPK
1.28*
1.33*
1.12
1.31*

HSP70
2.53**
3.18**
2.69**
2.60**

Grant Funding Source: Supported by National Basic Research Program of China (No. 2012CB126305), National Natural Science Foundation of China (No. 31372319 )
Intestinal permeability as measured by lactulose mannitol ratio continues to decrease during infancy after 3 months of age for both control infants and infants at high risk for autism spectrum disorders (LB751)
Authors: Penn Alexander , Lai Tiffany , Carver Leslie , Taylor Sharon , Schmid‐Schönbein Geert , Dobkins Karen
Published: June 2021

Approximately 40% of individuals with autism spectrum disorder (ASD) have hyperpermeable intestines as measured by lactulose/mannitol absorption ratio. This could be the cause or symptom of the gastrointestinal co‐morbidity associated with ASD. It is not known if this increased permeability is present prior to ASD diagnosis. The objective of this study was to determine intestinal permeability in both infants at high‐risk (HR) of developing ASD (due to having an older sibling with ASD their risk of developing ASD is increased from ~0.9% to ~20%) and in low‐risk (LR, no family history) controls, given that no studies of intestinal permeability have been done in infants greater than approximately 3 months of age. Using a commercial assay we measured lactulose/mannitol absorption ratio (L/M) in infants ranging from 3 months to 36 months of age (the majority less than 15 months) and determined their diet at the time by parental questionnaire. We observed a negative correlation between L/M and age (HR: ‐0.39, LR: ‐0.34) and a significantly higher L/M in infants between 3 and 7 months of age versus infants greater than 7 months for both HR and LR infants. Interestingly, HR infants had significantly lower L/M than LR infants, an effect that appeared to be driven by a significantly higher permeability in LR infants on a mixed diet of breast milk and other foods compared to HR infants on a mixed diet or LR infants on a no breast milk diet. Only one HR infant demonstrated hyperpermeability. These results suggest that intestinal permeability continues to decrease even after 3 months as part of typical development and that hyperpermeability is a later event in ASD and may not appear in the first year of life.

Grant Funding Source: NIH NS071580 and GM85072
Negative regulation of the adenosine‐generating ectoenzyme CD73 by a novel splice variant that is upregulated in hepatocellular carcinoma (LB752)
Authors: Snider Natasha , Altshuler Peter , Wan Shanshan , Welling Theodore , Omary M. Bishr
Published: June 2021

Background and aim: Ecto‐5’‐nucleotidase (CD73) is a ubiquitously‐expressed plasma membrane glycoprotein that functions as the major enzymatic source of extracellular adenosine. CD73 inhibition has been implicated as a potential approach in cancer immunotherapy, but the mechanisms of CD73 regulation are poorly understood. The aim of this study was to characterize a novel splice variant of CD73, which encodes a shorter protein. Methods: The activity and expression of canonical CD73, referred to as CD73‐long (CD73) and expression of the newly‐identified variant, CD73‐short (CD73S), were examined in human hepatocellular carcinoma (HCC) tumors and normal control livers, as well as HepG2 and Huh7 human HCC cell lines . A rabbit polyclonal antibody was made against CD73S to examine its expression by Western blot. The subcellular distribution and biochemical properties of CD73S were studied in an over‐expression cell culture system. Results: Basal expression of CD73S in normal liver is 400‐fold lower compared to CD73L. In contrast, CD73S mRNA levels increase 10 fold, whereas CD73L mRNA levels decrease 10‐fold in HCC relative to normal liver. CD73S protein is present in HCC surgical specimens, but not in normal human livers. Further studies revealed that CD73S is an endoplasmic reticulum (ER)‐associated glycoprotein that complexes with the ER chaperone calnexin and lacks enzymatic activity due to its inability to form a functional dimer. CD73S also associates with CD73L and decreases CD73L expression in a proteasome‐dependent manner. Conclusion: CD73S is a hitherto unappreciated variant isoform of CD73 that may modulate CD73 function in cancer.

Grant Funding Source: Supported by DK093776 and DK052951